Protease Subtype Research Articles

Mechanism of drug resistance in HIV-1 protease subtype C in the presence of Atazanavir

AIDS is one of the deadliest diseases in the history of humankind caused by HIV. Despite the technological development, curtailing the viral infection inside human host still remains a challenge. Therapies such as HAART uses a combination of drugs to inhibit the viral activity. One of the important targets includes HIV protease and inhibiting its activity will minimize the production of mature structural proteins. However, the genetic diversity and the occurrence of drug resistant mutations adds complexity to effective drug design. In this study, we aimed at understanding the drug binding mechanism of one such subtype, namely subtype C and its insertion variant L38HL. We performed multiple molecular dynamics simulations along with binding free energy analysis of wild-type and L38HL bound to Atazanavir (ATV). From the analysis, we revealed that the insertion alters the hydrogen bond and hydrophobic interaction networks. The alterations in the interaction networks increase flexibility at the hinge-fulcrum interface. Further, the effects of these changes affect flap tip curling. Moreover, the changes in the hinge-fulcrum-cantilever interface alters the concerted motion of the functional regions leading to change in the direction of flap movement thus causing a subtle change in the active site volume. Additionally, formation of intramolecular hydrogen bonds in the ATV docked to L38HL restricted the movement of R1 and R2 groups thereby altering the interactions. Overall, the changes in the flexibility of flap together with the changes in the active site volume and compactness of the ligand provide insights for increased binding affinity of ATV with L38HL.

Molecular alteration in drug susceptibility against subtype B and C-SA HIV-1 proteases: MD study

HIV-1 protease (HIV PR) is one of the most promising targets for anti-HIV drug discovery. Extensive anti-HIV drug discovery research was focused on HIV-1 subtype B protease though FDA-approved protease inhibitors (PIs) generally exhibit altered biological activities against other protease subtypes such as South African subtype C (C-SA) protease that is prevalent in sub-Saharan Africa. Recent experimental reports highlight the differences in dynamic behaviors between these two protease subtypes. Herein, the dynamics of three FDA approved PIs, namely atazanavir (ATV), darunavir (DRV), and ritonavir (RTV), were thoroughly analyzed for both subtypes B and C protease complexes using molecular dynamic (MD) simulation in explicit solvent. The comparative MD post-analyses through flap dynamics, cross-correlation analyses, principal component analyses (PCA), per residue decomposition analyses, and MM-GBSA binding free energy analyses, revealed the altered responses observed in the complexes of these PIs in these two protease subtypes. The calculated theoretical binding energy analyses are consistent with the experiment to a considerable extent. Theoretical binding energies combined with flap dynamics appear to correspond to altered drug susceptibility against subtype B and C-SA HIV-1 proteases. The current analyses provide useful insight on the mechanism of action of PIs against these two different HIV-1 protease subtypes which will be helpful in developing new anti-protease compounds.

Baseline prevalence and emergence of protease inhibitor resistance mutations following treatment in chronic HCV genotype-1-infected individuals.

The HCV NS3/4A serine protease inhibitors (PIs) boceprevir (BOC), telaprevir (TVR) and simeprevir (SMV) are approved for treatment of chronic hepatitis C infection in combination with pegylated interferon and ribavirin. The present study investigated the prevalence of HCV NS3 drug resistance mutations (DRMs) associated with HCV genotype-1-infected individuals at baseline and in viral breakthrough following BOC and TVR treatment. HCV genotype-1-infected individuals were enrolled in a multicentre, prospective outcomes study. The HCV NS3 viral protease was analysed for DRMs at baseline (n=164) and at viral breakthrough (n=18) following BOC/TVR treatment. Viral NS3 protease subtype analysis showed 65.2% (107/164) were HCV subtype-1a and 34.8% (57/164) were HCV subtype-1b infections. Naturally occurring PI DRMs in NS3 (V36L, T54S, V55A, Q80K/R and I132V) were identified in 57.3% (94/164) cases at baseline. The NS3 Q80K polymorphism was found in 43/107 (40.2%) of HCV subtype-1a and exclusively in clade 1 (43/82; 52.4%) versus clade 2 viruses (0/25; 0%, P<10(-6)). The pretreatment I132V variant was found in 78.9% (45/57) of subtype-1b. Of 18 patients who had viral breakthrough, the majority was subtype-1a (77.8%, 14/18). BOC/TVR-associated DRMs were detected in 94.4% (17/18), of which 64.7% (11/17) emerged on-treatment. To ensure the most appropriate direct-acting antiviral-based treatment regimen is employed, baseline reporting of clade and resistance mutations for HCV subtype-1a using nucleotide sequence-based analysis is warranted prior to commencement of therapy.

