Molecular Analysis of the HIV-1 Resistance Development: Enzymatic Activities, Crystal Structures, and Thermodynamics of Nelfinavir-resistant HIV Protease Mutants

Kožíšek, Milan; Bray, Jenelle K.; Řezáčová, P.; Šašková, Klára Grantz; Brynda, J.; Pokorná, Jana; Mammano, Fabrizio; Rulisek, Lubomfr; Konvalinka, Jan

doi:10.1016/j.jmb.2007.09.083

Cited by 78 publications

(58 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We also showed that D30 not only is key for inhibitor binding but also is essential for recognition of the p1-p6 cleavage site (8,15,22). Consequently, the D30N mutation, which lowers affinity for NFV (23), also likely compromises p1-p6 recognition, leading to coevolution of this cleavage site (15). Such p1-p6 cleavage site mutations commonly observed with D30N/N88D PR are L449F, S451N, and P453L (14), and they may help restoring the fit of the substrate in the consensus substrate envelope (24).…”

Section: H Uman Immunodeficiency Virus Type 1 (Hiv-1) Protease (Pr)mentioning

confidence: 76%

HIV-1 Protease-Substrate Coevolution in Nelfinavir Resistance

Kolli

Özen

KurtYilmaz

et al. 2014

J Virol

View full text Add to dashboard Cite

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. IMPORTANCEResistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations.

show abstract

Section: H Uman Immunodeficiency Virus Type 1 (Hiv-1) Protease (Pr)mentioning

confidence: 76%

HIV-1 Protease-Substrate Coevolution in Nelfinavir Resistance

Kolli

Özen

KurtYilmaz

et al. 2014

J Virol

View full text Add to dashboard Cite

show abstract

“…Therefore, a huge number of experimental studies in the fields of molecular biology, biochemistry and biophysics have been performed since the AIDS pandemics burst out which contributed to revealing the structure and enzymological properties of the enzyme. As a result of this effort, nine inhibitors have been brought to the stage of commercially produced and therapeutically used drugs during this period 1,2 .…”

Section: Introductionmentioning

confidence: 99%

Pressure induced structural changes and dimer destabilization of HIV-1 protease studied by molecular dynamics simulations

Kutálková

Hrnčiřík

Ingr

2014

Phys. Chem. Chem. Phys.

View full text Add to dashboard Cite

High-pressure methods became an attractive tool of investigation of structural stability of proteins.Besides protein unfolding, dimerization can be studied this way, too. HIV-1 protease is a convenient target of experimental and theoretical high-pressure studies. In this study molecular-dynamics simulations are used to predict the response of HIV-1 protease to the pressure of 0.1 to 600 MPa.The protease conformation of both monomer and dimer is highly rigid changing insignificantly with growing pressure. Hydrophobicity of the protease decreases with increasing pressure. Water density inside the active-site cavity grows from 87% to 100% of the bulk water density within the pressure range. Dimer-dissociation volume change is negative values for most of the pressure range with the minimum of -105 ml/mol, except a short interval of positive values at low pressures. The dimer is thus slightly stabilized up to 160 MPa, but strongly destabilized by higher pressures.

show abstract

“…On the contrary, several X-ray crystal structures of PR complexed with more asymmetric FDAapproved inhibitors, like darunavir, nelfinavir, and saquinavir (SQV), have the catalytic site occupied by the inhibitor oriented in two almost equivalent positions related by a pseudo-2-fold symmetry. [7][8][9][10][11][12] To investigate this order/disorder phenomenon and its possible relationship with crystal packing, we have undertaken a systematic search for new crystal forms of wild-type PR in complex with SQV. Single crystals of the PR/SQV complex were obtained by a vapor diffusion technique and analyzed by X-ray diffraction.…”

mentioning

confidence: 99%

Carbamylation of N-Terminal Proline

Olajuyigbe

Demitri²,

Ajele

et al. 2010

ACS Med. Chem. Lett.

View full text Add to dashboard Cite

Protein carbamylation is of great concern both in vivo and in vitro. Here, we report the first structural characterization of a protein carbamylated at the N-terminal proline. The unexpected carbamylation of the R-amino group of the least reactive codified amino acid has been detected in high-resolution electron density maps of a new crystal form of the HIV-1 protease/saquinavir complex. The carbamyl group is found coplanar to the proline ring with a trans conformation. The reaction of N-terminal with cyanate ion derived from the chaotropic agent urea was confirmed by mass spectra analysis on protease single crystals. Implications of carbamylation process in vitro and in vivo are discussed.KEYWORDS Carbamylation, N-terminal proline, HIV-1 protease/saquinavir complex, single crystals T he HIV protease (PR) is an aspartic protease that shares sequence homology around the active site with other retroviral proteases, as the conserved Asp-ThrGly catalytic triad. 1 PR is required for proteolytic cleavage of viral Gag and Gag-Pol polyproteins into individual structural and functional proteins during viral maturation. 2 In the absence of this proteolysis, immature noninfectious virions are produced, thus making PR a prime target for structureassisted drug design in antiviral therapy. 3,4 The structure-assisted drug design and discovery process utilizes techniques such as protein crystallography, nuclear magnetic resonance (NMR), and computational biochemistry to guide synthesis of potential drugs. PR has been widely characterized biochemically and structurally, which has led to the discovery of HIV protease inhibitors (PIs). Their utilization in highly active antiretroviral therapy (HAART) has been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). 5 Recently, we have demonstrated that high-resolution X-ray crystallography can be used as a powerful method to identify the most potent PR inhibitor present in an epimeric mixture. 6 The pseudosymmetric peptidomimetic inhibitor based on a novel Phe-Pro isostere core matched the 2-fold symmetry of the homodimeric structure of the PR. Fourier maps obtained by high-resolution diffraction data (1.3 Å) clearly showed the catalytic site fully occupied by a single ordered stereoisomer. On the contrary, several X-ray crystal structures of PR complexed with more asymmetric FDAapproved inhibitors, like darunavir, nelfinavir, and saquinavir (SQV), have the catalytic site occupied by the inhibitor oriented in two almost equivalent positions related by a pseudo-2-fold symmetry. [7][8][9][10][11][12] To investigate this order/disorder phenomenon and its possible relationship with crystal packing, we have undertaken a systematic search for new crystal forms of wild-type PR in complex with SQV. Single crystals of the PR/SQV complex were obtained by a vapor diffusion technique and analyzed by X-ray diffraction. These crystals belong to a new monoclinic crystal form, which contains two dimers of the complex in the asymmetric unit. Other crystal...

show abstract

Molecular Analysis of the HIV-1 Resistance Development: Enzymatic Activities, Crystal Structures, and Thermodynamics of Nelfinavir-resistant HIV Protease Mutants

Cited by 78 publications

References 48 publications

HIV-1 Protease-Substrate Coevolution in Nelfinavir Resistance

HIV-1 Protease-Substrate Coevolution in Nelfinavir Resistance

Pressure induced structural changes and dimer destabilization of HIV-1 protease studied by molecular dynamics simulations

Carbamylation of N-Terminal Proline

Contact Info

Product

Resources

About