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Journal of Virology Jan 2016Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most...
UNLABELLED
Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2' pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2.
IMPORTANCE
Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine FDA-approved HIV-1 protease inhibitors (PI) are active against HIV-2. The underlying reasons for intrinsic PI resistance in HIV-2 are not known. We examined the contributions of four amino acids in the ligand-binding pocket of the enzyme that differ between HIV-1 and HIV-2 by constructing HIV-2 clones encoding the corresponding HIV-1 amino acids and testing the PI susceptibilities of the resulting viruses. We found that the HIV-2 clone containing all four changes (PRΔ4) was as susceptible as HIV-1 to all nine PI. We also modeled the PRΔ4 enzyme structure and compared it to existing crystallographic structures of HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir. Our findings demonstrate that four positions in the ligand-binding cleft of protease are the primary cause of HIV-2 PI resistance.
Topics: Amino Acid Substitution; Amino Acids; Binding Sites; Cell Line; Drug Resistance, Viral; HIV Protease; HIV Protease Inhibitors; Humans; Microbial Sensitivity Tests; Models, Molecular; Mutation, Missense; Protein Binding; Protein Conformation
PubMed: 26559830
DOI: 10.1128/JVI.01772-15 -
Antimicrobial Agents and Chemotherapy Feb 2022To date, there are no specific treatment regimens for HIV-1-related central nervous system (CNS) complications, such as HIV-1-associated neurocognitive disorders (HAND)....
Fluorine Modifications Contribute to Potent Antiviral Activity against Highly Drug-Resistant HIV-1 and Favorable Blood-Brain Barrier Penetration Property of Novel Central Nervous System-Targeting HIV-1 Protease Inhibitors .
To date, there are no specific treatment regimens for HIV-1-related central nervous system (CNS) complications, such as HIV-1-associated neurocognitive disorders (HAND). Here, we report that two newly generated CNS-targeting HIV-1 protease (PR) inhibitors (PIs), GRL-08513 and GRL-08613, which have a P1-3,5--fluorophenyl or P1--monofluorophenyl ring and P2-tetrahydropyrano-tetrahydrofuran (-THF) with a sulfonamide isostere, are potent against wild-type HIV-1 strains and multiple clinically isolated HIV-1 strains (50% effective concentration [EC]: 0.0001 to ∼0.0032 μM). As assessed with HIV-1 variants that had been selected to propagate at a 5 μM concentration of each HIV-1 PI (atazanavir, lopinavir, or amprenavir), GRL-08513 and GRL-08613 efficiently inhibited the replication of these highly PI-resistant variants (EC: 0.003 to ∼0.006 μM). GRL-08513 and GRL-08613 also maintained their antiviral activities against HIV-2 as well as severely multidrug-resistant clinical HIV-1 variants. Additionally, when we assessed with the blood-brain barrier (BBB) reconstruction system, GRL-08513 and GRL-08613 showed the most promising properties of CNS penetration among the evaluated compounds, including the majority of FDA-approved combination antiretroviral therapy (cART) drugs. In the crystallographic analysis of compound-PR complexes, it was demonstrated that the -THF rings at the P2 moiety of GRL-08513 and GRL-08613 form robust hydrogen bond interactions with the active site of HIV-1 PR. Furthermore, both the P1-3,5--fluorophenyl- and P1--monofluorophenyl rings sustain greater contact surfaces and form stronger van der Waals interactions with PR than is the case with darunavir-PR complex. Taken together, these results strongly suggest that GRL-08513 and GRL-08613 have favorable features for patients infected with wild-type/multidrug-resistant HIV-1 strains and might serve as candidates for a preventive and/or therapeutic agent for HAND and other CNS complications.
Topics: Blood-Brain Barrier; Central Nervous System; Fluorine; HIV Protease; HIV Protease Inhibitors; HIV-1; Humans; Virus Replication
PubMed: 34978889
DOI: 10.1128/AAC.01715-21 -
Scientific Reports Aug 2015A fluorogenic substrate for HIV-1 protease was designed and used as the basis for a hypersensitive assay. The substrate exhibits a kcat of 7.4 s(-1), KM of 15 μM,...
A fluorogenic substrate for HIV-1 protease was designed and used as the basis for a hypersensitive assay. The substrate exhibits a kcat of 7.4 s(-1), KM of 15 μM, and an increase in fluorescence intensity of 104-fold upon cleavage, thus providing sensitivity that is unmatched in a continuous assay of HIV-1 protease. These properties enabled the enzyme concentration in an activity assay to be reduced to 25 pM, which is close to the Kd value of the protease dimer. By fitting inhibition data to Morrison's equation, Ki values of amprenavir, darunavir, and tipranavir were determined to be 135, 10, and 82 pM, respectively. This assay, which is capable of measuring Ki values as low as 0.25 pM, is well-suited for characterizing the next generation of HIV-1 protease inhibitors.
