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1.
Nat Commun ; 12(1): 618, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504799

RESUMO

Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSß and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.


Assuntos
5-Metilcitosina/metabolismo , Análise de Sequência de DNA , Sulfitos/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Sequência de Bases , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Oxirredução , Piridinas/metabolismo
2.
Xenobiotica ; 51(1): 5-14, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32662714

RESUMO

MGV354 was being developed as a novel ocular therapy for lowering of intraocular pressure, a key modifiable risk factor for glaucoma. MGV354 is an activator of soluble guanylate cyclase, an enzyme known to be involved in the regulation of IOP. MGV354 has been shown to robustly lower IOP over 24 h after a single topical ocular drop in rabbit and monkey pharmacology models. However, MGV354 failed to produce similar results in patients with ocular hypertension or open-angle glaucoma. With an objective of explaining the lack of efficacy in the clinic, we attempted to study whether human metabolism was significantly different from animal metabolism. The present study documents the investigation of metabolism of MGV354 in an effort to understand potential differences in biotransformation pathways of MGV354 in rabbits, monkeys, and humans. Overall twenty-six metabolites, formed via oxidative and conjugative pathways, were identified in vitro and in vivo. In vitro hepatic metabolism was qualitatively similar across species, with minor but distinct differences. There were no observable interspecies differences in the hepatic and ocular metabolism of MGV354. Although ocular metabolism was not as extensive as hepatic, the results do not explain the lack of efficacy of MGV354 in clinical studies.


Assuntos
Anti-Hipertensivos/metabolismo , Piperidinas/metabolismo , Pirazóis/metabolismo , Piridinas/metabolismo , Animais , Anti-Hipertensivos/uso terapêutico , Glaucoma de Ângulo Aberto/tratamento farmacológico , Humanos , Pressão Intraocular/efeitos dos fármacos , Hipertensão Ocular/tratamento farmacológico , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Coelhos
3.
Xenobiotica ; 51(1): 61-71, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32813611

RESUMO

UR-1102, a novel uricosuric agent for treating gout, has been confirmed to exhibit a pharmacological effect in patients. We clarified its metabolic pathway, estimated the contribution of each metabolic enzyme, and assessed the impact of genetic polymorphisms using human in vitro materials. Glucuronide, sulfate and oxidative metabolites of UR-1102 were detected in human hepatocytes. The intrinsic clearance by glucuronidation or oxidation in human liver microsomes was comparable, but sulfation in the cytosol was much lower, indicating that the rank order of contribution was glucuronidation ≥ oxidation > sulfation. Recombinant UGT1A1 and UGT1A3 showed high glucuronidation of UR-1102. We took advantage of a difference in the inhibitory sensitivity of atazanavir to the UGT isoforms and estimated the fraction metabolised (fm) with UGT1A1 to be 70%. Studies using recombinant CYPs and CYP isoform-specific inhibitors showed that oxidation was mediated exclusively by CYP2C9. The effect of UGT1A1 and CYP2C9 inhibitors on UR-1102 metabolism in hepatocytes did not differ markedly between the wild type and variants.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Glucuronosiltransferase/metabolismo , Gota/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Glucuronídeos/metabolismo , Gota/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Oxazinas/metabolismo , Piridinas/metabolismo
4.
J Mol Model ; 26(12): 341, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33200284

RESUMO

HER-2 type breast cancer is one of the most aggressive malignancies found in women. Tucatinib is recently developed and approved as a potential medicine to fight this disease. In this manuscript, we present the gross structural features of this compound and its reactivity and wave function properties using computational simulations. Density functional theory was used to optimise the ground state geometry of the molecule and molecular docking was used to predict biological activity. As the electrons interact with electromagnetic radiations, electronic excitations between different energy levels are analysed in detail using time-dependent density functional theory. Various intermolecular and intermolecular interactions are analysed and reaction sites for attacking electrophiles and nucleophiles identified. Information entropy calculations show that the compound is inherently stable. Docking with COVID-19 proteins show docking score of - 9.42, - 8.93, - 8.45 and - 8.32 kcal/mol respectively indicating high interaction between the drug and proteins. Hence, this is an ideal candidate to study repurposing of existing drugs to combat the pandemic.


