RESUMO
Substituted tetrahydrofuran derivatives were designed and synthesized to serve as the P2 ligand for a series of potent HIV-1 protease inhibitors. Both enantiomers of the tetrahydrofuran derivatives were synthesized stereoselectivity in optically active forms using lipase-PS catalyzed enzymatic resolution as the key step. These tetrahydrofuran derivatives are designed to promote hydrogen bonding and van der Waals interactions with the backbone atoms in the S2 subsite of the HIV-1 protease active site. Several inhibitors displayed very potent HIV-1 protease inhibitory activity. A high-resolution X-ray crystal structure of an inhibitor-bound HIV-1 protease provided important insight into the ligand binding site interactions in the active site.
RESUMO
Structure-based design, synthesis, X-ray structural studies, and biological evaluation of a new series of potent HIV-1 protease inhibitors are described. These inhibitors contain various pyridyl-pyrimidine, aryl thiazole or alkylthiazole derivatives as the P2 ligands in combination with darunavir-like hydroxyethylamine sulfonamide isosteres. These heterocyclic ligands are inherent to kinase inhibitor drugs, such as nilotinib and imatinib. These ligands are designed to make hydrogen bonding interactions with the backbone atoms in the S2 subsite of HIV-1 protease. Various benzoic acid derivatives have been synthesized and incorporation of these ligands provided potent inhibitors that exhibited subnanomolar level protease inhibitory activity and low nanomolar level antiviral activity. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease were determined. These structures provided important ligand-binding site interactions for further optimization of this class of protease inhibitors.
Assuntos
Inibidores da Protease de HIV , HIV-1 , Inibidores da Protease de HIV/química , HIV-1/metabolismo , Mesilato de Imatinib/farmacologia , Ligantes , Raios X , Protease de HIV/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Relação Estrutura-AtividadeRESUMO
We report here the synthesis and biological evaluation of darunavir derived HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. The P2' 4-amino functionality was modified to make a number of amide derivatives to interact with residues in the S2' subsite of the HIV-1 protease active site. Several compounds exhibited picomolar enzyme inhibitory and low nanomolar antiviral activity. The X-ray crystal structure of the chloroacetate derivative bound to HIV-1 protease was determined. Interestingly, the active chloroacetate group converted to the acetate functionality during X-ray exposure. The structure revealed that the P2' carboxamide functionality makes enhanced hydrogen bonding interactions with the backbone atoms in the S2'-subsite.
Assuntos
Inibidores da Protease de HIV , HIV-1 , Darunavir/farmacologia , Amidas/farmacologia , Protease de HIV/metabolismo , Cloroacetatos/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Relação Estrutura-AtividadeRESUMO
We report our recent development of a conceptually new generation of exceptionally potent non-peptidic HIV-1 protease inhibitors that displayed excellent pharmacological and drug-resistance profiles. Our X-ray structural studies of darunavir and other designed inhibitors from our laboratories led us to create a variety of inhibitors incorporating fused ring polycyclic ethers and aromatic heterocycles to promote hydrogen bonding interactions with the backbone atoms of HIV-1 protease as well as van der Waals interactions with residues in the S2 and S2' subsites. We have also incorporated specific functionalities to enhance van der Waals interactions in the S1 and S1' subsites. The combined effects of these structural templates are critical to the inhibitors' exceptional potency and drug-like properties. We highlight here our molecular design strategies to promote backbone hydrogen bonding interactions to combat drug-resistance and specific design of polycyclic ether templates to mimic peptide-like bonds in the HIV-1 protease active site. Our medicinal chemistry and drug development efforts led to the development of new generation inhibitors significantly improved over darunavir and displaying unprecedented antiviral activity against multidrug-resistant HIV-1 variants.
Assuntos
Inibidores da Protease de HIV , HIV-1 , Darunavir/farmacologia , Darunavir/química , Inibidores da Protease de HIV/farmacologia , Éter/farmacologia , Desenho de Fármacos , Protease de HIV/química , Protease de HIV/farmacologia , Resistência a Medicamentos , Peptídeos/farmacologia , Cristalografia por Raios X , Farmacorresistência ViralRESUMO
Antiretroviral drug resistance is a therapeutic obstacle for people with HIV. HIV protease inhibitors darunavir and lopinavir are recommended for resistant infections. We characterized a protease mutant (PR10x) derived from a highly resistant clinical isolate including 10 mutations associated with resistance to lopinavir and darunavir. Compared to the wild-type protease, PR10x exhibits â¼3-fold decrease in catalytic efficiency and Ki values of 2-3 orders of magnitude worse for darunavir, lopinavir, and potent investigational inhibitor GRL-519. Crystal structures of the mutant were solved in a ligand-free form and in complex with GRL-519. The structures show altered interactions in the active site, flap-core interface, hydrophobic core, hinge region, and 80s loop compared to the corresponding wild-type protease structures. The ligand-free crystal structure exhibits a highly curled flap conformation which may amplify drug resistance. Molecular dynamics simulations performed for 1 µs on ligand-free dimers showed extremely large fluctuations in the flaps for PR10x compared to equivalent simulations on PR with a single L76V mutation or wild-type protease. This analysis offers insight about the synergistic effects of mutations in highly resistant variants.
