HIV protease (HIV PR) is a primary target for anti-HIV drug design. We have previously identified and characterized substituted metallacarboranes as a new class of HIV protease inhibitors. In a structure-guided drug design effort, we connected the two cobalt bis(dicarbollide) clusters with a linker to substituted ammonium group and obtained a set of compounds based on a lead formula [H(2)N-(8-(C(2)H(4)O)(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))-3,3'-Co)(2)]Na. We explored inhibition properties of these compounds with various substitutions, determined the HIV PR:inhibitor crystal structure, and computationally explored the conformational space of the linker. Our results prove the capacity of linker-substituted dual-cage cobalt bis(dicarbollides) as lead compounds for design of more potent inhibitors of HIV PR.

• MeSH
• elektrony MeSH
• HIV-1 enzymologie   účinky léků   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• inhibitory HIV-proteasy farmakologie   chemická syntéza   chemie   metabolismus   MeSH
• kobalt chemie   MeSH
• krystalografie rentgenová MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• racionální návrh léčiv MeSH
• sloučeniny boru chemická syntéza   chemie   farmakologie   metabolismus   MeSH
• uhlík chemie   MeSH
• Publikační typ
• práce podpořená grantem MeSH

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

• MeSH
• genové produkty env - virus lidské imunodeficience metabolismus   MeSH
• genové produkty gag - virus lidské imunodeficience metabolismus   MeSH
• HIV infekce virologie   MeSH
• HIV-1 izolace a purifikace   účinky léků   MeSH
• HIV-proteasa genetika   chemie   metabolismus   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• polyproteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• sulfonamidy farmakologie   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Proteases play a crucial role in the retroviral infection but so far the mechanism of their regulation remains unclear. Protease MIA-14 from murine intracisternal A-type particles, containing a C-terminal domain rich in glycines (G-patch), is responsible for binding of single-stranded oligonucleotides (both RNA and DNA) without inhibiting the proteolytic activity. For investigations of untill now poorly characterized protease-oligonucleotide interactions, assignments of the observed NMR frequencies are mandatory. An almost complete assignments of the main chain and (13)C(beta) side chain resonances of the 34 kDa homo-dimeric inMIA-14 PR is presented in this study.

• MeSH
• geny pro IAP elementy MeSH
• myši MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteasy genetika   chemie   metabolismus   MeSH
• Retroviridae MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

The project involves analysis of resistance development in HIV-positive patients treated by protease inhibitors in the CR, design of novel, non-infectious phenotypic resistance assays and design of novel inhibitors, active against resistant forms of HIV.

Projekt navrhuje sledování vzniku resistence u HIV-positivních pacientů v ČR, vývoj nových, jednoduchých neinfekčních metod testování resistence a návrh nových strukturních typů inhibitorů, aktivních vůči resistentním formám HIV.

• MeSH
• diagnostické techniky molekulární MeSH
• HIV infekce MeSH
• inhibitory HIV-proteasy MeSH
• syndromy imunologické nedostatečnosti MeSH
• virová léková rezistence MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• dermatovenerologie
• chemie, klinická chemie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K(i) ) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in K(i) in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2' subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel beta-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft.

• MeSH
• alanin metabolismus   MeSH
• Escherichia coli genetika   MeSH
• financování organizované MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy farmakologie   chemie   metabolismus   MeSH
• katalýza MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• molekulární modely MeSH
• náchylnost k nemoci MeSH
• pyrimidinony farmakologie   chemie   metabolismus   MeSH
• rekombinantní proteiny antagonisté a inhibitory   chemie   izolace a purifikace   MeSH
• sekundární struktura proteinů MeSH
• substituce aminokyselin MeSH
• virová léková rezistence genetika   MeSH
• vodíková vazba MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.

