scholarly journals Understanding The Mechanism of Mutant HIV-1 Protease Resistance Against Darunavir Using Molecular Dynamic Study

2021 ◽  
Author(s):  
Yaser Shabanpour ◽  
Esmaeil Behmard ◽  
Parviz Abdolmaleki ◽  
Amir Homayoun Keihan ◽  
Sharareh Sajjadi
Keyword(s):  
Active Site ◽  
Resistance Mechanism ◽  
Principal Component ◽  
Van Der Waals Interactions ◽  
Hiv Virus ◽  
Immunodeficiency Virus ◽  
Mean Square Fluctuation ◽  
Hiv 1 ◽  
Important Enzyme

Abstract The human immunodeficiency virus type 1 protease (HIV-1 PR) is an important enzyme in life cycle of the HIV virus. It cleaves inactive pre-proteins of the virus and changes them into active proteins. Darunavir suppresses the wild type HIV-1 PR (WT-Pr) activity, but can’t inhibit the mutant resistant forms (MUT-Pr). Increasing knowledge about the resistance mechanism can be helpful for designing of more effective inhibitors. In this study, the mechanism of resistance of Ile47val and Ile54Met MUT-Pr strain against Darunavir was investigated. For this purpose, complexes of Darunavir with WT-Pr (WT-Pr-D) and MUT-Pr (MUT-Pr-D) were simulated for 200 ns and structure, dynamic and energetic properties of both simulations were investigated based on essential dynamics (principal component analysis (PCA)), root mean square fluctuation (RMSF), radial distribution function (RDF), molecular mechanics/Poisson Boltzman surface area (MM/PBSA) energies and etc. Our data revealed that mutations increased the flap tips flexibility and increased the active-site space, probably due to the reduction in hydrophobic forces. So, the protease’s conformation changed from closed state to semi-open state. Formation of semi open structure along with a reduction in van der Waals interactions and hydrogen bonds with Darunavir facilitates disjunction of Darunavir from the active-site in MUT-Pr-D.

Dissection of HIV-1 Protease Subtype B Inhibitors Resistance Through Molecular Modeling Approaches

2018 ◽  
pp. 149-170
Author(s):  
Ameeruddin Nusrath Unissa ◽  
Luke Elizabeth Hanna
Keyword(s):  
Active Site ◽  
Tight Binding ◽  
Viral Gene ◽  
Enzyme Active Site ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Competitive Inhibitors ◽  
Hiv 1 ◽  
Important Enzyme

Protease (PR) is an important enzyme required for the posttranslational processing of the viral gene products of type-1 human immunodeficiency virus (HIV-1). Protease inhibitors (PI) act as competitive inhibitors that bind to the active site of PR. The I84V mutation contributes resistance to multiple PIs, and structurally, this mutation affects both sides of the enzyme active site. In order to get insights about this major resistance site to PR inhibitors using in silico approaches, in this chapter, the wild-type (WT) and mutant (MT) I84V of PR were modeled and docked with all PR inhibitors: Atazanavir, Darunavir, Indinavir, Lopinavir, Nelfinavir, Saquinavir, and Tipranavir. Docking results revealed that in comparison to the WT, the binding score was higher for the MT-I84V. Thus, it can be suggested that the high affinity towards inhibitors in the MT could be due to the presence of energetically favorable interactions, which may lead to tight binding of inhibitors with the MT protein, leading to the development of PR resistance against PIs in HIV-1 eventually.

scholarly journals Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

2004 ◽  
Vol 78 (6) ◽  
pp. 3123-3132 ◽  
Author(s):  
Bradley C. Logsdon ◽  
John F. Vickrey ◽  
Philip Martin ◽  
Gheorghe Proteasa ◽  
Jay I. Koepke ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Surface Plasmon Resonance ◽  
Protease Inhibitors ◽  
Surface Plasmon ◽  
Active Site ◽  
Multidrug Resistant ◽  
Immunodeficiency Virus ◽  
Active Site Cavity ◽  
Hiv 1

ABSTRACT The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (Å) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease “flaps” stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 Å. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1′, S3, and S3′ pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k off rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k on and k off data (Kd = k off/k on) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

scholarly journals Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA

2004 ◽  
Vol 78 (7) ◽  
pp. 3387-3397 ◽  
Author(s):  
Elena N. Peletskaya ◽  
Alex A. Kogon ◽  
Steven Tuske ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Cross Linking ◽  
Immunodeficiency Virus ◽  
Dntp Binding ◽  
Hiv 1

ABSTRACT Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the “open” configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

scholarly journals Effects of Mutations in Residues near the Active Site of Human Immunodeficiency Virus Type 1 Integrase on Specific Enzyme-Substrate Interactions

1998 ◽  
Vol 72 (6) ◽  
pp. 5046-5055 ◽  
Author(s):  
Jennifer L. Gerton ◽  
Sharron Ohgi ◽  
Mari Olsen ◽  
Joseph DeRisi ◽  
Patrick O. Brown
Keyword(s):  
Human Immunodeficiency Virus ◽  
Active Site ◽  
Catalytic Core Domain ◽  
Viral Dna ◽  
Catalytic Core ◽  
Core Domain ◽  
Immunodeficiency Virus ◽  
Close Proximity ◽  
Hiv 1

