RESUMO
High-throughput screening (HTS) of ~50,000 chemical compounds against phosphorylated and unphosphorylated c-Met, a tyrosine kinase receptor for hepatocyte growth factor (HGF), was carried out in order to compare hit rates, hit potencies and also to explore scaffolds that might serve as potential leads targeting only the unphosphorylated form of the enzyme. The hit rate and potency for the confirmed hit molecules were higher for the unphosphoryalted form of c-Met. While the target of small molecule inhibitor discovery efforts has traditionally been the phosphorylated form, there are now examples of small molecules that target unphosphorylated kinases. Screening for inhibitors of unphosphorylated kinases may represent a complementary approach for prioritizing chemical scaffolds for hit-to-lead follow ups.
RESUMO
PURPOSE: Deregulation of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is a hallmark for the Philadelphia chromosome-negative myeloproliferative diseases polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We tested the efficacy of a selective JAK1/2 inhibitor in cellular and in vivo models of JAK2-driven malignancy. EXPERIMENTAL DESIGN: A novel inhibitor of JAK1/2 was characterized using kinase assays. Cellular effects of this compound were measured in cell lines bearing the JAK2V617F or JAK1V658F mutation, and its antiproliferative activity against primary polycythemiavera patient cells was determined using clonogenic assays. Antineoplastic activity in vivo was determined using a JAK2V617F-driven xenograft model, and effects of the compound on survival, organomegaly, body weight, and disease-associated inflammatory markers were measured. RESULTS: INCB16562 potently inhibited proliferation of cell lines and primary cells from PV patients carrying the JAK2V617F or JAK1V658F mutation by blocking JAK-STAT signaling and inducing apoptosis. In vivo, INCB16562 reduced malignant cell burden, reversed splenomegaly and normalized splenic architecture, improved body weight gains, and extended survival in a model of JAK2V617F-driven hematologic malignancy. Moreover, these mice suffered from markedly elevated levels of inflammatory cytokines, similar to advanced myeloproliferative disease patients, which was reversed upon treatment. CONCLUSIONS: These data showed that administration of the dual JAK1/2 inhibitor INCB16562 reduces malignant cell burden, normalizes spleen size and architecture, suppresses inflammatory cytokines, improves weight gain, and extends survival in a rodent model of JAK2V617F-driven hematologic malignancy. Thus, selective inhibitors of JAK1 and JAK2 represent a novel therapy for the patients with myeloproliferative diseases and other neoplasms associated with JAK dysregulation.
Assuntos
Inibidores Enzimáticos/farmacologia , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Mutação , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Concentração Inibidora 50 , Cinética , Camundongos , Transplante de Neoplasias , Policitemia Vera/tratamento farmacológicoRESUMO
After finishing the primary high-throughput screening, the screening team is often faced with thousands of hits to be evaluated further. Effective filtering of these hits is crucial in identifying leads. Mode of inhibition (MOI) study is extremely useful in validating whether the observed compound activity is specific to the biological target. In this article, the authors describe a high-throughput MOI determination method for evaluating thousands of compounds using an existing screening infrastructure. Based on enzyme or receptor kinetics theory, the authors developed the method by measuring the ratio of IC(50) or percent inhibition at 2 carefully chosen substrate or ligand concentrations to define an inhibitor as competitive, uncompetitive, or noncompetitive. This not only facilitates binning of HTS hits according to their MOI but also greatly expands HTS utility in support of the medicinal chemistry team's lead optimization practice. Three case studies are presented to demonstrate how the method was applied successfully in 3 discovery programs targeting either an enzyme or a G-protein-coupled receptor.
Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Baculoviridae/genética , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Técnicas de Química Combinatória , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/genética , Histidina/química , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Ligação Proteica , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Spodoptera/citologia , Spodoptera/metabolismoRESUMO
Structural analyses of the protein-tyrosine phosphatase 1B (PTP1B) active site and inhibitor complexes have aided in optimization of a peptide inhibitor containing the novel (S)-isothiazolidinone (IZD) phosphonate mimetic. Potency and permeability were simultaneously improved by replacing the polar peptidic backbone of the inhibitor with nonpeptidic moieties. The C-terminal primary amide was replaced with a benzimidazole ring, which hydrogen bonds to the carboxylate of Asp(48), and the N terminus of the peptide was replaced with an aryl sulfonamide, which hydrogen bonds to Asp(48) and the backbone NH of Arg(47) via a water molecule. Although both substituents retain the favorable hydrogen bonding network of the peptide scaffold, their aryl rings interact weakly with the protein. The aryl ring of benzimidazole is partially solvent exposed and only participates in van der Waals interactions with Phe(182) of the flap. The aryl ring of aryl sulfonamide adopts an unexpected conformation and only participates in intramolecular pi-stacking interactions with the benzimidazole ring. These results explain the flat SAR for substitutions on both rings and the reason why unsubstituted moieties were selected as candidates. Finally, substituents ortho to the IZD heterocycle on the aryl ring of the IZD-phenyl moiety bind in a small narrow site adjacent to the primary phosphate binding pocket. The crystal structure of an o-chloro derivative reveals that chlorine interacts extensively with residues in the small site. The structural insights that have led to the discovery of potent benzimidazole aryl sulfonamide o-substituted derivatives are discussed in detail.
Assuntos
Inibidores Enzimáticos/química , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Tiazóis/química , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Técnicas In Vitro , Modelos Moleculares , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Eletricidade Estática , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacologiaRESUMO
Crystal structures of protein-tyrosine phosphatase 1B in complex with compounds bearing a novel isothiazolidinone (IZD) heterocyclic phosphonate mimetic reveal that the heterocycle is highly complementary to the catalytic pocket of the protein. The heterocycle participates in an extensive network of hydrogen bonds with the backbone of the phosphate-binding loop, Phe(182) of the flap, and the side chain of Arg(221). When substituted with a phenol, the small inhibitor induces the closed conformation of the protein and displaces all waters in the catalytic pocket. Saturated IZD-containing peptides are more potent inhibitors than unsaturated analogs because the IZD heterocycle and phenyl ring directly attached to it bind in a nearly orthogonal orientation with respect to each other, a conformation that is close to the energy minimum of the saturated IZD-phenyl moiety. These results explain why the heterocycle is a potent phosphonate mimetic and an ideal starting point for designing small nonpeptidic inhibitors.
Assuntos
Mimetismo Molecular , Organofosfonatos/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/química , Tiazóis/farmacologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/genética , Humanos , Ligação de Hidrogênio , Hidrólise , Concentração Inibidora 50 , Cinética , Modelos Moleculares , Estrutura Molecular , Conformação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/análise , Proteínas Tirosina Fosfatases/isolamento & purificação , Relação Estrutura-Atividade , Especificidade por Substrato , Água/químicaRESUMO
Structure-based design led to the discovery of novel (S)-isothiazolidinone ((S)-IZD) heterocyclic phosphotyrosine (pTyr) mimetics that when incorporated into dipeptides are exceptionally potent, competitive, and reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B). The crystal structure of PTP1B in complex with our most potent inhibitor 12 revealed that the (S)-IZD heterocycle interacts extensively with the phosphate binding loop precisely as designed in silico. Our data provide strong evidence that the (S)-IZD is the most potent pTyr mimetic reported to date.