RESUMEN
The 1.64 A structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogenase, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.
Asunto(s)
Lisina/análogos & derivados , NAD/metabolismo , Saccharomyces cerevisiae/enzimología , Sacaropina Deshidrogenasas/química , Alanina-Deshidrogenasa/química , Alanina-Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Saccharomyces cerevisiae/crecimiento & desarrollo , Sacaropina Deshidrogenasas/aislamiento & purificación , Sacaropina Deshidrogenasas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The nonnucleoside inhibitor binding pocket is a well-defined region in the p66 palm domain of the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). This binding pocket opens toward the interface of the p66/p51 heterodimer and we have investigated whether ligand binding at or near this site induces structural changes that have an impact on the dimeric structure of HIV-1 RT. 1-[2',5'-bis-O-(tert-butyldimethylsilyl]-3'-spiro-5' '-(4' '-amino-1' ',2' '-oxathiole-2' ',2' '-dioxide)-3-ethylthymine (TSAOe(3)T) was found to destabilize the subunit interactions of both the p66/p51 heterodimer and p66/p66 homodimer enzymes. The Gibbs free energy of dimer dissociation (DeltaG(D)(H)2(O)) is decreased with increasing concentrations of TSAOe(3)T, resulting in a loss in dimer stability of 4.0 and 3.2 kcal/mol for the p66/p51 and p66/p66 HIV-1 RT enzymes, respectively. This loss of energy is not sufficient to induce the dissociation of the subunits in the absence of denaturant. This destabilizing effect seems to be unique for TSAOe(3)T, since neither the tight-binding inhibitor UC781 nor nevirapine showed any effects on the stability of HIV-1 RT dimers. TSAOe(3)T was unable to destabilize the subunit interactions of the E138K mutant enzyme, which exhibits significant resistance to TSAOe(3)T inhibition. Molecular modeling of TSAOm(3)T into the nonnucleoside inhibitor binding pocket of wild-type RT suggests that it makes significant interactions with the p51 subunit of the enzyme, a feature that has not been observed with other types of nonnucleoside inhibitors. The observed destabilization of the dimeric HIV-1 RT may result from structural/conformational perturbations at the reverse transcriptase subunit interface.
Asunto(s)
Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Inhibidores de la Transcriptasa Inversa/farmacología , Compuestos de Espiro/farmacología , Timidina/análogos & derivados , Anilidas/farmacología , Fármacos Anti-VIH/química , Sitios de Unión , Cromatografía Líquida de Alta Presión , Dimerización , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Furanos/farmacología , Transcriptasa Inversa del VIH/química , VIH-1/efectos de los fármacos , Humanos , Modelos Moleculares , Nevirapina/farmacología , Desnaturalización Proteica , Inhibidores de la Transcriptasa Inversa/química , Compuestos de Espiro/química , Tioamidas , Timidina/química , Timidina/farmacología , UreaRESUMEN
The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.
