Evaluation of novel Saquinavir analogs for resistance mutation compatibility and potential as an HIV-Protease inhibitor drug

A fundamental issue related to therapy of HIV-1 infection is the emergence of viral mutations which severely limits the long term efficiency of the HIV-protease (HIV-PR) inhibitors. Development of new drugs is therefore continuously needed. Chemoinformatics enables to design and discover novel molecules analogous to established drugs using computational tools and databases. Saquinavir, an anti-HIV Protease drug is administered for HIV therapy. In this work chemoinformatics tools were used to design structural analogs of Saquinavir as ligand and molecular dockings at AutoDock were performed to identify potential HIV-PR inhibitors. The analogs S1 and S2 when docked with HIV-PR had binding energies of -4.08 and -3.07 kcal/mol respectively which were similar to that for Saquinavir. The molecular docking studies revealed that the changes at N2 of Saquinavir to obtain newly designed analogs S1 (having N2 benzoyl group at N1) and S2 (having 3-oxo-3phenyl propanyl group at N2) were able to dock with HIV-PR with similar affinity as that of Saquinavir. Docking studies and computationally derived pharmacodynamic and pharmacokinetic properties׳ comparisons at ACD/I-lab establish that analog S2 has more potential to evade the problem of drug resistance mutation against HIV-1 PR subtype-A. S2 can be further developed and tested clinically as a real alternative drug for HIV-1 PR across the clades in future.     

Modified solvent accessibility free energy prediction analysis of cyclic urea inhibitors binding to the HIV-1 protease

One of the most successful drug targets against AIDS in the last decade has been the HIV-1 protease (HIV-1 PR), an enzyme that processes the polyprotein gene products into active replicative viral proteins. In our quest for a wide-ranging, binding free energy function we have extended the solvent accessibility free energy predictor (SAFE_p) method, recently developed for peptidic HIV-1 PR inhibitors, to the study of the binding of cyclic urea (CU) HIV-1 PR inhibitors. Our results show that there is a need for a specific term depicting polar contacts to be added to the original SAFE_p analytical expression, an outcome not seen in our studies of HIV-1 PR peptidic inhibitors. Nevertheless, despite the higher profile of the electrostatic interactions in the binding of the CU inhibitors, our analysis indicates that CU inhibitor binding is still driven by the hydrophobic entropic contribution, as much as for the peptidic inhibitors.     

Litchi chinensis-derived terpenoid as anti-HIV-1 protease agent: structural design from molecular dynamics simulations

The molecular structures of the binding between human immunodeficiency virus-1 protease (HIV-1PR) and various inhibitors including existing extensive natural products extracts have been investigated for anti-HIV drug development. In this study, the binding of HIV-1PR and a terpenoid from Litchi chinensis extracts (3-oxotrirucalla-7,24-dien-21-oic acid) was investigated in order to clarify the inhibition effectiveness of this compound. Molecular dynamics (MD) simulations of HIV-1PR complex with 3-oxotrirucalla-7,24-dien-21-oic acid were performed including water molecules. The MD simulation results indicated the formation of hydrogen bonds between the oxygen atoms of the inhibitor and the catalytic aspartates, which are commonly found in inhibitors–protease complexes. On the other hand, no hydrogen bonding of this particular inhibitor to the flap region was found. In addition, the radial distribution function of water oxygens around the catalytic carboxylate nitrogens of Asp29 and Asp30 suggests that at least one or two water molecules are in the active site region whereas direct interaction of the inhibitor was found for catalytic carboxylate oxygen of Asp25. The results of this simulation, in comparison with the structures of other HIV-PR inhibitor complexes, could lead to a better understanding of the activity of 3-oxotrirucalla-7,24-dien-21-oic acid.     

Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases.

Human T-cell leukemia virus type-1 (HTLV-1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV-1) PR inhibitors, the proteinase (PR) of HTLV-1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV-1 PR was characterized and compared to that of HIV-1 PR. Two sets of substrates were used that contained single amino-acid substitutions in peptides representing naturally occurring cleavage sites in HIV-1 and HTLV-1. The original HIV-1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV-1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV-1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV-1 and HIV-1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV-1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV-1 substrate and on analysis of the modeled complex of HTLV-1 PR with substrate, the substrate binding site of the HTLV-1 PR appeared to be more extended than that of HIV-1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV-1 is evolutionarily optimized for rapid hydrolysis.     

Crystal Structures of Inhibitor Complexes of Human T-Cell Leukemia Virus (HTLV-1) Protease

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.     

