RESUMO
BACKGROUND: Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. OBJECTIVES: The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. METHODS: Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. RESULTS: We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. CONCLUSION: Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.
Assuntos
Receptores Androgênicos , Neoplasias de Mama Triplo Negativas , Masculino , Humanos , Receptores Androgênicos/metabolismo , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Ligação Proteica , RNA/metabolismoRESUMO
The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.
Assuntos
Proteólise , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Concentração Inibidora 50 , Cinética , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Ubiquitinação/efeitos dos fármacosRESUMO
A co-crystal structure of amide-containing compound (4) in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein and molecular modeling were utilized to design and discover a potent novel cyanoguanidine-containing inhibitor bearing a sulfone moiety (5, Nampt Biochemical IC50=2.5nM, A2780 cell proliferation IC50=9.7nM). Further SAR exploration identified several additional cyanoguanidine-containing compounds with high potency and good microsomal stability. Among these, compound 15 was selected for in vivo profiling and demonstrated good oral exposure in mice. It also exhibited excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model. The co-crystal structure of this compound in complex with the NAMPT protein was also determined.
Assuntos
Antineoplásicos/farmacologia , Citocinas/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Feminino , Guanidinas/administração & dosagem , Guanidinas/química , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Nicotinamida Fosforribosiltransferase/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained.
Assuntos
Amidas/química , Ácidos Carboxílicos/química , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/química , Niacinamida/análogos & derivados , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Pirazóis/química , Piridinas/química , Sulfonas/química , Amidas/síntese química , Amidas/farmacocinética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Citocinas/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Feminino , Meia-Vida , Haplorrinos , Humanos , Camundongos , Camundongos Nus , NAD/metabolismo , Niacinamida/sangue , Niacinamida/química , Niacinamida/farmacocinética , Nicotinamida Fosforribosiltransferase/metabolismo , Estrutura Terciária de Proteína , Pirazóis/sangue , Pirazóis/farmacocinética , Ratos , Retina/efeitos dos fármacos , Retina/metabolismo , Relação Estrutura-Atividade , Sulfonas/sangue , Sulfonas/farmacocinética , Transplante HeterólogoRESUMO
Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole moiety (7, Nampt BC IC50 = 9.0 nM, A2780 cell proliferation IC50 = 10 nM). The Nampt-7 cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (58) was a potent inhibitor of multiple cancer cell lines (IC50 <10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust efficacy in a U251 mouse xenograft model.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/farmacocinética , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound 5, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of 5, SAR exploration revealed that the corresponding urea compound 7 exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound 50 as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC50 = 0.007 µM; A2780 IC50 = 0.032 µM). Compound 50 also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17).
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Ureia/química , Ureia/farmacologia , Humanos , Concentração Inibidora 50 , Nicotinamida Fosforribosiltransferase/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
PURPOSE: Satraplatin is an orally available platinum analog. The purpose of this study was to better characterize satraplatin's preclinical antitumor efficacy in a variety of sensitive and resistant human tumor cell lines and in a prostate cancer xenograft model and to evaluate the effect of satraplatin on PSA expression and/or secretion in a prostate cancer cell line. METHODS: Satraplatin and its primary metabolite JM-118 were preclinically tested for their cytotoxic activity in a range of cancer cells including: human prostate, those forming the NCI drug screening panel, and those resistant to anti-cancer drugs. Also, the antiproliferative efficacy of satraplatin was tested in vivo in a human prostate cancer model. The effect of satraplatin and JM-118 on PSA transcription was measured by quantitative real time PCR. RESULTS: Satraplatin and JM-118 inhibited in vitro and in vivo the growth of prostate cancer cells in a dose-dependent fashion. The IC50 cytotoxicity values for satraplatin ranged from 1 to 3 microM for androgen-insensitive cells and was 11 microM for the androgen-sensitive cell line. Interestingly, JM-118 was up to 16-fold more potent than satraplatin. Oral administration of satraplatin to nude mouse PC-3 xenograft models inhibited the growth of these human tumors. Satraplatin had no direct effect on PSA transcription and the observed decrease in secreted PSA correlated with a decrease in cell number. When evaluated in the NCI drug-screening panel, satraplatin was most active in leukemia and small cell lung cancer cell lines. Both satraplatin and JM-118 were tested on cells resistant to chemotherapeutic agents. Satraplatin and JM-118 were equally active in the cisplatin-resistant A129cp80 ovarian carcinoma cell line, with activity comparable to that observed in the parent line. Neither expression of MDR1, BCRP, MRP1, nor altered tubulin or topoisomerase I were found to mediate resistance to satraplatin or JM-118. Although these resistance mechanisms contribute to drug resistance for a number of chemotherapeutics, they do not appear to play a role in satraplatin resistance. CONCLUSIONS: These results demonstrate that satraplatin and JM-118 have preclinical antitumor activity in human prostate cancer and other tumor types as well, including several cell lines displaying drug resistance to cisplatin, docetaxel and mitoxantrone. In addition, the results suggest that PSA should be further evaluated as a relevant marker of clinical response in patients with prostate cancer treated with satraplatin.
