RESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterial pathogen, continues to pose a serious threat to the current public health system in our society. The high level of resistance to ß-lactam antibiotics in MRSA is attributed to the expression of penicillin-binding protein 2a (PBP2a), which catalyzes cell wall cross-linking. According to numerous research reports, the activity of the PBP2a protein is known to be regulated by an allosteric site distinct from the active site where cell wall cross-linking occurs. Here, we conducted a screening of 113 compounds containing a 1,3,4-oxadiazole core to design new covalent inhibitors targeting the allosteric site of PBP2a and establish their structural-activity relationship. The stereochemically selective synthesis of sulfonyl oxadiazole compounds identified in the initial screening resulted in a maximum eightfold enhancement in cell inhibition activity. The sulfonyl oxadiazole-based compounds formulated as PEG-based ointments, with low toxicity test results on human cells (CC 50 : >78µM), demonstrated potent antimicrobial effects not only in a mouse skin wound infection model but also against oxacillin-resistant clinical isolate MRSA (IC 50 ≈ 1µM), as evidenced by the results. Furthermore, additional studies utilizing LC-MS/MS and in-silico approaches clearly support the allosteric site covalent binding mechanism through the nucleophilic aromatic substitution (S N Ar) reaction, as well as its association with the closure of the major active site of PBP2a.
RESUMEN
Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent bacterial threat to public health, with only a few treatment options and a >50% fatality rate. Although several resistance mechanisms are understood, the appearance of these mutations is generally considered stochastic. Recent reports have, however, begun to challenge this assumption. Here, we demonstrate that independent samples of Ab, exposed to different carbapenems with escalating concentrations, show concentration- and carbapenem-dependent trends in ß-lactamase-isoform expression. This result, based on the isoforms identified through label-free-quantification LC-MS/MS measurements of cell-free, gel-separated ß-lactamases, suggests that the appearance of antibiotic resistance may be somewhat non-stochastic. Specifically, several minor AmpC/ADC ß-lactamase-isoforms were found to exhibit both dose- and carbapenem-dependent expression, suggesting the possibility of non-stochastic mutations. Additionally, these also have high sequence similarity to major expressed isoforms, indicating a potential path over which resistance occurred in independent samples. Antibiotic resistance maybe somewhat antibiotic-directed by a hitherto unknown mechanism and further investigation may lead to new strategies for mitigating antibiotic resistance. Teaser: The emergence of antibiotic-resistant ß-lactamase proteins from mutations may exhibit patterns based on specific antibiotics.
RESUMEN
The bacterial metallo-ß-lactamases (MBLs) catalyze the inactivation of ß-lactam antibiotics. Identifying novel pharmacophores remains crucial for the clinical development of additional MBL inhibitors. Previously, 1-hydroxypyridine-2(1H)-thione-6-carboxylic acid, hereafter referred to as 1,2-HPT-6-COOH, was reported as a low cytotoxic nanomolar ß-lactamase inhibitor of Verona-integron-encoded metallo-ß-lactamase 2, capable of rescuing ß-lactam antibiotic activity. In this study, we explore its exact mechanism of inhibition and the extent of its activity through structural characterization of its binding to New Delhi metallo-ß-lactamase 4 (NDM-4) and its inhibitory activity against both NDM-1 and NDM-4. Of all the structure-validated MBL inhibitors available, 1,2-HPT-6-COOH is the first discovered compound capable of forming an octahedral coordination sphere with Zn2 of the binuclear metal center. This unexpected mechanism of action provides important insight for the further optimization of 1,2-HPT-6-COOH and the identification of additional pharmacophores for MBL inhibition.
RESUMEN
Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to ß-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in ß-lactamase expression that occurs in CRAb with different ß-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of ß-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC-MS-based quantitative proteomics. Thirteen proteins were identified and evaluated using a 1789 sequence database of Ab ß-lactamases from UniProt, the majority of which were Class C ß-lactamases (≥ 80%). Importantly, different antibiotics, even those of the same class (e.g. penicillin and amoxicillin), induced non-equivalent responses comprising various isoforms of Class C and D serine-ß-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on ß-lactamase expression.
