date_range Published on March 22, 2018
share Vedithi SC, Malhotra S, Das M, Daniel S, Kishore N, George A, Arumugam S, Rajan L, Ebenezer M, Ascher DB, Arnold E, Blundell TL (2018). Sci Rep 8 (1):5016.
The rpoB gene encodes the β subunit of RNA polymerase holoenzyme in Mycobacterium leprae (M. leprae). Missense mutations in the rpoB gene were identified as etiological factors for rifampin resistance in leprosy. In the present study, we identified mutations corresponding to rifampin resistance in relapsed leprosy cases from three hospitals in southern India which treat leprosy patients. DNA was extracted from skin biopsies of 35 relapse/multidrug therapy non-respondent leprosy cases, and PCR was performed to amplify the 276 bp rifampin resistance-determining region of the rpoB gene. PCR products were sequenced, and mutations were identified in four out of the 35 cases at codon positions D441Y, D441V, S437L and H476R. The structural and functional effects of these mutations were assessed in the context of three-dimensional comparative models of wild-type and mutant M. leprae RNA polymerase holoenzyme (RNAP), based on the recently solved crystal structures of RNAP of Mycobacterium tuberculosis, containing a synthetic nucleic acid scaffold and rifampin. The resistance mutations were observed to alter the hydrogen-bonding and hydrophobic interactions of rifampin and the 5′ ribonucleotide of the growing RNA transcript. This study demonstrates that rifampin-resistant strains of M. leprae among leprosy patients in southern India are likely to arise from mutations that affect the drug-binding site and stability of RNAP.Download cloud_download
date_range Published on March 16, 2018
share Trapero A, Pacitto A, Singh V, Sabbah M, Coyne AG, Mizrahi V, Blundell TL, Ascher DB, Abell C (2018) J Med Chem 61 (7):2806-2822.
Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5′-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors using fragment-based screening and structure-based design techniques. Screening of a fragment library for Mycobacterium thermoresistible (Mth) IMPDH ΔCBS inhibitors identified a low affinity phenylimidazole derivative. X-ray crystallography of the Mth IMPDH ΔCBS–IMP–inhibitor complex revealed that two molecules of the fragment were bound in the NAD binding pocket of IMPDH. Linking the two molecules of the fragment afforded compounds with more than 1000-fold improvement in IMPDH affinity over the initial fragment hit.Download cloud_download
date_range Published on April 30, 2018
share Rodrigues CH, Pires DE, Ascher DB (2018) DynaMut: predicting the impact of mutations on protein conformation, flexibility and stability. Nucleic Acids Res 46 (W1):W350-W355.
Proteins are highly dynamic molecules, whose function is intrinsically linked to their molecular motions. Despite the pivotal role of protein dynamics, their computational simulation cost has led to most structure-based approaches for assessing the impact of mutations on protein structure and function relying upon static structures. Here we present DynaMut, a web server implementing two distinct, well established normal mode approaches, which can be used to analyze and visualize protein dynamics by sampling conformations and assess the impact of mutations on protein dynamics and stability resulting from vibrational entropy changes. DynaMut integrates our graph-based signatures along with normal mode dynamics to generate a consensus prediction of the impact of a mutation on protein stability. We demonstrate our approach outperforms alternative approaches to predict the effects of mutations on protein stability and flexibility (P-value < 0.001), achieving a correlation of up to 0.70 on blind tests. DynaMut also provides a comprehensive suite for protein motion and flexibility analysis and visualization via a freely available, user friendly web server at http://biosig.unimelb.edu.au/dynamut/.Download cloud_download
date_range Published on May 21, 2018
share Rodrigues CH, Ascher DB, Pires DE (2018) Nucleic Acids Res 46 (W1):W127-W132.
Protein phosphorylation is tightly regulated due to its vital role in many cellular processes. While gain of function mutations leading to constitutive activation of protein kinases are known to be driver events of many cancers, the identification of these mutations has proven challenging. Here we present Kinact, a novel machine learning approach for predicting kinase activating missense mutations using information from sequence and structure. By adapting our graph-based signatures, Kinact represents both structural and sequence information, which are used as evidence to train predictive models. We show the combination of structural and sequence features significantly improved the overall accuracy compared to considering either primary or tertiary structure alone, highlighting their complementarity. Kinact achieved a precision of 87% and 94% and Area Under ROC Curve of 0.89 and 0.92 on 10-fold cross-validation, and on blind tests, respectively, outperforming well established tools (P < 0.01). We further show that Kinact performs equally well on homology models built using templates with sequence identity as low as 33%. Kinact is freely available as a user-friendly web server at http://biosig.unimelb.edu.au/kinact/.Download cloud_download
date_range Published on March 29, 2018
share Pires DEV, Kaminskas LM, Ascher DB (2018) Methods Mol Biol 1762:271-284.
A crucial factor for the approval and success of any drug is how it behaves in the body. Many drugs, however, do not reach the market due to poor efficacy or unacceptable side effects. It is therefore important to take these into consideration early in the drug development process, both in the prioritization of potential hits, and optimization of lead compounds. In silico approaches offer a cost and time-effective approach to rapidly screen and optimize pharmacokinetic and toxicity properties. Here we demonstrate the use of the comprehensive analysis system pkCSM, to allow early identification of potential problems, prioritization of hits, and optimization of leads.Download cloud_download
date_range Published on Aug. 15, 2018
share Karmakar M, Globan M, Fyfe JAM, Stinear TP, Johnson PDR, Holmes NE, Denholm JT, Ascher DB (2018). Am J Respir Crit Care Med. 198(4): 541-542.
This case study demonstrates the power of using structural information to quantitatively evaluate novel variants in real time, providing invaluable insight to help guide therapy. While existing recommendations may suggest continuing treatment of MDR-TB with pyrazinamide irrespective of phenotype testing, our approach suggests that using structural information to guide analysis of genomic sequencing may offer useful tools for clinicians to consider. These structural insights also assist in informing the mechanisms for drug activity and the development of resistance. Our approach is not limited only to analysis of variants in pncA but could be applied to any protein associated with resistance for infectious and non-infectious disease treatment.Download cloud_download
date_range Published on May 21, 2018
share Holt KE, McAdam P, Thai PVK, Thuong NTT, Ha DTM, Lan NN, Lan NH, Nhu NTQ, Hai HT, Ha VTN, Thwaites G, Edwards DJ, Nath AP, Pham K, Ascher DB, Farrar J, Khor CC, Teo YY, Inouye M, Caws M, Dunstan SJ (2018) Nat Genet 50 (6):849-856.
To examine the transmission dynamics of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients in Ho Chi Minh City, Vietnam, we sequenced the whole genomes of 1,635 isolates and compared these with 3,144 isolates from elsewhere. The data identify an underlying burden of disease caused by the endemic Mtb lineage 1 associated with the activation of long-term latent infection, and a threefold higher burden associated with the more recently introduced Beijing lineage and lineage 4 Mtb strains. We find that Beijing lineage Mtb is frequently transferred between Vietnam and other countries, and detect higher levels of transmission of Beijing lineage strains within this host population than the endemic lineage 1 Mtb. Screening for parallel evolution of Beijing lineage-associated SNPs in other Mtb lineages as a signal of positive selection, we identify an alteration in the ESX-5 type VII-secreted protein EsxW, which could potentially contribute to the enhanced transmission of Beijing lineage Mtb in Vietnamese and other host populations.Download cloud_download
date_range Published on Feb. 25, 2018
share Hnizda A, Fabry M, Moriyama T, Pachl P, Kugler M, Brinsa V, Ascher DB, Carroll WL, Novak P, Zaliova M, Trka J, Rezacova P, Yang JJ, Veverka V (2018). Leukemia 32 (6):1393-1403.
Activating mutations in NT5C2, a gene encoding cytosolic purine 5′-nucleotidase (cN-II), confer chemoresistance in relapsed acute lymphoblastic leukemia. Here we show that all mutants became independent of allosteric effects of ATP and thus constitutively active. Structural mapping of mutations described in patients demonstrates that 90% of leukemia-specific allelles directly affect two regulatory hotspots within the cN-II molecule—the helix A region: residues 355–365, and the intersubunit interface: helix B (232–242) and flexible interhelical loop L (400–418). Furthermore, analysis of hetero-oligomeric complexes combining wild-type (WT) and mutant subunits showed that the activation is transmitted from the mutated to the WT subunit. This intersubunit interaction forms structural basis of hyperactive NT5C2 in drug-resistant leukemia in which heterozygous NT5C2 mutation gave rise to hetero-tetramer mutant and WT proteins. This enabled us to define criteria to aid the prediction of NT5C2 drug resistance mutations in leukemia.Download cloud_download
date_range Published on March 16, 2018
share Hawkey J, Ascher DB, Judd LM, Wick RR, Kostoulias X, Cleland H, Spelman DW, Padiglione A, Peleg AY, Holt KE (2018). Microb Genom. 2018:4
Acinetobacter baumannii is a common causative agent of hospital-acquired infections and a leading cause of infection in burns patients. Carbapenem-resistant A. baumannii is considered a major public-health threat and has been identified by the World Health Organization as the top priority organism requiring new antimicrobials. The most common mechanism for carbapenem resistance in A. baumannii is via horizontal acquisition of carbapenemase genes. In this study, we sampled 20 A. baumannii isolates from a patient with extensive burns, and characterized the evolution of carbapenem resistance over a 45 day period via Illumina and Oxford Nanopore sequencing. All isolates were multidrug resistant, carrying two genomic islands that harboured several antibiotic-resistance genes. Most isolates were genetically identical and represented a single founder genotype. We identified three novel non-synonymous substitutions associated with meropenem resistance: F136L and G288S in AdeB (part of the AdeABC efflux pump) associated with an increase in meropenem MIC to ≥8 µg ml−1; and A515V in FtsI (PBP3, a penicillin-binding protein) associated with a further increase in MIC to 32 µg ml−1. Structural modelling of AdeB and FtsI showed that these mutations affected their drug-binding sites and revealed mechanisms for meropenem resistance. Notably, one of the adeB mutations arose prior to meropenem therapy but following ciprofloxacin therapy, suggesting exposure to one drug whose resistance is mediated by the efflux pump can induce collateral resistance to other drugs to which the bacterium has not yet been exposed.Download cloud_download
date_range Published on Jan. 31, 2018
share Andrews KA, Ascher DB, Pires DEV, Barnes DR, Vialard L, Casey RT, Bradshaw N, Adlard J, Aylwin S, Brennan P, Brewer C, Cole T, Cook JA, Davidson R, Donaldson A, Fryer A, Greenhalgh L, Hodgson SV, Irving R, Lalloo F, McConachie M, McConnell VPM, Morrison PJ, Murday V, Park SM, Simpson HL, Snape K, Stewart S, Tomkins SE, Wallis Y, Izatt L, Goudie D, Lindsay RS, Perry CG, Woodward ER, Antoniou AC, Maher ER (2018) J Med Genet 55 (6):384-394.
Background Germline pathogenic variants in SDHB/SDHC/SDHD are the most frequent causes of inherited phaeochromocytomas/paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. We estimate penetrance for symptomatic tumours and elucidate genotype–phenotype correlations in a large cohort of SDHB/SDHC/SDHD mutation carriers. Methods A retrospective survey of 1832 individuals referred for genetic testing due to a personal or family history of phaeochromocytoma/paraganglioma. 876 patients (401 previously reported) had a germline mutation in SDHB/SDHC/SDHD (n=673/43/160). Tumour risks were correlated with in silico structural prediction analyses. Results Tumour risks analysis provided novel penetrance estimates and genotype–phenotype correlations. In addition to tumour type susceptibility differences for individual genes, we confirmed that the SDHD:p.Pro81Leu mutation has a distinct phenotype and identified increased age-related tumour risks with highly destabilising SDHB missense mutations. By Kaplan-Meier analysis, the penetrance (cumulative risk of clinically apparent tumours) in SDHB and (paternally inherited) SDHD mutation-positive non-probands (n=371/67 with detailed clinical information) by age 60 years was 21.8% (95% CI 15.2% to 27.9%) and 43.2% (95% CI 25.4% to 56.7%), respectively. Risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4.2%(95% CI 1.1% to 7.2%). With retrospective cohort analysis to adjust for ascertainment, cumulative tumour risks for SDHB mutation carriers at ages 60 years and 80 years were 23.9% (95% CI 20.9% to 27.4%) and 30.6% (95% CI 26.8% to 34.7%). Conclusions Overall risks of clinically apparent tumours for SDHB mutation carriers are substantially lower than initially estimated and will improve counselling of affected families. Specific genotype–tumour risk associations provides a basis for novel investigative strategies into succinate dehydrogenase-related mechanisms of tumourigenesis and the development of personalised management for SDHB/SDHC/SDHD mutation carriers.Download cloud_download
date_range Published on Oct. 1, 2017
share Trezza A, Bernini A, Langella A, Ascher DB, Pires DEV, Sodi A, Passerini I, Pelo E, Rizzo S, Niccolai N, Spiga O (2017) Invest Ophthalmol Vis Sci 58 (12):5320-5328.
