Design and synthesis of potent HIV-1 protease inhibitors incorporating hydroxyprolinamides as novel P2 ligands

A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different hydroxyprolinamide P2 ligands were designed and synthesized. Variation of substitutions at the P2 significantly affected the enzyme inhibitory potency of the inhibitors. Compounds 2a and 2d showed excellent enzyme inhibitory activity with IC₅₀ values in the nanomolar range. An active site binding model for inhibitors 2a and 2d was suggested based upon the computational-docking results of the ligand with HIV-1 protease. This model offers molecular insights regarding ligand-binding site interactions of the hydroxyprolinamide-derived novel P2-ligand.     

Design,synthesis and biological evaluation of novel HIV-1 protease inhibitors with pentacyclic triterpenoids as P2-ligands

The design, synthesis and SAR study of a new series of HIV-1 protease inhibitors with pentacyclic triterpenoids as P2 ligands and phenylsulfonamide as P2′ ligands were discussed. These compounds exhibited micromolar inhibitory potency, among which compound T1c displayed HIV-1 protease inhibition with IC₅₀ values of 0.12?μM, which was 67 times the inhibitory activity of its raw material Ursolic acid (8.0?μM).     

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design,synthesis, and biological evaluation

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory K_i value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.     

Design and synthesis of N1-aryl-benzimidazoles 2-substituted as novel HIV-1 non-nucleoside reverse transcriptase inhibitors

A series of novel N₁-aryl-2-arylthioacetamido-benzimidazoles were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Some of them proved to be effective in inhibiting HIV-1 replication at submicromolar and nanomolar concentration acting as HIV-1 non-nucleoside RT inhibitors (NNRTIs), with low cytotoxicity. The preliminary structure–activity relationship (SAR) of these new derivatives was discussed and rationalized by docking studies.     

Design and synthesis of novel pyrimidone analogues as HIV-1 integrase inhibitors

A series of novel pyrimidone analogues have been designed and synthesized as HIV-1 integrase (IN) inhibitors. This study demonstrated that introducing a substituent in the N1-position of the pyrimidone scaffold does not significantly influence IN inhibitory activity. Molecular docking studies showed these compounds could occupy the IN active site and form pi–pi interactions with viral DNA nucleotides DC16 and DA17 to displace reactive viral DNA 3′OH and block intasome activity.     

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.     

Biosensor analysis of the interaction of potential dimerization inhibitors with HIV-1 protease

Inhibitors of protein-protein interactions are currently considered as perspective prototypes of a new generation of drugs. The most attractive targets for such inhibitors are the oligomeric enzymes which active sites are formed by amino acid residues from different subunits. HIV-1 protease (HIVp), which is active only as a homodimer form, is the classic example of such enzymes. We have developed a new approach for experimental screening of HIVp dimerization inhibitors. It is based on an original biosensor test-system for differential analysis of interaction of tested substances with HIVp dimers and monomers. Using this test-system we have analyzed the most perspective candidate substances predicted by the method of virtual screening, and also some derivatives of glycyrrhizin, triterpenic and steroid glycosides. In the results of this study we have found one compound, which preferentially interacts with HIVp monomers and inhibits in vitro activity of this enzyme with the IC₅₀ value of about 10^?6 M.     

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     

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.     

Design,synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2 million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC₅₀ = 53.02 nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus–cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.     

Characterizing the structural variability of HIV-2 protease upon the binding of diverse ligands using a structural alphabet approach

Abstract

The HIV-2 protease (PR2) is an important target for designing new drugs against the HIV-2 infection. In this study, we explored the structural backbone variability of all available PR2 structures complexed with various inhibitors using a structural alphabet approach. 77% of PR2 positions are structurally variable, meaning they exhibit different local conformations in PR2 structures. This variability was observed all along the structure, particularly in the elbow and flap regions. A part of these backbone changes observed between the 18 PR2 is induced by intrinsic flexibility, and ligand binding putatively induces others occurring in the binding pocket. These latter changes could be important for PR2 adaptation to diverse ligands and are accompanied by changes outside the binding pocket. In addition, the study of the link between structural variability of the pocket and PR2–ligand interactions allowed us to localize pocket regions important for ligand binding and catalytic function, regions important for ligand recognition that adjust their backbone in response to ligand binding and regions important for the pocket opening and closing that have large intrinsic flexibility. Finally, we suggested that differences in ligand effectiveness for PR2 could be partially explained by different backbone deformations induced by these ligands. To conclude, this study is the first characterization of the PR2 structural variability considering ligand diversity. It provides information about the recognition of PR2 to various ligands and its mechanisms to adapt its local conformation to bound ligands that could help understand the resistance of PR2 to its inhibitors, a major antiretroviral class.

Communicated by Ramaswamy H. Sarma     

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography

Recombinant wild-type protease of human immunodeficiency virus, type [(HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/μg. Two proteolytically inactive HIV-1 mutant proteases (Arg-87 → Lys; Asn-88 → Glu) were found to bind to pepstatin A agarose, and they were purified as the wild-type protease. A third mutant protease (Arg-87 → Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.     

Overcoming drug resistance in HIV-1 chemotherapy: the binding thermodynamics of Amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease

Amprenavir is one of six protease inhibitors presently approved for clinical use in the therapeutic treatment of AIDS. Biochemical and clinical studies have shown that, unlike other inhibitors, Amprenavir is severely affected by the protease mutation I50V, located in the flap region of the enzyme. TMC-126 is a second-generation inhibitor, chemically related to Amprenavir, with a reported extremely low susceptibility to existing resistant mutations including I50V. In this paper, we have studied the thermodynamic and molecular origin of the response of these two inhibitors to the I50V mutation and the double active-site mutation V82F/I84V that affects all existing clinical inhibitors. Amprenavir binds to the wild-type HIV-1 protease with high affinity (5.0 x 10(9) M(-1) or 200 pM) in a process equally favored by enthalpic and entropic contributions. The mutations I50V and V82F/I84V lower the binding affinity of Amprenavir by a factor of 147 and 104, respectively. TMC-126, on the other hand, binds to the wild-type protease with extremely high binding affinity (2.6 x 10(11) M(-1) or 3.9 pM) in a process in which enthalpic contributions overpower entropic contributions by almost a factor of 4. The mutations I50V and V82F/I84V lower the binding affinity of TMC-126 by only a factor of 16 and 11, respectively, indicating that the binding affinity of TMC-126 to the drug-resistant mutants is still higher than the affinity of Amprenavir to the wild-type protease. Analysis of the data for TMC-126 and KNI-764, another second-generation inhibitor, indicates that their low susceptibility to mutations is caused by their ability to compensate for the loss of interactions with the mutated target by a more favorable entropy of binding.     

Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease

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.     

Synthesis and SAR of acyclic HCV NS3 protease inhibitors with novel P4-benzoxaborole moieties

We have synthesized and evaluated a new series of acyclic P4-benzoxaborole-based HCV NS3 protease inhibitors. Structure-activity relationships were investigated, leading to the identification of compounds 5g and 17 with low nanomolar potency in the enzymatic and cell-based replicon assay. The linker-truncated compound 5j was found to exhibit improved absorption and oral bioavailability in rats, suggesting that further reduction of molecular weight and polar surface area could result in improved drug-like properties of this novel series.