Assuntos
Inibidores da Protease de HIV , Cristalografia por Raios X , Darunavir/farmacologia , Farmacorresistência Viral/genética , Protease de HIV/química , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Humanos , Lopinavir/farmacologia , Simulação de Dinâmica Molecular , MutaçãoRESUMO
The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2' ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2' ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high-resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.
Assuntos
Inibidores da Protease de HIV , HIV-1 , Cristalografia por Raios X , Desenho de Fármacos , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , HIV-1/metabolismo , Ligantes , Relação Estrutura-Atividade , Raios XRESUMO
d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) catalyzes the flavin-dependent oxidation of d-arginine and other d-amino acids. Here, we report the crystal structure at 1.29 Å resolution for PaDADH-Y249F expressed and co-crystallized with d-arginine. The overall structure of PaDADH-Y249F resembled PaDADH-WT, but the electron density for the flavin cofactor was ambiguous, suggesting the presence of modified flavins. Electron density maps and mass spectrometric analysis confirmed the presence of both N5-(4-guanidino-oxobutyl)-FAD and 6-OH-FAD in a single crystal of PaDADH-Y249F and helped with the further refinement of the X-ray crystal structure. The versatility of the reduced flavin is apparent in the PaDADH-Y249F structure and is evidenced by the multiple functions it can perform in the same active site.
Assuntos
Aminoácido Oxirredutases/química , Proteínas de Bactérias/química , Flavina-Adenina Dinucleotídeo/análogos & derivados , Guanidinas/química , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Arginina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Guanidinas/metabolismo , Ligação de Hidrogênio , Mutação , Ligação Proteica , Pseudomonas aeruginosa/enzimologia , Eletricidade EstáticaRESUMO
Drug resistance is a serious problem for controlling the HIV/AIDS pandemic. Current antiviral drugs show several orders of magnitude worse inhibition of highly resistant clinical variant PRS17 of HIV-1 protease compared with wild-type protease. We have analyzed the effects of a common resistance mutation G48V in the flexible flaps of the protease by assessing the revertant PRS17V48G for changes in enzyme kinetics, inhibition, structure, and dynamics. Both PRS17 and the revertant showed about 10-fold poorer catalytic efficiency than wild-type enzyme (0.55 and 0.39 µM-1min-1 compared to 6.3 µM-1min-1). Clinical inhibitors, amprenavir and darunavir, showed 2-fold and 8-fold better inhibition, respectively, of the revertant than of PRS17, although the inhibition constants for PRS17V48G were still 25 to 1,200-fold worse than for wild-type protease. Crystal structures of inhibitor-free revertant and amprenavir complexes with revertant and PRS17 were solved at 1.3-1.5 Å resolution. The amprenavir complexes of PRS17V48G and PRS17 showed no significant differences in the interactions with inhibitor, although changes were observed in the conformation of Phe53 and the interactions of the flaps. The inhibitor-free structure of the revertant showed flaps in an open conformation, however, the flap tips do not have the unusual curled conformation seen in inhibitor-free PRS17. Molecular dynamics simulations were run for 1 µs on the two inhibitor-free mutants and wild-type protease. PRS17 exhibited higher conformational fluctuations than the revertant, while the wild-type protease adopted the closed conformation and showed the least variation. The second half of the simulations captured the transition of the flaps of PRS17 from a closed to a semi-open state, whereas the flaps of PRS17V48G tucked into the active site and the wild-type protease retained the closed conformation. These results suggest that mutation G48V contributes to drug resistance by altering the conformational dynamics of the flaps.
