Solute Effects on Spin Labels at an Aqueous-Exposed Site in the Flap Region of HIV-1 Protease

ABSTRACTWe generated two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL-001-15 and GRL-003-15, which contain unique crown-like tetrahydropyranofuran (Crn-THF) and P2′-cyclopropyl-aminobenzothiazole (Cp-Abt) moieties as P2 and P2′ ligands, respectively. GRL-001-15 and GRL-003-15 havemeta-monofluorophenyl andpara-monofluorophenyl at the P1 site, respectively, exert highly potent activity against wild-type HIV-1 with 50% effective concentrations (EC50s) of 57 and 50 pM, respectively, and have favorable cytotoxicity profiles with 50% cytotoxic concentrations (CC50s) of 38 and 11 μM, respectively. The activity of GRL-001-15 against multi-PI-resistant HIV-1 variants was generally greater than that of GRL-003-15. The EC50of GRL-001-15 against an HIV-1 variant that was highly resistant to multiple PIs, including darunavir (DRV) (HIV-1DRVRP30), was 0.17 nM, and that of GRL-003-15 was 3.3 nM, while DRV was much less active, with an EC50of 216 nM. The emergence of HIV-1 variants resistant to GRL-001-15 and GRL-003-15 was significantly delayed compared to that of variants resistant to selected PIs, including DRV. Structural analyses of wild-type protease (PRWT) complexed with the novel PIs revealed that GRL-001-15’smeta-fluorine atom forms halogen bond interactions (2.9 and 3.0 Å) with Gly49 and Ile50, respectively, of the protease flap region and with Pro81′ (2.7 and 3.2 Å), which is located close to the protease active site, and that two fluorine atoms of GRL-142-13 form multiple halogen bond interactions with Gly49, Ile50, Pro81′, Ile82′, and Arg8′. In contrast, GRL-003-15 forms halogen bond interactions with Pro81′ alone, suggesting that the reduced antiviral activity of GRL-003-15 is due to the loss of the interactions with the flap region.

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Comparative analysis of the unbinding pathways of antiviral drug Indinavir from HIV and HTLV1 proteases by Supervised Molecular Dynamics simulation

10.1101/2021.03.27.437308 ◽

2021 ◽

Author(s):

Farzin Sohraby ◽

Hassan Aryapour

Keyword(s):

Inhibitory Activity ◽

Antiviral Drug ◽

Good Explanation ◽

Dynamics Simulation ◽

Stacking Interactions ◽

Pi Stacking ◽

Efficacious Treatment ◽

The Stability ◽

Flap Region ◽

Hiv 1

Determining the unbinding pathways of potential small molecule compounds from their target proteins is of great significance for designing efficacious treatment solutions. One of these potential compounds is the approved HIV-1 protease inhibitor, indinavir, which has a weak effect on the HTLV-1 protease. In this work, by employing SuMD method, we reconstructed the unbinding pathways of indinavir from HIV and HTLV-1 proteases in order to compare and to understand the mechanism of the unbinding, and also to discover the reasons for the lack of inhibitory activity of indinavir against the HTLV-1 protease. We achieved multiple unbinding events from both HIV and HTLV-1 proteases in which the RMSD values of indinavir reached over 4 nm. Also, we found that the mobility and the fluctuations of the flap region is higher in the HTLV-1 protease which makes the drug less stable. We realized that critically positioned aromatic residues such as Trp98/Trp98' and Phe67/Phe67' in the HTLV-1 protease can make strong pi-Stacking interactions with indinavir in the unbinding pathway which are unfavorable for the stability of indinavir in the active site. The details found in this study can make a good explanation for the lack of inhibitory activity of this drug against HTLV-1 protease. We believe the details discovered in this work can be a great assist for designing more effective and more selective inhibitors for the HTLV-1 protease.

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The HIV-1 central DNA flap region contains a “flapping” third strand

Biophysical Chemistry ◽

10.1016/j.bpc.2006.12.005 ◽

2007 ◽

Vol 127 (1-2) ◽

pp. 64-68 ◽

Cited By ~ 3

Author(s):

Besik I. Kankia ◽

Karin Musier-Forsyth

Keyword(s):

Flap Region ◽

Hiv 1

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Ninety-Nine Is Not Enough: Molecular Characterization of Inhibitor-Resistant Human Immunodeficiency Virus Type 1 Protease Mutants with Insertions in the Flap Region

Journal of Virology ◽

10.1128/jvi.02325-07 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5869-5878 ◽

Cited By ~ 28

Author(s):

Milan Kožíšek ◽

Klára Grantz Šašková ◽

Pavlína Řezáčová ◽

Jiří Brynda ◽

Noortje M. van Maarseveen ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Substrate Binding ◽

Viral Resistance ◽

Replication Capacity ◽

Immunodeficiency Virus ◽

Flap Region ◽

Hiv 1 ◽

Selection Of

ABSTRACT While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

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1PT102 Structural change at the flap region of HIV-1 protease associated with darunavir resistance(The 50th Annual Meeting of the Biophysical Society of Japan)

Seibutsu Butsuri ◽

10.2142/biophys.52.s85_4 ◽

2012 ◽

Vol 52 (supplement) ◽

pp. S85

Author(s):

Hirotaka Ode ◽

Koji Suzuki ◽

Masayuki Fujino ◽

Masami Maejima ◽

Yuki Kimura ◽

...