An in silico pharmacological approach toward the discovery of potent inhibitors to combat drug resistance HIV-1 protease variants

Protease inhibitors (PIs) are crucial drugs in highly active antiretroviral therapy for human immunodeficiency virus-1 (HIV-1) infections. However, resistance owing to mutations challenge the long-term efficacy in the medication of HIV-1-infected individuals. Lopinavir (LPV) and darunavir (DRV), two second-generation drugs are the most potent among PIs, hustling the drug resistance when mutations occur in the active and nonactive site of the protease (PR). Herein, we strive for compounds that can stifle the function of wild-type (WT) HIV-1 PR along with four major single mutants (I54M, V82T, I84V, and L90M) instigating resistance to the PIs using in silico approach. Six common compounds are retrieved from six databases using combined pharmacophore-based and structure-based virtual screening methodology. LPV and DRV are docked and the binding free energy is calculated to set the cut-off value for selecting compounds. Further, to gain insight into the stability of the complexes the molecular dynamics simulation (MDS) is carried out, which uncovers two lead molecules namely NCI-524545 and ZINC12866729. Both the lead molecules connect with WT and mutant HIV-1 PRs through strong and stable hydrogen bond interactions when compared with LPV and DRV throughout the trajectory analysis. Interestingly, NCI-524545 and ZINC12866729 exhibit direct interactions with I50/50′ by replacing the conserved water molecule as evidenced by MDS, which indicates the credible potency of these compounds. Hence, we concluded that NCI-524545 and ZINC12866729 have great puissant to restrain the role of drug resistance HIV-1 PR variants, which can also show better activity through in vivo and in vitro conditions.     

Establishment of a new cell line inducibly expressing HIV-1 protease for performing safe and highly sensitive screening of HIV protease inhibitors

The human immunodeficiency virus type 1 (HIV-1) protease (PR) plays an essential role in processing viral polyproteins into mature proteins. As a result, it is a major target for the development of drugs against AIDS. However, due to the rapid emergence of drug-resistant HIV, the development of novel HIV PR inhibitors is urgently needed. We recently established a new cell line E-PR293 which can be used as a safe, convenient and highly efficient assay system to screen HIV-1 PR inhibitors. In the cells, the HIV-1 PR is expressed in a chimeric protein with the green fluorescence protein (GFP). This assay measures the PR activity as a function of either the fluorescence of GFP or the cytotoxic activity of HIV-1 PR which is expressed in the cell. E-PR293 cells were maintained in the presence of doxycycline, which suppresses the expression of HIV-1 PR. The removal of doxycycline induces the expression of HIV-1 PR, which is used to screen HIV-1 PR inhibitors. In E-PR293 cells, the 50% inhibitory concentration of the cytotoxic effects by nelfinavir and saquinavir were as low as nanomolar levels, almost equal to those found in the HIV-infection assay.     

Molecular dynamics simulations of HIV-1 protease monomer: Assembly of N-terminus and C-terminus into beta-sheet in water solution

HIV-1 protease (HIV-PR) consists of two identical subunits that are united together through a four-stranded antiparallel beta-sheet formed of the peptide termini of each monomer. Since the active site exists only in the dimer, a strategy that is attracting more and more attention in inhibitor design and which may overcome the serious drug resistance caused by competitive inhibitors is to block the peptide termini of the monomer, thereby interfering with formation of the active dimer. In the present work, we performed several extensive molecular dynamics (MD) simulations of the HIV-PR monomer in water to illustrate its solvated conformation and dynamics behavior. We found that the peptide termini usually assembled into beta-sheet after several nanoseconds' simulation, and became much less flexible. This beta-sheet is stabilized by intramolecular interactions and is not easily disaggregated under the present MD simulation conditions. This transformation may be an important transition during the relaxing and equilibrating of the HIV-PR monomer in aqueous solution, and the terminal beta-sheet may be one of the major conformations of the solvated HIV-PR monomer termini in water. This work may provide new insights into the dynamics behavior and dimerization mechanism of HIV-PR, and more significantly, offer a more rational receptor model for the design and discovery of novel dimerization inhibitors than crystalline structures.     

Structural insights into the South African HIV-1 subtype C protease: impact of hinge region dynamics and flap flexibility in drug resistance

The HIV protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. Resistance of HIV protease to protease inhibitors (PIs) is problematic for the effective treatment of HIV infection. The South African HIV-1 subtype C protease (C-SA PR), which contains eight polymorphisms relative to the consensus HIV-1 subtype B protease, was expressed in Escherichia coli, purified, and crystallized. The crystal structure of the C-SA PR was resolved at 2.7?Å, which is the first crystal structure of a HIV-1 subtype C protease that predominates in Africa. Structural analyses of the C-SA PR in comparison to HIV-1 subtype B proteases indicated that polymorphisms at position 36 of the homodimeric HIV-1 protease may impact on the stability of the hinge region of the protease, and hence the dynamics of the flap region. Molecular dynamics simulations showed that the flap region of the C-SA PR displays a wider range of movements over time as compared to the subtype B proteases. Reduced stability in the hinge region resulting from the absent E35-R57 salt bridge in the C-SA PR, most likely contributes to the increased flexibility of the flaps which may be associated with reduced susceptibility to PIs.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:36     