• MeSH
• dimerizace MeSH
• financování organizované MeSH
• genetická variace MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 enzymologie   genetika   účinky léků   MeSH
• HIV-proteasa genetika   imunologie   účinky léků   MeSH
• imunoglobuliny - fragmenty farmakologie   imunologie   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• monoklonální protilátky farmakologie   imunologie   MeSH
• mutace MeSH
• rekombinantní proteiny farmakologie   imunologie   MeSH
• virová léková rezistence genetika   MeSH
• vysoce aktivní antiretrovirová terapie MeSH
• Check Tag
• lidé MeSH

HIV protease (PR) is a prime target for rational anti-HIV drug design. We have previously identified icosahedral metallacarboranes as a novel class of nonpeptidic protease inhibitors. Now we show that substituted metallacarboranes are potent and specific competitive inhibitors of drug-resistant HIV PRs prepared either by site-directed mutagenesis or cloned from HIV-positive patients. Molecular modeling explains the inhibition profile of metallacarboranes by their unconventional binding mode.

• MeSH
• financování organizované MeSH
• HIV-1 enzymologie   účinky léků   MeSH
• HIV-proteasa genetika   chemie   metabolismus   MeSH
• inhibitory HIV-proteasy farmakologie   chemie   MeSH
• kovy chemie   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• mutace genetika   MeSH
• sloučeniny boru farmakologie   chemie   MeSH
• virová léková rezistence účinky léků   MeSH

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.

• MeSH
• buněčné linie MeSH
• chemické modely MeSH
• difrakce rentgenového záření MeSH
• financování organizované MeSH
• HIV-1 enzymologie   fyziologie   genetika   MeSH
• HIV-proteasa genetika   chemie   izolace a purifikace   metabolismus   MeSH
• inhibitory reverzní transkriptasy chemie   MeSH
• inzerční mutageneze MeSH
• katalýza MeSH
• kinetika MeSH
• konsenzuální sekvence MeSH
• látky proti HIV terapeutické užití   MeSH
• ledviny cytologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• rekombinantní proteiny chemie   izolace a purifikace   metabolismus   MeSH
• replikace viru MeSH
• RNA virová analýza   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence MeSH
• Check Tag
• lidé MeSH

Human immunodeficiency virus (HIV) encodes an aspartic protease (PR) that cleaves viral polyproteins into mature proteins, thus leading to the formation of infectious particles. Protease inhibitors (PIs) are successful virostatics. However, their efficiency is compromised by antiviral resistance. In the PR sequence of viral variants resistant to the PI nelfinavir, the mutations D30N and L90M appear frequently. However, these two mutations are seldom found together in vivo, suggesting that there are two alternative evolutionary pathways leading to nelfinavir resistance. Here we analyze the proteolytic activities, X-ray structures, and thermodynamics of inhibitor binding to HIV-1 PRs harboring the D30N and L90M mutations alone and in combination with other compensatory mutations. Vitality values obtained for recombinant mutant proteases and selected PR inhibitors confirm the crucial role of mutations in positions 30 and 90 for nelfinavir resistance. The combination of the D30N and L90M mutations significantly increases the enzyme vitality in the presence of nelfinavir, without a dramatic decrease in the catalytic efficiency of the recombinant enzyme. Crystal structures, molecular dynamics simulations, and calorimetric data for four mutants (D30N, D30N/A71V, D30N/N88D, and D30N/L90M) were used to augment our kinetic data. Calorimetric analysis revealed that the entropic contribution to the mutant PR/nelfinavir interaction is less favorable than the entropic contribution to the binding of nelfinavir by wild-type PR. This finding is supported by the structural data and simulations; nelfinavir binds most strongly to the wild-type protease, which has the lowest number of protein-ligand hydrogen bonds and whose structure exhibits the greatest degree of fluctuation upon inhibitor binding.

• MeSH
• aktivace enzymů MeSH
• financování organizované MeSH
• HIV-1 enzymologie   genetika   MeSH
• HIV-proteasa genetika   chemie   MeSH
• inhibitory HIV-proteasy chemie   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• mutace MeSH
• nelfinavir MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• virová léková rezistence MeSH