RESUMO
Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult.
Assuntos
Trifosfato de Adenosina/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Células Hep G2 , Humanos , Cinética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Piridazinas/química , Piridazinas/metabolismo , Piridazinas/farmacologia , Homologia de Sequência de Aminoácidos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
Compound 1 was identified as a HCV replication inhibitor from screening/early SAR triage. Potency improvement was achieved via modulation of substituent on the 5-azo linkage. Due to potential toxicological concern, the 5-azo linkage was replaced with 5-alkenyl or 5-alkynyl moiety. Analogs containing the 5-alkynyl linkage were found to be potent inhibitors of HCV replication. Further evaluation identified compounds 53 and 63 with good overall profile, in terms of replicon potency, selectivity and in vivo characteristics. Initial target engagement studies suggest that these novel carbanucleoside-like derivatives may inhibit the HCV replication complex (replicase).
Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Pirimidinas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Introduction of nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzofuran inhibitor 2, resulted in the discovery of the more potent pyridofuran analogue 5. Subsequent introduction of small alkyl and alkoxy ligands into the pyridine ring resulted in further improvements in replicon potency. Replacement of the 4-chloro moiety on the pyrimidine core with a methyl group, and concomitant monoalkylation of the C-2 amino moiety resulted in the identification of several inhibitors with desirable characteristics. Inhibitor 41, from the monosubstituted pyridofuran and inhibitor 50 from the disubstituted series displayed excellent potency, selectivity (GAPDH/MTS CC(50)) and PK parameters in all species studied, while the selectivity in the thymidine incorporation assay (DNA·CC(50)) was low.
Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Furanos/química , Hepacivirus/enzimologia , Nucleosídeos de Pirimidina/química , Pirimidinas/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/farmacocinética , Benzofuranos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Furanos/síntese química , Furanos/farmacocinética , Meia-Vida , Fígado/metabolismo , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/farmacocinética , Pirimidinas/síntese química , Pirimidinas/farmacocinética , RNA Polimerase Dependente de RNA/metabolismo , Ratos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
The installation of geminal substitution at the C5' position of the carbosugar in our pyrimidine-derived hepatitis C inhibitor series is reported. SAR studies around the C5' position led to the installation of the dimethyl group as the optimal functionality. An improved route was subsequently designed to access these substitutions. Expanded SAR at the C2 amino position led to the utilization of C2 ethers. These compounds exhibited good potency, high selectivity, and excellent plasma exposure and bioavailability in rodent as well as in higher species.
Assuntos
Antivirais/síntese química , Carboidratos/química , Pirimidinas/química , Animais , Antivirais/química , Antivirais/farmacocinética , Disponibilidade Biológica , Cães , Meia-Vida , Haplorrinos , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R(1), R(2) or R(3) positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC(50)), PK parameters in all species studied, and cross genotype activity.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacocinética , Cães , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Pirimidinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacocinética , Tiazóis/farmacologiaRESUMO
Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A benzothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of compound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ concentration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzothiazole moiety resulted in an efficient direct C-H arylation protocol.
Assuntos
Antivirais/química , Benzotiazóis/química , Hepacivirus/efeitos dos fármacos , Pirimidinas/química , Replicação Viral/efeitos dos fármacos , Animais , Cães , Haplorrinos , Hepacivirus/fisiologia , Metilação , Roedores , Especificidade da EspécieRESUMO
Forodesine HCl is a potent inhibitor of the enzyme purine nucleoside phosphorylase (PNP) and is currently in clinical trials for the treatment of leukemia and lymphoma. Animal models indicated that forodesine HCl would have low oral bioavailability in humans and it was initially developed as an intravenous formulation. We were interested in identifying analogs of forodesine HCl with improved oral bioavailability. The 2'-deoxy analog (BCX-3040) was synthesized and its pharmacokinetic and pharmacodynamic properties compared with forodesine HCl.
Assuntos
Inibidores Enzimáticos/farmacocinética , Hexosaminas/farmacocinética , Leucemia/tratamento farmacológico , Linfoma/tratamento farmacológico , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Administração Oral , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , Hexosaminas/administração & dosagem , Hexosaminas/síntese química , Injeções Intravenosas , Leucemia/enzimologia , Linfoma/enzimologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
A series of naltrexone-derived pyridomorphinans possessing various substituents at the 5'-position on the pyridine ring were synthesized and evaluated for opioid receptor binding in rodent brain membranes and functional activity in smooth muscle preparations. While the introduction of aromatic 1-pyrrolyl group (6h) improved the delta affinity and delta antagonist potency of the parent compound (3), the introduction of guanidine group (6i) transformed it to a kappa selective ligand in opioid receptor binding and [35S]GTP-gamma-S functional assays.