Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System

Mahdi, Mohamed; Szojka, Zsófia Ilona; Mótyán, János András; Tőzsér, József

doi:10.3390/v7122931

Cited by 14 publications

(11 citation statements)

References 50 publications

(62 reference statements)

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“…This will require the construction of mutant structures complexed with all PIs and knowledge of the resistance profile of each mutant. However, the resistance profile is particularly difficult to obtain for all mutants because there are few studies that have tested the effect of PIs against HIV-2 mutants using enzymatic or phenotypic susceptibility assays [9,16,17,19,23] and some of these studies have led to opposing results [1].…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the studied drug resistance mutations do not modify the structural asymmetry of these positions, reinforcing the important role of the structural asymmetry of these positions. These overrepresented asymmetric positions could be important for PR2 structure or activity, particularly the four residues belonging to the dimerization region (4, 5, 97, and 98) and the five pocket residues (23,32,47,50,80).…”

Section: Characterization Of Structural Asymmetry In the Pr2 Mutant Setmentioning

confidence: 99%

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Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease

Laville

Fartek

Cerisier

et al. 2020

BMC Mol and Cell Biol

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Background: Drug resistance is a severe problem in HIV treatment. HIV protease is a common target for the design of new drugs for treating HIV infection. Previous studies have shown that the crystallographic structures of the HIV-2 protease (PR2) in bound and unbound forms exhibit structural asymmetry that is important for ligand recognition and binding. Here, we investigated the effects of resistance mutations on the structural asymmetry of PR2. Due to the lack of structural data on PR2 mutants, the 3D structures of 30 PR2 mutants of interest have been modeled using an in silico protocol. Structural asymmetry analysis was carried out with an in-house structural-alphabet-based approach. Results: The systematic comparison of the asymmetry of the wild-type structure and a large number of mutants highlighted crucial residues for PR2 structure and function. In addition, our results revealed structural changes induced by PR2 flexibility or resistance mutations. The analysis of the highlighted structural changes showed that some mutations alter protein stability or inhibitor binding. Conclusions: This work consists of a structural analysis of the impact of a large number of PR2 resistant mutants based on modeled structures. It suggests three possible resistance mechanisms of PR2, in which structural changes induced by resistance mutations lead to modifications in the dimerization interface, ligand recognition or inhibitor binding.

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Section: Discussionmentioning

confidence: 99%

Section: Characterization Of Structural Asymmetry In the Pr2 Mutant Setmentioning

confidence: 99%

Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease

Laville

Fartek

Cerisier

et al. 2020

BMC Mol and Cell Biol

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“…Ty1 PR-His 6 activity was measured in the presence of various HIV-1 protease inhibitors. Atazanavir, nelfinavir, saquinavir, darunavir, amprenavir, lopinavir, tipranavir [28], acetylpepstatin, and pepstatin A [29], and DMP-323 [30] were in-house stocks. To study effect of inhibitors on proteolytic activity, reaction mixtures contained 5 μl purified Ty1 PR-His 6 (400-1600 nM), 10 μl peptide buffer A, 4.8 μl oligopeptide substrate (VPTIN � NVHTS, 0.44 mM), and 0.2 μl inhibitor.…”

Section: Inhibition Studymentioning

confidence: 99%

Biochemical characterization of Ty1 retrotransposon protease

et al. 2020

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Ty1 is one of the many transposons in the budding yeast Saccharomyces cerevisiae. The life-cycle of Ty1 shows numerous similarities with that of retroviruses, e.g. the initially synthesized polyprotein precursor undergoes proteolytic processing by the protease. The retroviral proteases have become important targets of current antiretroviral therapies due to the critical role of the limited proteolysis of Gag-Pol polyprotein in the replication cycle and they therefore belong to the most well-studied enzymes. Comparative analyses of retroviral and retroviral-like proteases can help to explore the key similarities and differences which may help understanding how resistance is developed against protease inhibitors, but the available information about the structural and biochemical characteristics of retroviral-like, and especially retrotransposon, proteases is limited. To investigate the main characteristics of Ty1 retrotransposon protease of Saccharomyces cerevisiae, untagged and His 6-tagged forms of Ty1 protease were expressed in E. coli. After purification of the recombinant proteins, activity measurements were performed using synthetic oligopeptide and fluorescent recombinant protein substrates, which represented the wild-type and the modified forms of naturally occurring cleavage sites of the protease. We investigated the dependence of enzyme activity on different reaction conditions (pH, temperature, ionic strength, and urea concentration), and determined enzyme kinetic parameters for the studied substrates. Inhibitory potentials of 10 different protease inhibitors were also tested. Ty1 protease was not inhibited by the inhibitors which have been designed against human immunodeficiency virus type 1 protease and are approved as antiretroviral therapeutics. A quaternary structure of homodimeric Ty1 protease was proposed based on homology modeling, and this structure was used to support interpretation of experimental results and to correlate some structural and biochemical characteristics with that of other retroviral proteases.

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“…Pepstatin A, acetyl-pepstatin [ 28 ], indinavir, tipranavir, saquinavir, nelfinavir, darunavir, lopinavir, and amprenavir [ 29 ] were in-house stocks. Acetyl-pepstatin was dissolved in acetic acid (50%), while all other inhibitors in dimethyl sulfoxide (DMSO).…”

Section: Methodsmentioning

confidence: 99%

Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

et al. 2020

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The human retroviral-like aspartic protease 1 (ASPRV1) is a mammalian retroviral-like enzyme that catalyzes a critical proteolytic step during epidermal differentiation; therefore, it is also referred to as skin-specific aspartic protease (SASPase). Neutrophil granulocytes were also found recently to express ASPRV1 that is involved in the progression of acute chronic inflammation of the central nervous system, especially in autoimmune encephalomyelitis. Thus, investigation of ASPRV1 is important due to its therapeutic or diagnostic potential. We investigated the structural characteristics of ASPRV1 by homology modeling; analysis of the proposed structure was used for interpretation of in vitro specificity studies. For in-vitro characterization, activities of SASP28 and SASP14 enzyme forms were measured using synthetic oligopeptide substrates. We demonstrated that self-processing of SASP28 precursor causes autoactivation of the protease. The highest activity was measured for GST-SASP14 at neutral pH and at high ionic strength, and we proved that pepstatin A and acetyl-pepstatin can also inhibit the protease. In agreement with the structural characteristics, the relatively lower urea dissociation constant implied lower dimer stability of SASP14 compared to that of HIV-1 protease. The obtained structural and biochemical characteristics support better understanding of ASPRV1 function in the skin and central nervous system.

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Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System

Cited by 14 publications

References 50 publications

Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease

Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease

Biochemical characterization of Ty1 retrotransposon protease

Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

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