Insight into binding mechanisms of inhibitors MKP56, MKP73, MKP86, and MKP97 to HIV-1 protease by using molecular dynamics simulation

HIV-1 protease (PR) has been a significant target for design of potent inhibitors curing acquired immunodeficiency syndrome. Molecular dynamics simulations coupled with molecular mechanics Poisson–Boltzmann surface area method were performed to study interaction modes of four inhibitors MKP56, MKP73, MKP86, and MKP97 with PR. The results suggest that the main force controlling interactions of inhibitors with PR should be contributed by van der Waals interactions between inhibitors and PR. The cross-correlation analyses based on MD trajectories show that inhibitor binding produces significant effect on the flap dynamics of PR. Hydrogen bond analyses indicate that inhibitors can form stable hydrogen bonding interactions with the residues from the catalytic strands of PR. The contributions of separate residues to inhibitor bindings are evaluated by using residue-based free energy decomposition method and the results demonstrate that the CH–π and CH–CH interactions between the hydrophobic groups of inhibitors with residues drive the associations of inhibitors with PR. We expect that this study can provide a significant theoretical aid for design of potent inhibitors targeting PR.     

An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity

A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.     

The role of cytochrome P450 2E1 on ethanol-mediated oxidative stress and HIV replication in human monocyte-derived macrophages

BackgroundAlcohol consumption is considered to be a major health problem among people living with HIV/AIDS. Our previous reports have shown that ethanol reduced intracellular concentrations of antiretroviral drugs elvitegravir and darunavir in the HIV-1-infected U1 cell line. Ethanol also increased HIV-1 replication despite the presence of elvitegravir. Our previous finding has also shown that the levels of cytochrome P450 enzyme 2E1 (CYP2E1) and oxidative stress in blood monocytes were induced, while the concentration of alcohol in the plasma was reduced in HIV-1-infected alcohol users compared to uninfected alcohol users. However, the role of CYP2E1 in ethanol-enhanced oxidative stress and HIV-1 replication is still unclear.MethodsThis study examined the chronic effects (14 days) of ethanol on HIV viral load, oxidative DNA damage, expression of CYP2E1, expression of antioxidant enzymes (AOEs), expression of reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM). Further, to evaluate the role of CYP2E1 in mediating ethanol-induced viral replication, CYP2E1 siRNA and CYP2E1 selective inhibitor were used in the HIV-1-infected U1 cell line following ethanol treatment.ResultsChronic ethanol exposure demonstrated an increase in oxidative DNA damage and CYP2E1 expression in both non-infected and HIV-1-infected MDM. Our results showed that ethanol chronic exposure increased HIV-1 replication by ~3-fold in HIV-1-infected MDM. This ethanol-enhanced HIV-1 replication was associated with an increased oxidative DNA damage, an increased expression of CYP2E1, and a decreased expression of antioxidant enzyme PRDX6. In HIV-1-infected U1 cell line, we observed a decreased viral replication (~30%) and a decreased DNA damage (~100%) after repression of CYP2E1 by siRNA, upon ethanol exposure. We also observed a decreased viral replication (~25%) after inhibition of CYP2E1 by using selective CYP2E1 inhibitor.ConclusionsThe data suggest that chronic ethanol exposure increases HIV-1 replication in MDM, at least in part, through CYP2E1-mediated oxidative stress. These results are clinically relevant to potentially find effective treatment strategies for HIV-1-infected alcohol users.     

Structural evidence for effectiveness of darunavir and two related antiviral inhibitors against HIV-2 protease

No drug has been targeted specifically for HIV-2 (human immunodeficiency virus type 2) infection despite its increasing prevalence worldwide. The antiviral HIV-1 (human immunodeficiency virus type 1) protease (PR) inhibitor darunavir and the chemically related GRL98065 and GRL06579A were designed with the same chemical scaffold and different substituents at P2 and P2′ to optimize polar interactions for HIV-1 PR (PR1). These inhibitors are also effective antiviral agents for HIV-2-infected cells. Therefore, crystal structures of HIV-2 PR (PR2) complexes with the three inhibitors have been solved at 1.2-Å resolution to analyze the molecular basis for their antiviral potency. Unusually, the crystals were grown in imidazole and zinc acetate buffer, which formed interactions with the PR2 and the inhibitors. Overall, the structures were very similar to the corresponding inhibitor complexes of PR1 with an RMSD of 1.1 Å on main-chain atoms. Most hydrogen-bond and weaker C-H…O interactions with inhibitors were conserved in the PR2 and PR1 complexes, except for small changes in interactions with water or disordered side chains. Small differences were observed in the hydrophobic contacts for the darunavir complexes, in agreement with relative inhibition of the two PRs. These near-atomic-resolution crystal structures verify the inhibitor potency for PR1 and PR2 and will provide the basis for the development of antiviral inhibitors targeting PR2.     

