RESUMO
Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2'-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2'-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.
Assuntos
Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Nucleotídeos/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/genética , Alanina/farmacologia , Antivirais/química , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Nucleotídeos/química , RNA , RNA Viral/biossíntese , Proteínas não Estruturais ViraisRESUMO
The field of small-molecule inhibitors of protein-protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.
Assuntos
Indóis/química , Indolizidinas/química , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Compostos de Espiro/química , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Imidazolinas/química , Indóis/uso terapêutico , Indóis/toxicidade , Indolizidinas/uso terapêutico , Indolizidinas/toxicidade , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirrolidinas/química , Compostos de Espiro/uso terapêutico , Compostos de Espiro/toxicidade , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , para-Aminobenzoatos/químicaRESUMO
The farnesoid X receptor (FXR) is a ligand-activated nuclear receptor. Activation of FXR significantly impacts the expressions of the pivotal genes involved in bile acid metabolism, inflammation, fibrosis, and homeostasis of lipid and glucose, leading to considerable interests in developing FXR agonists for the treatment of nonalcoholic steatohepatitis (NASH) or other FXR-relevant diseases. Herein, we describe the design, optimization, and characterization of a series of N-methylene-piperazinyl derivatives as the nonbile acid FXR agonists. Particularly, compound 23 (HPG1860), a potent full FXR agonist, shows high selectivity, favorable ADME and pharmacokinetics profile, along with favorable in vivo activities demonstrated in both rodent PD model and HFD-CCl4 model and is currently in clinical development in patients with NASH in phase II.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores Citoplasmáticos e Nucleares , Ácidos e Sais Biliares , Inflamação , Relação Estrutura-AtividadeRESUMO
A series of pyrimidodiazepines was identified as potent Polo-like kinase 1 (PLK1) inhibitors. The synthesis and SAR are discussed. The lead compound 7 (RO3280) has potent inhibitory activity against PLK1, good selectivity against other kinases, and excellent in vitro cellular potency. It showed strong antitumor activity in xenograft mouse models.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Azepinas/síntese química , Azepinas/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pirimidinas/síntese química , Pirimidinas/química , Estereoisomerismo , Relação Estrutura-Atividade , Quinase 1 Polo-LikeRESUMO
Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets Wolbachia, a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in L. sigmodontis, Brugia malayi, and Onchocerca ochengi in vivo preclinical models of filarial disease and in vitro selectivity against Loa loa (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.
Assuntos
Antibacterianos/uso terapêutico , Descoberta de Drogas , Filariose/tratamento farmacológico , Filariose/parasitologia , Filarioidea/fisiologia , Quinazolinas/uso terapêutico , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Modelos Animais de Doenças , Feminino , Filarioidea/efeitos dos fármacos , Filarioidea/microbiologia , Ensaios de Triagem em Larga Escala , Camundongos , Fenótipo , Quinazolinas/química , Quinazolinas/farmacologia , Bibliotecas de Moléculas Pequenas , Wolbachia/efeitos dos fármacosRESUMO
The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (K(i) = 1-3 nmol/L) and was inactive (K(i) > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC(50)s = 0.60 mumol/L. The growth-inhibitory activity is characterized by a cell cycle block at G(1) and G(2) phases and induction of apoptosis. R547 reduced phosphorylation of the cellular retinoblastoma protein at specific CDK phosphorylation sites at the same concentrations that induced cell cycle arrest, suggesting a potential pharmacodynamic marker for clinical use. In vivo, R547 showed antitumor activity in all of the models tested to date, including six human tumor xenografts and an orthotopic syngeneic rat model. R547 was efficacious with daily oral dosing as well as with once weekly i.v. dosing in established human tumor models and at the targeted efficacious exposures inhibited phosphorylation of the retinoblastoma protein in the tumors. The selective kinase inhibition profile and the preclinical antitumor activity of R547 suggest that it may be promising for development for use in the treatment of solid tumors. R547 is currently being evaluated in phase I clinical trials.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Quinases Ciclina-Dependentes/metabolismo , Feminino , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Genes MDR/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Ratos , Ratos Endogâmicos F344 , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Using the HIV-1 protease binding mode of MK-8718 and PL-100 as inspiration, a novel aspartate binding bicyclic piperazine sulfonamide core was designed and synthesized. The resulting HIV-1 protease inhibitor containing this core showed an 60-fold increase in enzyme binding affinity and a 10-fold increase in antiviral activity relative to MK-8718.
RESUMO
The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K(i) > 10 microM). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K(i) = 0.001, 0.003, and 0.001 microM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC(50) = 0.08 microM). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/farmacocinética , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Fenômenos Químicos , Físico-Química , Quinase 2 Dependente de Ciclina/química , Desenho de Fármacos , Feminino , Humanos , Indicadores e Reagentes , Cinética , Camundongos , Camundongos Nus , Modelos Moleculares , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The development of small-molecule MDM2 inhibitors to restore dysfunctional p53 activities represents a novel approach for cancer treatment. In a previous communication, the efforts leading to the identification of a non-imidazoline MDM2 inhibitor, RG7388, was disclosed and revealed the desirable in vitro and in vivo pharmacological properties that this class of pyrrolidine-based inhibitors possesses. Given this richness and the critical need for a wide variety of chemical structures to ensure success in the clinic, research was expanded to evaluate additional derivatives. Here we report two new potent, selective, and orally active p53-MDM2 antagonists, RO5353 and RO2468, as follow-ups with promising potential for clinical development.
