RESUMO
Utilizing the already described 3,4-bi-aryl pyridine series as a starting point, incorporation of a second ring system with a hydrogen bond donor and additional hydrophobic contacts yielded the azaindole series which exhibited potent, picomolar RSK2 inhibition and the most potent in vitro target modulation seen thus far for a RSK inhibitor. In the context of the more potent core, several changes at the phenol moiety were assessed to potentially find a tool molecule appropriate for in vivo evaluation.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Animais , Cromatografia Líquida , Desenho de Fármacos , Humanos , Espectrometria de Massas , Fenóis/farmacologia , Inibidores de Proteínas Quinases/química , Espectroscopia de Prótons por Ressonância Magnética , Relação Estrutura-AtividadeRESUMO
CSN5 is the zinc metalloprotease subunit of the COP9 signalosome (CSN), which is an important regulator of cullin-RING E3 ubiquitin ligases (CRLs). CSN5 is responsible for the cleavage of NEDD8 from CRLs, and blocking deconjugation of NEDD8 traps the CRLs in a hyperactive state, thereby leading to auto-ubiquitination and ultimately degradation of the substrate recognition subunits. Herein, we describe the discovery of azaindoles as a new class of CSN5 inhibitors, which interact with the active-site zinc ion of CSN5 through an unprecedented binding mode. The best compounds inhibited CSN5 with nanomolar potency, led to degradation of the substrate recognition subunit Skp2 in cells, and reduced the viability of HCT116 cells.
Assuntos
Complexo do Signalossomo COP9/antagonistas & inibidores , Indóis/metabolismo , Zinco/metabolismo , Sítios de Ligação , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Domínio Catalítico , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Células HCT116 , Humanos , Indóis/química , Indóis/farmacologia , Simulação de Acoplamento Molecular , Proteína NEDD8/química , Proteína NEDD8/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Quinases Associadas a Fase S/química , Proteínas Quinases Associadas a Fase S/metabolismo , Zinco/químicaRESUMO
2-Amino-7-substituted benzoxazole analogs were identified by HTS as inhibitors of RSK2. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of improving in vitro and target modulation potency and physicochemical properties.
Assuntos
Benzoxazóis/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Benzoxazóis/síntese química , Benzoxazóis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Relação Estrutura-AtividadeRESUMO
Drug development is hampered by poor target selection. Phenotypic screens using neurons differentiated from patient stem cells offer the possibility to validate known and discover novel disease targets in an unbiased fashion. To identify targets for managing hyperexcitability, a pathological feature of amyotrophic lateral sclerosis (ALS), we design a multi-step screening funnel using patient-derived motor neurons. High-content live cell imaging is used to evaluate neuronal excitability, and from a screen against a chemogenomic library of 2,899 target-annotated compounds, 67 reduce the hyperexcitability of ALS motor neurons carrying the SOD1(A4V) mutation, without cytotoxicity. Bioinformatic deconvolution identifies 13 targets that modulate motor neuron excitability, including two known ALS excitability modulators, AMPA receptors and Kv7.2/3 ion channels, constituting target validation. We also identify D2 dopamine receptors as modulators of ALS motor neuron excitability. This screen demonstrates the power of human disease cell-based phenotypic screens for identifying clinically relevant targets for neurological disorders.
Assuntos
Esclerose Lateral Amiotrófica/genética , Diferenciação Celular , Humanos , FenótipoRESUMO
The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin-proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Complexo do Signalossomo COP9/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Pirazóis/farmacologia , Ubiquitina-Proteína Ligases/genética , Animais , Antineoplásicos/síntese química , Azepinas/síntese química , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Feminino , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise/efeitos dos fármacos , Pirazóis/síntese química , Células THP-1 , Carga Tumoral/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
While the p90 ribosomal S6 kinase (RSK) family has been implicated in multiple tumor cell functions, the full understanding of this kinase family has been restricted by the lack of highly selective inhibitors. A bis-phenol pyrazole was identified from high-throughput screening as an inhibitor of the N-terminal kinase of RSK2. Structure-based drug design using crystallography, conformational analysis, and scaffold morphing resulted in highly optimized difluorophenol pyridine inhibitors of the RSK kinase family as demonstrated cellularly by the inhibition of YB1 phosphorylation. These compounds provide for the first time in vitro tools with an improved selectivity and potency profile to examine the importance of RSK signaling in cancer cells and to fully evaluate RSK as a therapeutic target.
Assuntos
Pirazóis/química , Piridinas/química , Pirimidinas/química , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Linhagem Celular , Cristalografia por Raios X , Humanos , Masculino , Camundongos , Modelos Moleculares , Fosforilação , Conformação Proteica , Pirazóis/síntese química , Pirazóis/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais , Relação Estrutura-Atividade , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
UNLABELLED: The p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases is expressed in a variety of cancers and its substrate phosphorylation has been implicated in direct regulation of cell survival, proliferation, and cell polarity. This study characterizes and presents the most selective and potent RSK inhibitors known to date, LJH685 and LJI308. Structural analysis confirms binding of LJH685 to the RSK2 N-terminal kinase ATP-binding site and reveals that the inhibitor adopts an unusual nonplanar conformation that explains its excellent selectivity for RSK family kinases. LJH685 and LJI308 efficiently inhibit RSK activity in vitro and in cells. Furthermore, cellular inhibition of RSK and its phosphorylation of YB1 on Ser102 correlate closely with inhibition of cell growth, but only in an anchorage-independent growth setting, and in a subset of examined cell lines. Thus, RSK inhibition reveals dynamic functional responses among the inhibitor-sensitive cell lines, underscoring the heterogeneous nature of RSK dependence in cancer. IMPLICATIONS: Two novel potent and selective RSK inhibitors will now allow a full assessment of the potential of RSK as a therapeutic target for oncology.