Purpose: The aim of this article is to report the investigation of the structural features of ABCA4, a protein associated with a genetic retinal disease. A new database collecting knowledge of ABCA4 structure may facilitate predictions about the possible functional consequences of gene mutations observed in clinical practice. Methods: In order to correlate structural and functional effects of the observed mutations, the structure of mouse P-glycoprotein was used as a template for homology modeling. The obtained structural information and genetic data are the basis of our relational database (ABCA4Database). Results: Sequence variability among all ABCA4-deposited entries was calculated and reported as Shannon entropy score at the residue level. The three-dimensional model of ABCA4 structure was used to locate the spatial distribution of the observed variable regions. Our predictions from structural in silico tools were able to accurately link the functional effects of mutations to phenotype. The development of the ABCA4Database gathers all the available genetic and structural information, yielding a global view of the molecular basis of some retinal diseases. Conclusions: ABCA4 modeled structure provides a molecular basis on which to analyze protein sequence mutations related to genetic retinal disease in order to predict the risk of retinal disease across all possible ABCA4 mutations. Additionally, our ABCA4 predicted structure is a good starting point for the creation of a new data analysis model, appropriate for precision medicine, in order to develop a deeper knowledge network of the disease and to improve the management of patients.Download cloud_download
date_range Published on Oct. 27, 2017
share Traynelis J, Silk M, Wang Q, Berkovic SF, Liu L, Ascher DB, Balding DJ, Petrovski S (2017) Genome Res 27 (10):1715-1729.
Gene panel and exome sequencing have revealed a high rate of molecular diagnoses among diseases where the genetic architecture has proven suitable for sequencing approaches, with a large number of distinct and highly penetrant causal variants identified among a growing list of disease genes. The challenge is, given the DNA sequence of a new patient, to distinguish disease-causing from benign variants. Large samples of human standing variation data highlight regional variation in the tolerance to missense variation within the protein-coding sequence of genes. This information is not well captured by existing bioinformatic tools, but is effective in improving variant interpretation. To address this limitation in existing tools, we introduce the missense tolerance ratio (MTR), which summarizes available human standing variation data within genes to encapsulate population level genetic variation. We find that patient-ascertained pathogenic variants preferentially cluster in low MTR regions (P < 0.005) of well-informed genes. By evaluating 20 publicly available predictive tools across genes linked to epilepsy, we also highlight the importance of understanding the empirical null distribution of existing prediction tools, as these vary across genes. Subsequently integrating the MTR with the empirically selected bioinformatic tools in a gene-specific approach demonstrates a clear improvement in the ability to predict pathogenic missense variants from background missense variation in disease genes. Among an independent test sample of case and control missense variants, case variants (0.83 median score) consistently achieve higher pathogenicity prediction probabilities than control variants (0.02 median score; Mann-Whitney U test, P < 1 × 10−16). We focus on the application to epilepsy genes; however, the framework is applicable to disease genes beyond epilepsy.Download cloud_download
date_range Published on March 20, 2017
share Soardi FC, Machado-Silva A, Linhares ND, Zheng G, Qu Q, Pena HB, Martins TMM, Vieira HGS, Pereira NB, Melo-Minardi RC, Gomes CC, Gomez RS, Gomes DA, Pires DEV, Ascher DB, Yu H, Pena SDJ (2017) NPJ Genom Med 2 (1):7.
We characterize a novel human cohesinopathy originated from a familial germline mutation of the gene encoding the cohesin subunit STAG2, which we propose to call STAG2-related X-linked Intellectual Deficiency. Five individuals carry a STAG2 p.Ser327Asn (c.980 G > A) variant that perfectly cosegregates with a phenotype of syndromic mental retardation in a characteristic X-linked recessive pattern. Although patient-derived cells did not show overt sister-chromatid cohesion defects, they exhibited altered cell cycle profiles and gene expression patterns that were consistent with cohesin deficiency. The protein level of STAG2 in patient cells was normal. Interestingly, STAG2 S327 is located at a conserved site crucial for binding to SCC1 and cohesin regulators. When expressed in human cells, the STAG2 p.Ser327Asn mutant is defective in binding to SCC1 and other cohesin subunits and regulators. Thus, decreased amount of intact cohesin likely underlies the phenotypes of STAG2-SXLID. Intriguingly, recombinant STAG2 p.Ser327Asn binds normally to SCC1, WAPL, and SGO1 in vitro, suggesting the existence of unknown in vivo mechanisms that regulate the interaction between STAG2 and SCC1.Download cloud_download
date_range Published on Jan. 13, 2017
share Singh V, Donini S, Pacitto A, Sala C, Hartkoorn RC, Dhar N, Keri G, Ascher DB, Mondesert G, Vocat A, Lupien A, Sommer R, Vermet H, Lagrange S, Buechler J, Warner DF, McKinney JD, Pato J, Cole ST, Blundell TL, Rizzi M, Mizrahi V (2017) ACS Infect Dis 3 (1)
Singh V, Donini S, Pacitto A, Sala C, Hartkoorn RC, Dhar N, Keri G, Ascher DB, Mondesert G, Vocat A, Lupien A, Sommer R, Vermet H, Lagrange S, Buechler J, Warner DF, McKinney JD, Pato J, Cole ST, Blundell TL, Rizzi M, Mizrahi V (2017) ACS Infect Dis 3 (1):5-17. VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.Download cloud_download
date_range Published on Feb. 3, 2017
share Sibanda BL, Chirgadze DY, Ascher DB, Blundell TL (2017) Science 355 (6324):520-524.
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128–amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.Download cloud_download
date_range Published on May 1, 2017
share Sharp JA, Brennan AJ, Polekhina G, Ascher DB, Lefevre C, Nicholas KR (2017) Cell Signal 33:86-97.
α-lactalbumin is a protein of dual function found in milk of most mammals. α-lactalbumin binds β-1,4-galactosyltransferase to form the regulatory subunit for lactose synthesis and has also been shown to cause cell death. This study shows, for the first time, that α-lactalbumin isolated in a rare 28 kDa dimeric form induces cell death, while 14 kDa monomeric α-lactalbumin is inactive. In contrast to the casein derived and chemically induced α-lactalbumin variants, MAL and HAMLET/BAMLET, the effects of 28 kDa α-lactalbumin are calcium independent and, unlike MAL and HAMLET, 28 kDa α-lactalbumin dimer causes cell death of primary mammary cells and a variety of immortalised cell lines, which are committed to cell death pathways within 1–4 h of exposure. Microarray analysis confirmed that cell death was the result of an apoptotic response. Functional assays determined that the mechanism by which 28 kDa α-lactalbumin kills cells involved inhibition of histone deacetylase activity mediated by NF-kB. We also show that 28 kDa α-lactalbumin occurs naturally in the milk of cows, goats and sheep, is low in concentration during mid-lactation, but accumulates during milk stasis, consistent with a role in involution.Download cloud_download
date_range Published on May 2, 2017
share Ramdzan YM, Trubetskov MM, Ormsby AR, Newcombe EA, Sui X, Tobin MJ, Bongiovanni MN, Gras SL, Dewson G, Miller JML, Finkbeiner S, Moily NS, Niclis J, Parish CL, Purcell AW, Baker MJ, Wilce JA, Waris S, Stojanovski D, Bocking T, Ang CS, Ascher DB, Reid GE, Hatters DM (2017). Cell Rep 19 (5):919-927.
Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.Download cloud_download
date_range Published on April 4, 2017
share Pires DEV, Ascher DB (2017). Nucleic Acids Res 45 (W1):W241-W246.
Over the past two decades, several computational methods have been proposed to predict how missense mutations can affect protein structure and function, either by altering protein stability or interactions with its partners, shedding light into potential molecular mechanisms giving rise to different phenotypes. Effectively and efficiently predicting consequences of mutations on protein–nucleic acid interactions, however, remained until recently a great and unmet challenge. Here we report an updated webserver for mCSM–NA, the only scalable method we are aware of capable of quantitatively predicting the effects of mutations in protein coding regions on nucleic acid binding affinities. We have significantly enhanced the original method by including a pharmacophore modelling and information of nucleic acid properties into our graph-based signatures, considering the reverse mutation and by using a refined, more reliable data set, based on a new release of the ProNIT database, which has significantly improved the reliability and applicability of the methodology. Our new predictive model was capable of achieving a correlation coefficient of up to 0.70 on cross-validation and 0.68 on blind-tests, outperforming its previous version. The server is freely available via a user-friendly web interface at: http://structure.bioc.cam.ac.uk/mcsm_na.Download cloud_download
date_range Published on Feb. 1, 2017
share Parker LJ, Bocedi A, Ascher DB, Aitken JB, Harris HH, Lo Bello M, Ricci G, Morton CJ, Parker MW (2017) Protein Sci 26 (2):317-326.
Arsenic‐based compounds are paradoxically both poisons and drugs. Glutathione transferase (GSTP1‐1) is a major factor in resistance to such drugs. Here we describe using crystallography, X‐ray absorption spectroscopy, mutagenesis, mass spectrometry, and kinetic studies how GSTP1‐1 recognizes the drug phenylarsine oxide (PAO). In conditions of cellular stress where glutathione (GSH) levels are low, PAO crosslinks C47 to C101 of the opposing monomer, a distance of 19.9 Å, and causes a dramatic widening of the dimer interface by approximately 10 Å. The GSH conjugate of PAO, which forms rapidly in cancerous cells, is a potent inhibitor (Ki = 90 nM) and binds as a di‐GSH complex in the active site forming part of a continuous network of interactions from one active site to the other. In summary, GSTP1‐1 can detoxify arsenic‐based drugs by sequestration at the active site and at the dimer interface, in situations where there is a plentiful supply of GSH, and at the reactive cysteines in conditions of low GSH.Download cloud_download
date_range Published on Jan. 1, 2017
share Park Y, Pacitto A, Bayliss T, Cleghorn LA, Wang Z, Hartman T, Arora K, Ioerger TR, Sacchettini J, Rizzi M, Donini S, Blundell TL, Ascher DB, Rhee K, Breda A, Zhou N, Dartois V, Jonnala SR, Via LE, Mizrahi V, Epemolu O, Stojanovski L, Simeons F, Osuna-Ca
Park Y, Pacitto A, Bayliss T, Cleghorn LA, Wang Z, Hartman T, Arora K, Ioerger TR, Sacchettini J, Rizzi M, Donini S, Blundell TL, Ascher DB, Rhee K, Breda A, Zhou N, Dartois V, Jonnala SR, Via LE, Mizrahi V, Epemolu O, Stojanovski L, Simeons F, Osuna-Cabello M, Ellis L, MacKenzie CJ, Smith AR, Davis SH, Murugesan D, Buchanan KI, Turner PA, Huggett M, Zuccotto F, Rebollo-Lopez MJ, Lafuente-Monasterio MJ, Sanz O, Diaz GS, Lelievre J, Ballell L, Selenski C, Axtman M, Ghidelli-Disse S, Pflaumer H, Bosche M, Drewes G, Freiberg GM, Kurnick MD, Srikumaran M, Kempf DJ, Green SR, Ray PC, Read K, Wyatt P, Barry CE, 3rd, Boshoff HI (2017) ACS Infect Dis 3 (1):18-33. A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH–IMP–inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi–pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time–kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5–2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.Download cloud_download
date_range Published on May 19, 2017
share Pandurangan AP, Ochoa-Montano B, Ascher DB, Blundell TL (2017) Nucleic Acids Res 45 (W1):W229-W235.
Here, we report a webserver for the improved SDM, used for predicting the effects of mutations on protein stability. As a pioneering knowledge-based approach, SDM has been highlighted as the most appropriate method to use in combination with many other approaches. We have updated the environment-specific amino-acid substitution tables based on the current expanded PDB (a 5-fold increase in information), and introduced new residue-conformation and interaction parameters, including packing density and residue depth. The updated server has been extensively tested using a benchmark containing 2690 point mutations from 132 different protein structures. The revised method correlates well against the hypothetical reverse mutations, better than comparable methods built using machine-learning approaches, highlighting the strength of our knowledge-based approach for identifying stabilising mutations. Given a PDB file (a Protein Data Bank file format containing the 3D coordinates of the protein atoms), and a point mutation, the server calculates the stability difference score between the wildtype and mutant protein. The server is available at http://structure.bioc.cam.ac.uk/sdm2Download cloud_download
date_range Published on April 15, 2017
share Pandurangan AP, Ascher DB, Thomas SE, Blundell TL (2017) Biochem Soc Trans 45 (2):303-311.