Topics: Carbamates; Darunavir; Fluorescence; Fluorescent Dyes; Furans; HIV Infections; HIV Protease; HIV Protease Inhibitors; HIV-1; Humans; Kinetics; Pyridines; Pyrones; Sulfonamides
PubMed: 26261098
DOI: 10.1038/srep11286 -
Anais Da Academia Brasileira de Ciencias 2020Fosamprenavir calcium is an amprenavir prodrug of the protease inhibitors class used in the treatment of patients with acquired immunodeficiency syndrome (AIDS)....
Fosamprenavir calcium is an amprenavir prodrug of the protease inhibitors class used in the treatment of patients with acquired immunodeficiency syndrome (AIDS). Different solid forms of this drug are described in patents, in this sense studies on the physico-chemical characterization and stability are relevant for the selection of a solid form with adequate features for pharmaceutical purposes. In the present work form I (commercial) and amorphous of fosamprenavir calcium were characterized by the techniques of Differential Scanning Calorimetry (DSC), Thermogravimetry (TGA), Powder X-ray Diffraction (PXRD), Fourier-Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). Furthermore, the chemical and polymorphic stability of the commercial form were evaluated by DSC, PXRD, FTIR and High-Performance Liquid Chromatography (HPLC). In the studies of characterization, thermal analyses allied to spectroscopic methods (PXRD and FTIR) demonstrated that the presence of water in the crystalline structure of Form I is fundamental for maintaining its crystallinity. In studies of accelerated stability the techniques of DSC, PXRD and FTIR showed that Form I does not suffer phase change when submitted to controlled conditions of temperature and humidity. Moreover, HPLC and FTIR proved the chemical stability of this solid form of fosamprenavir, thus demonstrating its suitability for pharmaceutical purposes.
Topics: Carbamates; Furans; Humidity; Microscopy, Electron, Scanning; Organophosphates; Pharmaceutical Preparations; Spectrum Analysis; Sulfonamides; Technology, Pharmaceutical; Temperature; Thermodynamics
PubMed: 32401841
DOI: 10.1590/0001-3765202020181021 -
ACS Omega May 2019We report the structural analysis of highly drug-resistant human immunodeficiency virus protease (PR) variant PR, rationally selected by machine learning, in complex...
We report the structural analysis of highly drug-resistant human immunodeficiency virus protease (PR) variant PR, rationally selected by machine learning, in complex with substrate analogues. Crystal structures were solved of inhibitor-free inactive PR-D25N, wild-type PR/CA-p2 complex, and PR 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 PR or approximately 3-fold better than for PR. CA-p2 is a better inhibitor of PR than are clinical inhibitors ( = 50-8390 nM) except for amprenavir ( = 11 nM). G48V resistance mutation induces curled flap tips in PR-D25N structure. The inner P2-P2' residues of substrate analogues in PR 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 β-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 PR 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 PR with substrate analogues agrees with their relative inhibition, suggesting that this mutant improves substrate binding while decreasing affinity for clinical inhibitors.
PubMed: 31172041
DOI: 10.1021/acsomega.9b00683 -
Scientific Reports Jan 2018The HIV-2 protease (PR2) is a homodimer of 99 residues with asymmetric assembly and binding various ligands. We propose an exhaustive study of the local structural...
The HIV-2 protease (PR2) is a homodimer of 99 residues with asymmetric assembly and binding various ligands. We propose an exhaustive study of the local structural asymmetry between the two monomers of all available PR2 structures complexed with various inhibitors using a structural alphabet approach. On average, PR2 exhibits asymmetry in 31% of its positions-i.e., exhibiting different backbone local conformations in the two monomers. This asymmetry was observed all along its structure, particularly in the elbow and flap regions. We first differentiated structural asymmetry conserved in most PR2 structures from the one specific to some PR2. Then, we explored the origin of the detected asymmetry in PR2. We localized asymmetry that could be induced by PR2's flexibility, allowing transition from the semi-open to closed conformations and the asymmetry potentially induced by ligand binding. This latter could be important for the PR2's adaptation to diverse ligands. Our results highlighted some differences between asymmetry of PR2 bound to darunavir and amprenavir that could explain their differences of affinity. This knowledge is critical for a better description of PR2's recognition and adaptation to various ligands and for a better understanding of the resistance of PR2 to most PR2 inhibitors, a major antiretroviral class.
Topics: Carbamates; Crystallography, X-Ray; Darunavir; Enzyme Inhibitors; Furans; HIV Protease; Protein Binding; Protein Conformation; Protein Subunits; Sulfonamides
PubMed: 29335428
DOI: 10.1038/s41598-017-18941-3 -
Antimicrobial Agents and Chemotherapy May 2018We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at...
GRL-079, a Novel HIV-1 Protease Inhibitor, Is Extremely Potent against Multidrug-Resistant HIV-1 Variants and Has a High Genetic Barrier against the Emergence of Resistant Variants.
We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2' cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (ECs) were 2.5 to 30 nM against wild-type HIV-1, 0.3 to 6.7 nM against HIV-2, and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIV). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIV), with ECs of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (ECs of >1,000 nM) against HIV Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2' Cp-Abt group forms strong hydrogen bonds with Asp30'. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIV strains (HIV), HIV became highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV failed to replicate at >0.08 μM and >0.2 μM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2' Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1 and a wide spectrum of HIV strains.
