RESUMO
KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Histonas/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Transdução de Sinais , Linhagem Celular TumoralRESUMO
Protein arginine methyltransferase 5 (PRMT5) overexpression in hematologic and solid tumors methylates arginine residues on cellular proteins involved in important cancer functions including cell-cycle regulation, mRNA splicing, cell differentiation, cell signaling, and apoptosis. PRMT5 methyltransferase function has been linked with high rates of tumor cell proliferation and decreased overall survival, and PRMT5 inhibitors are currently being explored as an approach for targeting cancer-specific dependencies due to PRMT5 catalytic function. Here, we describe the discovery of potent and selective S-adenosylmethionine (SAM) competitive PRMT5 inhibitors, with in vitro and in vivo characterization of clinical candidate PF-06939999. Acquired resistance mechanisms were explored through the development of drug resistant cell lines. Our data highlight compound-specific resistance mutations in the PRMT5 enzyme that demonstrate structural constraints in the cofactor binding site that prevent emergence of complete resistance to SAM site inhibitors. PRMT5 inhibition by PF-06939999 treatment reduced proliferation of non-small cell lung cancer (NSCLC) cells, with dose-dependent decreases in symmetric dimethyl arginine (SDMA) levels and changes in alternative splicing of numerous pre-mRNAs. Drug sensitivity to PF-06939999 in NSCLC cells associates with cancer pathways including MYC, cell cycle and spliceosome, and with mutations in splicing factors such as RBM10. Translation of efficacy in mouse tumor xenograft models with splicing mutations provides rationale for therapeutic use of PF-06939999 in the treatment of splicing dysregulated NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Resistência a Medicamentos , Feminino , Humanos , Neoplasias Pulmonares/patologia , CamundongosRESUMO
Therapeutically targeting aberrant intracellular kinase signaling is attractive from a biological perspective but drug development is often hindered by toxicities and inadequate efficacy. Predicting drug behaviors using cellular and animal models is confounded by redundant kinase activities, a lack of unique substrates, and cell-specific signaling networks. Cyclin-dependent kinase (CDK) drugs exemplify this phenomenon because they are reported to target common processes yet have distinct clinical activities. Tumor cell studies of ATP-competitive CDK drugs (dinaciclib, AG-024322, abemaciclib, palbociclib, ribociclib) indicate similar pharmacology while analyses in untransformed cells illuminates significant differences. To resolve this apparent disconnect, drug behaviors are described at the molecular level. Nonkinase binding studies and kinome interaction analysis (recombinant and endogenous kinases) reveal that proteins outside of the CDK family appear to have little role in dinaciclib/palbociclib/ribociclib pharmacology, may contribute for abemaciclib, and confounds AG-024322 analysis. CDK2 and CDK6 cocrystal structures with the drugs identify the molecular interactions responsible for potency and kinase selectivity. Efficient drug binding to the unique hinge architecture of CDKs enables selectivity toward most of the human kinome. Selectivity between CDK family members is achieved through interactions with nonconserved elements of the ATP-binding pocket. Integrating clinical drug exposures into the analysis predicts that both palbociclib and ribociclib are CDK4/6 inhibitors, abemaciclib inhibits CDK4/6/9, and dinaciclib is a broad-spectrum CDK inhibitor (CDK2/3/4/6/9). Understanding the molecular components of potency and selectivity also facilitates rational design of future generations of kinase-directed drugs. Mol Cancer Ther; 15(10); 2273-81. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Interações Medicamentosas , Resistencia a Medicamentos Antineoplásicos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Modelos Moleculares , Conformação Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/química , RatosRESUMO
We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lactamas Macrocíclicas/administração & dosagem , Neoplasias/tratamento farmacológico , Receptores Proteína Tirosina Quinases/genética , Aminopiridinas , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/farmacologia , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Lactamas , Lactamas Macrocíclicas/farmacologia , Camundongos , Mutação , Células NIH 3T3 , Neoplasias/genética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pirazóis , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although crizotinib demonstrates robust efficacy in anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinoma patients, progression during treatment eventually develops. Resistant patient samples revealed a variety of point mutations in the kinase domain of ALK, including the L1196M gatekeeper mutation. In addition, some patients progress due to cancer metastasis in the brain. Using structure-based drug design, lipophilic efficiency, and physical-property-based optimization, highly potent macrocyclic ALK inhibitors were prepared with good absorption, distribution, metabolism, and excretion (ADME), low propensity for p-glycoprotein 1-mediated efflux, and good passive permeability. These structurally unusual macrocyclic inhibitors were potent against wild-type ALK and clinically reported ALK kinase domain mutations. Significant synthetic challenges were overcome, utilizing novel transformations to enable the use of these macrocycles in drug discovery paradigms. This work led to the discovery of 8k (PF-06463922), combining broad-spectrum potency, central nervous system ADME, and a high degree of kinase selectivity.
Assuntos
Antineoplásicos/síntese química , Encéfalo/metabolismo , Lactamas Macrocíclicas/síntese química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Aminopiridinas , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos , Humanos , Lactamas , Lactamas Macrocíclicas/farmacocinética , Lactamas Macrocíclicas/farmacologia , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Mutação , Células NIH 3T3 , Pirazóis , Ratos , Receptores Proteína Tirosina Quinases/genética , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).