Asunto(s)
Inhibidores Enzimáticos/química , beta-Lactamasas/química , Alanina/química , Secuencia de Aminoácidos , Ácido Aspártico/química , Bacillus cereus/enzimología , Sitios de Unión , Escherichia coli/enzimología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Plásmidos , Staphylococcus aureus/enzimología , Inhibidores de beta-Lactamasas , beta-Lactamasas/biosíntesisRESUMEN
HIV-1 reverse transcriptase (RT) is multifunctional, with RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP), and ribonuclease H (RNase H) activities. N-(4-tert-Butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) inhibited both the polymerase and the RNase H activities of HIV-1 RT in vitro. IC50 values for inhibition of RDDP were 0.8-3.4 microM, depending on the template/primer (T/P) used in the assay. The IC50 for DDDP inhibition was about 12 microM, while that for inhibition of RNase H was 3.5 microM. EC50 for inhibition of HIV-1 replication in cord blood mononuclear cells was 1.5 microM. BBNH inhibition of RNase H in vitro was time-dependent, whereas inhibition of RT polymerase activities was immediate. BBNH was a linear mixed-type inhibitor of RT RDDP activity with respect to both T/P and to dNTP, whereas BBNH inhibition of RT RNase H activity was linear competitive. Protection experiments using an azidonevirapine photolabel showed that BBNH binds to the non-nucleoside RT inhibitor (NNRTI) binding pocket. Importantly, the compound inhibited recombinant RT containing mutations associated with high-level resistance to other NNRTI. While BBNH did not inhibit the DNA polymerase activities of other retroviral reverse transcriptases and DNA polymerases, the compound inhibited Escherichia coli RNase HI and the RNase H activity of murine leukemia virus RT. BBNH also inhibited HIV-1 RT RNase H in the presence of high concentrations of other non-nucleoside inhibitors with higher affinities for the NNRTI binding pocket, and of RT in which the NNRTI binding pocket had been irreversibly blocked by the azidonevirapine photolabel. We conclude that BBNH may therefore bind to two sites on HIV-1 RT. One site is the polymerase non-nucleoside inhibitor binding site and the second may be located in the RNase H domain. BBNH is therefore a promising lead compound for the development of multisite inhibitors of HIV-1 RT.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/fisiología , Hidrazonas/farmacología , Naftoles/farmacología , Inhibidores de la Síntesis del Ácido Nucleico , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/antagonistas & inhibidores , División Celular/efectos de los fármacos , Línea Celular , Sangre Fetal , VIH-1/efectos de los fármacos , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Mutación Puntual , Proteínas Recombinantes/antagonistas & inhibidoresRESUMEN
We compared the inhibition of HIV-1 reverse transcriptase (RT) by 1-[2',5'-bis-O-(t-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'- spiro-5"-(4"-amino-1", 2"-oxathiole-2",2"-dioxide)-3-ethylthymine (TSAOe3T) and the nonnucleoside RT inhibitor (NNRTI) 9-aminonevirapine (9-NH2N). Both compounds were equally effective against p51/p66 heterodimeric RT RNA-dependent DNA polymerase activity, although TSAOe3T was a much better inhibitor of the p51/p51 and p66/p66 RT homodimers. Inhibition by TSAOe3T and 9-NH2N combinations was essentially additive. TSAOe3T did not protect either free RT or the RT-template/ primer-deoxynucleoside triphosphate ternary complex from irreversible inactivation by the photolabel 9-azidonevirapine. Slight protection of the RT-template/primer binary complex was noted, but only at high TSAOe3T/photolabel ratios. Analysis of RT polymerization product profiles under both continuous- and single-processive cycle conditions showed that 9-NH2N prevented the formation of full-length product with a corresponding accumulation of smaller polymerization products. In contrast, all products formed in the absence of inhibitor, including full-length product, were noted in TSAOe3T-inhibited reactions, albeit at reduced levels. TSAOe3T thus inhibits HIV-1 RT by a different mechanism than NNRTI such as nevirapine. Our data suggest that TSAOe3T and 9-NH2N interact differently with HIV-1 RT, perhaps by binding to distinct sites on the enzyme.