Topics: Amino Acid Sequence; Atazanavir Sulfate; Carbamates; Cell Line, Tumor; Darunavir; Drug Resistance, Multiple, Viral; Furans; HIV Protease; HIV Protease Inhibitors; HIV-1; Humans; Lopinavir; Microbial Sensitivity Tests; Sulfonamides; Virus Replication
PubMed: 29463535
DOI: 10.1128/AAC.02060-17 -
Scientific Reports Sep 2017We report that GRL-09510, a novel HIV-1 protease inhibitor (PI) containing a newly-generated P2-crown-tetrahydrofuranylurethane (Crwn-THF), a P2'-methoxybenzene, and a...
GRL-09510, a Unique P2-Crown-Tetrahydrofuranylurethane -Containing HIV-1 Protease Inhibitor, Maintains Its Favorable Antiviral Activity against Highly-Drug-Resistant HIV-1 Variants in vitro.
We report that GRL-09510, a novel HIV-1 protease inhibitor (PI) containing a newly-generated P2-crown-tetrahydrofuranylurethane (Crwn-THF), a P2'-methoxybenzene, and a sulfonamide isostere, is highly active against laboratory and primary clinical HIV-1 isolates (EC: 0.0014-0.0028 μM) with minimal cytotoxicity (CC: 39.0 μM). Similarly, GRL-09510 efficiently blocked the replication of HIV-1 variants, which were capable of propagating at high-concentrations of atazanavir, lopinavir, and amprenavir (APV). GRL-09510 was also potent against multi-drug-resistant clinical HIV-1 variants and HIV-2. Under the selection condition, where HIV-1 rapidly acquired significant resistance to APV, an integrase inhibitor raltegravir, and a GRL-09510 congener (GRL-09610), no variants highly resistant against GRL-09510 emerged over long-term in vitro passage of the virus. Crystallographic analysis demonstrated that the Crwn-THF moiety of GRL-09510 forms strong hydrogen-bond-interactions with HIV-1 protease (PR) active-site amino acids and is bulkier with a larger contact surface, making greater van der Waals contacts with PR than the bis-THF moiety of darunavir. The present data demonstrate that GRL-09510 has favorable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants, that the newly generated P2-Crwn-THF moiety confers highly desirable anti-HIV-1 potency. The use of the novel Crwn-THF moiety sheds lights in the design of novel PIs.
Topics: Antiviral Agents; Cell Survival; Crystallography, X-Ray; Drug Resistance, Viral; Furans; HIV Protease; HIV Protease Inhibitors; HIV-1; Humans; Microbial Sensitivity Tests; Models, Molecular; Protein Binding; Serial Passage
PubMed: 28947797
DOI: 10.1038/s41598-017-12052-9 -
Acta Pharmaceutica (Zagreb, Croatia) Jun 2022The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently...
The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P--glycoprotein activity. Clone isolation the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.
Topics: Animals; Dogs; Madin Darby Canine Kidney Cells; Cell Membrane Permeability; Biological Transport; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily B; Permeability
PubMed: 36651516
DOI: 10.2478/acph-2022-0003 -
Laryngoscope Investigative... Feb 2024Approximately 25% of Americans suffer from laryngopharyngeal reflux (LPR), a disease for which no effective medical therapy exists. Pepsin is a predominant source of...
OBJECTIVES
Approximately 25% of Americans suffer from laryngopharyngeal reflux (LPR), a disease for which no effective medical therapy exists. Pepsin is a predominant source of damage during LPR and a key therapeutic target. Fosamprenavir (FOS) inhibits pepsin and prevents damage in an LPR mouse model. Inhaled FOS protects at a lower dose than oral; however, the safety of inhaled FOS is unknown and there are no inhalers for laryngopharyngeal delivery. A pre-Good Lab Practice (GLP) study of inhaled FOS was performed to assess safety and computational fluid dynamics (CFD) modeling used to predict the optimal particle size for a laryngopharyngeal dry powder inhaler (DPI).
METHODS
Aerosolized FOS, amprenavir (APR), or air (control) were provided 5 days/week for 4 weeks ( = 6) in an LPR mouse model. Organs (nasal cavity, larynx, esophagus, trachea, lung, liver, heart, and kidney) were assessed by a pathologist and bronchoalveolar lavage cytokines and plasma cardiotoxicity markers were assessed by Luminex assay. CFD simulations were conducted in a model of a healthy 49-year-old female.
RESULTS
No significant increase was observed in histologic lesions, cytokines, or cardiotoxicity markers in FOS or APR groups relative to the control. CFD predicted that laryngopharyngeal deposition was maximized with aerodynamic diameters of 8.1-11.5 μm for inhalation rates of 30-60 L/min.
CONCLUSIONS
A 4-week pre-GLP study supports the safety of inhaled FOS. A formal GLP assessment is underway to support a phase I clinical trial of an FOS DPI for LPR.
LEVEL OF EVIDENCE
NA.
PubMed: 38362183
DOI: 10.1002/lio2.1219