Assuntos
Antineoplásicos/química , Antivirais/química , Betacoronavirus/química , Elétrons , Oxazóis/química , Inibidores de Proteases/química , Piridinas/química , Quinazolinas/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antineoplásicos/metabolismo , Antivirais/metabolismo , Betacoronavirus/enzimologia , Sítios de Ligação , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Reposicionamento de Medicamentos , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oxazóis/metabolismo , Inibidores de Proteases/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Piridinas/metabolismo , Teoria Quântica , Quinazolinas/metabolismo , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
5.
Nat Commun ; 11(1): 5261, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067417

RESUMO

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.


Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/metabolismo , Mamíferos/metabolismo , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/genética , Inibidores Enzimáticos/química , Feminino , Mamíferos/genética , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Simulação de Dinâmica Molecular , Fosforilação Oxidativa , Piridinas/química , Piridinas/metabolismo
6.
Appl Environ Microbiol ; 86(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32737127

RESUMO

Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a "butterfly bend" conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products.IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.


Assuntos
Proteínas de Bactérias/química , Redes e Vias Metabólicas , Nicotina/metabolismo , Pseudomonas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mononucleotídeo de Flavina/metabolismo , Pseudomonas/enzimologia , Piridinas/metabolismo , Pirrolidinas/metabolismo , Alinhamento de Sequência
7.
Appl Environ Microbiol ; 86(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32709720

RESUMO

3-Hydroxypyridine (3HP) is an important natural pyridine derivative. Ensifer adhaerens HP1 can utilize 3HP as its sole sources of carbon, nitrogen, and energy to grow, but the genes responsible for the degradation of 3HP remain unknown. In this study, we predicted that a gene cluster, designated 3hpd, might be responsible for the degradation of 3HP. The analysis showed that the initial hydroxylation of 3HP in E. adhaerens HP1 was catalyzed by a four-component dehydrogenase (HpdA1A2A3A4) and led to the formation of 2,5-dihydroxypyridine (2,5-DHP). In addition, the SRPBCC component in HpdA existed as a separate subunit, which is different from other SRPBCC-containing molybdohydroxylases acting on N-heterocyclic aromatic compounds. Moreover, the results demonstrated that the phosphoenolpyruvate (PEP)-utilizing protein and pyruvate-phosphate dikinase were involved in the HpdA activity, and the presence of the gene cluster 3hpd was discovered in the genomes of diverse microbial strains. Our findings provide a better understanding of the microbial degradation of pyridine derivatives in nature and indicated that further research on the origin of the discovered four-component dehydrogenase with a separate SRPBCC domain and the function of PEP-utilizing protein and pyruvate-phosphate dikinase might be of great significance.IMPORTANCE 3-Hydroxypyridine is an important building block for the synthesis of drugs, herbicides, and antibiotics. Although the microbial degradation of 3-hydroxypyridine has been studied for many years, the molecular mechanisms remain unclear. Here, we show that 3hpd is responsible for the catabolism of 3-hydroxypyridine. The 3hpd gene cluster was found to be widespread in Actinobacteria, Rubrobacteria, Thermoleophilia, and Alpha-, Beta-, and Gammaproteobacteria, and the genetic organization of the 3hpd gene clusters in these bacteria shows high diversity. Our findings provide new insight into the catabolism of 3-hydroxypyridine in bacteria.