Asunto(s)
Antivirales/farmacología , Benzodiazepinas/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Síntesis del Ácido Nucleico , Piridinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Compuestos de Espiro/farmacología , Timidina/análogos & derivados , Benzodiazepinas/metabolismo , Sitios de Unión , ADN Polimerasa Dirigida por ADN/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Humanos , Cinética , Fotoquímica , Piridinas/metabolismo , ADN Polimerasa Dirigida por ARN/efectos de los fármacos , ADN Polimerasa Dirigida por ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/metabolismo , Compuestos de Espiro/metabolismo , Relación Estructura-Actividad , Timidina/metabolismo , Timidina/farmacologíaRESUMEN
Differences in specificities between the class A beta-lactamases for both substrate and inhibitors are known. The role of the nonconserved amino acid residue at position 167 of the class A enzyme, which forms a cis bond with the catalytically essential Glu-166 residue, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors, was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace thr-167 with the corresponding Staphylococcus aureus PC1 residue Ile. Kinetic data obtained from the purified Thr-167-Ile B. cereus 569/H beta-lactamase was compared to that obtained from the wild-type B. cereus and S. aureus enzymes and indicated that the replacement had little effect on the Michaelis parameters for the hydrolysis of S- and A-type penicillins. However, the Thr-167-Ile enzymes became more S. aureus PC1-like in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amidopenicillanic acid sulfone. A model for the role of this nonconserved residue at position 167 in the mechanism of inactivation by mechanism-based inhibitors is proposed.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de beta-Lactamasas , beta-Lactamasas/genética , Secuencia de Aminoácidos , Genes Bacterianos/genética , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Penicilinas/metabolismo , Plásmidos/genética , Especificidad por Sustrato , beta-Lactamasas/biosíntesisRESUMEN
The carboxanilides UC84 and UC38 are nonnucleoside inhibitors of both the RNA-dependent and DNA-dependent DNA polymerase activities of HIV-1 reverse transcriptase (RT). We have previously shown that UC84 and UC38 bind to the same site as nevirapine but interact with different RT mechanistic forms, with UC84 preferentially binding to the RT-primer/template complex and UC38 binding only to the RT-primer/template-dNTP ternary complex [Fletcher, R. S., et al. (1995) Biochemistry 34, 4346-4353]. Here we demonstrate that combinations of UC84 and UC38 inhibit RT DNA polymerase activity in vitro in a synergistic manner. This synergy was noted primarily in reactions containing high concentrations of primer/template and Km levels of dNTP substrate and was independent of both primer/template identity and the molar ratio of UC84:UC38. Combination indices were in the range of 0.4-0.6, indicating substantial synergy in the inhibition of RT activity. More importantly, combinations of UC84 and UC38 also showed a high degree of synergy in inhibiting HIV-1 replication in both MT-4 and cord blood mononuclear cells. We believe this to be the first example of synergistic inhibition of HIV-1 RT by combinations of structurally related nonnucleoside inhibitors.
Asunto(s)
Antivirales/farmacología , Benzoatos/farmacología , Carboxina/análogos & derivados , VIH-1/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico , Inhibidores de la Transcriptasa Inversa , Tiocarbamatos/farmacología , Secuencia de Bases , Carboxina/farmacología , Línea Celular , Sinergismo Farmacológico , Transcriptasa Inversa del VIH , VIH-1/enzimología , VIH-1/fisiología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Moldes Genéticos , Replicación Viral/efectos de los fármacosRESUMEN
Researchers at the National Cancer Institute first recognized the anti-HIV potential of the carboxanilide compound oxathiin carboxanilide (UC84) [Bader, J. P., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6740-6744]. We have compared the inhibitory effect of UC84 and a second-generation thiocarboxanilide derivative, UC38, on HIV-1 reverse transcriptase (RT) RNA-dependent DNA polymerase activity. UC38 was a much better inhibitor (IC50 = 0.8 microM) than UC84 (IC50 = 4.3 microM). Inhibition by UC84 was competitive with respect to primer/template (P/T), whereas that by UC38 was uncompetitive. Both compounds were mixed noncompetitive inhibitors with respect to deoxynucleoside triphosphate (dNTP). Both compounds protected RT from irreversible photoinactivation by an azido derivative of nevirapine, implying that UC84 and UC38 bind to the same region of RT as nevirapine. UC84 photoprotected both free RT and the RT-P/T binary complex, but did not protect the RT-P/T-dNTP ternary complex. In contrast, UC38 completely photoprotected the RT-P/T-dNTP ternary complex, but not free RT or the RT-P/T binary complex. UC84 and UC38 thus appear to bind to different mechanistic forms of RT in the polymerase reaction sequence.