ABSTRACT The phylogenetically conserved catalytic core domain of human immunodeficiency virus type 1 (HIV-1) integrase contains elements necessary for specific recognition of viral and target DNA features. In order to identify specific amino acids that determine substrate specificity, we mutagenized phylogenetically conserved residues that were located in close proximity to the active-site residues in the crystal structure of the isolated catalytic core domain of HIV-1 integrase. Residues composing the phylogenetically conserved DD(35)E active-site motif were also mutagenized. Purified mutant proteins were evaluated for their ability to recognize the phylogenetically conserved CA/TG base pairs near the viral DNA ends and the unpaired dinucleotide at the 5′ end of the viral DNA, using disintegration substrates. Our findings suggest that specificity for the conserved A/T base pair depends on the active-site residue E152. The phenotype of IN(Q148L) suggested that Q148 may be involved in interactions with the 5′ dinucleotide of the viral DNA end. The activities of some of the proteins with mutations in residues in close proximity to the active-site aspartic and glutamic acids were salt sensitive, suggesting that these mutations disrupted interactions with DNA.

scholarly journals Initial Cleavage of the Human Immunodeficiency Virus Type 1 GagPol Precursor by Its Activated Protease Occurs by an Intramolecular Mechanism

2004 ◽  
Vol 78 (16) ◽  
pp. 8477-8485 ◽  
Author(s):  
Steven C. Pettit ◽  
Lorraine E. Everitt ◽  
Sumana Choudhury ◽  
Ben M. Dunn ◽  
Andrew H. Kaplan
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Enzyme Activation ◽  
Particle Assembly ◽  
Precursor Processing ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.

scholarly journals Inhibition of Foamy Virus Reverse Transcriptase by Human Immunodeficiency Virus Type 1 RNase H Inhibitors

2014 ◽  
Vol 58 (7) ◽  
pp. 4086-4093 ◽  
Author(s):  
Angela Corona ◽  
Anna Schneider ◽  
Kristian Schweimer ◽  
Paul Rösch ◽  
Birgitta M. Wöhrl ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Foamy Virus ◽  
Rnase H ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTRNase H plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1). Therefore, it is a promising target for drug development. However, the identification of HIV-1 RNase H inhibitors (RHIs) has been hampered by the open morphology of its active site, the limited number of available RNase H crystal structures in complex with inhibitors, and the fact that, due to the high concentrations of Mg2+needed for protein stability, HIV-1 RNase H is not suitable for nuclear magnetic resonance (NMR) inhibitor studies. We recently showed that the RNase H domains of HIV-1 and prototype foamy virus (PFV) reverse transcriptases (RTs) exhibit a high degree of structural similarity. Thus, we examined whether PFV RNase H can serve as an HIV-1 RNase H model for inhibitor interaction studies. Five HIV-1 RHIs inhibited PFV RNase H activity at low-micromolar concentrations similar to those of HIV-1 RNase H, suggesting pocket similarity of the RNase H domains. NMR titration experiments with the PFV RNase H domain and the RHI RDS1643 (6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester) were performed to determine its binding site. Based on these results and previous data,in silicodocking analysis showed a putative RDS1643 binding region that reaches into the PFV RNase H active site. Structural overlays were performed with HIV-1 and PFV RNase H to propose the RDS1643 binding site in HIV-1 RNase H. Our results suggest that this approach can be used to establish PFV RNase H as a model system for HIV-1 RNase H in order to identify putative inhibitor binding sites in HIV-1 RNase H.

scholarly journals Molecular docking-based screening for novel inhibitors of the human immunodeficiency virus type 1 protease that effectively reduce the viral replication in human cells

2020 ◽  
Author(s):  
Carla Mavian ◽  
Roxana M Coman ◽  
Xinrui Zhang ◽  
Steve Pomeroy ◽  
David A. Ostrov ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Active Site ◽  
Ex Vivo ◽  
Human Cells ◽  
Immunodeficiency Virus ◽  
Hiv Type 1 ◽  
Therapeutic Pressure ◽  
Hiv 1

AbstractTherapeutic pressure by protease inhibitors (PIs) contributes to accumulation of mutations in the HIV type 1 (HIV-1) protease (PR) leading to development of drug resistance with subsequent therapy failure. Current PIs target the active site of PR in a competitive manner. Identification of molecules that exploit non-active site mechanisms of inhibition is essential to overcome resistance to current PIs. Potential non-active site HIV-1 protease (PR) inhibitors (PI) were identified by in silico screening of almost 140,000 molecules targeting the hinge region of PR. Inhibitory activity of best docking compounds was tested in an in vitro PR inhibition biochemical assay. Five compounds inhibited PR from multiple HIV-1 subtypes in vitro and reduced replicative capacity by PI-sensitive or multi-PI resistant HIV-1 variants in human cells ex vivo. Antiviral activity was boosted when combined with Ritonavir, potentially diminishing development of drug resistance, while providing effective treatment for drug resistant HIV-1 variants.