For over four decades structural biology has been used to understand the mechanisms of disease, and structure-guided approaches have demonstrated clearly that they can contribute to many aspects of early drug discovery, both computationally and experimentally. Structure can also inform our understanding of impacts of mutations in human genetic diseases and drug resistance in cancers and infectious diseases. We discuss the ways that structural insights might be useful in both repurposing off-licence drugs and guide the design of new molecules that might be less susceptible to drug resistance in the future.Download cloud_download
date_range Published on Feb. 1, 2017
share Liang S, Esswein SR, Ochi T, Wu Q, Ascher DB, Chirgadze D, Sibanda BL, Blundell TL (2017) Structural Chemistry 28 (1):161-171.
When double-strand breaks (DSBs) in DNA remain unrepaired, catastrophic loss of genes occurs, leading to translocations, mutations and carcinogenesis. If a sister chromatid is not available at the DNA DSB, non-homologous end joining (NHEJ) is used to join broken ends. The NHEJ pathway comprises synapsis, end processing and ligation. Here, we ask how DSBs in DNA are repaired efficiently. We suggest that colocation of proteins is achieved over time by the following components: stages, where the main actors are assembled, scaffolds that are erected quickly around broken parts to give access, and strings that tether proteins together. In NHEJ, a stage is provided by the Ku heterodimer interacting with DSBs and several other proteins including DNA-PKcs, APLF, BRCA1 and PAXX. A further stage, DNA-PKcs, links the kinase with DNA, Ku, PARP1, BRCA1 and Artemis. A temporary scaffold facilitates repair and is constructed from XRCC4/XLF filaments that bridge Ku bound at DSB ends. LigIV bound to XRCC4 C-termini likely terminates the scaffold, bringing LigIV close to the DNA broken ends. A string, provided by the Artemis C-terminal region, is intrinsically disordered but includes short linear “epitopes” that recognise DNA-PKcs, LigIV and PTIP, so keeping these components nearby. We show that these stages, scaffolds and strings facilitate colocation and efficient DSB repair. Understanding these processes provides insight into the biology of DNA repair and possible therapeutic intervention in cancer and other diseases.Download cloud_download
date_range Published on Sept. 1, 2017
share Jubb HC, Pandurangan AP, Turner MA, Ochoa-Montano B, Blundell TL, Ascher DB (2017) Prog Biophys Mol Biol 128:3-13.
Many essential biological processes including cell regulation and signalling are mediated through the assembly of protein complexes. Changes to protein-protein interaction (PPI) interfaces can affect the formation of multiprotein complexes, and consequently lead to disruptions in interconnected networks of PPIs within and between cells, further leading to phenotypic changes as functional interactions are created or disrupted. Mutations altering PPIs have been linked to the development of genetic diseases including cancer and rare Mendelian diseases, and to the development of drug resistance. The importance of these protein mutations has led to the development of many resources for understanding and predicting their effects. We propose that a better understanding of how these mutations affect the structure, function, and formation of multiprotein complexes provides novel opportunities for tackling them, including the development of small-molecule drugs targeted specifically to mutated PPIs.Download cloud_download
date_range Published on Feb. 3, 2017
share Jubb HC, Higueruelo AP, Ochoa-Montano B, Pitt WR, Ascher DB, Blundell TL (2017) J Mol Biol 429 (3):365-371.
Interactions between proteins and their ligands, such as small molecules, other proteins, and DNA, depend on specific interatomic interactions that can be classified on the basis of atom type and distance and angle constraints. Visualisation of these interactions provides insights into the nature of molecular recognition events and has practical uses in guiding drug design and understanding the structural and functional impacts of mutations. We present Arpeggio, a web server for calculating interactions within and between proteins and protein, DNA, or small-molecule ligands, including van der Waals', ionic, carbonyl, metal, hydrophobic, and halogen bond contacts, and hydrogen bonds and specific atom–aromatic ring (cation–π, donor–π, halogen–π, and carbon–π) and aromatic ring–aromatic ring (π–π) interactions, within user-submitted macromolecule structures. PyMOL session files can be downloaded, allowing high-quality publication images of the interactions to be generated. Arpeggio is implemented in Python and available as a user-friendly web interface at http://structure.bioc.cam.ac.uk/arpeggio/ and as a downloadable package at https://bitbucket.org/harryjubb/arpeggio.Download cloud_download
date_range Published on May 3, 2017
share Chirgadze DY, Ascher DB, Blundell TL, Sibanda BL (2017) Methods Enzymol 592:145-157.
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is central to the regulation of the DNA damage response and repair through nonhomologous end joining. The structure has proved challenging due to its large size and multiple HEAT repeats. We have recently reported crystals of selenomethionine-labeled DNA-PKcs complexed with native KU80ct194 (KU80 residues 539-732) diffracting to 4.3Å resolution. The novel use of crystals of selenomethionine-labeled protein expressed in HeLa cells has facilitated the use of single anomalous X-ray scattering of this 4128 amino acid, multiple HEAT-repeat structure. The monitoring of the selenomethionines in the anomalous-difference density map has allowed the checking of the amino acid residue registration in the electron density, and the labeling of the Ku-C-terminal moiety with selenomethionine has further allowed its identification in the structure of the complex with DNA-PKcs. The crystal structure defines a stage on which many of the components assemble and regulate the kinase activity through modulating the conformation and allosteric regulation of kinase activity.Download cloud_download
date_range Published on May 1, 2017
share Casey RT, Ascher DB, Rattenberry E, Izatt L, Andrews KA, Simpson HL, Challis B, Park SM, Bulusu VR, Lalloo F, Pires DEV, West H, Clark GR, Smith PS, Whitworth J, Papathomas TG, Taniere P, Savisaar R, Hurst LD, Woodward ER, Maher ER (2017) Mol Genet Genomic Med 5 (3):237-250.
PURPOSE: To evaluate the role of germline SDHA mutation analysis by (1) comprehensive literature review, (2) description of novel germline SDHA mutations and (3) in silico structural prediction analysis of missense substitutions in SDHA. PATIENTS AND METHODS: A systematic literature review and a retrospective review of the molecular and clinical features of patients identified with putative germline variants in UK molecular genetic laboratories was performed. To evaluate the molecular consequences of SDHA missense variants, a novel model of the SDHA/B/C/D complex was generated and the structural effects of missense substitutions identified in the literature, our UK novel cohort and a further 32 "control missense variants" were predicted by the mCSM computational platform. These structural predictions were correlated with the results of tumor studies and other bioinformatic predictions. RESULTS: Literature review revealed reports of 17 different germline SDHA variants in 47 affected individuals from 45 kindreds. A further 10 different variants in 15 previously unreported cases (seven novel variants in eight patients) were added from our UK series. In silico structural prediction studies of 11 candidate missense germline mutations suggested that most (63.7%) would destabilize the SDHA protomer, and that most (78.1%) rare SDHA missense variants present in a control data set (ESP6500) were also associated with impaired protein stability. CONCLUSION: The clinical spectrum of SDHA-associated neoplasia differs from that of germline mutations in other SDH-subunits. The interpretation of the significance of novel SDHA missense substitutions is challenging. We recommend that multiple investigations (e.g. tumor studies, metabolomic profiling) should be performed to aid classification of rare missense variants before genetic testing results are used to influence clinical management.Download cloud_download
date_range Published on June 1, 2017
share Albanaz ATS, Rodrigues CHM, Pires DEV, Ascher DB (2017) Expert Opin Drug Discov 12 (6):553-563.
Mutations introduce diversity into genomes, leading to selective changes and driving evolution. These changes have contributed to the emergence of many of the current major health concerns of the 21st century, from the development of genetic diseases and cancers to the rise and spread of drug resistance. The experimental systematic testing of all mutations in a system of interest is impractical and not cost-effective, which has created interest in the development of computational tools to understand the molecular consequences of mutations to aid and guide rational experimentation. Areas covered: Here, the authors discuss the recent development of computational methods to understand the effects of coding mutations to protein function and interactions, particularly in the context of the 3D structure of the protein. Expert opinion: While significant progress has been made in terms of innovative tools to understand and quantify the different range of effects in which a mutation or a set of mutations can give rise to a phenotype, a great gap still exists when integrating these predictions and drawing causality conclusions linking variants. This often requires a detailed understanding of the system being perturbed. However, as part of the drug development process it can be used preemptively in a similar fashion to pharmacokinetics predictions, to guide development of therapeutics to help guide the design and analysis of clinical trials, patient treatment and public health policy strategies.Download cloud_download
date_range Published on Nov. 21, 2016
share White RR, Ponsford AH, Weekes MP, Rodrigues RB, Ascher DB, Mol M, Selkirk ME, Gygi SP, Sanderson CM, Artavanis-Tsakonas K (2016) PLoS Pathog 12 (11):e1005977.
Trichinella spiralis is a muscle-specific parasitic worm that is uniquely intracellular. T. spiralis reprograms terminally differentiated skeletal muscle cells causing them to de-differentiate and re-enter the cell cycle, a process that cannot occur naturally in mammalian skeletal muscle cells, but one that holds great therapeutic potential. Although the host ubiquitin pathway is a common target for viruses and bacteria during infection, its role in parasite pathogenesis has been largely overlooked. Here we demonstrate that the secreted proteins of T. spiralis contain E2 Ub-conjugating and E3 Ub-ligase activity. The E2 activity is attributed to TsUBE2L3, a novel and conserved T. spiralis enzyme located in the secretory organ of the parasite during the muscle stages of infection. TsUBE2L3 cannot function with any T.spiralis secreted E3, but specifically binds to a panel of human RING E3 ligases, including the RBR E3 ARIH2 with which it interacts with a higher affinity than the mammalian ortholog UbcH7/UBE2L3. Expression of TsUBE2L3 in skeletal muscle cells causes a global downregulation in protein ubiquitination, most predominantly affecting motor, sarcomeric and extracellular matrix proteins, thus mediating their stabilization with regards to proteasomal degradation. This effect is not observed in the presence of the mammalian ortholog, suggesting functional divergence in the evolution of the parasite protein. These findings demonstrate the first example of host-parasite interactions via a parasite-derived Ub conjugating enzyme; an E2 that demonstrates a novel muscle protein stabilization function.Download cloud_download
date_range Published on July 28, 2016
share Silvino AC, Costa GL, Araujo FC, Ascher DB, Pires DE, Fontes CJ, Carvalho LH, Brito CF, Sousa TN (2016) PLoS One 11 (7):e0160172.
Although Plasmodium vivax relapses are classically associated with hypnozoite activation, it has been proposed that a proportion of these cases are due to primaquine (PQ) treatment failure caused by polymorphisms in cytochrome P-450 2D6 (CYP2D6). Here, we present evidence that CYP2D6 polymorphisms are implicated in PQ failure, which was reinforced by findings in genetically similar parasites, and may explain a number of vivax relapses. Using a computational approach, these polymorphisms were predicted to affect the activity of CYP2D6 through changes in the structural stability that could lead to disruption of the PQ-enzyme interactions. Furthermore, because PQ is co-administered with chloroquine (CQ), we investigated whether CQ-impaired metabolism by cytochrome P-450 2C8 (CYP2C8) could also contribute to vivax recurrences. Our results show that CYP2C8-mutated patients frequently relapsed early (<42 days) and had a higher proportion of genetically similar parasites, suggesting the possibility of recrudescence due to CQ therapeutic failure. These results highlight the importance of pharmacogenetic studies as a tool to monitor the efficacy of antimalarial therapy.Download cloud_download
date_range Published on Jan. 22, 2016
share Pires DE, Chen J, Blundell TL, Ascher DB (2016) Sci Rep 6:19848.
Despite interest in associating polymorphisms with clinical or experimental phenotypes, functional interpretation of mutation data has lagged behind generation of data from modern high-throughput techniques and the accurate prediction of the molecular impact of a mutation remains a non-trivial task. We present here an integrated knowledge-driven computational workflow designed to evaluate the effects of experimental and disease missense mutations on protein structure and interactions. We exemplify its application with analyses of saturation mutagenesis of DBR1 and Gal4 and show that the experimental phenotypes for over 80% of the mutations correlate well with predicted effects of mutations on protein stability and RNA binding affinity. We also show that analysis of mutations in VHL using our workflow provides valuable insights into the effects of mutations, and their links to the risk of developing renal carcinoma. Taken together the analyses of the three examples demonstrate that structural bioinformatics tools, when applied in a systematic, integrated way, can rapidly analyse a given system to provide a powerful approach for predicting structural and functional effects of thousands of mutations in order to reveal molecular mechanisms leading to a phenotype. Missense or non-synonymous mutations are nucleotide substitutions that alter the amino acid sequence of a protein. Their effects can range from modifying transcription, translation, processing and splicing, localization, changing stability of the protein, altering its dynamics or interactions with other proteins, nucleic acids and ligands, including small molecules and metal ions. The advent of high-throughput techniques including sequencing and saturation mutagenesis has provided large amounts of phenotypic data linked to mutations. However, one of the hurdles has been understanding and quantifying the effects of a particular mutation, and how they translate into a given phenotype. One approach to overcome this is to use robust, accurate and scalable computational methods to understand and correlate structural effects of mutations with disease.Download cloud_download
date_range Published on July 7, 2016
share Pires DE, Blundell TL, Ascher DB (2016) Sci Rep 6:29575.
