RESUMO
Herpesvirus infections are ubiquitous, with over 95% of the adult population infected by at least one strain. While most of these infections resolve without treatment in healthy individuals, they can cause significant morbidity and mortality in immunocompromised, stem cell, or organ transplant patients. Current nucleoside standards of care provide meaningful benefit but are limited due to poor tolerability, resistance, and generally narrow spectrum of activity. Herpesviruses share a conserved DNA polymerase, the inhibition of which is validated as an effective strategy to disrupt viral replication. By utilizing a non-nucleoside inhibitor of the viral DNA polymerase, we sought to develop agents covering multiple herpesviruses (e.g., CMV, VZV, HSV1/2, EBV, and HHV6). Herein is described the invention of an oxazolidinone class of broad-spectrum non-nucleoside herpes antiviral inhibitors. A lead compound (42) with potent biochemical and broad-spectrum cellular activity was found to be efficacious in murine models against both HSV-1 and CMV infection.
RESUMO
Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Lipopolissacarídeos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Camundongos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Transporte Biológico , Testes de Sensibilidade Microbiana , Humanos , Microscopia Crioeletrônica , Carbapenêmicos/farmacologia , Carbapenêmicos/metabolismo , Modelos Animais de Doenças , Feminino , Transportadores de Cassetes de Ligação de ATPRESUMO
Vorapaxar is an approved drug for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Subsequent to the discovery of Vorapaxar, medicinal chemistry efforts were continued to identify structurally differentiated leads. Toward this goal, extensive structure-activity relationship studies using a C-ring-truncated version of Vorapaxar culminated in the discovery of three leads, represented as 13, 14, and 23. Among these leads, compound 14 possessed favorable pharmacokinetic properties and an off-target profile, which supported additional profiling in an exploratory rat toxicology study.
Assuntos
Infarto do Miocárdio , Trombose , Animais , Humanos , Lactonas , Infarto do Miocárdio/tratamento farmacológico , Inibidores da Agregação Plaquetária , Ratos , Receptor PAR-1 , Receptores Ativados por Proteinase , Trombose/induzido quimicamente , Trombose/tratamento farmacológicoRESUMO
To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of E. coli DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of E. coli and Pseudomonas aeruginosa DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.
Assuntos
Antibacterianos/farmacologia , DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Fluoroquinolonas/farmacologia , Testes de Sensibilidade Microbiana , Domínios Proteicos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologiaRESUMO
High-throughput screening (HTS) has enabled millions of compounds to be assessed for biological activity, but challenges remain in the prioritization of hit series. While biological, absorption, distribution, metabolism, excretion, and toxicity (ADMET), purity, and structural data are routinely used to select chemical matter for further follow-up, the scarcity of historical ADMET data for screening hits limits our understanding of early hit compounds. Herein, we describe a process that utilizes a battery of in-house quantitative structure-activity relationship (QSAR) models to generate in silico ADMET profiles for hit series to enable more complete characterizations of HTS chemical matter. These profiles allow teams to quickly assess hit series for desirable ADMET properties or suspected liabilities that may require significant optimization. Accordingly, these in silico data can direct ADMET experimentation and profoundly impact the progression of hit series. Several prospective examples are presented to substantiate the value of this approach.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Preparações Farmacêuticas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Simulação por Computador , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Farmacologia , Relação Quantitativa Estrutura-AtividadeRESUMO
We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure-activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.
Assuntos
Lipídeos/química , Bibliotecas de Moléculas Pequenas , Animais , Feminino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Solubilidade , Relação Estrutura-AtividadeRESUMO
The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created an urgent need for new therapeutic agents capable of combating this threat. We have previously reported on the discovery of novel inhibitors targeting enzymes involved in the biosynthesis of wall teichoic acid (WTA) and demonstrated that these agents can restore ß-lactam efficacy against MRSA. In those previous reports pathway engagement of inhibitors was demonstrated by reduction in WTA levels measured by polyacrylamide gel electrophoresis. To enable a more rigorous analysis of these inhibitors we sought to develop a quantitative method for measuring whole-cell reductions in WTA. Herein we describe a robust methodology for hydrolyzing polymeric WTA to the monomeric component ribitol-N-acetylglucosamine coupled with measurement by LC-MS/MS. Critical elements of the protocol were found to include the time and temperature of hydrofluoric acid-mediated hydrolysis of polymeric WTA and optimization of these parameters is fully described. Most significantly, the assay enabled accurate and reproducible measurement of depletion EC50s for tunicamycin and representatives from the novel class of TarO inhibitors, the tarocins. The method described can readily be adapted to quantifying levels of WTA in tissue homogenates from a murine model of infection, highlighting the applicability for both in vitro and in vivo characterizations.