Assuntos
Inibidores da Protease de HIV , Preparações Farmacêuticas , Domínio Catalítico , Farmacorresistência Viral/genética , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , Mutação , Conformação ProteicaRESUMO
The retroviral protease of human immunodeficiency virus (HIV) is an excellent target for antiviral inhibitors for treating HIV/AIDS. Despite the efficacy of therapy, current efforts to control the disease are undermined by the growing threat posed by drug resistance. This review covers the historical background of studies on the structure and function of HIV protease, the subsequent development of antiviral inhibitors, and recent studies on drug-resistant protease variants. We highlight the important contributions of Dr. Stephen Oroszlan to fundamental knowledge about the function of the HIV protease and other retroviral proteases. These studies, along with those of his colleagues, laid the foundations for the design of clinical inhibitors of HIV protease. The drug-resistant protease variants also provide an excellent model for investigating the molecular mechanisms and evolution of resistance.
Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/história , HIV-1/efeitos dos fármacos , Pesquisa/história , Desenho de Fármacos , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , História do Século XX , História do Século XXI , Humanos , Modelos MolecularesRESUMO
The emergence of multidrug resistant (MDR) HIV strains severely reduces the effectiveness of antiretroviral therapy. Clinical inhibitor darunavir (1) has picomolar binding affinity for HIV-1 protease (PR), however, drug resistant variants like PRS17 show poor inhibition by 1, despite the presence of only two mutated residues in the inhibitor-binding site. Antiviral inhibitors that target MDR proteases like PRS17 would be valuable as therapeutic agents. Inhibitors 2 and 3 derived from 1 through substitutions at P1, P2 and P2' positions exhibit 3.4- to 500-fold better inhibition than clinical inhibitors for PRS17 with the exception of amprenavir. Crystal structures of PRS17/2 and PRS17/3 reveal how these inhibitors target the two active site mutations of PRS17. The substituted methoxy P2 group of 2 forms new interactions with G48V mutation, while the modified bis-fluoro-benzyl P1 group of 3 forms a halogen interaction with V82S mutation, contributing to improved inhibition of PRS17.
Assuntos
Darunavir/análogos & derivados , Darunavir/farmacologia , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , Domínio Catalítico/efeitos dos fármacos , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Protease de HIV/química , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Modelos Moleculares , Mutação Puntual/efeitos dos fármacosRESUMO
Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.
Assuntos
Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Flavinas/metabolismo , Fenilalanina/química , Mutação Puntual , Pseudomonas aeruginosa/enzimologia , Tirosina/química , Aminoácido Oxirredutases/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/genética , Fenilalanina/metabolismo , Conformação Proteica , Tirosina/genética , Tirosina/metabolismoRESUMO
BACKGROUND: Drug resistance is a critical problem limiting effective antiviral therapy for HIV/AIDS. Computational techniques for predicting drug resistance profiles from genomic data can accelerate the appropriate choice of therapy. These techniques can also be used to identify protease mutants for experimental studies of resistance and thereby assist in the development of next-generation therapies. Few studies, however, have assessed the evolution of resistance from genotype-phenotype data. RESULTS: The machine learning produced highly accurate and robust classification of resistance to HIV protease inhibitors. Genotype data were mapped to the enzyme structure and encoded using Delaunay triangulation. Estimates of evolutionary relationships, based on this encoding, and using Minimum Spanning Trees, showed clusters of mutations that closely resemble the wild type. These clusters appear to evolve uniquely to more resistant phenotypes. CONCLUSIONS: Using the triangulation metric and spanning trees results in paths that are consistent with evolutionary theory. The majority of the paths show bifurcation, namely they switch once from non-resistant to resistant or from resistant to non-resistant. Paths that lose resistance almost uniformly have far lower levels of resistance than those which either gain resistance or are stable. This strongly suggests that selection for stability in the face of a rapid rate of mutation is as important as selection for resistance in retroviral systems.
Assuntos
Farmacorresistência Viral/genética , Evolução Molecular , Protease de HIV/genética , Aprendizado de Máquina , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/patologia , Infecções por HIV/virologia , Inibidores da Protease de HIV/uso terapêutico , HIV-1/enzimologia , HIV-1/genética , Humanos , FenótipoRESUMO
The design, synthesis, biological evaluation, and X-ray structural studies are reported for a series of highly potent HIV-1 protease inhibitors. The inhibitors incorporated stereochemically defined amide-based bicyclic and tricyclic ether derivatives as the P2 ligands with (R)-hydroxyethylaminesulfonamide transition-state isosteres. A number of inhibitors showed excellent HIV-1 protease inhibitory and antiviral activity; however, ligand combination is critical for potency. Inhibitor 4h with a difluorophenylmethyl as the P1 ligand, crown-THF-derived acetamide as the P2 ligand, and a cyclopropylaminobenzothiazole P2'-ligand displayed very potent antiviral activity and maintained excellent antiviral activity against selected multidrug-resistant HIV-1 variants. A high resolution X-ray structure of inhibitor 4h-bound HIV-1 protease provided molecular insight into the binding properties of the new inhibitor.