Asunto(s)
Acinetobacter baumannii , Acinetobacter baumannii/metabolismo , Proteómica , Antibacterianos/farmacología , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Monobactamas , Pruebas de Sensibilidad Microbiana , Resistencia betalactámicaRESUMEN
Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to ß-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in ß-lactamase expression that occurs in CRAb with different ß-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of ß-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC-MS-based quantitative proteomics. Peptides were identified and evaluated using a 1789 sequence database of Ab ß-lactamases from UniProt. Importantly, we observed that different antibiotics, even those of the same class ( e.g. penicillin and amoxicillin), induce non-equivalent responses comprising various Class C and D serine-ß-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on ß-lactamase expression.
RESUMEN
Understanding the dynamical motions and ligand recognition motifs of heptosyltransferase I (HepI) can be critical to discerning the behavior of other glycosyltransferase (GT) enzymes. Prior studies in our lab have demonstrated that GTs in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conserved structural fold and dynamical motions, despite low sequence homology, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through molecular dynamics simulations and ligand binding free energy analysis of HepI in the apo and bound complexes (for all kinetically relevant combinations of the native substrates/products), we have determined the energetically favored enzymatic pathway for ligand binding and release. Our principal component, dynamic cross correlation, and network analyses of the simulations have revealed correlated motions involving residues within the N-terminal domain communicating with C-terminal domain residues via both proximal amino acid residues and also functional groups of the bound substrates. Analyses of the structural changes, energetics of substrate/product binding, and changes in pKa have elucidated a variety of inter and intradomain interactions that are critical for enzyme catalysis. These data corroborate our experimental observations of protein conformational changes observed in both presteady state kinetic and circular dichroism analyses of HepI. These simulations provided invaluable structural insights into the regions involved in HepI conformational rearrangement upon ligand binding. Understanding the specific interactions governing conformational changes is likely to enhance our efforts to develop novel dynamics disrupting inhibitors against GT-B structural enzymes in the future.
Asunto(s)
Glicosiltransferasas , Simulación de Dinámica Molecular , Glicosiltransferasas/química , Ligandos , Conformación ProteicaRESUMEN
Metallo-ß-lactamases (MBLs) are zinc-containing carbapenemases that inactivate a broad range of ß-lactam antibiotics. There is a lack of ß-lactamase inhibitors for restoring existing ß-lactam antibiotics arsenals against common bacterial infections. Fragment-based screening of a non-specific metal chelator library demonstrates 8-hydroxyquinoline as a broad-spectrum nanomolar inhibitor against VIM-2 and NDM-1. A hit-based substructure search provided an early structure-activity relationship of 8-hydroxyquinolines and identified 8-hydroxyquinoline-7-carboxylic acid as a low-cytotoxic ß-lactamase inhibitor that can restore ß-lactam activity against VIM-2-expressing E. coli. Molecular modeling further shed structural insight into its potential mode of binding within the dinuclear zinc active site. 8-Hydroxyquinoline-7-carboxylic acid is highly stable in human plasma and human liver microsomal study, making it an ideal lead candidate for further development.
Asunto(s)
Hidroxiquinolinas/química , Bibliotecas de Moléculas Pequeñas/química , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Escherichia coli/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Hidroxiquinolinas/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Unión Proteica , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-Actividad , Zinc/química , Inhibidores de beta-Lactamasas/metabolismoRESUMEN
Multidrug resistance and toxic side effects are the major challenges in cancer treatment with microtubule-targeting agents (MTAs), and thus, there is an urgent clinical need for new therapies. Chalcone, a common simple scaffold found in many natural products, is widely used as a privileged structure in medicinal chemistry. We have previously validated tubulin as the anticancer target for chalcone derivatives. In this study, an α-methyl-substituted indole-chalcone (FC77) was synthesized and found to exhibit an excellent cytotoxicity against the NCI-60 cell lines (average concentration causing 50% growth inhibition = 6 nM). More importantly, several multidrug-resistant cancer cell lines showed no resistance to FC77, and the compound demonstrated good selective toxicity against cancer cells versus normal CD34+ blood progenitor cells. A further mechanistic study demonstrated that FC77 could arrest cells that relate to the binding to tubulin and inhibit the microtubule dynamics. The National Cancer Institute COMPARE analysis and molecular modeling indicated that FC77 had a mechanism of action similar to that of colchicine. Overall, our data demonstrate that this indole-chalcone represents a novel MTA template for further development of potential drug candidates for the treatment of multidrug-resistant cancers.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Chalconas/química , Indoles/química , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
VIM-2 is one of the most common carbapenem-hydrolyzing metallo ß-lactamases (MBL) found in many drug-resistant Gram-negative bacterial strains. Currently, there is a lack of effective lead compounds with optimal therapeutic potential within our drug development pipeline. Here we report the discovery of 1-hydroxypyridine-2(1H)-thione-6-carboxylic acid (3) as a first-in-class metallo ß-lactamase inhibitor (MBLi) with a potent inhibition Ki of 13â nm against VIM-2 that corresponds to a remarkable 0.99 ligand efficiency. We further established that 3 can restore the antibiotic activity of amoxicillin against VIM-2-producing E. coli in a whole cell assay with an EC50 of 110â nm. The potential mode of binding of 3 from molecular modeling provided structural insights that could corroborate the observed changes in the biochemical activities. Finally, 3 possesses a low cytotoxicity (CC50 ) of 97â µm with a corresponding therapeutic index of 880, making it a promising lead candidate for further optimization in combination antibacterial therapy.
