3 research outputs found
Understanding molecular consequences of putative drug resistant mutations in Mycobacterium tuberculosis.
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
Efficient a Priori Identification of Drug Resistant Mutations Using Dead-End Elimination and MM-PBSA.
Active site mutations that disrupt drug binding are an
important
mechanism of drug resistance. Computational methods capable of predicting
resistance a priori are poised to become extremely useful tools in
the fields of drug discovery and treatment design. In this paper,
we describe an approach to predicting drug resistance on the basis
of Dead-End Elimination and MM-PBSA that requires no prior knowledge
of resistance. Our method utilizes a two-pass search to identify mutations
that impair drug binding while maintaining affinity for the native
substrate. We use our method to probe resistance in four drug-target
systems: isoniazid-enoyl-ACP reductase (tuberculosis), ritonavir-HIV
protease (HIV), methotrexate-dihydrofolate reductase (breast cancer
and leukemia), and gleevec-ABL kinase (leukemia). We validate our
model using clinically known resistance mutations for all four test
systems. In all cases, the model correctly predicts the majority of
known resistance mutations
Investigating the structural impact of hiv-1 integrase natural occurring polymorphisms and novel mutations identified among group m subtypes circulating in sub-Saharan Africa
Thesis (DScMedSc)--Stellenbosch University, 2020.Introduction HIV/AIDS remains a major health concern worldwide, with sub-Saharan Africa (SSA) carrying the largest burden.HIV is characterised by extremely high genetic diversity, with all the major groups and subtypes circulating in SSA. Combination antiretroviral therapy (cART) have substantially reduced HIV related deaths, but this is counteracted by the development of HIVdrug resistance, caused by certain drug resistance-associated mutations (RAMS). Integrase (IN) strand transferase inhibitors (INSTIs), the newest class of antiretroviral drugs,has a high genetic barrier and can be used in individuals that previously exhibited resistance to other classes of drugs. The World Health Organisation (WHO) approved the use of Dolutegravir (DTG) as part of first-line cART.
Methods
This is a descriptive experimental design study, which aimed to identify IN natural occurring polymorphisms (NOP) among different HIV-1 group M subtypes and Drug resistance mutations within the HIV-1 pol gene fragment of INSTI naïve patients from South Africa (SA) and Cameroon (CR), using the Stanford University genotypic resistance interpretation algorithm. Structural computational methods that included; homology modelling, molecular docking, molecular dynamics simulations and interaction analysis was performed to understand the structural impact of mutations from diverse HIV-1 subtypes on DTG drug binding. ResultsWe observed low-level RAMs against INSTIs in SA (2.2%) and CR sequences (5.4%). Through Fisher’sexact test we noted that the two NOPs occurred: VI72I and R269K, with p-values ≤0. 05, were statistically enriched. The impact of having these mutations are yet to be fully understood. Through molecular modelling and stability predictions, we observed a destabilizing effect of the known G140S mutant on the HIV-1C IN protein structure and simulation analysis showed that it affected structural stability and flexibility of the protein structure. Interactions analysis of different drug binding conformations to different HIV-1 IN subtypes reported differences in the number of binding interactions to different HIV-1 IN subtypes, but we did not observe any significant differences in binding affinity for each INSTIs.
