Kinetic and thermodynamic characterisation of HIV-protease inhibitors against E35D?G?S mutant in the South African HIV-1 subtype C protease.
Ontology highlight
ABSTRACT:
SUBMITTER: Maseko S
PROVIDER: S-EPMC6713120 | biostudies-literature | 2019 Dec
REPOSITORIES: biostudies-literature
Publications
Kinetic and thermodynamic characterisation of HIV-protease inhibitors against E35D↑G↑S mutant in the South African HIV-1 subtype C protease.
Maseko Sibusiso S Padayachee Eden E Maphumulo Siyabonga S Govender Thavendran T Sayed Yasien Y Maguire Glenn G Lin Johnson J Naicker Tricia T Baijnath Sooraj S Gerhardus Kruger Hendrik KH
Journal of enzyme inhibition and medicinal chemistry 20191201 1
Herein, we report the effect of nine FDA approved protease inhibitor drugs against a new HIV-1 subtype C mutant protease, E35D↑G↑S. The mutant has five mutations, E35D, two insertions, position 36 (G and S), and D60E. Kinetics, inhibition constants, vitality, Gibbs free binding energies are reported. The variant showed a decreased affinity for substrate and low catalytic efficiency compared to the wild type. There was a significant decrease in the binding of seven FDA approved protease inhibitor ...[more]
Similar Datasets
Project description:HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.
| S-EPMC3403440 | biostudies-literature
Project description:The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.
| S-EPMC2144115 | biostudies-other
Project description:BackgroundWhereas human immunodeficiency virus (HIV) subtype B-infected individuals generally progress to AIDS within 8-10 years, limited data exist for other clades, especially from Africa. We investigated rates of HIV disease progression of clade C-infected South African women.MethodsProspective seroincidence cohorts in KwaZulu-Natal were assessed for acute HIV infection monthly (n = 245) or every 3 months (n = 594) for up to 4 years. Rapid disease progression was defined as CD4 decline to <350 cells/µL by 2 years postinfection. Serial clinical and laboratory assessments were compared using survival analysis and logistic regression models.ResultsSixty-two women were identified at a median of 42 days postinfection (interquartile range, 34-59), contributing 282 person-years of follow-up. Mean CD4 count dropped by 39.6% at 3 months and 46.7% at 6 months postinfection in women with preinfection measurements. CD4 decline to <350 cells/µL occurred in 31%, 44%, and 55% of women at 1, 2, and 3 years postinfection, respectively, and to <500 cells/µL in 69%, 79%, and 81% at equivalent timepoints. Predictors of rapid progression were CD4 count at 3 months postinfection (hazard ratio [HR], 2.07; 95% confidence interval [CI], 1.31-3.28; P = .002), setpoint viral load (HR, 3.82; 95% CI, 1.51-9.67; P = .005), and hepatitis B coinfection (HR, 4.54; 95% CI, 1.31-15.69; P = .017). Conversely, presence of any of HLAB*1302, B*27, B*57, B*5801, or B*8101 alleles predicted non-rapid progression (HR, 0.19; 95% CI, .05-.74; P = .016).ConclusionsNearly half of subtype C-infected women progressed to a CD4 count <350 cells/µL within 2 years of infection. Implementing 2013 World Health Organization treatment guidelines (CD4 count <500 cells/µL) would require most individuals to start antiretroviral therapy within 1 year of HIV infection.
| S-EPMC4271037 | biostudies-literature
Project description:BackgroundProtein-protein interactions (PPIs) play important roles in biological functions. Studies of the effects of mutants on protein interactions can provide further understanding of PPIs. Currently, many databases collect experimental mutants to assess protein interactions, but most of these databases are old and have not been updated for several years.ResultsTo address this issue, we manually curated a kinetic and thermodynamic database of mutant protein interactions (dbMPIKT) that is freely accessible at our website. This database contains 5291 mutants in protein interactions collected from previous databases and the literature published within the last three years. Furthermore, some data analysis, such as mutation number, mutation type, protein pair source and network map construction, can be performed online.ConclusionOur work can promote the study on PPIs, and novel information can be mined from the new database. Our database is available in http://DeepLearner.ahu.edu.cn/web/dbMPIKT/ for use by all, including both academics and non-academics.