Assuntos
Proteínas de Bactérias/genética , Família Multigênica , Oxirredutases/genética , Piridinas/metabolismo , Rhizobiaceae/genética , Proteínas de Bactérias/metabolismo , Catálise , Oxirredutases/metabolismo , Rhizobiaceae/metabolismo
8.
Sci Rep ; 10(1): 7623, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376995

RESUMO

Small-molecule agonism of peroxisome proliferator-activated receptor α (PPARα), a ligand-activated transcriptional factor involved in regulating fatty acid metabolism, is an important approach for treating dyslipidemia. Here, we determined the structures of the ligand-binding domain (LBD) of PPARα in complex with 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives, which were recently identified as PPARα-selective activators with markedly different structures from those of the well-known PPARα agonists fibrates. The crystal structures of the complexes showed that they form a canonical hydrogen-bond network involving helix 12 in the LBD, which is thought to be essential for PPARα activation, as also observed for fibrates. However, the phenyl side chain of the compounds occupies a small cavity between Ile272 and Ile354, which is rarely accessed by fibrates. This unique feature may be essential for subtype selectivity and combine with the well-characterized binding mode of fibrates to improve activity. These findings demonstrate the advantage of using 1H-pyrazolo-[3,4-b]pyridine as a skeleton of PPARα agonists and provide insight into the design of molecules for treating dyslipidemia.


Assuntos
PPAR alfa/metabolismo , Pirazóis/química , Piridinas/química , Piridinas/farmacologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , PPAR alfa/química , Domínios Proteicos , Piridinas/metabolismo
9.
Appl Environ Microbiol ; 86(15)2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32471913

RESUMO

Pyridine and its derivatives constitute the majority of heterocyclic aromatic compounds that occur largely as a result of human activities and contribute to environmental pollution. It is known that they can be degraded by various bacteria in the environment; however, the degradation of unsubstituted pyridine has not yet been completely resolved. In this study, we present data on the pyridine catabolic pathway in Arthrobacter sp. strain 68b at the level of genes, enzymes, and metabolites. The pyr gene cluster, responsible for the degradation of pyridine, was identified in a catabolic plasmid, p2MP. The pathway of pyridine metabolism consisted of four enzymatic steps and ended by the formation of succinic acid. The first step in the degradation of pyridine proceeds through a direct ring cleavage catalyzed by a two-component flavin-dependent monooxygenase system, encoded by pyrA (pyridine monooxygenase) and pyrE genes. The genes pyrB, pyrC, and pyrD were found to encode (Z)-N-(4-oxobut-1-enyl)formamide dehydrogenase, amidohydrolase, and succinate semialdehyde dehydrogenase, respectively. These enzymes participate in the subsequent steps of pyridine degradation. The metabolites of these enzymatic reactions were identified, and this allowed us to reconstruct the entire pyridine catabolism pathway in Arthrobacter sp. 68b.IMPORTANCE The biodegradation pathway of pyridine, a notorious toxicant, is relatively unexplored, as no genetic data related to this process have ever been presented. In this paper, we describe the plasmid-borne pyr gene cluster, which includes the complete set of genes responsible for the degradation of pyridine. A key enzyme, the monooxygenase PyrA, which is responsible for the first step of the catabolic pathway, performs an oxidative cleavage of the pyridine ring without typical activation steps such as reduction or hydroxylation of the heterocycle. This work provides new insights into the metabolism of N-heterocyclic compounds in nature.


Assuntos
Arthrobacter/metabolismo , Genes Bacterianos , Família Multigênica , Piridinas/metabolismo , Biodegradação Ambiental
10.
Eur J Med Chem ; 197: 112308, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32339853