Asunto(s)
Ácidos Picolínicos/síntesis química , Ácidos Picolínicos/farmacología , Tionas/síntesis química , Tionas/farmacología , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/farmacología , Supervivencia Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Ácidos Picolínicos/toxicidad , Pseudomonas aeruginosa/efectos de los fármacos , Tionas/toxicidad , Inhibidores de beta-Lactamasas/toxicidad , beta-Lactamasas/metabolismoRESUMEN
DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases η and κ. DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50-100-fold lower than for native G. Both human polymerase (hPol) κ and hPol η inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol η. Molecular dynamics simulation of an hPol κ ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.
Asunto(s)
Aductos de ADN/química , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Guanina/análogos & derivados , Péptidos/química , Proteínas/química , Aminación , Secuencia de Aminoácidos , Secuencia de Bases , Aductos de ADN/genética , Guanina/química , Humanos , Simulación de Dinámica Molecular , Oxidación-Reducción , Proteínas Recombinantes/metabolismoRESUMEN
Histone deacetylase (HDAC) is a validated target for pursuing anticancer agents. However, obtaining a selective inhibitor against a given HDAC member remains a significant challenge. We report here the use of 1-hydroxypyridine-2-thione (1HPT) as a key pharmacophore for zinc-binding can result in highly selective HDAC inhibitors. 1HPT-6-carboxylic acid exhibits selective inhibition of HDAC6 with an IC50 of 150 nM that corresponds to a remarkable 0.9 ligand efficiency. Two analogs with simple amino acids shows nearly 600-fold selectivity among the eleven zinc-dependent HDACs. At low micromolar concentration these compounds inhibit the growth of HDAC8-overexpressing chronic myelogenous leukemia cells and specific form of acute myelogenous leukemia cells. Their potential mode of binding was examined by molecular docking and their stability was assessed in mouse and human plasma. Together the results suggest 1HPT analogs exhibit promising therapeutic potential for further development as anticancer agents to treat leukemia.
Asunto(s)
Descubrimiento de Drogas , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Piridinas/farmacología , Tionas/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , Tionas/síntesis química , Tionas/químicaRESUMEN
The p53 and nuclear factor κB (NF-κB) pathways play crucial roles in human cancer development. Simultaneous targeting of both pathways is an attractive therapeutic strategy against cancer. In this study, we report an antitumor molecule that bears a pyrrolo[3,4-c]pyrazole scaffold and functions as an enantiomeric inhibitor against both the p53-MDM2 interaction and the NF-κB activation. It is a first-in-class enantiomeric inhibitor with dual efficacy for cancer therapy. Synergistic effect was observed in vitro and in vivo. Docking and molecular dynamics simulation studies further provided insights into the nature of stereoselectivity.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , FN-kappa B/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Pirazoles/farmacología , Pirrolidinonas/farmacología , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , FN-kappa B/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazoles/síntesis química , Pirazoles/química , Pirrolidinonas/síntesis química , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
VanX is an induced zinc metallo d-Ala-d-Ala dipeptidase involved in the viable remodeling of bacterial cell wall that is essential for the development of VREF. Here we report two cyclic thiohydroxamic acid-based peptide analogs that were designed, synthesized and investigated as vancomycin re-sensitizing agents. These compounds exhibit low micromolar inhibitory activity against vanX, with low cytotoxicity and were shown to increase vancomycin sensitivity against VREF. The improved pharmacological properties of these novel inhibitors over previous transition state mimics should provide an enhanced platform for designing potent vanX inhibitors for overcoming vancomycin resistance.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/antagonistas & inhibidores , Vancomicina/farmacología , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Ácidos Hidroxámicos/química , Estructura Molecular , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , Relación Estructura-Actividad , Vancomicina/síntesis química , Vancomicina/químicaRESUMEN