The Impact of Active Site Mutations of South African HIV PR on Drug Resistance: Insight from Molecular Dynamics Simulations, Binding Free Energy and Per-Residue Footprints

Molecular dynamics simulations and binding free energy calculations were used to provide an understanding of the impact of active site drug-resistant mutations of the South African HIV protease subtype C (C-SA HIV PR), V82A and V82F/I84V on drug resistance. Unique per-residue interaction energy 'footprints' were developed to map the overall drug-binding profiles for the wild type and mutants. Results confirmed that these mutations altered the overall binding landscape of the amino acid residues not only in the active site region but also in the flaps as well. Four FDA-approved drugs were investigated in this study; these include ritonavir (RTV), saquinavir (SQV), indinavir (IDV), and nelfinavir (NFV). Computational results compared against experimental findings were found to be complementary. Against the V82F/I84V variant, saquinavir, indinavir, and nelfinavir lose remarkable entropic contributions relative to both wild-type and V82A C-SA HIV PRs. The per-residue energy 'footprints' and the analysis of ligand-receptor interactions for the drug complexes with the wild type and mutants have also highlighted the nature of drug interactions. The data presented in this study will prove useful in the design of more potent inhibitors effective against drug-resistant HIV strains.

Targeting Dynamic Pockets of HIV-1 Protease by Structure-Based Computational Screening for Allosteric Inhibitors

We present the discovery of low molecular weight inhibitors of human immunodeficiency virus 1 (HIV-1) protease subtype B that were identified by structure-based virtual screening as ligands of an allosteric surface cavity. For pocket identification and prioritization, we performed a molecular dynamics simulation and observed several flexible, partially transient surface cavities. For one of these presumable ligand-binding pockets that are located in the so-called "hinge region" of the identical protease chains, we computed a receptor-derived pharmacophore model, with which we retrieved fragment-like inhibitors from a screening compound pool. The most potent hit inhibited protease activity in vitro in a noncompetitive mode of action. Although attempts failed to crystallize this ligand bound to the enzyme, the study provides proof-of-concept for identifying innovative tool compounds for chemical biology by addressing flexible protein models with receptor pocket-derived pharmacophore screening.

Identification of bistable populations of Porphyromonas gingivalis that differ in epithelial cell invasion

Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen Porphyromonas gingivalis exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct subtypes within a population of P. gingivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly invasive subtype invades cells at 10-30-fold higher levels than the poorly invasive subtype and remains highly invasive for approximately 12-16 generations. Analysis of the gingipain activity of these subtypes revealed that the highly invasive type had reduced cell-associated arginine-specific protease activity. The role of Arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an Arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition, a population of ΔrgpAB bacteria did not contain a hyperinvasive subtype. Screening of the protease activity of wild-type populations of both strains identified high and low protease subtypes which also showed a corresponding reduction or enhancement, respectively, of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that includes oxidative stress resistance and iron transport genes, and which might be critical to invasion of or survival within epithelial cells.