Keyword(s):

Structural Change ◽

Annual Meeting ◽

Biophysical Society ◽

Flap Region ◽

Hiv 1

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Structural Adaptation of Darunavir Analogs Against Primary Resistance Mutations in HIV-1 Protease

10.1101/406637 ◽

2018 ◽

Author(s):

Gordon J. Lockbaum ◽

Florian Leidner ◽

Linah N. Rusere ◽

Mina Henes ◽

Klajdi Kosovrasti ◽

...

Keyword(s):

Protease Inhibitor ◽

Crystal Structures ◽

Active Site ◽

Active Site Residues ◽

Resistance Mutations ◽

Structural Adaptation ◽

Primary Resistance ◽

Principle Component ◽

Flap Region ◽

Hiv 1

AbstractHIV-1 protease is one of the prime targets of agents used in antiretroviral therapy against HIV. However, under selective pressure of protease inhibitors, primary mutations at the active site weaken inhibitor binding to confer resistance. Darunavir (DRV) is the most potent HIV-1 protease inhibitor in clinic; resistance is limited, as DRV fits well within the substrate envelope. Nevertheless, resistance is observed due to hydrophobic changes at residues including I50, V82 and I84 that line the S1/S1’ pocket within the active site. Through enzyme inhibition assays and a series of 12 crystal structures, we interrogated susceptibility of DRV and two potent analogs to primary S1’ mutations. The analogs had modifications at the hydrophobic P1’ moiety to better occupy the unexploited space in the S1’ pocket where the primary mutations were located. Considerable losses of potency were observed against protease variants with I84V and I50V mutations for all three inhibitors. The crystal structures revealed an unexpected conformational change in the flap region of I50V protease bound to the analog with the largest P1’ moiety, indicating interdependency between the S1’ subsite and the flap region. Collective analysis of protease-inhibitor van der Waals (vdW) interactions in the crystal structures using principle component analysis indicated I84V mutation underlying the largest variation in the vdW contacts. Interestingly, the principle components were able to distinguish inhibitor identity and relative potency solely based on vdW interactions of active site residues in the crystal structures. Our results reveal the interplay between inhibitor P1’ moiety and primary S1’ mutations, as well as suggesting a novel method for distinguishing the interdependence of resistance through principle component analyses.

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Structure of the unbound form of HIV-1 subtype A protease: comparison with unbound forms of proteases from other HIV subtypes

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444909054298 ◽

2010 ◽

Vol 66 (3) ◽

pp. 233-242 ◽

Cited By ~ 21

Author(s):

Arthur H. Robbins ◽

Roxana M. Coman ◽

Edith Bracho-Sanchez ◽

Marty A. Fernandez ◽

C. Taylor Gilliland ◽

...

Keyword(s):

Crystal Structure ◽

Hiv Protease ◽

Space Groups ◽

Subtype B ◽

Closed Position ◽

Crystal Space ◽

Subtype A ◽

Flap Region ◽

Hiv 1 ◽

Hiv Subtypes

The crystal structure of the unbound form of HIV-1 subtype A protease (PR) has been determined to 1.7 Å resolution and refined as a homodimer in the hexagonal space groupP61to anRcrystof 20.5%. The structure is similar in overall shape and fold to the previously determined subtype B, C and F PRs. The major differences lie in the conformation of the flap region. The flaps in the crystal structures of the unbound subtype B and C PRs, which were crystallized in tetragonal space groups, are either semi-open or wide open. In the present structure of subtype A PR the flaps are found in the closed position, a conformation that would be more anticipated in the structure of HIV protease complexed with an inhibitor. The amino-acid differences between the subtypes and their respective crystal space groups are discussed in terms of the differences in the flap conformations.

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Comparative analysis of the unbinding pathways of antiviral drug Indinavir from HIV and HTLV1 proteases by supervised molecular dynamics simulation

PLoS ONE ◽

10.1371/journal.pone.0257916 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0257916

Author(s):

Farzin Sohraby ◽

Hassan Aryapour

Keyword(s):

Active Site ◽

Inhibitory Activity ◽

Antiviral Drug ◽

Dynamics Simulation ◽

Stacking Interactions ◽

Aromatic Residues ◽

Efficacious Treatment ◽

The Stability ◽

Flap Region ◽

Hiv 1

Determining the unbinding pathways of potential small molecule compounds from their target proteins is of great significance for designing efficacious treatment solutions. One of these potential compounds is the approved HIV-1 protease inhibitor, Indinavir, which has a weak effect on the HTLV-1 protease. In this work, by employing the SuMD method, we reconstructed the unbinding pathways of Indinavir from HIV and HTLV-1 proteases to compare and understand the mechanism of the unbinding and to discover the reasons for the lack of inhibitory activity of Indinavir against the HTLV-1 protease. We achieved multiple unbinding events from both HIV and HTLV-1 proteases in which the RMSD values of Indinavir reached over 40 Å. Also, we found that the mobility and fluctuations of the flap region are higher in the HTLV-1 protease, making the drug less stable. We realized that critically positioned aromatic residues such as Trp98/Trp98′ and Phe67/Phe67′ in the HTLV-1 protease could make strong π-Stacking interactions with Indinavir in the unbinding pathway, which are unfavorable for the stability of Indinavir in the active site. The details found in this study can make a reasonable explanation for the lack of inhibitory activity of this drug against HTLV-1 protease. We believe the details discovered in this work can help design more effective and selective inhibitors for the HTLV-1 protease.

Download Full-text