RESUMO
Coronaviruses infect many animals, including humans, due to interspecies transmission. Three of the known human coronaviruses: MERS, SARS-CoV-1, and SARS-CoV-2, the pathogen for the COVID-19 pandemic, cause severe disease. Improved methods to predict host specificity of coronaviruses will be valuable for identifying and controlling future outbreaks. The coronavirus S protein plays a key role in host specificity by attaching the virus to receptors on the cell membrane. We analyzed 1238 spike sequences for their host specificity. Spike sequences readily segregate in t-SNE embeddings into clusters of similar hosts and/or virus species. Machine learning with SVM, Logistic Regression, Decision Tree, Random Forest gave high average accuracies, F1 scores, sensitivities and specificities of 0.95-0.99. Importantly, sites identified by Decision Tree correspond to protein regions with known biological importance. These results demonstrate that spike sequences alone can be used to predict host specificity.
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Biologia Computacional/métodos , Coronavirus/patogenicidade , Especificidade de Hospedeiro , Aprendizado de Máquina , Glicoproteína da Espícula de Coronavírus , Animais , Humanos , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
We describe here design, synthesis, and biological evaluation of a series of highly potent HIV-1 protease inhibitors containing stereochemically defined and unprecedented tricyclic furanofuran derivatives as P2 ligands in combination with a variety of sulfonamide derivatives as P2' ligands. These inhibitors were designed to enhance the ligand-backbone binding and van der Waals interactions in the protease active site. A number of inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2' ligand and a difluorophenylmethyl as the P1 ligand, displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized efficiently in an optically active form using enzymatic desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insights into the binding properties of these new inhibitors.
Assuntos
Furanos/farmacologia , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Substituição de Aminoácidos , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Furanos/síntese química , Furanos/metabolismo , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/metabolismo , HIV-1/enzimologia , Compostos Heterocíclicos de Anel em Ponte/síntese química , Compostos Heterocíclicos de Anel em Ponte/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ligação Proteica , EstereoisomerismoRESUMO
HIV-1 protease is an essential therapeutic target for the design and development of antiviral inhibitors to treat AIDS. We used room temperature neutron crystallography to accurately determine hydrogen atom positions in several protease complexes with clinical drugs, amprenavir and darunavir. Hydrogen bonding interactions were carefully mapped to provide an unprecedented picture of drug binding to the protease target. We demonstrate that hydrogen atom positions within the enzyme catalytic site can be altered by introducing drug resistant mutations and by protonating surface residues that trigger proton transfer reactions between the catalytic Asp residues and the hydroxyl group of darunavir. When protein perdeuteration is not feasible, we validate the use of initial H/D exchange with unfolded protein and partial deuteration in pure D2O with hydrogenous glycerol to maximize deuterium incorporation into the protein, with no detrimental effects on the growth of quality crystals suitable for neutron diffraction experiments.
Assuntos
Difração de Nêutrons , Preparações Farmacêuticas , Sítios de Ligação , Cristalografia por Raios X , Protease de HIV/genética , Ligação de Hidrogênio , PrótonsRESUMO
Drug-resistance is a serious problem for treatment of the HIV/AIDS pandemic. Potent clinical inhibitors of HIV-1 protease show several orders of magnitude worse inhibition of highly drug-resistant variants. Hence, the structure and enzyme activities were analyzed for HIV protease mutant HIV-1 protease (EC 3.4.23.16) (PR) with 22 mutations (PRS5B) from a clinical isolate that was selected by machine learning to represent high-level drug-resistance. PRS5B has 22 mutations including only one (I84V) in the inhibitor binding site; however, clinical inhibitors had poor inhibition of PRS5B activity with kinetic inhibition value (Ki ) values of 4-1000 nm or 18- to 8000-fold worse than for wild-type PR. High-resolution crystal structures of PRS5B complexes with the best inhibitors, amprenavir (APV) and darunavir (DRV) (Ki ~ 4 nm), revealed only minor changes in protease-inhibitor interactions. Instead, two distinct clusters of mutations in distal regions induce coordinated conformational changes that decrease favorable internal interactions across the entire protein subunit. The largest structural rearrangements are described and compared to other characterized resistant mutants. In the protease hinge region, the N83D mutation eliminates a hydrogen bond connecting the hinge and core of the protease and increases disorder compared to highly resistant mutants PR with 17 mutations and PR with 20 mutations with similar hinge mutations. In a distal ß-sheet, mutations G73T and A71V coordinate with accessory mutations to bring about shifts that propagate throughout the subunit. Molecular dynamics simulations of ligand-free dimers show differences consistent with loss of interactions in mutant compared to wild-type PR. Clusters of mutations exhibit both coordinated and antagonistic effects, suggesting PRS5B may represent an intermediate stage in the evolution of more highly resistant variants. DATABASES: Structural data are available in Protein Data Bank under the accession codes 6P9A and 6P9B for PRS5B/DRV and PRS5B/APV, respectively.
