RESUMO
We previously disclosed the identification of cereblon modulator 3 (CC-885), with potent antitumor activity mediated through the degradation of GSPT1. We describe herein the structure-activity relationships for analogs of 3 with exploration of the structurally related dioxoisoindoline class. The observed activity of protein degradation could in part be rationalized through docking into the previously disclosed 3-CRBN-GSPT1 cocrystal ternary complex. For SAR that could not be rationalized through the cocrystal complex, we sought to predict SAR through a QSAR model developed in house. Through these analyses, selective protein degradation could be achieved between the two proteins of interest, GSPT1 and Aiolos.
Assuntos
Fator de Transcrição Ikaros/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteólise/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/química , Antineoplásicos/farmacologia , Técnicas de Química Sintética , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Fator de Transcrição Ikaros/química , Fator de Transcrição Ikaros/genética , Simulação de Acoplamento Molecular , Mieloma Múltiplo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Ftalimidas/química , Piperidonas/químicaRESUMO
The drugs lenalidomide and pomalidomide bind to the protein cereblon, directing the CRL4-CRBN E3 ligase toward the transcription factors Ikaros and Aiolos to cause their ubiquitination and degradation. Here we describe CC-220 (compound 6), a cereblon modulator in clinical development for systemic lupus erythematosis and relapsed/refractory multiple myeloma. Compound 6 binds cereblon with a higher affinity than lenalidomide or pomalidomide. Consistent with this, the cellular degradation of Ikaros and Aiolos is more potent and the extent of substrate depletion is greater. The crystal structure of cereblon in complex with DDB1 and compound 6 reveals that the increase in potency correlates with increased contacts between compound 6 and cereblon away from the modeled binding site for Ikaros/Aiolos. These results describe a new cereblon modulator which achieves greater substrate degradation via tighter binding to the cereblon E3 ligase and provides an example of the effect of E3 ligase binding affinity with relevance to other drug discovery efforts in targeted protein degradation.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Fator de Transcrição Ikaros/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Humanos , Lenalidomida/química , Lenalidomida/metabolismo , Morfolinas , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Ftalimidas , Piperidonas , Ligação Proteica , Ubiquitina-Proteína LigasesRESUMO
Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.
Assuntos
Antineoplásicos/farmacologia , Peptídeo Hidrolases/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Compostos de Fenilureia/farmacologia , Talidomida/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Antineoplásicos/química , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Fator de Transcrição Ikaros/química , Fator de Transcrição Ikaros/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/química , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/deficiência , Compostos de Fenilureia/química , Ligação Proteica , Proteólise/efeitos dos fármacos , Especificidade por Substrato , Talidomida/química , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas de Ligação a DNA/metabolismo , Peptídeo Hidrolases/metabolismo , Talidomida/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Animais , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Humanos , Lenalidomida , Camundongos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Talidomida/química , Talidomida/farmacologia , Ubiquitina-Proteína LigasesRESUMO
Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Mieloma Múltiplo/tratamento farmacológico , Peptídeo Hidrolases/imunologia , Isoformas de RNA , Talidomida/uso terapêutico , Ubiquitina-Proteína LigasesRESUMO
Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Escherichia coli ENR yielded four structurally distinct classes of hits. Several members of one of these, the 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles ("thiopyridines"), inhibited both purified ENR (50% inhibitory concentration [IC(50)] = 3 to 25 micro M) and the growth of Staphylococcus aureus and Bacillus subtilis (MIC = 1 to 64 micro g/ml). The effect on cell growth is due in part to inhibition of fatty acid biosynthesis as judged by inhibition of incorporation of [(14)C]acetate into fatty acids and by the increased sensitivity of cells that underexpress an ENR-encoding gene (four- to eightfold MIC shift). Synthesis of a variety of compounds in this chemical series revealed a correlation between IC(50) and MIC, and the results provided initial structure-activity relationships. Preliminary structure-activity relationships, potency on purified ENR, and activity on bacterial cells indicate that members of the thiopyridine chemical series are effective fatty acid biosynthesis inhibitors suitable for further antibacterial development.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Oxirredutases/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Escherichia coli/efeitos dos fármacos , Ácidos Graxos/biossíntese , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Cinética , Óperon Lac/genética , Testes de Sensibilidade Microbiana , Oxirredutases/biossíntese , Oxirredutases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Staphylococcus/metabolismo , Relação Estrutura-AtividadeRESUMO
3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO-8-P) synthase catalyzes the aldol-type condensation of phosphoenolpyruvate and D-arabinose-5-phosphate (A-5-P) to produce KDO-8-P and inorganic phosphate. All KDO-8-P synthases, as exemplified by the enzyme from Escherichia coli, were believed not to require a metal cofactor for catalytic activity. However, recent studies have demonstrated that the KDO-8-P synthase from Aquifex aeolicus is a metalloenzyme. Moreover, sequence alignments and phylogenetic analysis of KDO-8-P synthase protein sequences strongly suggested that there is a whole subfamily of KDO-8-P synthases that are also metalloenzymes. One of these putative metalloenzymes is the ortholog from the human pathogen Helicobacter pylori. In order to test this model, we have cloned the kdsa gene encoding H. pylori KDO-8-P synthase, and overexpressed and purified the protein. This enzyme was found to bind one mol Zn/mol monomer, and the removal of this metal by treatment with 2,6-pyridine dicarboxylic acid abolished enzymatic activity. The Zn(2+) in the enzyme could be quantitatively replaced by Cd(2+), which increased the observed k(cat) by approximately 2-fold, and decreased the apparent K(m)(A-5-P) by approximately 6.5-fold. Furthermore, removal of the Zn(2+) from the enzyme did not greatly perturb its circular dichroism spectra. Thus, the divalent metal most likely serves as cofactor directly involved in catalysis.