RESUMO
Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.
Assuntos
Reparo do DNA por Junção de Extremidades , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Ligantes , DNA/metabolismo , DNA Polimerase tetaRESUMO
Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.
Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Amidas/química , Amidas/metabolismo , Animais , Células CACO-2 , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais/efeitos dos fármacos , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Recombinação Homóloga/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Organoides/efeitos dos fármacos , Neoplasias Ovarianas/genética , Ratos , Mutações Sintéticas Letais/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , DNA Polimerase tetaRESUMO
KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for â¼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.This article is highlighted in the In This Issue feature, p. 995.
Assuntos
Neoplasias Colorretais/genética , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Inibidores de MTOR/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Animais , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
A novel series of EP4 agonists and antagonists have been identified, and then used to validate their potential in the treatment of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical models of pain, ultimately leading to the identification of the EP4 partial agonist GSK726701A.
Assuntos
Anti-Inflamatórios/química , Isoindóis/química , Receptores de Prostaglandina E Subtipo EP4/agonistas , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/uso terapêutico , Células Sanguíneas/citologia , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , Dinoprostona/química , Dinoprostona/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Concentração Inibidora 50 , Isoindóis/farmacocinética , Isoindóis/uso terapêutico , Lipopolissacarídeos/farmacologia , Dor/tratamento farmacológico , Dor/patologia , Dor/veterinária , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The discovery and optimisation of novel, potent and selective small molecule inhibitors of the α-isoform of type III phosphatidylinositol-4-kinase (PI4Kα) are described. Lead compounds show cellular activity consistent with their PI4Kα potency inhibiting the accumulation of IP1 after PDGF stimulation and reducing cellular PIP, PIP2 and PIP3 levels. Hence, these compounds are useful in vitro tools to delineate the complex biological pathways involved in signalling through PI4Kα.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Humanos , Fosfatos de Inositol/metabolismo , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Fator de Crescimento Derivado de Plaquetas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
Two series of inhibitors of type III phosphatidylinositol-4-kinase were identified by high throughput screening and optimised to derive probe compounds that independently and selectively inhibit the α- and the ß-isoforms with no significant activity towards related kinases in the pathway. In a cellular environment, inhibition of the α- but not the ß-subtype led to a reduction in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate concentration, causing inhibition of inositol-1-phosphate formation and inhibition of proliferation in a panel of cancer cell lines.