RESUMO

A series of novel isocombretapyridines were designed and synthesized based on a lead compound isocombretastatin A-4 (isoCA-4) by replacing 3,4,5-trimethoxylphenyl with substituent pyridine nucleus. The MTT assay results showed that compound 20a possessed the most potent activities against all tested cell lines with IC50 values at nanomolar concentration ranges. Moreover, 20a inhibited tubulin polymerization at a micromolar level and also displayed potent anti-vascular activity in vitro. Further mechanistic studies were conducted to demonstrate that compound 20a could bind to the colchicine site of tubulin,and disrupte the cell microtubule networks, induce G2/M phase arrest, promote apoptosis and depolarize mitochondria of K562 cells in a dose-dependent manner. Notably, 20a exhibited more potent tumor growth inhibition activity with 68.7% tumor growth inhibition than that of isoCA-4 in H22 allograft mouse model without apparent toxicity. The present results suggested that compound 20a may serve as a promising potent microtubule-destabilizing agent candidate for the development of therapeutics to treat cancer.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias/tratamento farmacológico , Piridinas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Piridinas/síntese química , Piridinas/metabolismo , Piridinas/farmacologia , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologia
11.
J Med Chem ; 63(10): 5458-5476, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32329342

RESUMO

SIS3 is a specific inhibitor of Smad3 that inhibits the TGFß1-induced phosphorylation of Smad3. In this article, a variety of SIS3 derivatives were designed and synthesized to discover potential inhibitors against P-glycoprotein-mediated multidrug resistance aided by late-stage functionalization of a 2-(4-(pyridin-2-yl)phenoxy)pyridine analogue. A novel class of potent P-gp reversal agents were investigated, and a lead compound 37 was identified as a potent P-gp reversal agent with strong bioactivity and outstanding affinity for P-gp.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Descoberta de Drogas/métodos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Isoquinolinas/química , Isoquinolinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Resistência a Múltiplos Medicamentos/fisiologia , Humanos , Isoquinolinas/metabolismo , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular/métodos , Piridinas/metabolismo , Pirróis/metabolismo , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
12.
J Med Chem ; 63(10): 5585-5623, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32324999

RESUMO

The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clinical development for oncology bind to each of the eight bromodomains with similar affinities. We hypothesized that it may be possible to achieve an improved therapeutic index by selectively targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger degree of divergence (∼40% identity), suggesting selectivity between BD1 and BD2 of all family members would be more straightforward to achieve. Exploiting the Asp144/His437 and Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of compound 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1. Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clinical development compound 46 (ABBV-744).


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Descoberta de Drogas/métodos , Piridinas/química , Piridinas/metabolismo , Pirróis/química , Pirróis/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Animais , Feminino , Células HeLa , Humanos , Camundongos , Camundongos SCID , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Piridinas/farmacologia , Pirróis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
13.
J Med Chem ; 63(9): 4603-4616, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32223240

RESUMO

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cytotoxic treatments. As the cytosolic enzyme aldehyde dehydrogenase 1A3 turns out to be overexpressed in these kinds of cells, playing a key role for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM. In this work, we describe a novel class of imidazo[1,2-a]pyridine derivatives as aldehyde dehydrogenase 1A3 inhibitors, reporting the evidence of their significance as novel drug candidates for the treatment of GBM. Besides showing an interesting functional profile, in terms of activity against the target enzyme and selectivity toward highly homologous isoenzymes, representative examples of the series also showed a nanomolar to picomolar efficacy against patient-derived GBM stem-like cells, thus proving the concept that targeting aldehyde dehydrogenase might represent a novel and promising way to combat GBM by striking its ability to divide immortally.


Assuntos
Aldeído Oxirredutases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Piridinas/farmacologia , Aldeído Oxirredutases/química , Aldeído Oxirredutases/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Glioblastoma/tratamento farmacológico , Humanos , Imidazóis/síntese química , Imidazóis/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Piridinas/síntese química , Piridinas/metabolismo , Relação Estrutura-Atividade
14.
J Med Chem ; 63(10): 5488-5500, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32337993