Molecular dynamics simulations exploring drug resistance in HIV-1 proteases

Although HIV-1 subtype B still dominates the epidemic AIDS in developed countries, an increasing number of people in developing countries are suffering from an epidemic of non-subtype B viruses. What is worse, the efficacy of the combinational use of antiretroviral drugs is gradually compromised by the rapid development of drug resistance. To gain an insight into drug resistance, 10-ns MD simulations were simultaneously conducted on the complexes of the TL-3 inhibitor with 4 different proteases (B wt, B mut, F wt and F mut), among which the complex of the B wt protease with the TL-3 inhibitor was treated as the control group. Detailed analyses of MD data indicated that the drug resistance of B mut against TL-3 mainly derived from loss of an important hydrogen bond and that of F wt was caused by the decrease of hydrophobic interactions in S1/S1’ pocket, while both of the two reasons mentioned above were the cause of the F mut protease’s resistance. These results are in good agreement with the previous experiments, revealing a possible mechanism of drug resistance for the aforementioned protease subtypes against the TL-3 inhibitor. Additionally, another indication was obtained that the mutations of M36I, V82A and L90M may induce structural transforms so as to alter the inhibitor’s binding mode.

Analysis of correlated mutations in HIV-1 protease using spectral clustering

Motivation: The ability of human immunodeficiency virus-1 (HIV-1) protease to develop mutations that confer multi-drug resistance (MDR) has been a major obstacle in designing rational therapies against HIV. Resistance is usually imparted by a cooperative mechanism that can be elucidated by a covariance analysis of sequence data. Identification of such correlated substitutions of amino acids may be obscured by evolutionary noise.Results: HIV-1 protease sequences from patients subjected to different specific treatments (set 1), and from untreated patients (set 2) were subjected to sequence covariance analysis by evaluating the mutual information (MI) between all residue pairs. Spectral clustering of the resulting covariance matrices disclosed two distinctive clusters of correlated residues: the first, observed in set 1 but absent in set 2, contained residues involved in MDR acquisition; and the second, included those residues differentiated in the various HIV-1 protease subtypes, shortly referred to as the phylogenetic cluster. The MDR cluster occupies sites close to the central symmetry axis of the enzyme, which overlap with the global hinge region identified from coarse-grained normal-mode analysis of the enzyme structure. The phylogenetic cluster, on the other hand, occupies solvent-exposed and highly mobile regions. This study demonstrates (i) the possibility of distinguishing between the correlated substitutions resulting from neutral mutations and those induced by MDR upon appropriate clustering analysis of sequence covariance data and (ii) a connection between global dynamics and functional substitution of amino acids.Contact: bahar@ccbb.pitt.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Bicyclic carbamates as inhibitors of papain-like cathepsin proteases

A 6-oxa-1-aza-bicyclo[3.2.1]octan-7-one system inhibits the proteolytic activity of several cysteine proteases belonging to the papain family. In vitro mechanistic studies and in silico calculations suggest that the minimal π-overlap between the bridgehead nitrogen and the carbonyl leads to a considerable weakening of the urethane system, making it susceptible to nucleophilic attack from the active site thiol group. The resulting covalent adduct is slowly hydrolyzed, releasing the hydroxypiperidine product of the inhibitor. Synthesis and testing of a set of analogs led to variable protease subtype selectivities ranging from micromolar to nanomolar potencies.

HIV-1 subtype C reverse transcriptase and protease genotypes in Zimbabwean patients failing antiretroviral therapy.

HIV-1 drug resistance mutations have been identified and characterized mostly in subtype B HIV-1 infection. The extent to which antiretroviral drugs select for drug resistance mutations in non-subtype B HIV-1 is not known. We obtained HIV-1 reverse transcriptase (RT) and protease sequences from 21 Zimbabwean patients failing antiretroviral drug therapy. We compared these sequences with 56 published RT and protease subtype C sequences from untreated patients, 990 RT and 1140 protease subtype B sequences from treated patients, and 340 RT and 907 protease subtype B sequences from untreated patients and identified four mutation categories of subtype C HIV-1. Seventeen of the 21 patients (81%) had known drug resistance mutations. Mutations at 15 RT and 11 protease positions were more common in subtype C isolates than in subtype B isolates. HIV-1 subtype C-infected individuals receiving antiretroviral therapy develop many of the known subtype B drug resistance mutations. Comparison of subtype C RT and protease sequences with a large database of subtype B sequences identified subtype C-specific polymorphisms and candidate drug resistance mutations.

