RESUMEN
Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/ß-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/ß-catenin pathway-related cancers, such as colorectal cancers.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasa-1/genética , Tanquirasas/genética , Antineoplásicos/farmacología , Bencimidazoles/farmacología , Sitios de Unión/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Daño del ADN/efectos de los fármacos , Reparación del ADN/genética , Femenino , Humanos , Indazoles/farmacología , Ligandos , Ftalazinas/farmacología , Piperidinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (3a and 4a) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.
RESUMEN
In human pathogens, the sulfate assimilation pathway provides reduced sulfur for biosynthesis of essential metabolites, including cysteine and low-molecular-weight thiol compounds. Sulfonucleotide reductases (SRs) catalyze the first committed step of sulfate reduction. In this reaction, activated sulfate in the form of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is reduced to sulfite. Gene knockout, transcriptomic and proteomic data have established the importance of SRs in oxidative stress-inducible antimicrobial resistance mechanisms. In previous work, we focused on rational and high-throughput design of small-molecule inhibitors that target the active site of SRs. However, another critical goal is to discover functionally important regions in SRs beyond the traditional active site. As an alternative to conservation analysis, we used directed evolution to rapidly identify functional sites in PAPS reductase (PAPR). Four new regions were discovered that are essential to PAPR function and lie outside the substrate binding pocket. Our results highlight the use of directed evolution as a tool to rapidly discover functionally important sites in proteins.
Asunto(s)
Adenosina Fosfosulfato/metabolismo , Evolución Molecular Dirigida , Oxidorreductasas/metabolismo , Azufre/metabolismo , Adenosina Fosfosulfato/química , Humanos , Modelos Moleculares , Oxidorreductasas/química , Oxidorreductasas/genéticaRESUMEN
Epidermal growth factor receptor (EGFR) is a target of signal-derived H2O2, and oxidation of active-site cysteine 797 to sulfenic acid enhances kinase activity. Although a major class of covalent drugs targets C797, nothing is known about its catalytic importance or how S-sulfenylation leads to activation. Here, we report the first detailed functional analysis of C797. In contrast to prior assumptions, mutation of C797 diminishes catalytic efficiency in vitro and cells. The experimentally determined pKa and reactivity of C797 toward H2O2 correspondingly distinguish this residue from the bulk of the cysteinome. Molecular dynamics simulation of reduced versus oxidized EGFR, reinforced by experimental testing, indicates that sulfenylation of C797 allows new electrostatic interactions to be formed with the catalytic loop. Finally, we show that chronic oxidative stress yields an EGFR subpopulation that is refractory to the FDA-approved drug afatinib. Collectively, our data highlight the significance of redox biology to understanding kinase regulation and drug pharmacology.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas Quinasas/metabolismo , Afatinib , Activación Enzimática , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Humanos , Simulación de Dinámica Molecular , Mutación , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38â¯350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional ΔAPSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.
Asunto(s)
Antituberculosos/farmacología , Evaluación Preclínica de Medicamentos , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/antagonistas & inhibidores , Azufre/metabolismo , Animales , Antituberculosos/síntesis química , Antituberculosos/química , Modelos Animales de Enfermedad , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Estructura Molecular , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Reproducibilidad de los Resultados , Azufre/química , Compuestos de Azufre/metabolismo , Tuberculosis/tratamiento farmacológicoRESUMEN
Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx-protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx-target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these protein-protein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx-target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition.
Asunto(s)
Entropía , Tiorredoxinas/metabolismo , Sitios de Unión , Escherichia coli/enzimología , Oxidación-Reducción , Oxidorreductasas/metabolismo , Conformación Proteica , Tiorredoxinas/químicaRESUMEN
Production of the Gag-Pol polyprotein in human immunodeficiency virus (HIV) requires a -1 ribosomal frameshift, which is directed by a highly conserved RNA stem-loop. Building on our discovery of a set of disulfide-containing peptides that bind this RNA, we describe medicinal chemistry efforts designed to begin to understand the structure-activity relationships and RNA sequence-selectivity relationships associated with these compounds. Additionally, we have prepared analogues incorporating an olefin or saturated hydrocarbon bioisostere of the disulfide moiety, as a first step toward enhancing biostability. The olefin-containing compounds exhibit affinity comparable to the lead disulfide and, importantly, have no discernible toxicity when incubated with human fibroblasts at concentrations up to 1 mM.
Asunto(s)
Disulfuros/química , Sistema de Lectura Ribosómico , VIH-1/genética , Oligopéptidos/química , Quinolinas/química , ARN Viral/genética , Alquenos/síntesis química , Alquenos/química , Alquenos/farmacología , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Secuencia de Bases , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Disulfuros/síntesis química , Disulfuros/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Ligandos , Mutación , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Quinolinas/síntesis química , Quinolinas/farmacología , Relación Estructura-ActividadRESUMEN
Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is an RNA-mediated disease. Dramatically expanded (CUG) repeats accumulate in nuclei and sequester RNA-binding proteins such as the splicing regulator MBNL1. We have employed resin-bound dynamic combinatorial chemistry (RBDCC) to identify the first examples of compounds able to inhibit MBNL1 binding to (CUG) repeat RNA. Screening an RBDCL with a theoretical diversity of 11 325 members yielded several molecules with significant selectivity for binding to (CUG) repeat RNA over other sequences. These compounds were also able to inhibit the interaction of GGG-(CUG)(109)-GGG RNA with MBNL1 in vitro, with K(i) values in the low micromolar range.