scholarly journals Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase

2005 ◽  
Vol 79 (4) ◽  
pp. 2493-2505 ◽  
Author(s):  
Richard Lu ◽  
Ana Limón ◽  
Hina Z. Ghory ◽  
Alan Engelman
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Class I ◽  
Catalytic Core Domain ◽  
Catalytic Core ◽  
Core Domain ◽  
Target Dna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The catalytic core domain (CCD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) harbors the enzyme active site and binds viral and chromosomal DNA during integration. Thirty-five CCD mutant viruses were constructed, paying particular attention to conserved residues in the Phe139-Gln146 flexible loop and abutting Ser147-Val165 amphipathic alpha helix that were implicated from previous in vitro work as important for DNA binding. Defective viruses were typed as class I mutants (specifically blocked at integration) or pleiotropic class II mutants (additional particle assembly and/or reverse transcription defects). Whereas HIV-1P145A and HIV-1Q146K grew like the wild type, HIV-1N144K and HIV-1Q148L were class I mutants, reinforcing previous results that Gln-148 is important for DNA binding and uncovering for the first time an important role for Asn-144 in integration. HIV-1Q62K, HIV-1H67E, HIV-1N120K, and HIV-1N155K were also class I mutants, supporting findings that Gln-62 and Asn-120 interact with viral and target DNA, respectively, and suggesting similar integration-specific roles for His-67 and Asn-155. Although results from complementation analyses established that IN functions as a multimer, the interplay between active-site and CCD DNA binding functions was unknown. By using Vpr-IN complementation, we determined that the CCD protomer that catalyzes integration also preferentially binds to viral and target DNA. We additionally characterized E138K as an intramolecular suppressor of Gln-62 mutant virus and IN. The results of these analyses highlight conserved CCD residues that are important for HIV-1 replication and integration and define the relationship between DNA binding and catalysis that occurs during integration in vivo.

scholarly journals Cross-Linking of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Template-Primer

2001 ◽  
Vol 75 (19) ◽  
pp. 9435-9445 ◽  
Author(s):  
Elena N. Peletskaya ◽  
Paul L. Boyer ◽  
Alex A. Kogon ◽  
Patrick Clark ◽  
Heiko Kroth ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Cross Linking ◽  
Cysteine Residues ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Cross-linking experiments were performed with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Two approaches were used—photoaffinity cross-linking and disulfide chemical cross-linking (using an oligonucleotide that contained an N2-modified dG with a reactive thiol group). In the former case, cross-linking can occur to any nucleotide in either DNA strand, and in the latter case, a specific cross-link is produced between the template and the enzyme. Neither the introduction of the unique cysteine residues into the fingers nor the modification of these residues with photocross-linking reagents caused a significant decrease in the enzymatic activities of RT. We were able to use this model system to investigate interactions between specific points on the fingers domain of RT and double-stranded DNA (dsDNA). Photoaffinity cross-linking of the template to the modified RTs with Cys residues in positions 65, 67, 70, and 74 of the fingers domain of the p66 subunit was relatively efficient. Azide-modified Cys residues produced 10 to 25% cross-linking, whereas diazirine modified residues produced 5 to 8% cross-linking. Disulfide cross-linking yields were up to 90%. All of the modified RTs preferentially photocross-linked to the 5′ extended template strand of the dsDNA template-primer substrate. The preferred sites of interactions were on the extended template, 5 to 7 bases beyond the polymerase active site. HIV-1 RT is quite flexible. There are conformational changes associated with substrate binding. Cross-linking was used to detect intramolecular movements associated with binding of the incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreases the efficiency of cross-linking, but causes only modest changes in the preferred positions of cross-linking. This suggests that the interactions between the fingers of p66 and the extended template involve the “open” configuration of the enzyme with the fingers away from the active site rather than the closed configuration with the fingers in direct contact with the incoming dNTP. This experimental approach can be used to measure distances between any site on the surface of the protein and an interacting molecule.

scholarly journals Crystal Structure of Lysine Sulfonamide Inhibitor Reveals the Displacement of the Conserved Flap Water Molecule in Human Immunodeficiency Virus Type 1 Protease

2007 ◽  
Vol 81 (17) ◽  
pp. 9512-9518 ◽  
Author(s):  
Madhavi N. L. Nalam ◽  
Anik Peeters ◽  
Tim H. M. Jonckers ◽  
Inge Dierynck ◽  
Celia A. Schiffer
Keyword(s):  
Crystal Structure ◽  
Human Immunodeficiency Virus ◽  
Water Molecule ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Hiv Protease ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) protease has been continuously evolving and developing resistance to all of the protease inhibitors. This requires the development of new inhibitors that bind to the protease in a novel fashion. Most of the inhibitors that are on the market are peptidomimetics, where a conserved water molecule mediates hydrogen bonding interactions between the inhibitors and the flaps of the protease. Recently a new class of inhibitors, lysine sulfonamides, was developed to combat the resistant variants of HIV protease. Here we report the crystal structure of a lysine sulfonamide. This inhibitor binds to the active site of HIV-1 protease in a novel manner, displacing the conserved water and making extensive hydrogen bonds with every region of the active site.

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