In this study, rapid structure-based virtual screening and hit-based substructure search were utilized to identify small molecules that disrupt the interaction of Keap1-Nrf2. Special emphasis was placed toward maximizing the exploration of chemical diversity of the initial hits while economically establishing informative structure-activity relationship (SAR) of novel scaffolds. Our most potent noncovalent inhibitor exhibits three times improved cellular activation in Nrf2 activation than the most active noncovalent Keap1 inhibitor known to date.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Amidas/química , Amidas/farmacología , Animales , Derivados del Benceno/química , Derivados del Benceno/farmacología , Núcleo Celular/metabolismo , Simulación por Computador , Bases de Datos de Compuestos Químicos , Diseño de Fármacos , Furanos/química , Furanos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch , Simulación del Acoplamiento Molecular , Naftalenos/química , Naftalenos/farmacología , Células PC12 , Unión Proteica , Pirroles/química , Pirroles/farmacología , ARN Mensajero/metabolismo , Ratas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
Breast cancer resistance protein (BCRP), an ATP-dependent efflux transporter, confers drug resistance to many chemotherapy agents. BCRP is overexpressed in tumors exposed to an acidic environment; therefore, it is important to establish the effect of low pH on BCRP transport activity. It has recently been reported that BCRP transports substrates more efficiently in an acidic microenvironment. In the study presented here, we examine the pH dependence of BCRP using methothrexate (MTX), pemetrexed (PMX), and estrone sulfate (ES) as model substrates. Our study revealed an increase of approximately 40-fold in the BCRP-mediated transport of PMX and MTX when the pH was decreased from 7.4 to 5.5. In contrast, only a 2-fold increase was observed for ES. These results indicate a mechanism of transport that is directly dependent on the effective ionization state of the substrates and BCRP. For ES, which retains a constant ionization state throughout the applied pH, the observed mild increase in activity is attributable to the overall changes in the effective ionization state and conformation of BCRP. For MTX and PMX, the marked increase in BCRP transport activity was likely due to the change in ionization state of MTX and PMX at lowered pH and their intermolecular interactions with BCRP. To further rationalize the molecular basis of the pH dependence, molecular modeling and docking studies were carried out using a homology model of BCRP, which has previously been closely examined in structural and site-directed mutagenesis studies (Am J Physiol Cell Physiol 299:C1100-C1109, 2010). On the basis of docking studies, all model compounds were found to associate with arginine 482 (Arg482) by direct salt-bridge interactions via their negatively charged carboxylate or sulfate groups. However, at lower pH, protonated MTX and PMX formed an additional salt-bridge interaction between their positively charged moieties and the nearby negatively charged aspartic acid 477 (Asp477) carboxylate side chain. The formation of this "salt-bridge triad" is expected to increase the overall electrostatic interactions between MTX and PMX with BCRP, which can form a rational basis for the pH dependence of the observed enhanced binding selectivity and transport activity. Removal of Arg482 in site-directed mutagenesis studies eliminated this pH dependence, which lends further support to our binding model. These results shed light on the importance of electrostatic interactions in transport activity and may have important implications in the design of ionizable chemotherapeutics intended for tumors in the acidic microenvironment.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Glutamatos/farmacocinética , Guanina/análogos & derivados , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Antineoplásicos/metabolismo , Línea Celular Transformada , Estrona/análogos & derivados , Estrona/farmacocinética , Femenino , Guanina/farmacocinética , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Metotrexato/farmacocinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida/métodos , Proteínas de Neoplasias/genética , Pemetrexed , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Vesículas Transportadoras/metabolismo , Microambiente Tumoral/fisiologíaRESUMEN
The HIV-1 protease is a validated drug target for the design of antiretroviral drugs to combat AIDS. We previously established the sulfoximine functionality as a valid transition state mimetic (TSM) in the HIV-1 protease inhibitors (PI) design and have identified a lead pseudosymmetric compound with nanomolar enzymatic inhibitory activity. Here, we report the asymmetric synthesis of this compound and its application in the synthesis of sulfoximine-based peptidomimetic HIV-1 protease inhibitors. Molecular modeling revealed the potential mode of binding of the sulfoximine inhibitor as a TSM. The predicted absolute binding free energies suggested similar inhibitory effect as observed in our enzymatic inhibitory studies.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Inhibidores de la Proteasa del VIH/síntesis química , Proteasa del VIH/química , Iminas/química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Simulación por Computador , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Iminas/síntesis química , Iminas/farmacología , Estereoisomerismo , Replicación Viral/efectos de los fármacosRESUMEN
