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1.
Phys Chem Chem Phys ; 21(10): 5661-5673, 2019 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-30794275

RESUMEN

Transient and fuzzy intermolecular interactions are fundamental to many biological processes. Despite their importance, they are notoriously challenging to characterize. Effects induced by paramagnetic ligands in the NMR spectra of interacting biomolecules provide an opportunity to amplify subtle manifestations of weak intermolecular interactions observed for diamagnetic ligands. Here, we present an approach to characterizing dynamic interactions between a partially flexible dimeric protein, HIV-1 protease, and a metallacarborane-based ligand, a system for which data obtained by standard NMR approaches do not enable detailed structural interpretation. We show that for the case where the experimental data are significantly averaged to values close to zero the standard fitting of pseudocontact shifts cannot provide reliable structural information. We based our approach on generating a large ensemble of full atomic models, for which the experimental data can be predicted, ensemble averaged and finally compared to the experiment. We demonstrate that a combination of paramagnetic NMR experiments, quantum chemical calculations, and molecular dynamics simulations offers a route towards structural characterization of dynamic protein-ligand complexes.


Asunto(s)
Boranos/química , Proteasa del VIH/química , Metales/química , Simulación de Dinámica Molecular , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Unión Proteica , Conformación Proteica , Teoría Cuántica
2.
J Virol ; 88(6): 3586-90, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24371077

RESUMEN

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.


Asunto(s)
Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/química , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Mutagénesis Insercional , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Proteasa del VIH/química , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/química , VIH-1/genética , Humanos , Modelos Moleculares , Organofosfonatos/análisis
3.
iScience ; 27(10): 110893, 2024 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-39328932

RESUMEN

DDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the transcription factor TCF11/NRF1 (NFE2L1), crucial for maintaining proteostasis in mammalian cells, enabling the expression of rescue factors, including proteasome subunits. Here, we describe the consequences of DDI2 ablation in vivo and in cells. DDI2 knock-out (KO) in mice caused embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of embryos showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates and induction of the unfolded protein response (UPR) and cell death pathways. In DDI2 surrogate KO cells, proteotoxic stress activated the integrated stress response (ISR) and induced a type I interferon (IFN) signature and IFN-induced proliferative signaling, possibly ensuring survival. These results indicate an important role for DDI2 in the cell-tissue proteostasis network and in maintaining a balanced immune response.

4.
Cancers (Basel) ; 12(5)2020 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-32344880

RESUMEN

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.

5.
DNA Repair (Amst) ; 80: 45-51, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31276951

RESUMEN

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.


Asunto(s)
Replicación del ADN , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ADN/metabolismo , Daño del ADN , Reparación del ADN , Hidroxiurea/toxicidad , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal
6.
J Med Chem ; 59(2): 545-58, 2016 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-26685880

RESUMEN

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.


Asunto(s)
Fármacos Anti-VIH/farmacología , Cápside/metabolismo , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Quinazolinas/farmacología , Fármacos Anti-VIH/metabolismo , Cápside/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Quinazolinas/síntesis química , Quinazolinas/química , Proteínas Recombinantes/biosíntesis , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Termodinámica , Replicación Viral/efectos de los fármacos
7.
FEBS J ; 281(7): 1834-47, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24785545

RESUMEN

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).


Asunto(s)
Farmacorresistencia Viral/genética , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/química , Simulación del Acoplamiento Molecular , Mutación , Sulfonamidas/farmacología , Secuencia de Aminoácidos , Darunavir , Proteasa del VIH/genética , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/química , Datos de Secuencia Molecular , Unión Proteica , Sulfonamidas/química , Termodinámica
8.
J Med Chem ; 55(22): 10130-5, 2012 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-23050738

RESUMEN

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.


Asunto(s)
Benzodiazepinas/química , Diseño de Fármacos , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/síntesis química , Proteasa del VIH/química , VIH-1/efectos de los fármacos , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Infecciones por VIH/enzimología , Infecciones por VIH/virología , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Fragmentos de Péptidos/farmacología , Conformación Proteica , Relación Estructura-Actividad
9.
J Med Chem ; 52(22): 7132-41, 2009 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-19874035

RESUMEN

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.


Asunto(s)
Compuestos de Boro/química , Compuestos de Boro/farmacología , Carbono/química , Cobalto/química , Diseño de Fármacos , Proteasa del VIH/metabolismo , Compuestos de Boro/síntesis química , Compuestos de Boro/metabolismo , Cristalografía por Rayos X , Electrones , Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , Modelos Moleculares , Conformación Molecular
10.
J Med Chem ; 51(15): 4839-43, 2008 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-18598016

RESUMEN

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.


Asunto(s)
Compuestos de Boro/química , Compuestos de Boro/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/metabolismo , Metales/química , Cristalografía por Rayos X , Proteasa del VIH/química , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Modelos Moleculares , Estructura Molecular , Mutación/genética
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