RESUMO
Restoration of p53 activity by inhibition of the p53-MDM2 interaction has been considered an attractive approach for cancer treatment. However, the hydrophobic protein-protein interaction surface represents a significant challenge for the development of small-molecule inhibitors with desirable pharmacological profiles. RG7112 was the first small-molecule p53-MDM2 inhibitor in clinical development. Here, we report the discovery and characterization of a second generation clinical MDM2 inhibitor, RG7388, with superior potency and selectivity.
Assuntos
Antineoplásicos/síntese química , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirrolidinas/síntese química , Proteína Supressora de Tumor p53/antagonistas & inibidores , para-Aminobenzoatos/síntese química , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Descoberta de Drogas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirrolidinas/farmacologia , Pirrolidinas/uso terapêutico , para-Aminobenzoatos/farmacologia , para-Aminobenzoatos/uso terapêuticoRESUMO
Two libraries of hMC4R agonists, X-Y-DPhe(7)-Arg(8)-2-Nal(9)-Z-NH(2) and X-Y-DPhe(7)-Arg(8)-Trp(9)-Z-NH(2), totaling 185 peptides were prepared using Irori radiofrequency tagging technology and Argonaut Quest 210 Synthesizer, where X stands for N-caps, Y for His(6) surrogates and Z for Gly(10) surrogates. As a result of this study, His-modified pentapeptides with Trp were found to be more hMC4R potent than the corresponding 2-Nal analogs, novel N-caps and Gly surrogates were identified and 19 new peptides which are potent hMC4R agonists (EC(50) 1-15nM) and selective against hMC1R were discovered.
Assuntos
Oligopeptídeos/síntese química , Biblioteca de Peptídeos , Receptor Tipo 4 de Melanocortina/agonistas , Sequência de Aminoácidos , Glicina , Humanos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
Linear pentapeptides (Penta-cis-Apc-DPhe-Arg-Trp-Gly-NH2) containing 1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc) and substituted Apc are potent hMC4R agonists and they are inactive or weakly active in hMC1R, hMC3R, and hMC5R agonist assays. This study, together with our earlier report on 5-BrAtc, demonstrated the importance of replacing His6 with phenyl-containing rigid templates in achieving good hMC4R agonist potency and selectivity against hMC1R in linear pentapeptides.
Assuntos
Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Cicloexanos/química , Cicloexanos/farmacologia , Receptor Tipo 1 de Melanocortina/agonistas , Receptor Tipo 4 de Melanocortina/agonistas , Sequência de Aminoácidos , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structures for a series of potent agonists for the human melanocortin-4 receptor (hMC4R), based on the cyclic peptide MT-II [Ac-Nle-cyclo-(Asp-Lys) (Asp-His-(D)Phe-Arg-Trp-Lys)-NH2]. Members of this series were designed to improve selectivity for MC4R versus the other melanocortin receptors, and to reduce the flexibility of the side chains. The most selective and rigid analog [penta-cyclo(D-K)-Asp-Apc-(D)Phe-Arg-(2S,3S)-beta-methylTrp-Lys-NH2] was found to be a full agonist of hMC4R with an EC50 of 11nM against hMC4R, and to exhibit 65-fold selectivity against hMC1R. This compound represents the most constrained hMC4R peptide agonist described to date. A beta-turn structure was conserved among all of the cyclic peptides studied. The rigidity of the analogs allowed an exceptionally well-defined pharmacophore model to be derived. This model was used to perform a virtual screen using a library of 1000 drug-like compounds, to which a small set of known potent ligands had been intentionally added. The utility of the model was validated by its ability to identify the known ligands from among this large library.
Assuntos
Modelos Moleculares , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Receptor Tipo 4 de Melanocortina/agonistas , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Ligação Proteica , Soluções , Relação Estrutura-AtividadeRESUMO
Systematic substitution of His(6) residue using non-selective hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)) as the template led to the identification of Bu-Atc(6)(2-aminotetraline-2-carboxylic acid)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which showed moderate selectivity towards hMC4R over hMC1R. Further SAR studies resulted in the discovery of Penta-5-BrAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) and Penta-5-Me(2)NAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which are potent hMC4R agonists and are inactive in hMC1R, hMC3R and hMC5R agonist assays.
Assuntos
Oligopeptídeos/síntese química , Receptores da Corticotropina/agonistas , Sequência de Aminoácidos , Substituição de Aminoácidos , AMP Cíclico/biossíntese , Histidina , Humanos , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Ligação Proteica , Receptor Tipo 4 de Melanocortina , Receptores da Corticotropina/metabolismo , Receptores de Melanocortina , Relação Estrutura-AtividadeRESUMO
A series of MT-II related cyclic peptides, based on potent but non-selective hMC4R agonist (Penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2)) was prepared in which His(6) residue was systematically substituted. Two of the most interesting peptides identified in this study are Penta-c[Asp-5-ClAtc-DPhe-Arg-Trp-Lys]-NH(2) and Penta-c[Asp-5-ClAtc-DPhe-Cit-Trp-Lys]-NH(2) which are potent hMC4R agonists and are either inactive or weak partial agonists (not tested for their antagonist activities) in hMC1R, hMC3R and hMC5R agonist assays.
Assuntos
Histidina/química , Receptores da Corticotropina/agonistas , Substituição de Aminoácidos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Receptor Tipo 4 de Melanocortina , Receptores de Melanocortina , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
A series of pentapeptides, based on hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)), was prepared in which either DPhe(7) or Trp(9) residue was systematically substituted. A number of interesting DPhe surrogates (D-Thi, D-3-CF(3)Phe, D-2-Nal and D-3,4-diClPhe) as well as Trp surrogates (2-Nal and Bta) were identified in this study.