RESUMO
The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.
Assuntos
Doença de Huntington , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Proteína Huntingtina/metabolismo , Doença de Huntington/tratamento farmacológico , Camundongos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rhoRESUMO
Using an iterative structure-activity relationship driven approach, we identified a CNS-penetrant 5-(trifluoromethyl)-1,2,4-oxadiazole (TFMO, 12) with a pharmacokinetic profile suitable for probing class IIa histone deacetylase (HDAC) inhibition in vivo. Given the lack of understanding of endogenous class IIa HDAC substrates, we developed a surrogate readout to measure compound effects in vivo, by exploiting the >100-fold selectivity compound 12 exhibits over class I/IIb HDACs. We achieved adequate brain exposure with compound 12 in mice to estimate a class I/IIb deacetylation EC50, using class I substrate H4K12 acetylation and global acetylation levels as a pharmacodynamic readout. We observed excellent correlation between the compound 12 in vivo pharmacodynamic response and in vitro class I/IIb cellular activity. Applying the same relationship to class IIa HDAC inhibition, we estimated the compound 12 dose required to inhibit class IIa HDAC activity, for use in preclinical models of Huntington's disease.
RESUMO
We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and â¼150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC50 levels for â¼8â¯h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The resurgence of interest in caspases (Csp) as therapeutic targets for the treatment of neurodegenerative diseases prompted us to examine the suitability of published nonpeptidic Csp-3 and Csp-6 inhibitors for our medicinal chemistry programs. To support this effort, fluorescence-based Csp-2, Csp-3, and Csp-6 enzymatic assays were optimized for robustness against apparent enzyme inhibition caused by redox-cycling or aggregating compounds. The data obtained under these improved conditions challenge the validity of previously published data on Csp-3 and Csp-6 inhibitors for all but one series, namely, the isatins. Furthermore, in this series, it was observed that the nature of the rhodamine-labeled substrate, typically used to measure caspase activity, interfered with the pharmacological sensitivity of the Csp-2 assay. As a result, a liquid chromatography/tandem mass spectrometry-based assay that eliminates label-dependent assay interference was developed for Csp-2 and Csp-3. In these label-free assays, the activity values of the Csp-2 and Csp-3 reference inhibitors were in agreement with those obtained with the fluorogenic substrates. However, isatin 10a was 50-fold less potent in the label-free Csp-2 assay compared with the rhodamine-based fluorescence format, thus proving the need for an orthogonal readout to validate inhibitors in this class of targets highly susceptible to artifactual inhibition.
Assuntos
Inibidores de Caspase/farmacologia , Ensaios Enzimáticos/métodos , Caspase 2/metabolismo , Caspase 3/metabolismo , Caspase 6/metabolismo , Inibidores de Caspase/química , Cromatografia Líquida/métodos , Desenho de Fármacos , Isatina/química , Isatina/farmacologia , Doenças Neurodegenerativas/metabolismo , Oligopeptídeos/farmacologia , Espectrometria de Massas em Tandem/métodosRESUMO
Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and â¼200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD.
Assuntos
Inibidores de Caspase , Inibidores de Cisteína Proteinase/síntese química , Desenho de Fármacos , Sítios de Ligação , Caspase 2/metabolismo , Caspase 3/metabolismo , Domínio Catalítico , Linhagem Celular , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Isoquinolinas/química , Simulação de Dinâmica Molecular , Piperidinas/química , Prolina/química , Especificidade por SubstratoRESUMO
The design and synthesis of a novel series of potent and cell permeable peptidomimetic inhibitors of the human beta-secretase (BACE) are described. These inhibitors feature a hydroxyethyl secondary amine isostere and a novel aromatic ring replacement for the C-terminus. The crystal structure of BACE in complex with this hydroxyethyl secondary amine isostere inhibitor is also presented.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Modelos Moleculares , Peptídeos/química , Ácidos Ftálicos/síntese química , Precursor de Proteína beta-Amiloide/química , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Mimetismo Molecular , Estrutura Molecular , Ácidos Ftálicos/química , Ácidos Ftálicos/farmacologia , EstereoisomerismoRESUMO
5'-Ether derivatives of the potent adenosine agonist N6-cyclopentyladenosine (CPA) were designed as "caged" ligands for the activation of A1-adenosine receptors following in situ photolysis. The synthesis involved a 2',3'-diol protection scheme using the acid labile ethoxymethynyl group. Generation of CPA was demonstrated chromatographically and in a bioassay measuring the inhibition of synaptic potentials in the rat hippocampus.