Asunto(s)
Carboxina/análogos & derivados , VIH-1/enzimología , Inhibidores de la Transcriptasa Inversa , Marcadores de Afinidad/farmacología , Benzodiazepinonas/metabolismo , Benzodiazepinonas/farmacología , Sitios de Unión , Unión Competitiva , Carboxina/metabolismo , Carboxina/farmacología , Cartilla de ADN/metabolismo , Transcriptasa Inversa del VIH , Cinética , Nevirapina , Fotoquímica , Piridinas/metabolismo , Piridinas/farmacología , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Moldes Genéticos , Nucleótidos de Timina/farmacología , Rayos UltravioletaRESUMEN
The effects of zymosan on lipid metabolism in mouse peritoneal macrophages (MPM) in vitro and in vivo were studied with special reference to the following parameters: i) 14C-oleate incorporation into cholesteryl esters (CE), triglycerides (TG), and phospholipids (PL) in MPM incubated with low density lipoproteins (LDL) and acetylated LDL; ii) cholesteryl-14C-oleate-acetyl LDL uptake and 125I-acetyl LDL degradation; iii) oxidative modification of LDL. Zymosan administered to mice caused significant stimulation of 14C-oleate incorporation into CE, TG, and PL with no effect on 3H-cholesterol (Ch) incorporation into CE or 3H-glycerol incorporation into TG and PL in MPM. The 14C-oleate incorporation into cellular lipids was unaffected by 18-hour incubation of MPM with zymosan (100-500 micrograms/ml) but increased after incubation of unstimulated MPM with blood serum and peritoneal fluid harvested harvested from zymosan-treated mice. One possible explanation of this phenomenon is oleyl-CoA formation induction in cytokine-stimulated MPM in vivo. Zymosan decreased the Ch-14C-oleate-acetyl LDL uptake, 125I-acetyl LDL degradation, and Ch esterification in the presence of acetyl LDL in MPM both in vitro and in vivo. An increase in Ch esterification after incubation of MPM with zymosan for 6-18 hours in the presence of LDL was accompanied by an increase in lipid peroxidation of LDL and its electrophoretic mobility. The data obtained suggest that the macrophage acetyl LDL receptor pathway may be inhibited by zymosan and that cytokines released from zymosan-stimulated cells may influence the generation of foam cells.
Asunto(s)
Lípidos/biosíntesis , Lipoproteínas LDL/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Zimosan/farmacología , Acetilación , Animales , Líquido Ascítico , Sangre , Técnicas In Vitro , Peroxidación de Lípido , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ácido Oléico , Ácidos Oléicos/metabolismo , Cavidad Peritoneal/citologíaRESUMEN
6-beta-(Trifluoromethane sulfonyl)amidopenicillanic acid sulfone and its N-methyl derivative were found to be potent inhibitors of Bacillus cereus 569/H beta-lactamase I. The rate of the inactivation of the enzyme by both compounds was found to increase with the decreasing pH of the reaction medium. The reaction of the enzyme with 6-beta-(trifluoromethane sulfonyl)amidopenicillanic acid sulfone was found to be irreversible at the pH values investigated. In contrast, the reaction with the N-methyl derivative at neutral pH was consistent with the partitioning of the acyl enzyme intermediate in three pathways which included (a) deacylation to yield active enzyme, (b) conversion to a transiently inhibited species, and (c) conversion to an irreversibly inactive form. The amino acid composition of the chromophoric peptide obtained from the enzyme inactivated by either of the compounds was consistent with the occurrence of an initial acylation of serine-70 of the protein.
Asunto(s)
Bacillus cereus/enzimología , Ácido Penicilánico/farmacología , Sulbactam/análogos & derivados , Inhibidores de beta-Lactamasas , Aminoácidos/análisis , Concentración de Iones de Hidrógeno , Cinética , Tripsina/metabolismoRESUMEN
beta-Lactamase I was isolated from Bacillus cereus 569/H. Treatment with ampicillin in the presence of sodium nitrite at pH 4 or 5 resulted in the inactivation of the enzyme presumably by modification of a carboxyl group in the active site. However, this inactivation was rapidly, reversible at neutral pH and the available evidence points to the participation of a second carboxyl group which is involved in the reactivation process.