The ability to predict how a mutation affects ligand binding is an essential step in understanding, anticipating and improving the design of new treatments for drug resistance, and in understanding genetic diseases. Here we present mCSM-lig, a structure-guided computational approach for quantifying the effects of single-point missense mutations on affinities of small molecules for proteins. mCSM-lig uses graph-based signatures to represent the wild-type environment of mutations, and small-molecule chemical features and changes in protein stability as evidence to train a predictive model using a representative set of protein-ligand complexes from the Platinum database. We show our method provides a very good correlation with experimental data (up to ρ = 0.67) and is effective in predicting a range of chemotherapeutic, antiviral and antibiotic resistance mutations, providing useful insights for genotypic screening and to guide drug development. mCSM-lig also provides insights into understanding Mendelian disease mutations and as a tool for guiding protein design. mCSM-lig is freely available as a web server at http://structure.bioc.cam.ac.uk/mcsm_lig.Download cloud_download
date_range Published on July 8, 2016
share Pires DE, Ascher DB (2016) Nucleic Acids Res 44 (W1):W557-561
Determining the affinity of a ligand for a given protein is a crucial component of drug development and understanding their biological effects. Predicting binding affinities is a challenging and difficult task, and despite being regarded as poorly predictive, scoring functions play an important role in the analysis of molecular docking results. Here, we present CSM-Lig (http://structure.bioc.cam.ac.uk/csm_lig), a web server tailored to predict the binding affinity of a protein-small molecule complex, encompassing both protein and small-molecule complementarity in terms of shape and chemistry via graph-based structural signatures. CSM-Lig was trained and evaluated on different releases of the PDBbind databases, achieving a correlation of up to 0.86 on 10-fold cross validation and 0.80 in blind tests, performing as well as or better than other widely used methods. The web server allows users to rapidly and automatically predict binding affinities of collections of structures and assess the interactions made. We believe CSM-lig would be an invaluable tool for helping assess docking poses, the effects of multiple mutations, including insertions, deletions and alternative splicing events, in protein-small molecule affinity, unraveling important aspects that drive protein-compound recognition.Download cloud_download
date_range Published on July 8, 2016
share Pires DE, Ascher DB (2016) Nucleic Acids Res 44 (W1):W469-473.
Computational methods have traditionally struggled to predict the effect of mutations in antibody–antigen complexes on binding affinity. This has limited their usefulness during antibody engineering and development, and their ability to predict biologically relevant escape mutations. Here we present mCSM-AB, a user-friendly web server for accurately predicting antibody–antigen affinity changes upon mutation which relies on graph-based signatures. We show that mCSM-AB performs better than comparable methods that have been previously used for antibody engineering. mCSM-AB web server is available at http://structure.bioc.cam.ac.uk/mcsm_ab.Download cloud_download
date_range Published on March 23, 2016
share Phelan J, Coll F, McNerney R, Ascher DB, Pires DE, Furnham N, Coeck N, Hill-Cawthorne GA, Nair MB, Mallard K, Ramsay A, Campino S, Hibberd ML, Pain A, Rigouts L, Clark TG (2016). BMC Med 14 (1):31.
Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance mutations to improve the design of tuberculosis control measures, such as diagnostics, and inform patient management.Download cloud_download
date_range Published on Jan. 1, 2016
share Nemethova M, Radvanszky J, Kadasi L, Ascher DB, Pires DE, Blundell TL, Porfirio B, Mannoni A, Santucci A, Milucci L, Sestini S, Biolcati G, Sorge F, Aurizi C, Aquaron R, Alsbou M, Lourenco CM, Ramadevi K, Ranganath LR, Gallagher JA, van Kan C, Hall AK, Olsson B, Sireau N, Ayoob H, Timmis OG, Sang KH, Genovese F, Imrich R, Rovensky J, Srinivasaraghavan R, Bharadwaj SK, Spiegel R, Zatkova A (2016) Eur J Hum Genet 24 (1):66-72.
Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650-85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.Download cloud_download
date_range Published on Dec. 13, 2016
share Kano FS, Souza-Silva FA, Torres LM, Lima BA, Sousa TN, Alves JR, Rocha RS, Fontes CJ, Sanchez BA, Adams JH, Brito CF, Pires DE, Ascher DB, Sell AM, Carvalho LH (2016) PLoS Negl Trop Dis 10 (12):e0005177.
Background The human malaria parasite Plasmodium vivax infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of P. vivax-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked. Methodology/Principal Findings A community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (DRB1, DQA1 and DQB1 loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII–erythrocyte binding). The results demonstrated an increased susceptibility of the DRB1*13:01 carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype DRB1*14:02-DQA1*05:03-DQB1*03:01 were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (DRB1*07:01, DQA1*02:01 and DQB1*02:02) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses. Conclusions/Significance The current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase knowledge of the binding affinities of DBPII peptides for class II molecules linked with good or poor antibody responses might lead to the development of strategies for controlling the type of helper T cells activated in response to DBPII.Download cloud_download
date_range Published on Aug. 15, 2016
share Coelho MB, Ascher DB, Gooding C, Lang E, Maude H, Turner D, Llorian M, Pires DE, Attig J, Smith CW (2016) Biochem Soc Trans 44 (4):1058-1065.
Polypyrimidine tract binding protein (PTBP1) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that plays roles in most stages of the life-cycle of pre-mRNA and mRNAs in the nucleus and cytoplasm. PTBP1 has four RNA binding domains of the RNA recognition motif (RRM) family, each of which can bind to pyrimidine motifs. In addition, RRM2 can interact via its dorsal surface with proteins containing short peptide ligands known as PTB RRM2 interacting (PRI) motifs, originally found in the protein Raver1. Here we review our recent progress in understanding the interactions of PTB with RNA and with various proteins containing PRI ligands.Download cloud_download
date_range Published on April 4, 2016
share Chan LJ, Ascher DB, Yadav R, Bulitta JB, Williams CC, Porter CJ, Landersdorfer CB, Kaminskas LM (2016) Mol Pharm 13 (4):1229-1241.
The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab′ as a model. Specifically, the Fab′ was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3–4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab′ displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab′. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab′s against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.Download cloud_download
date_range Published on Feb. 25, 2016
share Andrews KA, Vialard L, Ascher DB, Pires DEV, Bradshaw N, Cole T, Cook J, Irving R, Kumar A, Lalloo F, Izatt L, Goudie D, Woodward ER, Maher ER, Grp USC (2016) Lancet 387:19-19.
Background Germline mutations in the succinate dehydrogenase subunit genes SDHB, SDHC, and SDHD are the most frequent causes of inherited phaeochromocytomas and paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. Our aim was to provide estimates of penetrance and genotype–phenotype correlations in a large cohort of succinate dehydrogenase mutation carriers. Methods We undertook a retrospective survey of 800 individuals in the UK (401 previously reported) with germline mutations in SDHB (620), SDHC (31), and SDHD (149). We estimated and compared tumour risks for each gene according to age using survival analysis and Cox proportional hazards modelling with the statistical programming language R. DUET, a computational approach for predicting the effects of mutations on protein stability, was used to evaluate the functional effects of SDHB and SDHD mutations. Findings Analysis of age-related tumour risks provided novel estimates of penetrance. In addition to tumour-specific differences in risk for individual genes, we confirmed that the SDHD p.Pro81Leu mutation had a distinct phenotype, with a low risk of phaeochromocytoma and extra-adrenal paraganglioma (only one case in 55 patients), and found evidence suggesting higher penetrance with SDHB p.Ile127Ser mutations. Comparison of age-dependent penetrance of disease in p.Ile127Ser carriers versus other SDHB missense mutation carriers showed that p.Ile127Ser was associated with a higher overall penetrance of phaeochromocytoma and paraganglioma (χ 2=4·49, p=0·034). SDHB p.Ile127Ser was predicted by DUET to be the most destabilising SDHB missense mutation, through disruption of key intramolecular hydrophobic interactions by the introduction of a polar serine. The penetrance in SDHB and SDHD mutation-positive non-probands by age 60 years was 22·1% (95% CI 15·6–28·3) and 47·5% (29·3–61·1), respectively, and the risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4·2% (1·1–7·2). Interpretation Increased knowledge of the lifetime tumour risks is crucial to long-term surveillance and management. Knowledge of the molecular basis of the phenotypic variability commonly observed in individuals with germline SDHB, SDHC, and SDHD mutations will facilitate the development of personalised management based on gene-specific and mutation-specific tumour risks.Download cloud_download
date_range Published on Feb. 15, 2015
share Usher JL, Ascher DB, Pires DE, Milan AM, Blundell TL, Ranganath LR (2015) JIMD Rep 24:3-11.
Alkaptonuria (AKU) is a rare autosomal recessive disorder with incidence ranging from 1:100,000 to 1:250,000. The disorder is caused by a deficiency of the enzyme homogentisate 1,2-dioxygenase (HGD), which results from defects in the HGD gene. This enzyme converts homogentisic acid to maleylacetoacetate and has a major role in the catabolism of phenylalanine and tyrosine. To elucidate the mutation spectrum of the HGD gene in patients with alkaptonuria from 42 patients attending the National Alkaptonuria Centre, 14 exons of the HGD gene and the intron–exon boundaries were analysed by PCR-based sequencing. A total of 34 sequence variants was observed, confirming the genetic heterogeneity of AKU. Of these mutations, 26 were missense substitutions and four splice site mutations. There were two deletions and one duplication giving rise to frame shifts and one substitution abolishing the translation termination codon (no stop). Nine of the mutations were previously unreported novel variants. Using computational approaches based on the 3D structure, these novel mutations are predicted to affect the activity of the protein complex through destabilisation of the individual protomer structure or through disruption of protomer–protomer interactions.Download cloud_download
date_range Published on July 31, 2015
share Sigurdardottir AG, Winter A, Sobkowicz A, Fragai M, Chirgadze D, Ascher DB, Blundell TL, Gherardi E (2015) Chemical Science 6 (11):6147-6157.
The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, constitute a signalling system essential for embryogenesis and for tissue/organ regeneration in post-natal life. HGF/SF-MET signalling, however, also plays a key role in the onset of metastasis of a large number of human tumours. Both HGF/SF and MET are high molecular weight proteins that bury an extensive interface upon complex formation and thus constitute a challenging target for the development of low molecular weight inhibitors. Here we have used surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and X-ray crystallography to screen a diverse fragment library of 1338 members as well as a range of piperazine-like compounds. Several small molecules were found to bind in the lysine-binding pocket of the kringle 1 domain of HGF/SF and its truncated splice variant NK1. We have defined the binding mode of these compounds, explored their biological activity and we show that selected fragments inhibit MET downstream signalling. Thus we demonstrate that targeting the lysine-binding pocket of NK1 is an effective strategy to generate MET receptor antagonists and we offer proof of concept that the HGF/SF-MET interface may be successfully targeted with small molecules. These studies have broad implications for the development of HGF/SF-MET therapeutics and cancer treatment.Download cloud_download
date_range Published on Jan. 1, 2015
share Pires DE, Blundell TL, Ascher DB (2015). Nucleic Acids Res 43 (Database issue):D387-391.
Drug resistance is a major challenge for the treatment of many diseases and a significant concern throughout the drug development process. The ability to understand and predict the effects of mutations on protein-ligand affinities and their roles in the emergence of resistance would significantly aid treatment and drug design strategies. In order to study and understand the impacts of missense mutations on the interaction of ligands with the proteome, we have developed Platinum (http://structure.bioc.cam.ac.uk/platinum). This manually curated, literature-derived database, comprising over 1000 mutations, associates for the first time experimental information on changes in affinity with three-dimensional structures of protein-ligand complexes. To minimize differences arising from experimental techniques and to directly compare binding affinities, Platinum considers only changes measured by the same group and with the same amino-acid sequence used for structure determination, providing a direct link between protein structure, how a ligand binds and how mutations alter the affinity of the ligand of the protein. We believe Platinum will be an invaluable resource for understanding the effects of mutations that give rise to drug resistance, a major problem emerging in pandemics including those caused by the influenza virus, in infectious diseases such as tuberculosis, in cancer and in many other life-threatening illnesses.Download cloud_download
date_range Published on May 14, 2015
share Pires DE, Blundell TL, Ascher DB (2015). J Med Chem 58 (9):4066-4072.