| S-EPMC6260753 | biostudies-literature
Project description:Understanding the mechanism of action of compounds capable of inhibiting amyloid-fibril formation is critical to the development of potential therapeutics against protein-misfolding diseases. A fundamental challenge for progress is the range of possible target species and the disparate timescales involved, since the aggregating proteins are simultaneously the reactants, products, intermediates, and catalysts of the reaction. It is a complex problem, therefore, to choose the states of the aggregating proteins that should be bound by the compounds to achieve the most potent inhibition. We present here a comprehensive kinetic theory of amyloid-aggregation inhibition that reveals the fundamental thermodynamic and kinetic signatures characterizing effective inhibitors by identifying quantitative relationships between the aggregation and binding rate constants. These results provide general physical laws to guide the design and optimization of inhibitors of amyloid-fibril formation, revealing in particular the important role of on-rates in the binding of the inhibitors.
| S-EPMC7533883 | biostudies-literature
Project description:BACKGROUND:Modest efficacy was reported for the HIV vaccine tested in the RV144 trial, which comprised a canarypox vector (ALVAC) and envelope (env) glycoprotein (gp120). These vaccine components were adapted to express HIV-1 antigens from strains circulating in South Africa, and the adjuvant was changed to increase immunogenicity. Furthermore, 12-month immunisation was added to improve durability. In the HIV Vaccine Trials Network (HVTN) 100 trial, we aimed to assess this new regionally adapted regimen for advancement to efficacy testing. METHODS:HVTN 100 is a phase 1/2, randomised controlled, double-blind trial at six community research sites in South Africa. We randomly allocated adults (aged 18-40 years) without HIV infection and at low risk of HIV infection to either the vaccine regimen (intramuscular injection of ALVAC-HIV vector [vCP2438] at 0, 1, 3, 6, and 12 months plus bivalent subtype C gp120 and MF59 adjuvant at 3, 6, and 12 months) or placebo, in a 5:1 ratio. Randomisation was done by computer-generated list. Participants, investigators, and those assessing outcomes were masked to random assignments. Primary outcomes included safety and immune responses associated with correlates of HIV risk in RV144, 2 weeks after vaccination at 6 months (month 6·5). We compared per-protocol participants (ie, those who completed the first four vaccinations and provided samples at month 6·5) from HVTN 100 with stored RV144 samples assayed contemporaneously. This trial is registered with the South African National Clinical Trials Registry (DOH-27-0215-4796) and ClinicalTrials.gov (NCT02404311). FINDINGS:Between Feb 9, 2015, and May 26, 2015, 252 participants were enrolled, of whom 210 were assigned vaccine and 42 placebo. 222 participants were included in the per-protocol analysis (185 vaccine and 37 placebo). 185 (100%) vaccine recipients developed IgG binding antibodies to all three vaccine-matched gp120 antigens with significantly higher titres (3·6-8·8 fold; all p<0·0001) than the corresponding vaccine-matched responses of RV144. The CD4+ T-cell response to the ZM96.C env protein in HVTN 100 was 56·4% (n=102 responders), compared with a response of 41·4% (n=79 responders) to 92TH023.AE in RV144 (p=0·0050). The IgG response to the 1086.C variable loops 1 and 2 (V1V2) env antigen in HVTN 100 was 70·5% (95% CI 63·5-76·6; n=129 responders), lower than the response to V1V2 in RV144 (99·0%, 95% CI 96·4-99·7; n=199 responders). INTERPRETATION:Although the IgG response to the HVTN 100 vaccine was lower than that reported in RV144, it exceeded the predicted 63% threshold needed for 50% vaccine efficacy using a V1V2 correlate of protection model. Thus, the subtype C HIV vaccine regimen qualified for phase 2b/3 efficacy testing, a critical next step of vaccine development. FUNDING:US National Institute of Allergy and Infectious Diseases (NIAID), and Bill & Melinda Gates Foundation.
| S-EPMC6028742 | biostudies-literature
Project description:Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric beta-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR(*), was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.