Human immunodeficiency virus type 1 reverse-transcriptase and protease subtypes: classification, amino acid mutation patterns, and prevalence in a northern California clinic-based population.

Phylogenetic analysis of the reverse transcriptase (RT) and protease of 117 published complete human immunodeficiency virus (HIV) type 1 genome sequences demonstrated that these genes cluster into distinct subtypes. There was a slightly higher proportion of informative sites in the RT (40.4%) than in the protease (34.8%; P= .03). Although most variation between subtypes was due to synonymous nucleotide substitutions, several subtype-specific amino acid patterns were observed. In the protease, the subtype-specific variants included 7 positions associated with drug resistance. Variants at positions 10, 20, 36, and 82 were more common in non-B isolates, whereas variants at positions 63, 77, and 93 were more common in subtype B isolates. In the RT, the subtype-specific mutations did not include positions associated with anti-retroviral drug resistance. RT and protease sequences from 2246 HIV-infected persons in northern California were also examined: 99.4% of the sequences clustered with subtype B, whereas 0.6% clustered with subtype A, C, or D.

Genotypic and phenotypic evidence of different drug-resistance mutation patterns between B and non-B subtype isolates of human immunodeficiency virus type 1 found in Brazilian patients failing HAART.

We have investigated the phenotypic and genotypic susceptibility of 14 HIV-1 strains isolated from individuals failing HAART therapy to protease inhibitors (PI). Proviral and plasma viral pol gene fragment were amplified, sequenced and subtyped. Nine samples clustered with protease subtype B reference strains and the remaining samples were classified as non-B subtype corresponding to subtype F (n = 4) and subtype A (n = 1). Although all patients were treated with similar P1 drug regimen, the non-B subtype isolates did not present the L90M and 184V mutations and used mainly G48V and V82A/F to achieve drug resistance. A strong cross-resistance phenotype among all four PI was associated with the mutation L90M in the subtype-B isolates, and with G48V and V82A/F in the non-B counterparts. This observation revealed that the non-B viruses tested had specific genotypic characteristics contrasting with the subtype-B isolates.

Genetic variation and susceptibilities to protease inhibitors among subtype B and F isolates in Brazil.

The genetic variation of the human immunodeficiency virus type 1 (HIV-1) protease gene (prt) permits the classification of HIV-1 strains into five distinct protease subtypes, which follow the gag subtyping patterns. The susceptibilities of non-B-subtype strains to protease inhibitors (PIs) and other antiretroviral drugs remain largely unknown. Subtype F is the main non-B strain contributing to the Brazilian epidemic, accounting for 15 to 20% of these infections. In this work, we report the findings on 81 isolates from PI-naive Brazilian patients collected between 1993 and 1997. In addition, the relevant PI resistance mutations and their phenotypes were determined in vitro for 15 of these patients (B = 9 and F = 6). Among these, the subtype F samples evidenced high sensitivities in vitro to ritonavir and indinavir, with MICs at which 50 and 90% of the isolates are inhibited similar to those of both the Brazilian and the U.S. subtype B isolates. Analysis of the 81 Brazilian prt sequences demonstrated that the subtype F consensus sequence differs from the U.S. and Brazilian subtype B consensus in eight positions (I15V, E35D, M36I, R41K, R57K, Q61N, L63P, and L89M). The frequency of critical PI resistance substitutions (amino acid changes D30N, V82A/F/T, I84V, N88D, and L90M) among Brazilian isolates is very low (mean, 2.5%), and the associated secondary substitutions (amino acid positions 10L, 20K, 36M, 46M, 48G, 54I, 63P, 71A, and 77A) are infrequent. These observations document the relative rarity of resistance to PIs in the treatment of patients infected with HIV-1 subtype F in South America.