Drug development has a high attrition rate, with poor pharmacokinetic and safety properties a significant hurdle. Computational approaches may help minimize these risks. We have developed a novel approach (pkCSM) which uses graph-based signatures to develop predictive models of central ADMET properties for drug development. pkCSM performs as well or better than current methods. A freely accessible web server (http://structure.bioc.cam.ac.uk/pkcsm), which retains no information submitted to it, provides an integrated platform to rapidly evaluate pharmacokinetic and toxicity properties.Download cloud_download
date_range Published on Dec. 2, 2015
share Pacitto A, Ascher DB, Wong LH, Blaszczyk BK, Nookala RK, Zhang N, Dokudovskaya S, Levine TP, Blundell TL (2015) Open Biol 5 (12):150174.
The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt-Hogg-Dubé (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.Download cloud_download
date_range Published on May 1, 2015
share Landersdorfer CB, Caliph SM, Shackleford DM, Ascher DB, Kaminskas LM (2015) J Pharm Sci 104 (5):1848-1855.
Gender and immune status can considerably impact on the pharmacokinetics (PK) of macromolecular and small molecule drugs. However, these effects are often not considered in drug development. We aimed to quantitatively evaluate effects of gender and immune status on the PK of PEGylated interferon in frequently used murine models. Chronically cannulated female athymic nude and female and male immunocompetent C57Bl/6J mice (n = 24 in total) received a single intravenous or subcutaneous (s.c.) dose of PEGylated interferon. Serial blood samples were taken for 48 h. Noncompartmental analysis and population PK modeling with covariate analysis were performed to evaluate the data. The PK of PEGylated interferon followed a three compartment disposition model with two sequential compartments for s.c. absorption. Female nude mice had significantly higher plasma clearance than C57Bl/6J mice (0.503 vs. 0.397 mL/h). Male mice had a slower absorption rate constant (0.138 h(-1)) and extent (46.2%) of s.c. absorption than female mice (0.274 in C57Bl/6J and 0.374 h(-1) in nude, 60.8% in both). Thus, gender and immune status significantly impacted on important PK parameters of PEGylated interferon in murine models commonly utilized in drug development. It is critical to take into account these differences when choosing animal models and conducting translational pharmacology research.Download cloud_download
date_range Published on Feb. 2, 2015
share Kaminskas LM, McLeod VM, Ascher DB, Ryan GM, Jones S, Haynes JM, Trevaskis NL, Chan LJ, Sloan EK, Finnin BA, Williamson M, Velkov T, Williams ED, Kelly BD, Owen DJ, Porter CJ (2015) Mol Pharm 12 (2):432-443.
The current study sought to explore whether the subcutaneous administration of lymph targeted dendrimers, conjugated with a model chemotherapeutic (methotrexate, MTX), was able to enhance anticancer activity against lymph node metastases. The lymphatic pharmacokinetics and antitumor activity of PEGylated polylysine dendrimers conjugated to MTX [D-MTX(OH)] via a tumor-labile hexapeptide linker was examined in rats and compared to a similar system where MTX was α-carboxyl O-tert-butylated [D-MTX(OtBu)]. The latter has previously been shown to exhibit longer plasma circulation times. D-MTX(OtBu) was well absorbed from the subcutaneous injection site via the lymph, and 3 to 4%/g of the dose was retained by sentinel lymph nodes. In contrast, D-MTX(OH) showed limited absorption from the subcutaneous injection site, but absorption was almost exclusively via the lymph. The retention of D-MTX(OH) by sentinel lymph nodes was also significantly elevated (approximately 30% dose/g). MTX alone was not absorbed into the lymph. All dendrimers displayed lower lymph node targeting after intravenous administration. Despite significant differences in the lymph node retention of D-MTX(OH) and D-MTX(OtBu) after subcutaneous and intravenous administration, the growth of lymph node metastases was similarly inhibited. In contrast, the administration of MTX alone did not significantly reduce lymph node tumor growth. Subcutaneous administration of drug-conjugated dendrimers therefore provides an opportunity to improve drug deposition in downstream tumor-burdened lymph nodes. In this case, however, increased lymph node biodistribution did not correlate well with antitumor activity, possibly suggesting constrained drug release at the site of action.Download cloud_download
date_range Published on Oct. 1, 2015
share Jubb H, Blundell TL, Ascher DB (2015) Prog Biophys Mol Biol 119 (1):2-9.
The transient assembly of multiprotein complexes mediates many aspects of cell regulation and signalling in living organisms. Modulation of the formation of these complexes through targeting protein-protein interfaces can offer greater selectivity than the inhibition of protein kinases, proteases or other post-translational regulatory enzymes using substrate, co-factor or transition state mimetics. However, capitalising on protein-protein interaction interfaces as drug targets has been hindered by the nature of interfaces that tend to offer binding sites lacking the well-defined large cavities of classical drug targets. In this review we posit that interfaces formed by concerted folding and binding (disorder-to-order transitions on binding) of one partner and other examples of interfaces where a protein partner is bound through a continuous epitope from a surface-exposed helix, flexible loop or chain extension may be more tractable for the development of "orthosteric", competitive chemical modulators; these interfaces tend to offer small-volume but deep pockets and/or larger grooves that may be bound tightly by small chemical entities. We discuss examples of such protein-protein interaction interfaces for which successful chemical modulators are being developed.Download cloud_download
date_range Published on July 1, 2015
share Jafri M, Wake NC, Ascher DB, Pires DE, Gentle D, Morris MR, Rattenberry E, Simpson MA, Trembath RC, Weber A, Woodward ER, Donaldson A, Blundell TL, Latif F, Maher ER (2015) Cancer Discov 5 (7):723-729.
Familial renal cell carcinoma (RCC) is genetically heterogeneous and may be caused by mutations in multiple genes, including VHL, MET, SDHB, FH, FLCN, PTEN, and BAP1. However, most individuals with inherited RCC do not have a detectable germline mutation. To identify novel inherited RCC genes, we undertook exome resequencing studies in a familial RCC kindred and identified a CDKN2B nonsense mutation that segregated with familial RCC status. Targeted resequencing of CDKN2B in individuals (n = 82) with features of inherited RCC then revealed three candidate CDKN2B missense mutations (p.Pro40Thr, p.Ala23Glu, and p.Asp86Asn). In silico analysis of the three-dimensional structures indicated that each missense substitution was likely pathogenic through reduced stability of the mutant or reduced affinity for cyclin-dependent kinases 4 and 6, and in vitro studies demonstrated that each of the mutations impaired CDKN2B-induced suppression of proliferation in an RCC cell line. These findings identify germline CDKN2B mutations as a novel cause of familial RCC.Download cloud_download
date_range Published on Feb. 1, 2015
share Hermans SJ, Ascher DB, Hancock NC, Holien JK, Michell BJ, Chai SY, Morton CJ, Parker MW (2015). Protein Sci 24 (2):190-199.
Insulin-regulated aminopeptidase (IRAP or oxytocinase) is a membrane-bound zinc-metallopeptidase that cleaves neuroactive peptides in the brain and produces memory enhancing effects when inhibited. We have determined the crystal structure of human IRAP revealing a closed, four domain arrangement with a large, mostly buried cavity abutting the active site. The structure reveals that the GAMEN exopeptidase loop adopts a very different conformation from other aminopeptidases, thus explaining IRAP's unique specificity for cyclic peptides such as oxytocin and vasopressin. Computational docking of a series of IRAP-specific cognitive enhancers into the crystal structure provides a molecular basis for their structure-activity relationships and demonstrates that the structure will be a powerful tool in the development of new classes of cognitive enhancers for treating a variety of memory disorders such as Alzheimer's disease.Download cloud_download
date_range Published on March 2, 2015
share Chan LJ, Bulitta JB, Ascher DB, Haynes JM, McLeod VM, Porter CJ, Williams CC, Kaminskas LM (2015) Mol Pharm 12 (3):794-809.
The lymphatic system plays a major role in the metastatic dissemination of cancer and has an integral role in immunity. PEGylation enhances drainage and lymphatic uptake following subcutaneous (sc) administration of proteins and protein-like polymers, but the impact of PEGylation of very large proteins (such as antibodies) on subcutaneous and lymphatic pharmacokinetics is unknown. This study therefore aimed to evaluate the impact of PEGylation on the sc absorption and lymphatic disposition of the anti-HER2 antibody trastuzumab in rats. PEG-trastuzumab was generated via the conjugation of a single 40 kDa PEG-NHS ester to trastuzumab. PEG-trastuzumab showed a 5-fold reduction in HER2 binding affinity, however the in vitro growth inhibitory effects were preserved as a result of changes in cellular trafficking when compared to native trastuzumab. The lymphatic pharmacokinetics of PEG-trastuzumab was evaluated in thoracic lymph duct cannulated rats after iv and sc administration and compared to the pharmacokinetics of native trastuzumab. The iv pharmacokinetics and lymphatic exposure of PEG-trastuzumab was similar when compared to trastuzumab. After sc administration, initial plasma pharmacokinetics and lymphatic exposure were also similar between PEG-trastuzumab and trastuzumab, but the absolute bioavailability of PEG-trastuzumab was 100% when compared to 86.1% bioavailability for trastuzumab. In contrast to trastuzumab, PEG-trastuzumab showed accelerated plasma clearance beginning approximately 7 days after sc, but not iv, administration, presumably as a result of the generation of anti-PEG IgM. This work suggests that PEGylation does not significantly alter the lymphatic disposition of very large proteins, and further suggests that it is unlikely to benefit therapy with monoclonal antibodies.Download cloud_download
date_range Published on March 1, 2015
share Caliph SM, Shackleford DM, Ascher DB, Kaminskas LM (2015) J Pharm Sci 104 (3):1207-1209.
Cannulation of the thoracic lymph duct in experimental animals allows direct measurement of the lymphatic exposure of lymph-targeted drugs. When coupled with recent advances in genetically modified and diseased mouse models, this presents further opportunities to define changes in biological processes and disease in response to drug treatment. Although cannulation of the thoracic lymph duct in mice is inherently challenging because of the small size and delicate nature of the duct, it can be further confounded by anatomical variations between animals. In this communication, we present our observations on the anatomical features of the thoracic lymph duct between mice of different strains and genders, and discuss the impact of these features on the "cannulatability" of the duct. We also provide some technical tips to help guide other investigators to deliver higher experimental success rates.Download cloud_download
date_range Published on Sept. 1, 2015
share Blaszczyk M, Harmer NJ, Chirgadze DY, Ascher DB, Blundell TL (2015) Prog Biophys Mol Biol 118 (3):103-111.
How is information communicated both within and between cells of living systems with high signal to noise? We discuss transmembrane signaling models involving two receptor tyrosine kinases: the fibroblast growth factor receptor (FGFR) and the MET receptor. We suggest that simple dimerization models might occur opportunistically giving rise to noise but cooperative clustering of the receptor tyrosine kinases observed in these systems is likely to be important for signal transduction. We propose that this may be a more general prerequisite for high signal to noise in transmembrane receptor signaling.Download cloud_download
date_range Published on Feb. 10, 2015
share Ascher DB, Jubb HC, Pires DE, Ochi T, Higueruelo A, Blundell TL (2015) NATO Science for Peace and Security Series A: Chemistry and Biology. Springer Netherlands, pp 141-163.
While protein-protein interfaces have promised a range of benefits over conventional sites in drug discovery, they present unique challenges. Here we describe recent developments that facilitate many aspects of the drug discovery process – including characterization and classification of interfaces, identifying druggable sites and strategies for inhibitor development.Download cloud_download
date_range Published on June 1, 2014
share Watt AD, Crespi GA, Down RA, Ascher DB, Gunn A, Perez KA, McLean CA, Villemagne VL, Parker MW, Barnham KJ, Miles LA (2014) Acta Neuropathol 127 (6):803-810.
Reducing amyloid-β peptide (Aβ) burden at the pre-symptomatic stages of Alzheimer's disease (AD) is currently the advocated clinical strategy for treating this disease. The most developed method for targeting Aβ is the use of monoclonal antibodies including bapineuzumab, solanezumab and crenezumab. We have synthesized these antibodies and used surface plasmon resonance (SPR) and mass spectrometry to characterize and compare the ability of these antibodies to target Aβ in transgenic mouse tissue as well as human AD tissue. SPR analysis showed that the antibodies were able to bind Aβ with high affinity. All of the antibodies were able to bind Aβ in mouse tissue. However, significant differences were observed in human brain tissue. While bapineuzumab was able to capture a variety of N-terminally truncated Aβ species, the Aβ detected using solanezumab was barely above detection limits while crenezumab did not detect any Aβ. None of the antibodies were able to detect any Aβ species in human blood. Immunoprecipitation experiments using plasma from AD subjects showed that both solanezumab and crenezumab have extensive cross-reactivity with non-Aβ related proteins. Bapineuzumab demonstrated target engagement with brain Aβ, consistent with published clinical data. Solanezumab and crenezumab did not, most likely as a result of a lack of specificity due to cross-reactivity with other proteins containing epitope overlap. This lack of target engagement raises questions as to whether solanezumab and crenezumab are suitable drug candidates for the preventative clinical trials for AD.Download cloud_download
date_range Published on Oct. 1, 2014
share Watt AD, Crespi GA, Down RA, Ascher DB, Gunn A, Perez KA, McLean CA, Villemagne VL, Parker MW, Barnham KJ, Miles LA (2014) Acta Neuropathol 128 (4):611-614.