Asunto(s)
Plomo/química , Distrofia Miotónica/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/química , ARN/metabolismo , Secuencias Repetidas en Tándem , Técnicas Químicas Combinatorias , Dimerización , Ligandos , Distrofia Miotónica/genética , Distrofia Miotónica/patología , Proteínas de Unión al ARN/genéticaRESUMEN
The design and synthesis of receptors capable of selective, noncovalent recognition of carbohydrates continues to be a signature challenge in bioorganic chemistry. We report a new generation of tripodal receptors incorporating three pyridine (compound 2) or quinoline (compound 3) rings around a central cyclohexane core for use in molecular recognition of monosaccharides in apolar and polar protic solvents. These tripodal receptors were investigated using (1)H NMR, UV, and fluorescence titrations in order to determine their binding abilities toward a set of octyl glycosides. Receptor 2 displayed the highest binding affinity reported to date for noncovalent 1:1 binding of an alpha-glucopyranoside in chloroform (Ka = 212,000 +/- 27,000 M(-1)) and an approximately 8-fold selectivity for the alpha anomer over the beta anomer of the glucopyranoside. Most importantly, 2 retained its micromolar range of affinities toward monosaccharides in a polar and highly competitive solvent (methanol). The quinoline variant 3 also displayed micromolar binding affinities for selected monosaccharides in methanol (as measured by fluorescence) that were generally smaller than those of 2. Compound 3 was found to follow a selectivity pattern similar to that of 2, displaying higher affinities for glucopyranosides than for other monosaccharides. The binding stoichiometry was estimated to be 1:1 for the complexes formed by both 2 and 3 with glucopyranosides, as determined by Job plots. Nuclear Overhauser effect spectroscopy allowed for the derivation of a binding model consistent with the observed selectivities.
Asunto(s)
Ciclohexanos/química , Glucósidos/química , Piridinas/química , Quinolinas/química , Conformación de Carbohidratos , Cristalografía por Rayos X , Ciclohexanos/síntesis química , Cinética , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Piridinas/síntesis química , Quinolinas/síntesis química , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Additional structural modifications of the new chemical entity, 2,8-dicyclopentyl-4-methylquinoline (DCMQ; MIC=6.25 microg/mL, M. tuberculosis H37Rv) resulted in the synthesis of four new series of the ring-substituted quinolinecarbohydrazides (series 1-4) constituting 22 analogues. All new derivatives were evaluated for in vitro antimycobacterial activities against drug-sensitive M. tuberculosis H37Rv strain. Certain ring-substituted-2-quinolinecarbohydrazide analogues described herein showed good inhibitory activity. In particular, analogues 4-(1-adamantyl)-2-quinolinecarbohydrazide (2d), 4,5-dicyclopentyl-2-quinolinecarbohydrazide (2e), 4,8-dicyclopentyl-2-quinolinecarbohydrazide (2f), and 4,5-dicyclohexyl-2-quinolinecarbohydrazide (2g) have exhibited the MIC value of 6.25 microg/mL. Further investigation of the most suitable lead prototype, 4-(1-adamantyl)-2-quinolinecarbohydrazide (2d, series 1) led to the synthesis of N2-alkyl/N2,N2-dialkyl/N2-aryl-4-(1-adamantyl)-2-quinolinecarboxamides (series 5) consisting of 13 analogues. Some of the synthesized carboxamides 7a, 7h, and 7m reported herein have exhibited excellent antimycobacterial activities in the range of 6.25-3.125 microg/mL against drug-sensitive and drug-resistant M. tuberculosis H37Rv strains.
Asunto(s)
Antibacterianos/síntesis química , Mycobacterium tuberculosis/efectos de los fármacos , Ácidos Quinolínicos/síntesis química , Amidas , Antibacterianos/farmacología , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana , Ácidos Quinolínicos/farmacología , Relación Estructura-ActividadRESUMEN
Structural optimization of recently discovered new chemical entity, 2,8-dicyclopentyl-4-methylquinoline (DCMQ; MIC= 6.25 microg/mL, M. tuberculosis H37Rv) resulted in the synthesis of four new series of ring-substituted quinolinecarboxylic acids/esters constituting 45 analogues. All new derivatives were evaluated for in vitro antimycobacterial activities against M. tuberculosis H37Rv. Certain ring-substituted-2-quinolinecarboxylic acid ester and ring-substituted-2-quinoline acetic acid ester analogues described herein showed moderate to good inhibitory activity. In particular, three analogues methyl 4,5-dicyclopentyl-2-quinolinecarboxylate (3b), methyl 4,8-dicyclopentyl-2-quinolinecarboxylate (3c) and ethyl 2-(2,8-dicyclopentyl-4-quinolyl)acetate (14g) exhibited excellent MIC values of 1.00, 2.00 and 4.00microg/mL, respectively. Results obtained indicate that substitution of the quinoline ring with dicyclopentyl substituent presumably enhances the antimycobacterial activities in the quinoline analogues described herein.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Quinolinas/síntesis química , Quinolinas/farmacología , Antibacterianos/química , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Diseño de Fármacos , Ésteres/síntesis química , Ésteres/química , Ésteres/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Quinolinas/química , Relación Estructura-ActividadRESUMEN
We report synthesis and anti-tuberculosis activities of a series of novel ring-substituted quinolines. The most effective compound of the series 3d (MIC=6.25 microg/mL, Mycobacterium tuberculosis H37Rv strain) was synthesized in one step; thus is an attractive lead molecule for anti-tuberculosis drug development. The results of this study represent the discovery of ring-substituted 4-methylquinolines as new class of potential anti-tuberculosis agents.