Selective and facile assay of human immunodeficiency virus protease activity by a novel fluorogenic reaction

A highly selective and facile assay of human immunodeficiency virus protease (HIV-PR) has been required for the screening of medicinal inhibitors and also for classifying the subtypes of HIV in the therapeutic treatment of acquired immune deficiency syndrome (AIDS). This article describes a novel assay method of HIV-PR based on the selective fluorogenic reaction of peptides. A peptide fragment generated from a substrate by the enzymatic digestion with HIV-PR could be selectively quantified by the spectrofluorometric detection after the fluorogenic reaction with catechol in the presence of sodium periodate and sodium borate (pH 7.0). This assay system uses an N-terminal acetyl peptide as the substrate and crude extracts from Escherichia coli expressing recombinant HIV-PR. The activity obtained by the proposed assay correlated with that obtained by a conventional HIV-PR assay based on fluorescence resonance energy transfer detection.     

195 In silico study on HIV-PRIs substructures to terminate proteolytic activity in HTLV

Human T-lymphotropic virus (HTLV) is RNA retrovirus, which causes CD3?+?and CD4?+?T-cell type leukemia and demyelinating diseases, like tropical spastic myelopathy. The replicative stage of the virus is one of the critical stages for the development of the disease. At present, there are no approved therapeutic agents targeting HTLV. The HTLV mechanism of malignant cell growth in adult T-cell leukemia (ATL)/lymphoma, and the HTLV-PR has been an attractive target for anticancer drug design. In comparison with other retroviruses, HTLV also encodes protease (PR) enzyme which is essential for maturation. Both the HIV and HTLV proteases show high structural similarity but known inhibitors of HIV-PR are not able to inhibit the HTLV-PR, while comparing the binding pocket of both proteases, MET37 of HTLV shows repulsive role with known HIV inhibitors. Functional analysis of M37A mutation clearly shows that MET37 is highly important for the protease function. Available inhibitors were tested against the HTLV-PR binding pocket and failed to interact with MET37. Screening of similar libraries of known compounds provides better interactions with MET37 and further validation with in vivo and in vitro studies on these screened compounds will provide more strength in discovering potent inhibitor for HTLV-PR.     

Insights from atomic-resolution X-ray structures of chemically synthesized HIV-1 protease in complex with inhibitors

The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.     

Exploration of the effects of sequence variations between HIV-1 and HIV-2 proteases on their three-dimensional structures

Abstract

HIV protease inhibitors (PIs) approved by the FDA (US Food and Drug Administration) are a major class of antiretroviral. HIV-2 protease (PR2) is naturally resistant to most of them as PIs were designed for HIV-1 protease (PR1). In this study, we explored the impact of amino-acid substitutions between PR1 and PR2 on the structure of protease (PR) by comparing the structural variability of 13 regions using 24 PR1 and PR2 structures complexed with diverse ligands. Our analyses confirmed structural rigidity of the catalytic region and highlighted the important role of three regions in the conservation of the catalytic region conformation. Surprisingly, we showed that the flap region, corresponding to a flexible region, exhibits similar conformations in PR1 and PR2. Furthermore, we identified regions exhibiting different conformations in PR1 and PR2, which could be explained by the intrinsic flexibility of these regions, by crystal packing, or by PR1 and PR2 substitutions. Some substitutions induce structural changes in the R2 and R4 regions that could have an impact on the properties of PI-binding site and could thus modify PI binding mode. Substitutions involved in structural changes in the elbow region could alter the flexibility of the PR2 flap regions relative to PR1, and thus play a role in the transition from the semi-open form to the closed form, and have an impact on ligand binding. These results improve the understanding of the impact of sequence variations between PR1 and PR2 on the natural resistance of HIV-2 to commercially available PIs.

Communicated by Ramaswamy H. Sarma     

Solvation effects are responsible for the reduced inhibitor affinity of some HIV-1 PR mutants.

The formulation of HIV-1 PR inhibitors as anti-viral drugs has been hindered by the appearance of protease strains that present drug resistance to these compounds. The mechanism by which the HIV-1 PR mutants lower their affinity for the inhibitor is not yet fully understood. We have applied a modified Poisson-Boltzmann method to the evaluation of the molecular interactions that contribute to the lowering of the inhibitor affinity to some polar mutants at position 82. These strains present drug resistance behavior and hence are ideally suited for these studies. Our results indicate that the reduction in binding affinity is due to the solvation effects that penalize the binding to the more polar mutants. The inhibitor binding ranking of the different mutants can be explained from the analysis of the different components of our free energy scoring function.