RESUMO

Neprilysin (NEP) and angiotensin-converting enzyme (ACE) are two key zinc-dependent metallopeptidases in the natriuretic peptide and kinin systems and renin-angiotensin-aldosterone system, respectively. They play an important role in blood pressure regulation and reducing the risk of heart failure. Vasopeptidase inhibitors omapatrilat and sampatrilat possess dual activity against these enzymes by blocking the ACE-dependent conversion of angiotensin I to the potent vasoconstrictor angiotensin II while simultaneously halting the NEP-dependent degradation of vasodilator atrial natriuretic peptide. Here, we report crystal structures of omapatrilat, sampatrilat, and sampatrilat-ASP (a sampatrilat analogue) in complex with NEP at 1.75, 2.65, and 2.6 Å, respectively. A detailed analysis of these structures and the corresponding structures of ACE with these inhibitors has provided the molecular basis of dual inhibitor recognition involving the catalytic site in both enzymes. This new information will be very useful in the design of safer and more selective vasopeptidase inhibitors of NEP and ACE for effective treatment in hypertension and heart failure.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Desenho de Fármacos , Mesilatos/metabolismo , Neprilisina/metabolismo , Peptidil Dipeptidase A/metabolismo , Piridinas/metabolismo , Tiazepinas/metabolismo , Tirosina/análogos & derivados , Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Cristalografia por Raios X/métodos , Mesilatos/química , Neprilisina/química , Peptidil Dipeptidase A/química , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Piridinas/química , Tiazepinas/química , Tirosina/química , Tirosina/metabolismo
15.
J Med Chem ; 63(9): 4880-4895, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32298120

RESUMO

Due to their role in many important signaling pathways, phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive targets for the development of experimental therapeutics for cancer, metabolic, and immunological disorders. Recent efforts to develop small molecule inhibitors for these lipid kinases resulted in compounds with low- to sub-micromolar potencies. Here, we report the identification of CVM-05-002 using a high-throughput screen of PI5P4Kα against our in-house kinase inhibitor library. CVM-05-002 is a potent and selective inhibitor of PI5P4Ks, and a 1.7 Å X-ray structure reveals its binding interactions in the ATP-binding pocket. Further investigation of the structure-activity relationship led to the development of compound 13, replacing the rhodanine-like moiety present in CVM-05-002 with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kα and -ß by the inhibitors in HEK 293T cells.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Sulfonamidas/farmacologia , Tiazolidinas/farmacologia , Cristalografia por Raios X , Descoberta de Drogas , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Piridinas/síntese química , Piridinas/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/metabolismo , Tiazolidinas/síntese química , Tiazolidinas/metabolismo
16.
Bull Cancer ; 107(5): 574-585, 2020 May.
Artigo em Francês | MEDLINE | ID: mdl-32252973

RESUMO

Direct oral anticoagulants, anti-IIa or anti-Xa, are widely used in the treatment and prevention of venous thromboembolic disease as well as in nonvalvular atrial fibrillation. Direct oral anticoagulants are characterized by a rapid onset of activity, a predictable response and a relatively wide therapeutic window. Nevertheless, theoretical drug interactions exist since direct oral anticoagulants are substrates of the transport protein P-glycoprotein and/or of isoforms of cytochromes P450 pathway. Direct oral anticoagulants do not have a marketing authorization for the treatment of cancer-associated thrombosis unlike low-molecular-weight heparins which remain the gold standard treatment today. However, recent studies have compared low-molecular-weight heparins to direct oral anticoagulants in the treatment of cancer-associated thrombosis. Results of these studies showed a non-inferiority of direct oral anticoagulants in the prevention of recurrent thromboembolic events but at the cost of an increased hemorrhagic risk, in particular for patients with gastrointestinal and urogenital cancers. Thus, international guidelines, unlike French guidelines, integrate them in first line of the therapeutic strategy of cancer patients. We are certainly entering an era of personalized therapy for cancer-associated thrombosis, considering cancer type and also the theoretical risk of drug interactions with anti-cancer treatments or supportive care.