We have read with interest the commentary by Siemers et al.  regarding our paper describing the ability of the three anti-Aβ antibodies, bapineuzumab, crenezumab and solanezumab to engage Aβ in both a synthetic and a biological setting. We appreciate the opportunity to clarify any misunderstandings and here provide a brief response to their concerns.Download cloud_download
date_range Published on Feb. 1, 2014
share Pires DE, Ascher DB, Blundell TL (2014) Bioinformatics 30 (3):335-342.
MOTIVATION: Mutations play fundamental roles in evolution by introducing diversity into genomes. Missense mutations in structural genes may become either selectively advantageous or disadvantageous to the organism by affecting protein stability and/or interfering with interactions between partners. Thus, the ability to predict the impact of mutations on protein stability and interactions is of significant value, particularly in understanding the effects of Mendelian and somatic mutations on the progression of disease. Here, we propose a novel approach to the study of missense mutations, called mCSM, which relies on graph-based signatures. These encode distance patterns between atoms and are used to represent the protein residue environment and to train predictive models. To understand the roles of mutations in disease, we have evaluated their impacts not only on protein stability but also on protein-protein and protein-nucleic acid interactions. RESULTS: We show that mCSM performs as well as or better than other methods that are used widely. The mCSM signatures were successfully used in different tasks demonstrating that the impact of a mutation can be correlated with the atomic-distance patterns surrounding an amino acid residue. We showed that mCSM can predict stability changes of a wide range of mutations occurring in the tumour suppressor protein p53, demonstrating the applicability of the proposed method in a challenging disease scenario. AVAILABILITY AND IMPLEMENTATION: A web server is available at http://structure.bioc.cam.ac.uk/mcsm.Download cloud_download
date_range Published on July 1, 2014
share Pires DE, Ascher DB, Blundell TL (2014) Nucleic Acids Res 42:W314-319.
Cancer genome and other sequencing initiatives are generating extensive data on non-synonymous single nucleotide polymorphisms (nsSNPs) in human and other genomes. In order to understand the impacts of nsSNPs on the structure and function of the proteome, as well as to guide protein engineering, accurate in silicomethodologies are required to study and predict their effects on protein stability. Despite the diversity of available computational methods in the literature, none has proven accurate and dependable on its own under all scenarios where mutation analysis is required. Here we present DUET, a web server for an integrated computational approach to study missense mutations in proteins. DUET consolidates two complementary approaches (mCSM and SDM) in a consensus prediction, obtained by combining the results of the separate methods in an optimized predictor using Support Vector Machines (SVM). We demonstrate that the proposed method improves overall accuracy of the predictions in comparison with either method individually and performs as well as or better than similar methods. The DUET web server is freely and openly available at http://structure.bioc.cam.ac.uk/duet.Download cloud_download
date_range Published on Feb. 20, 2014
share Feil SC, Ascher DB, Kuiper MJ, Tweten RK, Parker MW (2014) J Mol Biol 426 (4):785-792.
Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation-pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane.Download cloud_download
date_range Published on March 1, 2014
share Crespi GA, Ascher DB, Parker MW, Miles LA (2014) Acta Crystallogr F Struct Biol Commun 70 (Pt 3):374-377.
Bapineuzumab (AAB-001) and its derivative (AAB-003) are humanized versions of the anti-Aβ murine antibody 3D6 and are immunotherapy candidates in Alzheimer's disease. The common Fab fragment of these immunotherapies has been expressed, purified and crystallized in complex with β-amyloid peptides (residues 1-8 and 1-28). Diffraction data at high resolution were acquired from crystals of Fab-Aβ8 (2.0 Å) and Fab-Aβ28 (2.2 Å) complexes at the Australian Synchrotron. Both crystal forms belonged to the primitive orthorhombic space group P21221.Download cloud_download
date_range Published on April 23, 2014
share Ascher DB, Wielens J, Nero TL, Doughty L, Morton CJ, Parker MW (2014) Sci Rep 4:4765.
Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA.Download cloud_download
date_range Published on March 1, 2013
share Polekhina G, Ascher DB, Kok SF, Beckham S, Wilce M, Waltham M (2013) Acta Crystallogr D Biol Crystallogr 69 (Pt 3):333-344.
Thioredoxin-interacting protein (TXNIP) is one of the six known α-arrestins and has recently received considerable attention owing to its involvement in redox signalling and metabolism. Various stress stimuli such as high glucose, heat shock, UV, H2O2 and mechanical stress among others robustly induce the expression of TXNIP, resulting in the sequestration and inactivation of thioredoxin, which in turn leads to cellular oxidative stress. While TXNIP is the only α-arrestin known to bind thioredoxin, TXNIP and two other α-arrestins, Arrdc4 and Arrdc3, have been implicated in metabolism. Furthermore, owing to its roles in the pathologies of diabetes and cardiovascular disease, TXNIP is considered to be a promising drug target. Based on their amino-acid sequences, TXNIP and the other α-arrestins are remotely related to β-arrestins. Here, the crystal structure of the N-terminal domain of TXNIP is reported. It provides the first structural information on any of the α-arrestins and reveals that although TXNIP adopts a β-arrestin fold as predicted, it is structurally more similar to Vps26 proteins than to β-arrestins, while sharing below 15% pairwise sequence identity with either.Download cloud_download
date_range Published on June 10, 2013
share Kaminskas LM, Ascher DB, McLeod VM, Herold MJ, Le CP, Sloan EK, Porter CJ (2013) J Control Release 168 (2):200-208.
The efficacy of protein-based therapeutics with indications in the treatment of lymphatic diseases is expected to be improved by enhancing lymphatic disposition. This study was therefore aimed at examining whether PEGylation can usefully be applied to improve the lymphatic uptake of interferon α2 and whether this ultimately translates into improved therapeutic efficacy against lymph-resident cancer. The lymphatic pharmacokinetics of interferon α2b (IFN, 19kDa) and PEGylated interferon α2b (IFN-PEG12, 31kDa) or α2a (IFN-PEG40, 60kDa) was examined in thoracic lymph duct cannulated rats. IFN was poorly absorbed from the SC injection site (Fabs 36%) and showed little uptake into lymph after SC or IV administration (≤1%). In contrast, IFN-PEG12 was efficiently absorbed from the SC injection site (Fabs 82%) and approximately 20% and 8% of the injected dose was recovered in thoracic lymph over 30h after SC or IV administration respectively. IFN-PEG40, however, was incompletely absorbed from the SC injection site (Fabs 23%) and showed similar lymphatic access after SC administration to IFN-PEG12 (21%). The recovery of IFN-PEG40 in thoracic lymph after IV administration, however, was significantly greater (29%) when compared to IV IFN-PEG12. The anti-tumour efficacy of interferon against axillary metastases of a highly lymph-metastatic variant of human breast MDA-MB-231 carcinoma was significantly increased by SC administration of lymph-targeted IFN-PEG12 when compared to the administration of IFN on the ipsilateral side to the axillary metastasis. Optimal PEGylation may therefore represent a viable approach to improving the lymphatic disposition and efficacy of therapeutic proteins against lymphatic diseases.Download cloud_download
date_range Published on Oct. 1, 2012
share Parker LJ, Ascher DB, Gao C, Miles LA, Harris HH, Parker MW (2012) J Inorg Biochem 115:138-147.
In this mini-review we focus on metal interactions with proteins with a particular emphasis on the evident synergism between different biophysical approaches toward understanding metallobiology. We highlight three recent examples from our own laboratory. Firstly, we describe metallodrug interactions with glutathione S-transferases, an enzyme family known to attack commonly used anti-cancer drugs. We then describe a protein target for memory enhancing drugs called insulin-regulated aminopeptidase in which zinc plays a role in catalysis and regulation. Finally we describe our studies on a protein, amyloid precursor protein, that appears to play a central role in Alzheimer's disease. Copper ions have been implicated in playing both beneficial and detrimental roles in the disease by binding to different regions of this protein.Download cloud_download
date_range Published on March 28, 2012
share Ascher DB, Polekhina G, Parker MW (2012) Acta Crystallogr Sect F Struct Biol Cryst Commun 68 (Pt 4):468-471.
Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the ‘molecular-ruler’ mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure–function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures.Download cloud_download
date_range Published on May 1, 2011
share Polekhina G, Ascher DB, Kok SF, Waltham M (2011) Acta Crystallogr Sect F Struct Biol Cryst Commun 67 (Pt 5):613-617.
Thioredoxin-interacting protein (TXNIP) is a negative regulator of thioredoxin and its roles in the pathologies of diabetes and cardiovascular diseases have marked it out as a potential drug target. Expression of TXNIP is robustly induced under various stress conditions such as high glucose, heat shock, UV, H(2)O(2) and mechanical stress amongst others. Elevated levels of TXNIP result in the sequestration and inactivation of thioredoxin, leading to cellular oxidative stress. For some time, this was the only known function of TXNIP; however, more recently the protein has been shown to play a role in regulation of glucose uptake and activation of the inflammasome. Based on the primary sequence, TXNIP is remotely related to β-arrestins, which include the visual arrestins. TXNIP has thus been classified as a member of the α-arrestin family, which to date includes five other members. None of the other α-arrestins are known to interact with thioredoxin, although curiously one has been implicated in glucose uptake. In order to gain insight into the structure-function relationships of the α-arrestin protein family, and particularly that of TXNIP, the N-terminal domain of TXNIP has been crystallized. The crystals belonged to a monoclinic space group and diffracted to 3 Å resolution using synchrotron radiation.Download cloud_download
date_range Published on July 4, 2011
share Parker LJ, Italiano LC, Morton CJ, Hancock NC, Ascher DB, Aitken JB, Harris HH, Campomanes P, Rothlisberger U, De Luca A, Lo Bello M, Ang WH, Dyson PJ, Parker MW (2011) Chemistry 17 (28):7806-7816.
Platinum-based cancer drugs, such as cisplatin, are highly effective chemotherapeutic agents used extensively for the treatment of solid tumors. However, their effectiveness is limited by drug resistance, which, in some cancers, has been associated with an overexpression of pi class glutathione S-transferase (GST P1-1), an important enzyme in the mercapturic acid detoxification pathway. Ethacraplatin (EA-CPT), a trans-Pt(IV) carboxylate complex containing ethacrynate ligands, was designed as a platinum cancer metallodrug that could also target cytosolic GST enzymes. We previously reported that EA-CPT was an excellent inhibitor of GST activity in live mammalian cells compared to either cisplatin or ethacrynic acid. In order to understand the nature of the drug-protein interactions between EA-CPT and GST P1-1, and to obtain mechanistic insights at a molecular level, structural and biochemical investigations were carried out, supported by molecular modeling analysis using quantum mechanical/molecular mechanical methods. The results suggest that EA-CPT preferentially docks at the dimer interface at GST P1-1 and subsequent interaction with the enzyme resulted in docking of the ethacrynate ligands at both active sites (in the H-sites), with the Pt moiety remaining bound at the dimer interface. The activation of the inhibitor by its target enzyme and covalent binding accounts for the strong and irreversible inhibition of enzymatic activity by the platinum complex.Download cloud_download
date_range Published on April 5, 2011
share Ascher DB, Cromer BA, Morton CJ, Volitakis I, Cherny RA, Albiston AL, Chai SY, Parker MW (2011) Biochemistry 50 (13):2611-2622.
The development of inhibitors of insulin-regulated aminopeptidase (IRAP), a membrane-bound zinc metallopeptidase, is a promising approach for the discovery of drugs for the treatment of memory loss such as that associated with Alzheimer's disease. There is, however, no consensus in the literature about the mechanism by which inhibition occurs. Sequence alignments, secondary structure predictions, and homology models based on the structures of recently determined related metallopeptidases suggest that the extracellular region consists of four domains. Partial proteolysis and mass spectrometry reported here confirm some of the domain boundaries. We have produced purified recombinant fragments of human IRAP on the basis of these data and examined their kinetic and biochemical properties. Full-length extracellular constructs assemble as dimers with different nonoverlapping fragments dimerizing as well, suggesting an extended dimer interface. Only recombinant fragments containing domains 1 and 2 possess aminopeptidase activity and bind the radiolabeled hexapeptide inhibitor, angiotensin IV (Ang IV). However, fragments lacking domains 3 and 4 possess reduced activity, although they still bind a range of inhibitors with the same affinity as longer fragments. In the presence of Ang IV, IRAP is resistant to proteolysis, suggesting significant conformational changes occur upon binding of the inhibitor. We show that IRAP has a second Zn(2+) binding site, not associated with the catalytic region, which is lost upon binding Ang IV. Modulation of activity caused by domains 3 and 4 is consistent with a conformational change regulating access to the active site of IRAP.Download cloud_download
date_range Published on June 1, 2008
share Ye S, Chai SY, Lew RA, Ascher DB, Morton CJ, Parker MW, Albiston AL (2008) Biochem Cell Biol 86 (3):251-261.
Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the 'entrance' of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys1-vasopressin (2-9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine1-angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH2-terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.Download cloud_download
date_range Published on May 1, 2008
share Wun KS, Miles LA, Crespi GA, Wycherley K, Ascher DB, Barnham KJ, Cappai R, Beyreuther K, Masters CL, Parker MW, McKinstry WJ (2008Acta Crystallogr Sect F Struct Biol Cryst Commun 64 (Pt 5):438-441.
The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid beta peptide (Abeta) associated with Alzheimer's disease. This region of Abeta has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Abeta peptides Abeta(1-16) and Abeta(1-28) are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO(4); they belonged to the orthorhombic space group P2(1)2(1)2(1) and diffracted to 1.6 A resolution. The complexes of WO2 Fab with either Abeta(1-16) or Abeta(1-28) were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2(1)2(1)2(1), and diffracted to 1.6 A resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Abeta(1-42) in PEG 550 MME. This second form belonged to space group P2(1) and diffracted to 1.9 A resolution.Download cloud_download
date_range Published on Dec. 3, 2008
share Chai SY, Yeatman HR, Parker MW, Ascher DB, Thompson PE, Mulvey HT, Albiston AL (2008) BMC Neurosci 9 Suppl 2:S14.
The peptides angiotensin IV and LVV-hemorphin 7 were found to enhance memory in a number of memory tasks and reverse the performance deficits in animals with experimentally induced memory loss. These peptides bound specifically to the enzyme insulin-regulated aminopeptidase (IRAP), which is proposed to be the site in the brain that mediates the memory effects of these peptides. However, the mechanism of action is still unknown but may involve inhibition of the aminopeptidase activity of IRAP, since both angiotensin IV and LVV-hemorphin 7 are competitive inhibitors of the enzyme. IRAP also has another functional domain that is thought to regulate the trafficking of the insulin-responsive glucose transporter GLUT4, thereby influencing glucose uptake into cells. Although the exact mechanism by which the peptides enhance memory is yet to be elucidated, IRAP still represents a promising target for the development of a new class of cognitive enhancing agents.Download cloud_download
date_range Published on Dec. 5, 2008
share Ascher DB, Crespi GA, Ng HL, Morton CJ, Parker MW (2008) J Proteomics Bioinform 1:464-476.
Cognitive decline most commonly associated with Alzheimer’s dementia can also result from other conditions including cerebral ischemia or brain trauma. One quarter of people over the age of 65 are estimated to suffer some form of cognitive impairment underscoring the need for effective classes of cognitive-enhancing agents. In this mini-review we highlight recent work from our laboratory on the structural biology of Alzheimers disease and other memory disorders that was presented at the recent PRICPS – AOHUPO 2008 Conference held in Cairns, Australia. Our current work is focused on two proteins, amyloid precursor protein and insulin-regulated aminopeptidase, that are promising targets for the development of anti-Alzheimer’s drugs and as cognitive enhancers. In both cases structures determined by X-ray crystallography are being used to discover promising lead compounds by structure-based drug design.Download cloud_download
date_range Published on Dec. 1, 2008
share Albiston AL, Morton CJ, Ng HL, Pham V, Yeatman HR, Ye S, Fernando RN, De Bundel D, Ascher DB, Mendelsohn FA, Parker MW, Chai SY (2008) FASEB J 22 (12):4209-4217.
Approximately one-quarter of people over the age of 65 are estimated to suffer some form of cognitive impairment, underscoring the need for effective cognitive-enhancing agents. Insulin-regulated aminopeptidase (IRAP) is potentially an innovative target for the development of cognitive enhancers, as its peptide inhibitors exhibit memory-enhancing effects in both normal and memory-impaired rodents. Using a homology model of the catalytic domain of IRAP and virtual screening, we have identified a class of nonpeptide, small-molecule inhibitors of IRAP. Structure-based computational development of an initial "hit" resulted in the identification of two divergent families of compounds. Subsequent medicinal chemistry performed on the highest affinity compound produced inhibitors with nanomolar affinities (K(i) 20-700 nM) for IRAP. In vivo efficacy of one of these inhibitors was demonstrated in rats with an acute dose (1 nmol in 1 microl) administered into the lateral ventricles, improving performance in both spatial working and recognition memory paradigms. We have identified a family of specific IRAP inhibitors that is biologically active which will be useful both in understanding the physiological role of IRAP and potentially in the development of clinically useful cognitive enhancers. Notably, this study also provides unequivocal proof of principal that inhibition of IRAP results in memory enhancement.Download cloud_download
date_range Published on Sept. 5, 2018
share Abayakoon P, Jin Y, Lingford JP, Petricevic M, John A, Ryan E, Wai-Ying Mui J, Pires DEV, Ascher DB, Davies GJ, Goddard-Borger ED, Williams SJ. (2018). ACS Central Science, 4, 1266-1273.
An estimated 10 billion tonnes of sulfoquinovose (SQ) are produced and degraded each year. Prokaryotic sulfoglycolytic pathways catabolize sulfoquinovose (SQ) liberated from plant sulfolipid, or its delipidated form α-d-sulfoquinovosyl glycerol (SQGro), through the action of a sulfoquinovosidase (SQase), but little is known about the capacity of SQ glycosides to support growth. Structural studies of the first reported SQase (Escherichia coli YihQ) have identified three conserved residues that are essential for substrate recognition, but crossover mutations exploring active-site residues of predicted SQases from other organisms have yielded inactive mutants casting doubt on bioinformatic functional assignment. Here, we show that SQGro can support the growth of E. coli on par with d-glucose, and that the E. coli SQase prefers the naturally occurring diastereomer of SQGro. A predicted, but divergent, SQase from Agrobacterium tumefaciens proved to have highly specific activity toward SQ glycosides, and structural, mutagenic, and bioinformatic analyses revealed the molecular coevolution of catalytically important amino acid pairs directly involved in substrate recognition, as well as structurally important pairs distal to the active site. Understanding the defining features of SQases empowers bioinformatic approaches for mapping sulfur metabolism in diverse microbial communities and sheds light on this poorly understood arm of the biosulfur cycle.Download cloud_download
date_range Published on Oct. 18, 2018
share Portelli S, Phelan JE, Ascher DB, Clark TG, Furnham N (2018) Scientific Reports 8 (1):15356.
Genomic studies of Mycobacterium tuberculosis bacteria have revealed loci associated with resistance to anti-tuberculosis drugs. However, the molecular consequences of polymorphism within these candidate loci remain poorly understood. To address this, we have used computational tools to quantify the effects of point mutations conferring resistance to three major anti-tuberculosis drugs, isoniazid (n = 189), rifampicin (n = 201) and D-cycloserine (n = 48), within their primary targets, katG, rpoB, and alr. Notably, mild biophysical effects brought about by high incidence mutations were considered more tolerable, while different structural effects brought about by haplotype combinations reflected differences in their functional importance. Additionally, highly destabilising mutations such as alr Y388, highlighted a functional importance of the wildtype residue. Our qualitative analysis enabled us to relate resistance mutations onto a theoretical landscape linking enthalpic changes with phenotype. Such insights will aid the development of new resistance-resistant drugs and, via an integration into predictive tools, in pathogen surveillance.Download cloud_download
date_range Published on April 4, 2019
share Pires DEV, Rodrigues CHM, Albanaz ATS, Karmakar M, Myung Y, Xavier J, Michanetzi E-M, Portelli S, Ascher DB (2019) Exploring Protein Supersecondary Structure Through Changes in Protein Folding, Stability, and Flexibility. In: Kister AE (ed) Protein Supersecondary Structures: Methods and Protocols. Springer New York, New York, NY, pp 173-185.
The ability to predict how mutations affect protein structure, folding, and flexibility can elucidate the molecular mechanisms leading to disruption of supersecondary structures, the emergence of phenotypes, as well guiding rational protein engineering. The advent of fast and accurate computational tools has enabled us to comprehensively explore the landscape of mutation effects on protein structures, prioritizing mutations for rational experimental validation.Here we describe the use of two complementary web-based in silico methods, DUET and DynaMut, developed to infer the effects of mutations on folding, stability, and flexibility and how they can be used to explore and interpret these effects on protein supersecondary structures.Download cloud_download
date_range Published on April 28, 2020
share Jatana N, Ascher DB, Pires DEV, Gokhale RS, Thukral L (2019) Human LC3 and GABARAP subfamily members achieve functional specificity via specific structural modulations. Autophagy:1-17.
Autophagy is a conserved adaptive cellular pathway essential to maintain a variety of physiological functions. Core components of this machinery are the six human Atg8 orthologs that initiate formation of appropriate protein complexes. While these proteins are routinely used as indicators of autophagic flux, it is presently not possible to discern their individual biological functions due to our inability to predict specific binding partners. In our attempts towards determining downstream effector functions, we developed a computational pipeline to define structural determinants of human Atg8 family members that dictate functional diversity. We found a clear evolutionary separation between human LC3 and GABARAP subfamilies and also defined a novel sequence motif responsible for their specificity. By analyzing known protein structures, we observed that functional modules or microclusters reveal a pattern of intramolecular network, including distinct hydrogen bonding of key residues (F52/Y49; a subset of HP2) that may directly modulate their interaction preferences. Multiple molecular dynamics simulations were performed to characterize how these proteins interact with a common protein binding partner, PLEKHM1. Our analysis showed remarkable differences in binding modes via intrinsic protein dynamics, with PLEKHM1-bound GABARAP complexes showing less fluctuations and higher number of contacts. We further mapped 373 genomic variations and demonstrated that distinct cancer-related mutations are likely to lead to significant structural changes. Our findings present a quantitative framework to establish factors underlying exquisite specificity of human Atg8 proteins, and thus facilitate the design of precise modulators. Abbreviations: Atg: autophagy-related; ECs: evolutionary constraints; GABARAP: GABA type A receptor-associated protein; HsAtg8: human Atg8; HP: hydrophobic pocket; KBTBD6: kelch repeat and BTB domain containing 6; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MD: molecular dynamics; HIV-1 Nef: human immunodeficiency virus type 1 negative regulatory factor; PLEKHM1: pleckstrin homology and RUN domain containing M1; RMSD: root mean square deviation; SQSTM1/p62: sequestosome 1; WDFY3/ALFY: WD repeat and FYVE domain containing 3.Download cloud_download
date_range Published on May 29, 2019
share Karmakar M, Rodrigues CHM, Holt KE, Dunstan SJ, Denholm J, Ascher DB (2019) Empirical ways to identify novel Bedaquiline resistance mutations in AtpE. PLOS ONE 14 (5):e0217169.
Clinical resistance against Bedaquiline, the first new anti-tuberculosis compound with a novel mechanism of action in over 40 years, has already been detected in Mycobacterium tuberculosis. As a new drug, however, there is currently insufficient clinical data to facilitate reliable and timely identification of genomic determinants of resistance. Here we investigate the structural basis for M. tuberculosis associated bedaquiline resistance in the drug target, AtpE. Together with the 9 previously identified resistance-associated variants in AtpE, 54 non-resistance-associated mutations were identified through comparisons of bedaquiline susceptibility across 23 different mycobacterial species. Computational analysis of the structural and functional consequences of these variants revealed that resistance associated variants were mainly localized at the drug binding site, disrupting key interactions with bedaquiline leading to reduced binding affinity. This was used to train a supervised predictive algorithm, which accurately identified likely resistance mutations (93.3% accuracy). Application of this model to circulating variants present in the Asia-Pacific region suggests that current circulating variants are likely to be susceptible to bedaquiline. We have made this model freely available through a user-friendly web interface called SUSPECT-BDQ, StrUctural Susceptibility PrEdiCTion for bedaquiline (http://biosig.unimelb.edu.au/suspect_bdq/). This tool could be useful for the rapid characterization of novel clinical variants, to help guide the effective use of bedaquiline, and to minimize the spread of clinical resistance.Download cloud_download
date_range Published on Feb. 8, 2019
share Ascher DB, Spiga O, Sekelska M, Pires DEV, Bernini A, Tiezzi M, Kralovicova J, Borovska I, Soltysova A, Olsson B, Galderisi S, Cicaloni V, Ranganath L, Santucci A, Zatkova A (2019) Homogentisate 1,2-dioxygenase (HGD) gene variants, their analysis and genotype–phenotype correlations in the largest cohort of patients with AKU. European Journal of Human Genetics 27 (6):888-902.