Assuntos
Espectrometria de Massas/métodos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Ácidos Teicoicos/metabolismo , Cromatografia Líquida/métodos , Staphylococcus aureus Resistente à Meticilina/química , Ácidos Teicoicos/química , Tunicamicina/farmacologiaRESUMO
A series of benzimidazole analogs have been synthesized to improve the profile of the previous lead compounds tarocin B and 1. The syntheses, structure-activity relationships, and selected biochemical data of these analogs are described. The optimization efforts allowed the identification of 21, a fluoro-substituted benzimidazole, exhibiting potent TarO inhibitory activity and typical profile for a wall teichoic acid (WTA) biosynthesis inhibitor. Compound 21 displayed a potent synergistic and bactericidal effect in combination with imipenem against diverse methicillin-resistant Staphylococci.
Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Ácidos Teicoicos/antagonistas & inibidores , Animais , Antibacterianos/química , Benzimidazóis/química , Testes de Sensibilidade Microbiana , Ratos , Relação Estrutura-Atividade , Ácidos Teicoicos/biossínteseRESUMO
The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to be chemically synergistic with ß-lactams. These previously disclosed inhibitors, termed tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad spectrum ß-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci. The synthesis and structure-activity relationships (SAR) of a tarocin series will be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant Staphylococci.
Assuntos
Antibacterianos/química , Ácidos Teicoicos/metabolismo , beta-Lactamas/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Avaliação Pré-Clínica de Medicamentos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , beta-Lactamas/metabolismoRESUMO
Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro ß-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the ß-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore ß-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.
Assuntos
Proteínas de Bactérias/metabolismo , Racemases e Epimerases/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus epidermidis/metabolismo , Ácidos Teicoicos/biossíntese , Animais , Proteínas de Bactérias/química , Biofilmes/crescimento & desenvolvimento , Parede Celular/metabolismo , Cristalografia por Raios X , Modelos Animais de Doenças , Staphylococcus aureus Resistente à Meticilina , Camundongos , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência , Ressonância Magnética Nuclear Biomolecular , Racemases e Epimerases/química , Infecções Estafilocócicas/metabolismoRESUMO
The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with ß-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum ß-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant staphylococci.
Assuntos
Proteínas de Bactérias/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Parede Celular/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Ácidos Teicoicos/biossíntese , beta-Lactamas/farmacologia , Animais , Parede Celular/efeitos dos fármacos , Dicloxacilina/farmacologia , Dicloxacilina/uso terapêutico , Feminino , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Modelos Moleculares , Fenótipo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Resultado do TratamentoRESUMO
Nonessential enzymes in the staphylococcal wall teichoic acid (WTA) pathway serve as highly validated ß-lactam potentiation targets. MnaA (UDP-GlcNAc 2-epimerase) plays an important role in an early step of WTA biosynthesis by providing an activated form of ManNAc. Identification of a selective MnaA inhibitor would provide a tool to interrogate the contribution of the MnaA enzyme in the WTA pathway as well as serve as an adjuvant to restore ß-lactam activity against methicillin-resistant Staphylococcus aureus (MRSA). However, development of an epimerase functional assay can be challenging since both MnaA substrate and product (UDP-GlcNAc/UDP-ManNAc) share an identical molecular weight. Herein, we developed a nuclear magnetic resonance (NMR) functional assay that can be combined with other NMR approaches to triage putative MnaA inhibitors from phenotypic cell-based screening campaigns. In addition, we determined that tunicamycin, a potent WTA pathway inhibitor, inhibits both S. aureus MnaA and a functionally redundant epimerase, Cap5P.