Assuntos
Inibidores do Fator Xa/uso terapêutico , Neoplasias/complicações , Trombose/tratamento farmacológico , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Anticoagulantes/uso terapêutico , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Sistema Enzimático do Citocromo P-450/metabolismo , Dabigatrana/metabolismo , Dabigatrana/uso terapêutico , Interações Medicamentosas , Inibidores do Fator Xa/efeitos adversos , Inibidores do Fator Xa/metabolismo , Hemorragia/induzido quimicamente , Heparina de Baixo Peso Molecular/uso terapêutico , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Pirazóis/metabolismo , Pirazóis/uso terapêutico , Piridinas/metabolismo , Piridinas/uso terapêutico , Piridonas/metabolismo , Piridonas/uso terapêutico , Recidiva , Rivaroxabana/metabolismo , Rivaroxabana/uso terapêutico , Prevenção Secundária , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Trombose/etiologia , Tromboembolia Venosa/prevenção & controle
17.
J Med Chem ; 63(6): 3327-3347, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32129996

RESUMO

Cyclin-dependent kinases (CDKs) are promising therapeutic targets for cancer therapy. Herein, we describe our efforts toward the discovery of a series of 5-chloro-N4-phenyl-N2-(pyridin-2-yl)pyrimidine-2,4-diamine derivatives as dual CDK6 and 9 inhibitors. Intensive structural modifications lead to the identification of compound 66 as the most active dual CDK6/9 inhibitor with balancing potency against these two targets and good selectivity over CDK2. Further biological studies revealed that compound 66 was directly bound to CDK6/9, resulting in suppression of their downstream signaling pathway and inhibition of cell proliferation by blocking cell cycle progression and inducing cellular apoptosis. More importantly, compound 66 significantly inhibited tumor growth in a xenograft mouse model with no obvious toxicity, indicating the promising therapeutic potential of CDK6/9 dual inhibitors for cancer treatment. Therefore, the above results are of great importance in the development of dual CDK6/9 inhibitors for cancer therapy.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/química , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Piridinas/administração & dosagem , Piridinas/síntese química , Piridinas/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Pirimidinas/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-Atividade
18.
Neurology ; 94(15): e1592-e1604, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32139504

RESUMO

OBJECTIVE: In vivo glycolysis-related glucose metabolism and electron transport chain-related mitochondrial activity may be different regionally in the brains of patients with Alzheimer disease (AD). To test this hypothesis regarding AD pathophysiology, we measured the availability of mitochondrial complex-I (MC-I) with the novel PET probe [18F]2-tert- butyl-4-chloro-5-2H- pyridazin-3-one ([18F]BCPP-EF), which binds to MC-I, and compared [18F]BCPP-EF uptake with 18F-fluorodeoxyglucose ([18F]FDG) uptake in the living AD brain. METHODS: First, the total distribution volume (VT) of [18F]BCPP-EF from 10 normal controls (NCs) was quantified using arterial blood samples and then tested to observe whether VT could substitute for the standard uptake value relative to the global count (SUVRg). Eighteen NCs and 14 different NCs underwent PET with [18F]BCPP-EF or [18F]FDG, respectively. Second, 32 patients with AD were scanned semiquantitatively with double PET tracers. Interparticipant and intraparticipant comparisons of the levels of MC-I activity ([18F]BCPP-EF) and glucose metabolism ([18F]FDG) were performed. RESULTS: The [18F]BCPP-EF VT was positively correlated with the [18F]BCPP-EF SUVRg, indicating that the use of the SUVRg was sufficient for semiquantitative evaluation. The [18F]BCPP-EF SUVRg, but not the [18F]FDG SUVRg, was significantly lower in the parahippocampus in patients with AD, highlighting the prominence of oxidative metabolic failure in the medial temporal cortex. Robust positive correlations between the [18F]BCPP-EF SUVRg and [18F]FDG SUVRg were observed in several brain regions, except the parahippocampus, in early-stage AD. CONCLUSIONS: Mitochondrial dysfunction in the parahippocampus was shown in early-stage AD. Mitochondria-related energy failure may precede glycolysis-related hypometabolism in regions with pathologically confirmed early neurodegeneration in AD.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Glicólise/fisiologia , Mitocôndrias/metabolismo , Idoso , Feminino , Fluordesoxiglucose F18/metabolismo , Humanos , Masculino , Piridinas/metabolismo , Compostos Radiofarmacêuticos/metabolismo
19.
J Med Chem ; 63(6): 3298-3316, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32125159