Alkaptonuria (AKU) is a rare metabolic disorder caused by a deficient enzyme in the tyrosine degradation pathway, homogentisate 1,2-dioxygenase (HGD). In 172 AKU patients from 39 countries, we identified 28 novel variants of the HGD gene, which include three larger genomic deletions within this gene discovered via self-designed multiplex ligation-dependent probe amplification (MLPA) probes. In addition, using a reporter minigene assay, we provide evidence that three of eight tested variants potentially affecting splicing cause exon skipping or cryptic splice-site activation. Extensive bioinformatics analysis of novel missense variants, and of the entire HGD monomer, confirmed mCSM as an effective computational tool for evaluating possible enzyme inactivation mechanisms. For the first time for AKU, a genotype-phenotype correlation study was performed for the three most frequent HGD variants identified in the Suitability Of Nitisinone in Alkaptonuria 2 (SONIA2) study. We found a small but statistically significant difference in urinary homogentisic acid (HGA) excretion, corrected for dietary protein intake, between variants leading to 1% or >30% residual HGD activity. There was, interestingly, no difference in serum levels or absolute urinary excretion of HGA, or clinical symptoms, indicating that protein intake is more important than differences in HGD variants for the amounts of HGA that accumulate in the body of AKU patients.Download cloud_download
date_range Published on May 22, 2019
share Rodrigues CHM, Myung Y, Pires DEV, Ascher DB (2019) mCSM-PPI2: predicting the effects of mutations on protein–protein interactions. Nucleic Acids Research 47 (W1):W338-W344.
Protein-protein Interactions are involved in most fundamental biological processes, with disease causing mutations enriched at their interfaces. Here we present mCSM-PPI2, a novel machine learning computational tool designed to more accurately predict the effects of missense mutations on protein-protein interaction binding affinity. mCSM-PPI2 uses graph-based structural signatures to model effects of variations on the inter-residue interaction network, evolutionary information, complex network metrics and energetic terms to generate an optimised predictor. We demonstrate that our method outperforms previous methods, ranking first among 26 others on CAPRI blind tests. mCSM-PPI2 is freely available as a user friendly webserver at http://biosig.unimelb.edu.au/mcsm_ppi2/.Download cloud_download
date_range Published on June 6, 2019
share Silk M, Petrovski S, Ascher DB (2019) MTR-Viewer: identifying regions within genes under purifying selection. Nucleic Acids Research 47 (W1):W121-W126.
Advances in genomic sequencing have enormous potential to revolutionize personalized medicine, however distinguishing disease-causing from benign variants remains a challenge. The increasing number of human genome and exome sequences available has revealed areas where unfavourable variation is removed through purifying selection. Here, we present the MTR-Viewer, a web-server enabling easy visualization at the gene or variant level of the Missense Tolerance Ratio (MTR), a measure of regional intolerance to missense variation calculated using variation from 240 000 exome and genome sequences. The MTR-Viewer enables exploration of MTR calculations, using different sliding windows, for over 18 000 human protein-coding genes and 85 000 alternative transcripts. Users can also view MTR scores calculated for specific ethnicities, to enable easy exploration of regions that may be under different selective pressure. The spatial distribution of population and known disease variants is also displayed on the protein's domain structure. Intolerant regions were found to be highly enriched for ClinVar pathogenic and COSMIC somatic missense variants (Mann-Whitney U test P < 2.2 × 10-16). As the MTR is not biased by known domains and protein features, it can highlight functionally important regions within genes overlooked or inaccessible by traditional methods. MTR-Viewer is freely available via a user friendly web-server at http://biosig.unimelb.edu.au/mtr-viewer/.Download cloud_download
date_range Published on July 15, 2019
share Singh V, Pacitto A, Donini S, Ferraris DM, Boros S, Illyés E, Szokol B, Rizzi M, Blundell TL, Ascher DB, Pato J, Mizrahi V (2019) Synthesis and Structure–Activity relationship of 1-(5-isoquinolinesulfonyl)piperazine analogues as inhibitors of Mycobacterium tuberculosis IMPDH. European Journal of Medicinal Chemistry 174:309-329.
Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.Download cloud_download
date_range Published on Sept. 11, 2019
share Sernee MF, Ralton JE, Nero TL, Sobala LF, Kloehn J, Vieira-Lara MA, Cobbold SA, Stanton L, Pires DEV, Hanssen E, Males A, Ward T, Bastidas LM, van der Peet PL, Parker MW, Ascher DB, Williams SJ, Davies GJ, McConville MJ (2019) A Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites. Cell Host & Microbe 26 (3):385-399.e389.
Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.Download cloud_download
date_range Published on Oct. 10, 2019
share Copoiu L, Torres PHM, Ascher DB, Blundell TL, Malhotra S (2019) ProCarbDB: a database of carbohydrate-binding proteins. Nucleic Acids Research.
Carbohydrate-binding proteins play crucial roles across all organisms and viruses. The complexity of carbohydrate structures, together with inconsistencies in how their 3D structures are reported, has led to difficulties in characterizing the protein-carbohydrate interfaces. In order to better understand protein-carbohydrate interactions, we have developed an open-access database, ProCarbDB, which, unlike the Protein Data Bank (PDB), clearly distinguishes between the complete carbohydrate ligands and their monomeric units. ProCarbDB is a comprehensive database containing over 5200 3D X-ray crystal structures of protein-carbohydrate complexes. In ProCarbDB, the complete carbohydrate ligands are annotated and all their interactions are displayed. Users can also select any protein residue in the proximity of the ligand to inspect its interactions with the carbohydrate ligand and with other neighbouring protein residues. Where available, additional curated information on the binding affinity of the complex and the effects of mutations on the binding have also been provided in the database. We believe that ProCarbDB will be an invaluable resource for understanding protein-carbohydrate interfaces. The ProCarbDB web server is freely available at http://www.procarbdb.science/procarb.Download cloud_download
date_range Published on Oct. 26, 2019
share Myung Y, Rodrigues CHM, Ascher DB, Pires DEV (2019) mCSM-AB2: guiding rational antibody design using graph-based signatures. Bioinformatics.
MOTIVATION: A lack of accurate computational tools to guide rational mutagenesis has made affinity maturation a recurrent challenge in antibody development. We previously showed that graph-based signatures can be used to predict the effects of mutations on antibody binding affinity. RESULTS: Here we present an updated and refined version of this approach, mCSM-AB2, capable of accurately modelling the effects of mutations on antibody-antigen binding affinity, through the inclusion of evolutionary and energetic terms. Using a new and expanded database of over 1800 mutations with experimental binding measurements and structural information, mCSM-AB2 achieved a Pearson's correlation of 0.73 and 0.77 across training and blind tests, respectively, outperforming available methods currently used for rational antibody engineering. AVAILABILITY AND IMPLEMENTATION: mCSM-AB2 is available as a user-friendly and freely-accessible web server providing rapid analysis of both individual mutations or the entire binding interface to guide rational antibody affinity maturation at http://biosig.unimelb.edu.au/mcsm_ab2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.Download cloud_download
date_range Published on Oct. 28, 2019
share Karpiyevich M, Adjalley S, Mol M, Ascher DB, Mason B, van der Heden van Noort GJ, Laman H, Ovaa H, Lee MCS, Artavanis-Tsakonas K (2019) Nedd8 hydrolysis by UCH proteases in Plasmodium parasites. PLOS Pathogens 15 (10):e1008086.
Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design.Download cloud_download
date_range Published on Oct. 29, 2019
share Kaminskas LM, Pires DEV, Ascher DB (2019) dendPoint: a web resource for dendrimer pharmacokinetics investigation and prediction. Scientific Reports 9 (1):15465.
Nanomedicine development currently suffers from a lack of efficient tools to predict pharmacokinetic behavior without relying upon testing in large numbers of animals, impacting success rates and development costs. This work presents dendPoint, the first in silico model to predict the intravenous pharmacokinetics of dendrimers, a commonly explored drug vector, based on physicochemical properties. We have manually curated the largest relational database of dendrimer pharmacokinetic parameters and their structural/physicochemical properties. This was used to develop a machine learning-based model capable of accurately predicting pharmacokinetic parameters, including half-life, clearance, volume of distribution and dose recovered in the liver and urine. dendPoint successfully predicts dendrimer pharmacokinetic properties, achieving correlations of up to r = 0.83 and Q2 up to 0.68. dendPoint is freely available as a user-friendly web-service and database at http://biosig.unimelb.edu.au/dendpoint . This platform is ultimately expected to be used to guide dendrimer construct design and refinement prior to embarking on more time consuming and expensive in vivo testing.Download cloud_download
date_range Published on Nov. 9, 2019
share Trapero A, Pacitto A, Chan DS-H, Abell C, Blundell TL, Ascher DB, Coyne AG (2019) Covalent inactivation of Mycobacterium thermoresistibile inosine-5′-monophosphate dehydrogenase (IMPDH). Bioorganic & Medicinal Chemistry Letters:126792.
Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in nucleotide biosynthesis. Because of its critical role in purine biosynthesis, IMPDH is a drug design target for immunosuppressive, anticancer, antiviral and antimicrobial chemotherapy. In this study, we use mass spectrometry and X-ray crystallography to show that the inhibitor 6-Cl-purine ribotide forms a covalent adduct with the Cys-341 residue of Mycobacterium thermoresistibile IMPDH.Download cloud_download
date_range Published on Oct. 1, 2019
share Karmakar M, Trauer JM, Ascher DB, Denholm JT (2019) Hyper transmission of Beijing lineage Mycobacterium tuberculosis: Systematic review and meta-analysis. J Infect 79 (6):572-581.
OBJECTIVES: The globally distributed "Beijing" lineage of Mycobacterium tuberculosis has been associated with outbreaks worldwide. Laboratory based studies have suggested that Beijing lineage may have increased fitness; however, it has not been established whether these differences are of epidemiological significance with regards to transmission. Therefore, we undertook a systematic review of epidemiological studies of tuberculosis clustering to compare the transmission dynamics of Beijing lineages versus the non-Beijing lineages. METHODS: We systematically searched Embase and MEDLINE before 31st December 2018, for studies which provided information on the transmission dynamics of the different M. tuberculosis lineages. We included articles that conducted population-based cross-sectional or longitudinal molecular epidemiological studies reporting information about extent of transmission of different lineages. The protocol for this systematic review was prospectively registered with PROSPERO (CDR42018088579). RESULTS: Of 2855 records identified by the search, 46 were included in the review, containing 42,700 patients from 27 countries. Beijing lineage was the most prevalent and highly clustered strain in 72.4% of the studies and had a higher likelihood of transmission than non-Beijing lineages (OR 1·81 [95% 1·28-2·57], I2 = 94·0%, τ2 = 0·59, p < 0·01). CONCLUSIONS: Despite considerable heterogeneity across epidemiological contexts, Beijing lineage appears to be more transmissible than other lineages.Download cloud_download
date_range Published on Sept. 6, 2019
share Bayley JP, Bausch B, Rijken JA, van Hulsteijn LT, Jansen JC, Ascher D, Pires DEV, Hes FJ, Hensen EF, Corssmit EPM, Devilee P, Neumann HPH (2019) Variant type is associated with disease characteristics in SDHB, SDHC and SDHD-linked phaeochromocytoma–paraganglioma. Journal of Medical Genetics:jmedgenet-2019-106214.
BACKGROUND: Pathogenic germline variants in subunits of succinate dehydrogenase (SDHB, SDHC and SDHD) are broadly associated with disease subtypes of phaeochromocytoma-paraganglioma (PPGL) syndrome. Our objective was to investigate the role of variant type (ie, missense vs truncating) in determining tumour phenotype. METHODS: Three independent datasets comprising 950 PPGL and head and neck paraganglioma (HNPGL) patients were analysed for associations of variant type with tumour type and age-related tumour risk. All patients were carriers of pathogenic germline variants in the SDHB, SDHC or SDHD genes. RESULTS: Truncating SDH variants were significantly over-represented in clinical cases compared with missense variants, and carriers of SDHD truncating variants had a significantly higher risk for PPGL (p<0.001), an earlier age of diagnosis (p<0.0001) and a greater risk for PPGL/HNPGL comorbidity compared with carriers of missense variants. Carriers of SDHB truncating variants displayed a trend towards increased risk of PPGL, and all three SDH genes showed a trend towards over-representation of missense variants in HNPGL cases. Overall, variant types conferred PPGL risk in the (highest-to-lowest) sequence SDHB truncating, SDHB missense, SDHD truncating and SDHD missense, with the opposite pattern apparent for HNPGL (p<0.001). CONCLUSIONS: SDHD truncating variants represent a distinct group, with a clinical phenotype reminiscent of but not identical to SDHB. We propose that surveillance and counselling of carriers of SDHD should be tailored by variant type. The clinical impact of truncating SDHx variants is distinct from missense variants and suggests that residual SDH protein subunit function determines risk and site of disease.Download cloud_download
date_range Published on Oct. 29, 2019
share Brown P, Consortium R, Zhou Y (2019) Large expert-curated database for benchmarking document similarity detection in biomedical literature search. Database 2019.
Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Download cloud_download