Proteasome inhibitors are the backbone of multiple myeloma therapy. However, disease progression or early relapse occur due to development of resistance to the therapy. One important cause of resistance to proteasome inhibition is the so-called bounce-back response, a recovery pathway driven by the TCF11/Nrf1 transcription factor, which activates proteasome gene re-synthesis upon impairment of the proteasome function. Thus, inhibiting this recovery pathway potentiates the cytotoxic effect of proteasome inhibitors and could benefit treatment outcomes. DDI2 protease, the 3D structure of which resembles the HIV protease, serves as the key player in TCF11/Nrf1 activation. Previous work found that some HIV protease inhibitors block DDI2 in cell-based experiments. Nelfinavir, an oral anti-HIV drug, inhibits the proteasome and/or pAKT pathway and has shown promise for treatment of relapsed/refractory multiple myeloma. Here, we describe how nelfinavir inhibits the TCF11/Nrf1-driven recovery pathway by a dual mode of action. Nelfinavir decreases the total protein level of TCF11/Nrf1 and inhibits TCF11/Nrf1 proteolytic processing, likely by interfering with the DDI2 protease, and therefore reduces the TCF11/Nrf1 protein level in the nucleus. We propose an overall mechanism that explains nelfinavir's effectiveness in the treatment of multiple myeloma.

• Publikační typ
• časopisecké články MeSH

Genome integrity and cell survival are dependent on proper replication stress response. Multiple repair pathways addressing obstacles generated by replication stress arose during evolution, and a detailed understanding of these processes is crucial for treatment of numerous human diseases. Here, we investigated the strong negative genetic interaction between two proteases involved in the DNA replication stress response, yeast Wss1 and Ddi1. While Wss1 proteolytically acts on DNA-protein crosslinks, mammalian DDI1 and DDI2 proteins remove RTF2 from stalled forks via a proposed proteasome shuttle hypothesis. We show that the double-deleted Δddi1, Δwss1 yeast strain is hypersensitive to the replication drug hydroxyurea and that this phenotype can be complemented only by catalytically competent Ddi1 protease. Furthermore, our data show the key involvement of the helical domain preceding the Ddi1 protease domain in response to replication stress caused by hydroxyurea, offering the first suggestion of this domain's biological function. Overall, our study provides a basis for a novel dual protease-based mechanism enabling yeast cells to counteract DNA replication stress.

• MeSH
• DNA metabolismus   MeSH
• hydroxymočovina toxicita   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• replikace DNA * MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae účinky léků   enzymologie   genetika   metabolismus   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations.

• MeSH
• chinazoliny chemická syntéza   chemie   farmakologie   MeSH
• HIV-1 účinky léků   metabolismus   MeSH
• kapsida účinky léků   metabolismus   MeSH
• knihovny malých molekul MeSH
• látky proti HIV metabolismus   farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• rekombinantní proteiny biosyntéza   MeSH
• replikace viru účinky léků   MeSH
• reprodukovatelnost výsledků MeSH
• rychlé screeningové testy MeSH
• termodynamika MeSH
• viabilita buněk účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.

• MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 chemie   účinky léků   enzymologie   genetika   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• inzerční mutageneze * MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• organofosfonáty analýza   MeSH
• sekvence aminokyselin MeSH
• vazebná místa MeSH
• virová léková rezistence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

UNLABELLED: We report enzymologic, thermodynamic and structural analyses of a series of six clinically derived mutant HIV proteases (PR) resistant to darunavir. As many as 20 mutations in the resistant PRs decreased the binding affinity of darunavir by up to 13 000-fold, mostly because of a less favorable enthalpy of binding that was only partially compensated by the entropic contribution. X-ray structure analysis suggested that the drop in enthalpy of darunavir binding to resistant PR species was mostly the result of a decrease in the number of hydrogen bonds and a loosening of the fit between the inhibitor and the mutated enzymes. The favorable entropic contribution to darunavir binding to mutated PR variants correlated with a larger burial of the nonpolar solvent-accessible surface area upon inhibitor binding. We show that even very dramatic changes in the PR sequence leading to the loss of hydrogen bonds with the inhibitor could be partially compensated by the entropy contribution as a result of the burial of the larger nonpolar surface area of the mutated HIV PRs. DATABASE: Atomic coordinates and structure factors for the crystal structures PRwt-DRV and PRDRV2-DRV complex have been deposited in the Protein Data Bank under accession codes 4LL3 and 3TTP, respectively. STRUCTURED DIGITAL ABSTRACT: • PR and PR bind by x-ray crystallography (View interaction).

• MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• molekulární sekvence - údaje MeSH
• mutace * MeSH
• sekvence aminokyselin MeSH
• simulace molekulového dockingu * MeSH
• sulfonamidy chemie   farmakologie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• virová léková rezistence genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

HIV protease is a primary target for the design of virostatics. Screening of libraries of non-peptide low molecular weight compounds led to the identification of several new compounds that inhibit HIV PR in the low micromolar range. X-ray structure of the complex of one of them, a dibenzo[b,e][1,4]diazepinone derivative, showed that two molecules of the inhibitor bind to the PR active site. Covalent linkage of two molecules of such a compound by a two-carbon linker led to a decrease of the inhibition constant of the resulting compound by 3 orders of magnitude. Molecular modeling shows that these dimeric inhibitors form two crucial hydrogen bonds to the catalytic aspartates that are responsible for their improved activity compared to the monomeric parental building blocks. Dibenzo[b,e][1,4]diazepinone analogues might represent a potential new class of HIV PIs.

• MeSH
• benzodiazepiny chemie   MeSH
• HIV infekce farmakoterapie   enzymologie   virologie   MeSH
• HIV-1 účinky léků   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• inhibitory HIV-proteasy chemická syntéza   farmakologie   MeSH
• katalytická doména MeSH
• katalýza MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• peptidové fragmenty farmakologie   MeSH
• racionální návrh léčiv * MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

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