Assuntos
Farmacorresistência Viral/genética , Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , Protease de HIV/metabolismo , Mutação , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Protease de HIV/química , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Drug-resistance threatens effective treatment of HIV/AIDS. Clinical inhibitors, including darunavir (1), are ineffective for highly resistant protease mutant PR20, however, antiviral compound 2 derived from 1 with fused tricyclic group at P2, extended amino-benzothiazole P2' ligand and two fluorine atoms on P1 shows 16-fold better inhibition of PR20 enzyme activity. Crystal structures of PR20 and wild-type PR complexes reveal how the extra groups of 2 counteract the expanded ligand-binding pocket, dynamic flaps, and faster dimer dissociation of PR20.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , Antivirais/química , Cristalografia por Raios X , Inibidores da Protease de HIV/química , Humanos , Cinética , Modelos Moleculares , Conformação Molecular , MutaçãoRESUMO
We report the synthesis and biological evaluation of phenylcarboxylic acid and phenylboronic acid containing HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. Inhibitors bearing bis-THF ligand as P2 ligand and phenylcarboxylic acids and carboxamide as the P2' ligands, showed very potent HIV-1 protease inhibitory activity. However, carboxylic acid containing inhibitors showed very poor antiviral activity relative to carboxamide-derived inhibitors which showed good antiviral IC50 value. Boronic acid derived inhibitor with bis-THF as the P2 ligand showed very potent enzyme inhibitory activity, but it showed lower antiviral activity than darunavir in the same assay. Boronic acid containing inhibitor with a P2-Crn-THF ligand also showed potent enzyme Ki but significantly decreased antiviral activity. We have evaluated antiviral activity against a panel of highly drug-resistant HIV-1 variants. One of the inhibitors maintained good antiviral activity against HIVDRVRP20 and HIVDRVRP30 viruses. We have determined high resolution X-ray structures of two synthetic inhibitors bound to HIV-1 protease and obtained molecular insight into the ligand-binding site interactions.
Assuntos
Fármacos Anti-HIV/farmacologia , Ácidos Borônicos/farmacologia , Ácidos Carboxílicos/farmacologia , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Ácidos Borônicos/síntese química , Ácidos Borônicos/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Linhagem Celular , Cristalografia por Raios X , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/química , Humanos , Ligantes , Modelos Moleculares , Conformação MolecularRESUMO
We report the structural analysis of highly drug-resistant human immunodeficiency virus protease (PR) variant PRS17, rationally selected by machine learning, in complex with substrate analogues. Crystal structures were solved of inhibitor-free inactive PRS17-D25N, wild-type PR/CA-p2 complex, and PRS17 in complex with substrate analogues, CA-p2 and p2-NC. Peptide analogues p2-NC and CA-p2 exhibit inhibition constants of 514 and 22 nM, respectively, for PRS17 or approximately 3-fold better than for PR. CA-p2 is a better inhibitor of PRS17 than are clinical inhibitors (K i = 50-8390 nM) except for amprenavir (K i = 11 nM). G48V resistance mutation induces curled flap tips in PRS17-D25N structure. The inner P2-P2' residues of substrate analogues in PRS17 complexes maintain similar conformations to those of wild-type complex, while significant conformational changes are observed in the peripheral residues P3, P4' of CA-p2 and P3, P4, and P3' of p2-NC. The loss of ß-branched side chain by V82S mutation initiates a shift in 80's loop and reshapes the S3/S3' subsite, which enhances substrate binding with new hydrogen bonds and van der Waals interactions that are absent in the wild-type structures. The steric hindrance caused by G48V mutation in the flap of PRS17 contributes to altered binding interactions of P3 Arg, P4' norleucine of CA-p2, and P4 and P3' of p2-NC with the addition of new hydrogen bonds and van der Waals contacts. The enhanced interaction of PRS17 with substrate analogues agrees with their relative inhibition, suggesting that this mutant improves substrate binding while decreasing affinity for clinical inhibitors.