Diadenosine disulfide (5) was reported to inhibit NAD kinase from Listeria monocytogenes and the crystal structure of the enzyme-inhibitor complex has been solved. We have synthesized tiazofurin adenosine disulfide (4) and the disulfide 5, and found that these compounds were moderate inhibitors of human NAD kinase (IC(50)=110 microM and IC(50)=87 microM, respectively) and Mycobacterium tuberculosis NAD kinase (IC(50)=80 microM and IC(50)=45 microM, respectively). We also found that NAD mimics with a short disulfide (-S-S-) moiety were able to bind in the folded (compact) conformation but not in the common extended conformation, which requires the presence of a longer pyrophosphate (-O-P-O-P-O-) linkage. Since majority of NAD-dependent enzymes bind NAD in the extended conformation, selective inhibition of NAD kinases by disulfide analogues has been observed. Introduction of bromine at the C8 of the adenine ring restricted the adenosine moiety of diadenosine disulfides to the syn conformation making it even more compact. The 8-bromoadenosine adenosine disulfide (14) and its di(8-bromoadenosine) analogue (15) were found to be the most potent inhibitors of human (IC(50)=6 microM) and mycobacterium NAD kinase (IC(50)=14-19 microM reported so far. None of the disulfide analogues showed inhibition of lactate-, and inosine monophosphate-dehydrogenase (IMPDH), enzymes that bind NAD in the extended conformation.
Asunto(s)
Adenosina/química , Adenosina/farmacología , Disulfuros/química , Disulfuros/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Ribavirina/análogos & derivados , Adenosina/síntesis química , Sitios de Unión , Disulfuros/síntesis química , Humanos , Modelos Moleculares , Conformación Molecular , Mycobacterium tuberculosis/enzimología , NAD/análogos & derivados , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ribavirina/síntesis química , Ribavirina/química , Ribavirina/farmacologíaRESUMEN
A new class of potent sulfoximine inhibitors for HIV-1 protease has been designed and synthesized. Substitution of the sulfoximine moiety into different parent compounds yields different inhibition effects. While our previously studied sulfoximine-based inhibitors display potency of 2.5 nM (IC(50)) against HIV-1 protease, introduction of the sulfoximine moiety into the asymmetric Indinavir yielded only micromolar inhibition. Docking studies showed structural variations in their modes of binding which explains this unexpected observation. The implication of these observations in the development of other sulfoximine inhibitors is discussed.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/síntesis química , Proteasa del VIH/química , Iminas/química , Sulfóxidos/química , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Enlace de Hidrógeno , Indinavir/química , Concentración 50 Inhibidora , Modelos Químicos , Estructura Molecular , Unión Proteica , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Temperature induced unfolding of Escherichia coli dihydrofolate reductase was carried out by using molecular dynamic simulations. The simulations show that the unfolding generally involves an initial end-to-end collapse of the adenine binding domain into partially extended loops, followed by a gradual breakdown of the remaining beta sheet core structure. The core, which consists of beta strands 5-7, was observed to be the most resistant to thermal unfolding. This region, which is made up of part of the N terminus domain and part of the large domain of the E. coli dihydrofolate reductase, may constitute the nucleation site for protein folding and may be important for the eventual formation of both domains. The unfolding of different domains at different stages of the unfolding process suggests that protein domains vary in stability and that the rate at which they unfold can affect the overall outcome of the unfolding pathway. This observation is compared with the recently proposed hierarchical folding model. Finally, the results of the simulation were found to be consistent with a previous experimental study (Frieden, Proc Natl Acad Sci USA 1990;87:4413-4416) which showed that the folding process of E. coli dihydrofolate reductase involves sequential formation of the substrate binding sites.