This implies no significant alteration to the binding site in the wild type IN, which may not prevent INSTIs drug binding. In addition, all accessory mutations that resulted in a change in the number of interactions encompassing residues were found within the stable alpha-helix secondary structure element and not in close proximity to the drug active site.ConclusionThe study data indicate that RAMS against INSTIs remain low both in SA and in CR.Subtype C in SA and CRF02_AG in CR continues to be the driving force ofthe epidemic. We further reported on the impact of various NOPs on drug susceptibility. The analyses suggested that NOPs does not have an impact on IN protein structure and stability,and does not affect drug binding in the WT IN, but the known mutation G140S affect DTG binding. The study support recommendations made by the WHO to use DTG as part of salvage therapy in patients with RAM’s and accessory mutations. Data obtained from thisstudy can help to tailor effective treatment strategies in the African population, where diverse HIV subtypes circulate.InleidingMIV/vigs bly wĂŞreldwyd ’n ernstige gesondheidskwessie, en Afrika suid van die Saharadra die swaarste las. MIV word deur uiters hoĂ« genetiese diversiteit gekenmerk, waarvan al die vernaamste groepe en subtipes in Afrika suid van die Sahara in omloop is. Kombinasie-antiretrovirale terapie (kART) het ’n aansienlike vermindering in MIV-verwante sterftes tot gevolg, hoewel dĂt teengewerk word deur die ontwikkeling van MIV-middelweerstandigheid vanweĂ« sekere middelweerstandigheidsverwante mutasies (oftewel RAM’s). Integrasestringtransferase-inhibitors (INSTI’s), die jongste klas antiretrovirale middels, het ’n hoĂ« genetiese skans en kan gebruik word by individue wat voorheen weerstandigheid teen ander klasse middels getoon het. Die WĂŞreldgesondheidsorganisasie (WGO) het die gebruik van dolutegravir (DTG) as deel van eerstelinie-kART goedgekeur.
MetodesHierdie studie gebruik ’n beskrywende proefondervindelike ontwerp om natuurlike integrase-(IN-) polimorfismes (NOP’s) in verskillende MIV-1-groep-M-subtipes en middelweerstandige mutasies in die MIV-1-pol-geenfragment van INSTI-naïewe pasiënte van Suid-Afrika (SA) en Kameroen (KR) te identifiseer. Dit word met behulp van die Universiteit van Stanford se algoritme vir genotipiese weerstandigheidsvertolking gedoen. Strukturele berekeningsmetodes soos homologiemodellering, molekulêre koppeling, molekulêre dinamikasimulasies en interaksieontleding is uitgevoer om die strukturele impak van mutasies uit diverse MIV-1-subtipes op DTG-middelbinding te verstaan.
ResultateLaevlak-RAM’s teen INSTI’s is in reekse van SA (2,2%) Ă©n KR (5,4%) opgemerk. Fisher se eksakte toets het twee NOP’s opgespoor –VI72I en R269K –met p-waardes van ≤0,05, wat statisties verryk was. Die impak van hierdie mutasies is nog nie ten volle duidelik nie. Deur molekulĂŞre modellering en stabiliteitsvoorspellings het ons bepaal dat die bekende G140S-mutant ’n destabiliseringsuitwerking het op die MIV-1C-IN-proteĂŻenstruktuur. Simulasieontleding het getoon dat dĂt die strukturele stabiliteit en buigbaarheid van die proteĂŻenstruktuur beĂŻnvloed. Interaksieontleding van middelbindingskonformasies met MIV-1-IN-subtipes het verskille in die getal bindingsinteraksies met verskillende subtipes opgelewer, maar geen beduidende verskille in bindingsaffiniteit is vir enige van die INSTI’s opgemerk nie. DĂt impliseer dat daar geen beduidende aanpassing is in die bindingsetel by die wilde-tipe IN wat INSTI-middelbinding kan verhoed nie. Daarbenewens is alle bykomstige mutasies wat ’n verandering in die getal interaksies in residu’s veroorsaak het in die stabiele alfaheliks-sekondĂŞre struktuurelement aangetref, en nie naby die aktiewe setel van die middel nie.GevolgtrekkingDie studiedata toon dat RAM’s teen INSTI’s steeds laag is in sowel SA as KR. Subtipe C in SA en CRF02_AG in KR bly die dryfkrag agter dieepidemie. Daarbenewens is daar oor die impak van verskillende NOP’s op middelvatbaarheid verslag gedoen. Die ontledings toon dat NOP’s nie ’n impak op IN-proteĂŻenstruktuur en -stabiliteit het nie, en ook nie middelbinding in die WT-IN beĂŻnvloed nie. Nogtans het die bekende mutasie G140S wĂ©l ’n invloed op DTG-binding. Die studie ondersteun die WGO se aanbeveling dat DTG as deel van reddingsbehandeling by pasiĂ«nte met RAM’s en bykomstige mutasies gebruik word. Die data uit hierdie studie kan doeltreffende behandelingstrategieĂ« help ontwikkel vir die bevolking van Afrika, waar diverse MIV-suptipes in omloop is.Doctora