RESUMO
BACE-1 (beta-site amyloid precursor protein cleaving enzyme), a prominent target in Alzheimer's disease drug discovery efforts, was surveyed using Tethering technology to discover small molecule fragment ligands that bind to the enzyme active site. Screens of a library of >15000 thiol-containing fragments versus a panel of BACE-1 active site cysteine mutants under redox-controlled conditions revealed several novel amine-containing fragments that could be selectively captured by subsets of the tethering sites. For one such hit class, defined by a central aminobenzylpiperidine (ABP) moiety, X-ray crystal structures of BACE mutant-disulfide conjugates revealed that the fragment bound by engaging both catalytic aspartates with hydrogen bonds. The affinities of ABP fragments were improved by structure-guided chemistry, first for conjugation as thiol-containing fragments and then for stand-alone, noncovalent inhibition of wild-type (WT) BACE-1 activity. Crystallography confirmed that the inhibitors bound in exactly the same mode as the disulfide-conjugated fragments that were originally selected from the screen. The ABP ligands represent a new type of nonpeptidic BACE-1 inhibitor motif that has not been described in the aspartyl protease literature and may serve as a starting point for the development of BACE-1-directed Alzheimer's disease therapeutics.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Biocatálise , Domínio Catalítico , Cisteína , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Mutação , Peptídeos/química , Piperidinas/química , Piperidinas/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Química Farmacêutica/métodos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirazóis/síntese química , Piridinas/síntese química , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Ciclo Celular , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Modelos Químicos , Conformação Molecular , Fenótipo , Pirazóis/química , Pirazóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Relação Estrutura-Atividade , Quinase 1 Polo-LikeRESUMO
Caspase-1 is a key endopeptidase responsible for the post-translational processing of the IL-1beta and IL-18 cytokines and small-molecule inhibitors that modulate the activity of this enzyme are predicted to be important therapeutic treatments for many inflammatory diseases. A fragment-assembly approach, accompanied by structural analysis, was employed to generate caspase-1 inhibitors. With the aid of Tethering with extenders (small molecules that bind to the active-site cysteine and contain a free thiol), two novel fragments that bound to the active site and made a disulfide bond with the extender were identified by mass spectrometry. Direct linking of each fragment to the extender generated submicromolar reversible inhibitors that significantly reduced secretion of IL-1beta but not IL-6 from human peripheral blood mononuclear cells. Thus, Tethering with extenders facilitated rapid identification and synthesis of caspase-1 inhibitors with cell-based activity and subsequent structural analyses provided insights into the enzyme's ability to accommodate different inhibitor-binding modes in the active site.
Assuntos
Inibidores de Caspase , Técnicas de Química Combinatória/métodos , Inibidores de Cisteína Proteinase/química , Sítios de Ligação/efeitos dos fármacos , Caspase 1/química , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Desenho de Fármacos , Humanos , Interleucina-1/antagonistas & inibidores , Interleucina-1/sangue , Interleucina-6/antagonistas & inibidores , Interleucina-6/sangue , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/metabolismo , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , SolubilidadeRESUMO
Disulfide Tethering was applied to the active site of human caspase-1, resulting in the discovery of a novel, tricyclic molecular fragment that selectively binds in S4. This fragment was developed into a class of potent inhibitors of human caspase-1. Several key analogues determined the optimal distance of the tricycle from the catalytic residues, the relative importance of various features of the tricycle, and the importance of the linker.