RESUMO
Inhibitors of the cancer-related cysteine isopeptidase human ubiquitin-specific proteases 7 (USP7) and 47 (USP47) are considered to have potential as cancer therapeutics, owing to their ability to stabilize the tumor suppressor p53 and to decrease DNA polymerase ß (Polß), both of which are potential anticancer effects. A new class of dual small molecule inhibitors of these enzymes has been discovered. Compound 1, a selective inhibitor of USP7 and USP47 with moderate potency, demonstrates inhibition of USP7 in cells and induces elevated p53 and apoptosis in cancer cell lines. Compound 1 has been shown to demonstrate modest activity in human xenograft multiple myeloma and B-cell leukemia in vivo models. This activity may be the result of dual inhibition of USP7 and USP47. To address issues regarding potency and developability, analogues of compound 1 have been synthesized and tested, leading to improvements in potency, solubility, and metabolic reactivity profile. Further optimization is expected to yield preclinical candidates and, ultimately, clinical candidates for the treatment of multiple myeloma, prostate cancer, and other cancers.
RESUMO
The deubiquitylating enzyme USP7 (HAUSP) sits at a critical node regulating the activities of numerous proteins broadly characterized as tumor suppressors, DNA repair proteins, immune responders, viral proteins, and epigenetic modulators. Aberrant USP7 activity may promote oncogenesis and viral disease making it a compelling target for therapeutic intervention. Disclosed drug discovery programs have identified inhibitors of USP7 such as P005091 with cellular proof of concept and anti-proliferative activity in cancer models. Taken together, USP7 inhibitors hold promise as a new strategy for the treatment of disease.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores de Proteases/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Indenos/farmacologia , Pirazinas/farmacologia , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de UbiquitinaRESUMO
Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Musculares/antagonistas & inibidores , Atrofia Muscular/prevenção & controle , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Biocatálise/efeitos dos fármacos , Western Blotting , Linhagem Celular , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Glucocorticoides/farmacologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas , Especificidade por Substrato , Proteínas com Motivo Tripartido , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)-compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain's inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform's broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.
Assuntos
Enzimas Reparadoras do DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Descoberta de Drogas , Humanos , Luminescência , Proteínas do Tecido Nervoso/metabolismo , Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.
Assuntos
Rim/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , Apoptose , Linhagem Celular , Proliferação de Células , Humanos , LigantesRESUMO
Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor kappaB (NF-kappaB) transcription factor activation triggered by proteolysis of its inhibitor IkappaB. IkappaB degradation is dependent upon its phosphorylation by the IkappaB kinase (IKK) complex and subsequent ubiquitination facilitated by beta-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF(V600E) exhibit enhanced beta-Trcp expression, increased IKK activity and a concomitant increase in the rate of IkappaBalpha degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAF(V600E) expression by RNA interference in human melanoma cells leads to decreased IKK activity and beta-Trcp expression, stabilization of IkappaB, inhibition of NF-kappaB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-kappaB activity and to increased survival of melanoma cells.