Resistance mechanism revealed by crystal structures of unliganded nelfinavir-resistant HIV-1 protease non-active site mutants N88D and N88S

Bihani, Subhash C.; Das, Amit Kumar; Prashar, Vishal; Ferrer, Jean‐Luc; Hosur, M.V.

doi:10.1016/j.bbrc.2009.08.138

Cited by 10 publications

(10 citation statements)

References 39 publications

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“…It has been suggested that substitution of asparagine with serine creates a hydrogen bond with the residue D30, which in turn affects the interaction between D30 and the inhibitor NFV [55]. A similar observation with regard to the N88S effect on the interaction with D30 has been previously made for L10F/ N88S mutant with NFV as well as the unliganded N88S protease [56,57]. Another mutation at this site that occurs in patients treated with NFV, N88D, has been reported to co-occur with mutation D30N, which coincides with losing water molecules that mediate this site's interactions with residues T31 and T74 [58].…”

Section: Table 2 Change Of the Binding Free Energy ( G ) Of Inhibitorsupporting

confidence: 69%

Non-active site mutants of HIV-1 protease influence resistance and sensitisation towards protease inhibitors

et al. 2020

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Background: HIV-1 can develop resistance to antiretroviral drugs, mainly through mutations within the target regions of the drugs. In HIV-1 protease, a majority of resistance-associated mutations that develop in response to therapy with protease inhibitors are found in the protease's active site that serves also as a binding pocket for the protease inhibitors, thus directly impacting the protease-inhibitor interactions. Some resistance-associated mutations, however, are found in more distant regions, and the exact mechanisms how these mutations affect protease-inhibitor interactions are unclear. Furthermore, some of these mutations, e.g. N88S and L76V, do not only induce resistance to the currently administered drugs, but contrarily induce sensitivity towards other drugs. In this study, mutations N88S and L76V, along with three other resistance-associated mutations, M46I, I50L, and I84V, are analysed by means of molecular dynamics simulations to investigate their role in complexes of the protease with different inhibitors and in different background sequence contexts. Results: Using these simulations for alchemical calculations to estimate the effects of mutations M46I, I50L, I84V, N88S, and L76V on binding free energies shows they are in general in line with the mutations' effect on IC 50 values. For the primary mutation L76V, however, the presence of a background mutation M46I in our analysis influences whether the unfavourable effect of L76V on inhibitor binding is sufficient to outweigh the accompanying reduction in catalytic activity of the protease. Finally, we show that L76V and N88S changes the hydrogen bond stability of these residues with residues D30/K45 and D30/T31/T74, respectively. Conclusions: We demonstrate that estimating the effect of both binding pocket and distant mutations on inhibitor binding free energy using alchemical calculations can reproduce their effect on the experimentally measured IC 50 values. We show that distant site mutations L76V and N88S affect the hydrogen bond network in the protease's active site, which offers an explanation for the indirect effect of these mutations on inhibitor binding. This work thus provides valuable insights on interplay between primary and background mutations and mechanisms how they affect inhibitor binding.

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Section: Table 2 Change Of the Binding Free Energy ( G ) Of Inhibitorsupporting

confidence: 69%

Non-active site mutants of HIV-1 protease influence resistance and sensitisation towards protease inhibitors

et al. 2020

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“…DEER profiles are significantly shifted to favor the closed state with the addition of active-site PR inhibitors(29). Of 270 structures of HIV protease in the PDB, the only apo structures in the closed conformation are of tethered dimers(32–36). The only other known apo-PR crystal structures either have flaps in an open or semi-open conformation stabilized by lattice contacts, or the flaps are disordered(18).…”

Section: Resultsmentioning

confidence: 99%

Small Molecule Regulation of Protein Conformation by Binding in the Flap of HIV Protease

et al. 2013

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The fragment indole-6-carboxylic acid (1F1), previously identified as a flap site binder in a fragment-based screen against HIV protease (PR), has been co-crystallized with pepstatin-inhibited PR and with apo-PR. Another fragment, 3-indolepropionic acid (1F1-N), predicted by AutoDock calculations and confirmed in a novel ‘inhibition of nucleation’ crystallization assay, exploits the same interactions in the flap site in two crystal structures. Both 1F1 and 1F1-N bind to the closed form of apo-PR and to pepstatin:PR. In solution, 1F1 and 1F1-N raise the Tm of apo-PR by 3.5–5 °C as assayed by differential scanning fluorimetry (DSF), and show equivalent low-micromolar binding constants to both apo-PR and pepstatin:PR, assayed by backscattering interferometry (BSI). The observed signal intensities in BSI are greater for each fragment upon binding to apo-PR than to pepstatin-bound PR, consistent with greater conformational change in the former binding event. Together, these data indicate that fragment binding in the flap site favors a closed conformation of HIV PR.

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“…In both cases, the mutation increases the mobility of the Asp30 side chain, thus decreasing interactions with the inhibitor. The dynamic side chain position of Asp30 has been observed in various D30N 27 single mutants and double mutants such as D30N/N88D and D30N/N88S 34 . This mutation has significantly reduced the effectiveness of the clinical PIs as observed from the kinetic studies.…”

Section: Discussionmentioning

confidence: 99%

Potent Antiviral HIV-1 Protease Inhibitor GRL-02031 Adapts to the Structures of Drug Resistant Mutants with Its P1′-Pyrrolidinone Ring

Chang

Zhang

et al. 2012

J. Med. Chem.

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GRL-02031 (1) is an HIV-1 protease (PR) inhibitor containing a novel P1′ (R)-aminomethyl-2-pyrrolidinone group. Crystal structures at resolutions of 1.25 to 1.55 Å were analyzed for complexes of 1 with the PR containing major drug resistant mutations, PRI47V, PRL76V, PRV82A and PRN88D. Mutations of I47V and V82A alter residues in the inhibitor-binding site, while L76V and N88D are distal mutations having no direct contact with the inhibitor. Substitution of a smaller amino acid in PRI47V and PRL76V, and the altered charge of PRN88D are associated with significant local structural changes compared to the wild-type PRWT, while substitution of alanine in PRV82A increases the size of the S1′ subsite. The P1′ pyrrolidinone group of 1 accommodates to these local changes by assuming two different conformations. Overall, the conformation and interactions of 1 with PR mutants resemble those of PRWT with similar inhibition constants in good agreement with the antiviral potency on multidrug resistant HIV-1.

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Resistance mechanism revealed by crystal structures of unliganded nelfinavir-resistant HIV-1 protease non-active site mutants N88D and N88S

Cited by 10 publications

References 39 publications

Non-active site mutants of HIV-1 protease influence resistance and sensitisation towards protease inhibitors

Non-active site mutants of HIV-1 protease influence resistance and sensitisation towards protease inhibitors

Small Molecule Regulation of Protein Conformation by Binding in the Flap of HIV Protease

Potent Antiviral HIV-1 Protease Inhibitor GRL-02031 Adapts to the Structures of Drug Resistant Mutants with Its P1′-Pyrrolidinone Ring

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