Assuntos
Parede Celular/efeitos dos fármacos , Espectroscopia de Ressonância Magnética/métodos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Carboidratos Epimerases/antagonistas & inibidores , Carboidratos Epimerases/química , Parede Celular/química , Humanos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Ácidos Teicoicos/química , Ácidos Teicoicos/metabolismo , Açúcares de Uridina Difosfato/química , Açúcares de Uridina Difosfato/metabolismo , Resistência beta-Lactâmica/efeitos dos fármacos , beta-Lactamases/química , beta-Lactamases/efeitos dos fármacosRESUMO
The ribonucleotide reductase (RNR) enzyme is a heteromer of RRM1 and RRM2 subunits. The active enzyme catalyzes de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. Complexity in the generation of physiologically relevant, active RRM1/RRM2 heterodimers was perceived as limiting to the identification of selective RRM1 inhibitors by high-throughput screening of compound libraries and led us to seek alternative methods to identify lead series. In short, we found that gemcitabine, as its diphosphate metabolite, represents one of the few described active site inhibitors of RRM1. We herein describe the identification of novel 5'-amino gemcitabine analogs as potent RRM1 inhibitors through in-cell phenotypic screening.
Assuntos
Desoxicitidina/análogos & derivados , Proteínas Supressoras de Tumor/antagonistas & inibidores , Linhagem Celular Tumoral , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ribonucleosídeo Difosfato Redutase , Relação Estrutura-Atividade , GencitabinaRESUMO
The synthesis and hit-to-lead SAR development from a pyrazolo[1,5-a]pyrimidine-derived hit 5 to the identification of a series of potent, pan-Pim inhibitors such as 11j are described.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , Humanos , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-pim-1/química , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/síntese química , Relação Estrutura-AtividadeRESUMO
Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a ß-lactam potentiation screen to identify small molecules that augment the activity of ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the ß-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive ß-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Peptidoglicano Glicosiltransferase/metabolismo , Pirazóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Esteróis/farmacologia , Simulação por Computador , Farmacorresistência Bacteriana , Inibidores Enzimáticos/farmacologia , Humanos , Microscopia de Fluorescência , Modelos Moleculares , Pirazóis/química , Staphylococcus aureus/enzimologia , Esteróis/químicaRESUMO
Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Óxidos N-Cíclicos , Quinases Ciclina-Dependentes/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Histonas/metabolismo , Humanos , Immunoblotting , Indolizinas , Camundongos , Camundongos Nus , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/administração & dosagem , Pirazóis/química , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/química , Interferência de RNA , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.
Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Pirazóis/química , Pirimidinas/química , Sítios de Ligação , Domínio Catalítico , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/síntese química , Pirazóis/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The synthesis and hit-to-lead SAR development of a pyrazolo[1,5-a]pyrimidine hit 4 is described leading to a series of potent, selective CHK1 inhibitors such as compound 17r. In the Letter, the further utility of the pyrazolo[1,5-a]pyrimidine template for the development of potent, selective kinase inhibitors is detailed.
Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Pirazóis/química , Pirimidinas/química , Sítios de Ligação , Domínio Catalítico , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/síntese química , Pirazóis/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The nature and the size of the benzylic substituent are shown to be the key to controlling receptor selectivity (CCR5 vs M1, M2) and potency in the title compounds. Optimization of the lead benzylic methyl compound 3 led to the methoxymethyl analogue 30, which had excellent receptor selectivity and oral bioavailability in rats and monkeys. Compound 30 (Sch-417690/Sch-D), a potent inhibitor of HIV-1 entry into target cells, is currently in clinical trials.
Assuntos
Fármacos Anti-HIV/síntese química , Antagonistas dos Receptores CCR5 , HIV-1/efeitos dos fármacos , Piperazinas/síntese química , Piperidinas/síntese química , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Pirimidinas/síntese química , Administração Oral , Animais , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/farmacologia , Disponibilidade Biológica , Encéfalo/efeitos dos fármacos , Proteínas de Transporte de Cátions/efeitos dos fármacos , Sistema Digestório/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go , HIV-1/isolamento & purificação , Humanos , Técnicas In Vitro , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Macaca fascicularis , Piperazinas/efeitos adversos , Piperazinas/química , Piperazinas/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Canais de Potássio/efeitos dos fármacos , Pirimidinas/efeitos adversos , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E. coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.