| S-EPMC2756696 | biostudies-literature
Project description:The South African national combination antiretroviral therapy (cART) roll-out program started in 2006, with over 4.4 million people accessing treatment since it was first introduced. HIV-1 drug resistance can hamper the success of cART. This study determined the patterns of HIV-1 drug-resistance associated mutations (RAMs) in People Living with HIV-1 (PLHIV-1). Receiving first (for children below 3 years of age) and second-line (for adults) cART regimens in South Africa. During 2017 and 2018, 110 patients plasma samples were selected, 96 samples including those of 17 children and infants were successfully analyzed. All patients were receiving a boosted protease inhibitor (bPI) as part of their cART regimen. The viral sequences were analyzed for RAMs through genotypic resistance testing. We performed genotypic resistance testing (GRT) for Protease inhibitors (PIs), Reverse transcriptase inhibitors (RTIs) and Integrase strand transfer inhibitors (InSTIs). Viral sequences were subtyped using REGAv3 and COMET. Based on the PR/RT sequences, HIV-1 subtypes were classified as 95 (99%) HIV-1 subtype C (HIV-1C) while one sample as 02_AG. Integrase sequencing was successful for 89 sequences, and all the sequences were classified as HIV-1C (99%, 88/89) except one sequence classified CRF02_AG, as observed in PR/RT. Of the 96 PR/RT sequences analyzed, M184V/I (52/96; 54%) had the most frequent RAM nucleoside reverse transcriptase inhibitor (NRTI). The most frequent non-nucleoside reverse transcriptase inhibitor (NNRTI) RAM was K103N/S (40/96, 42%). Protease inhibitor (PI) RAMs M46I and V82A were present in 12 (13%) of the sequences analyzed. Among the InSTI major RAM two (2.2%) sequences have Y143R and T97A mutations while one sample had T66I. The accessory RAM E157Q was identified in two (2.2%). The data indicates that the majority of the patients failed on bPIs didn't have any mutation; therefore adherence could be major issue in these groups of individuals. We propose continued viral load monitoring for better management of infected PLHIV.
| S-EPMC7099763 | biostudies-literature
Project description:BACKGROUND: Bacterial vaginosis (BV), which is highly prevalent in the African population, is one of the most common vaginal syndromes affecting women in their reproductive age placing them at increased risk for sexually transmitted diseases including infection by human immunodeficiency virus-1. The vaginal microbiota of a healthy woman is often dominated by the species belonging to the genus Lactobacillus namely L. crispatus, L. gasseri, L. jensenii and L. iners, which have been extensively studied in European populations, albeit less so in South African women. In this study, we have therefore identified the vaginal Lactobacillus species in a group of 40 African women from Soweto, a township on the outskirts of Johannesburg, South Africa. METHODS: Identification was done by cultivating the lactobacilli on Rogosa agar, de Man-Rogosa-Sharpe (MRS) and Blood agar plates with 5% horse blood followed by sequencing of the 16S ribosomal DNA. BV was diagnosed on the basis of Nugent scores. Since some of the previous studies have shown that the lack of vaginal hydrogen peroxide (H2O2) producing lactobacilli is associated with bacterial vaginosis, the Lactobacillus isolates were also characterised for their production of H2O2. RESULTS: Cultivable Lactobacillus species were identified in 19 out of 21 women without BV, in three out of five women with intermediate microbiota and in eight out of 14 women with BV. We observed that L. crispatus, L. iners, L. jensenii, L. gasseri and L. vaginalis were the predominant species. The presence of L. crispatus was associated with normal vaginal microbiota (P = 0.024). High level of H2O2 producing lactobacilli were more often isolated from women with normal microbiota than from the women with BV, although not to a statistically significant degree (P = 0.064). CONCLUSION: The vaginal Lactobacillus species isolated from the cohort of South African women are similar to those identified in European populations. In accordance with the other published studies, L. crispatus is related to a normal vaginal microbiota. Hydrogen peroxide production was not significantly associated to the BV status which could be attributed to the limited number of samples or to other antimicrobial factors that might be involved.
| S-EPMC3600991 | biostudies-literature
Project description:Human immunodeficiency virus type 1 protease (HIV-1 PR) is an essential enzyme in the HIV-1 life cycle. As such, this protein represents a major drug target in AIDS therapy, but emerging resistance to antiretroviral inhibitor cocktails, caused by high viral mutation rates, represents a significant challenge in AIDS treatment. Many mutations are not located within the active site or binding pocket, nor they do significantly modify the three-dimensional structural organization of the enzyme; hence, the mechanism(s) by which they alter inhibitor affinity for the protease remains uncertain. In this article, we present an all-atom computational analysis of the dynamic residue-residue coordination between the active site residues and the rest of the protein and of the energetic properties of different HIV-1 PR complexes. We analyze both the wild-type form and mutated forms that induce drug resistance. In particular, the results show differences between the wild type and the mutants in their mechanism of dynamic coordination, in the signal propagation between the active site residues and the rest of the protein, and in the energy networks responsible for the stabilization of the bound inhibitor conformation. Finally, we propose a dynamic and energetic explanation for HIV-1 protease drug resistance, and, through this model, we identify a possible new site that could be helpful in the design of a new family of HIV-1 PR allosteric inhibitors.
| S-EPMC2868114 | biostudies-literature