RESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/genética , Tanquirases/genética , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Sítios de Ligação/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Feminino , Humanos , Indazóis/farmacologia , Ligantes , Ftalazinas/farmacologia , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
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.
Assuntos
Entropia , Tiorredoxinas/metabolismo , Sítios de Ligação , Escherichia coli/enzimologia , Oxirredução , Oxirredutases/metabolismo , Conformação Proteica , Tiorredoxinas/químicaRESUMO
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.
Assuntos
Adenosina Fosfossulfato/metabolismo , Evolução Molecular Direcionada , Oxirredutases/metabolismo , Enxofre/metabolismo , Adenosina Fosfossulfato/química , Humanos , Modelos Moleculares , Oxirredutases/química , Oxirredutases/genéticaRESUMO
Protein arginine methyltransferase 5 (PRMT5) overexpression in hematologic and solid tumors methylates arginine residues on cellular proteins involved in important cancer functions including cell-cycle regulation, mRNA splicing, cell differentiation, cell signaling, and apoptosis. PRMT5 methyltransferase function has been linked with high rates of tumor cell proliferation and decreased overall survival, and PRMT5 inhibitors are currently being explored as an approach for targeting cancer-specific dependencies due to PRMT5 catalytic function. Here, we describe the discovery of potent and selective S-adenosylmethionine (SAM) competitive PRMT5 inhibitors, with in vitro and in vivo characterization of clinical candidate PF-06939999. Acquired resistance mechanisms were explored through the development of drug resistant cell lines. Our data highlight compound-specific resistance mutations in the PRMT5 enzyme that demonstrate structural constraints in the cofactor binding site that prevent emergence of complete resistance to SAM site inhibitors. PRMT5 inhibition by PF-06939999 treatment reduced proliferation of non-small cell lung cancer (NSCLC) cells, with dose-dependent decreases in symmetric dimethyl arginine (SDMA) levels and changes in alternative splicing of numerous pre-mRNAs. Drug sensitivity to PF-06939999 in NSCLC cells associates with cancer pathways including MYC, cell cycle and spliceosome, and with mutations in splicing factors such as RBM10. Translation of efficacy in mouse tumor xenograft models with splicing mutations provides rationale for therapeutic use of PF-06939999 in the treatment of splicing dysregulated NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Resistência a Medicamentos , Feminino , Humanos , Neoplasias Pulmonares/patologia , CamundongosRESUMO
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.
RESUMO
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.
Assuntos
Cicloexanos/química , Glucosídeos/química , Piridinas/química , Quinolinas/química , Configuração de Carboidratos , Cristalografia por Raios X , Cicloexanos/síntese química , Cinética , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Piridinas/síntese química , Quinolinas/síntese química , Espectrometria de Fluorescência , TermodinâmicaRESUMO
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.
Assuntos
Chumbo/química , Distrofia Miotônica/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , Sequências de Repetição em Tandem , Técnicas de Química Combinatória , Dimerização , Ligantes , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Proteínas de Ligação a RNA/genéticaRESUMO
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.
Assuntos
Antituberculosos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Enxofre/metabolismo , Animais , Antituberculosos/síntese química , Antituberculosos/química , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Reprodutibilidade dos Testes , Enxofre/química , Compostos de Enxofre/metabolismo , Tuberculose/tratamento farmacológicoRESUMO
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.
Assuntos
Receptores ErbB/metabolismo , Proteínas Quinases/metabolismo , Afatinib , Ativação Enzimática , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Humanos , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
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.
Assuntos
Dissulfetos/química , Mudança da Fase de Leitura do Gene Ribossômico , HIV-1/genética , Oligopeptídeos/química , Quinolinas/química , RNA Viral/genética , Alcenos/síntese química , Alcenos/química , Alcenos/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dissulfetos/síntese química , Dissulfetos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Ligantes , Mutação , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Quinolinas/síntese química , Quinolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Antituberculosos/síntese química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Quinolinas/síntese química , Quinolinas/farmacologia , 4-Quinolonas , Desenho de Fármacos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Complexo Mycobacterium avium/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Quinolinas/síntese química , Quinolinas/farmacologia , Antibacterianos/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Desenho de Fármacos , Ésteres/síntese química , Ésteres/química , Ésteres/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
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.