RESUMO

Cruzain, an essential cysteine protease of the parasitic protozoan, Trypanosoma cruzi, is an important drug target for Chagas disease. We describe here a new series of reversible but time-dependent inhibitors of cruzain, composed of a dipeptide scaffold appended to vinyl heterocycles meant to provide replacements for the irreversible reactive "warheads" of vinyl sulfone inactivators of cruzain. Peptidomimetic vinyl heterocyclic inhibitors (PVHIs) containing Cbz-Phe-Phe/homoPhe scaffolds with vinyl-2-pyrimidine, vinyl-2-pyridine, and vinyl-2-(N-methyl)-pyridine groups conferred reversible, time-dependent inhibition of cruzain (Ki* = 0.1-0.4 µM). These cruzain inhibitors exhibited moderate to excellent selectivity versus human cathepsins B, L, and S and showed no apparent toxicity to human cells but were effective in cell cultures of Trypanosoma brucei brucei (EC50 = 1-15 µM) and eliminated T. cruzi in infected murine cardiomyoblasts (EC50 = 5-8 µM). PVHIs represent a new class of cruzain inhibitors that could progress to viable candidate compounds to treat Chagas disease and human sleeping sickness.


Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Peptidomiméticos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Compostos de Vinila/farmacologia , Animais , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Desenho de Fármacos , Ensaios Enzimáticos , Humanos , Cinética , Camundongos , Simulação de Acoplamento Molecular , Mioblastos Cardíacos/efeitos dos fármacos , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Piridinas/síntese química , Piridinas/metabolismo , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Compostos de Vinila/síntese química , Compostos de Vinila/metabolismo
20.
J Med Chem ; 63(6): 3274-3289, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32011145

RESUMO

Trypsin and thrombin, structurally similar serine proteases, recognize different substrates; thrombin cleaves after Arg, whereas trypsin cleaves after Lys/Arg. Both recognize basic substrate headgroups via Asp189 at the bottom of the S1 pocket. By crystallography and isothermal titration calorimetry (ITC), we studied a series of d-Phe/d-DiPhe-Pro-(amino)pyridines. Identical ligand pairs show the same binding poses. Surprisingly, one ligand binds to trypsin in protonated state and to thrombin in unprotonated state at P1 along with differences in the residual solvation pattern. While trypsin binding is mediated by an ordered water molecule, in thrombin, water is scattered over three hydration sites. Although having highly similar S1 pockets, our results suggest different electrostatic properties of Asp189 possibly contributing to the selectivity determinant. Thrombin binds a specific Na+ ion next to Asp189, which is absent in trypsin. The electrostatic properties across the S1 pocket are further attenuated by charged Glu192 at the rim of S1 in thrombin, which is replaced by uncharged Gln192 in trypsin.


Assuntos
Antitrombinas/metabolismo , Dipeptídeos/metabolismo , Piridinas/metabolismo , Trombina/metabolismo , Inibidores da Tripsina/metabolismo , Tripsina/metabolismo , Animais , Antitrombinas/síntese química , Sítios de Ligação , Calorimetria , Bovinos , Cristalografia por Raios X , Dipeptídeos/síntese química , Humanos , Ligantes , Ligação Proteica , Prótons , Piridinas/síntese química , Termodinâmica , Trombina/química , Tripsina/química , Inibidores da Tripsina/síntese química , Água/metabolismo
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