Assuntos
Antineoplásicos/farmacologia , Compostos Organoplatínicos/farmacologia , Administração Oral , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Organic small molecules generally act by perturbing the function of one or more cellular target proteins, the identification of which is essential to an understanding of the molecular basis of drug action. Here we describe the application of methotrexate-linked small molecule ligands to a mammalian three-hybrid interaction trap for proteome-wide identification of small molecule targets, quantification of the targeting potency of unmodified small molecules for such targets in intact cells, and screening for inhibitors of small molecule-protein interactions. During the course of this study we also identified the pyrido[2,3-d]pyrimidine PD173955, a known SRC kinase inhibitor, as a potent inhibitor of several ephrin receptor tyrosine kinases. This finding could perhaps be exploited in the design of inhibitors for this kinase subfamily, members of which have been implicated in the pathogenesis of various diseases, including cancer.
Assuntos
Proteínas/química , Proteoma , Western Blotting , Linhagem Celular , DNA Complementar , Citometria de Fluxo , Humanos , Cinética , Piridonas/química , Piridonas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologiaRESUMO
The identification of molecular determinants of tumor cell survival is an important objective in cancer research. Here, we describe a small-molecule kinase inhibitor (RGB-286147), which, besides inhibiting tumor cell cycle progression, exhibits potent cytotoxic activity toward noncycling tumor cells, but not nontransformed quiescent fibroblasts. Extensive yeast three-hybrid (Y3H)-based proteome/kinome scanning with chemical dimerizers revealed CDK1/2/3/5/7/9 and the less well-characterized CDK-related kinases (CRKs) p42/CCRK, PCTK1/3, and PFTK1 as its predominant targets. Thus, RGB-286147 is a proteome-wide CDK/CRK-specific kinase inhibitor whose further study could yield new insight into molecular determinants of tumor cell survival. Our results also suggest that the [1, 3, 6]-tri-substituted-pyrazolo[3,4-d]-pyrimidine-4-one kinase inhibitor scaffold is a promising template for the rational design of kinase inhibitors with potential applications to disease indications other than cancer, such as neurodegeneration, cardiac hypertrophic growth, and AIDS.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Proteoma/efeitos dos fármacos , Pirimidinas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/classificação , Pirimidinas/classificaçãoRESUMO
The oncoprotein hdm2 ubiquitinates p53, resulting in the rapid degradation of p53 through the ubiquitin (Ub)-proteasome pathway. Hdm2-mediated destabilization and inactivation of p53 are thought to play a critical role in a number of human cancers. We have used an in vitro enzyme assay, monitoring hdm2-catalyzed Ub transfer from preconjugated Ub-Ubc4 to p53, to identify small molecule inhibitors of this enzyme. Three chemically distinct types of inhibitors were identified this way, each with potency in the micromolar range. All three types of compounds display selective inhibition of hdm2 E3 ligase activity, with little or no effect on other Ub-using enzymes. Most strikingly, these compounds do not inhibit the autoubiquitination activity of hdm2. Steady-state analysis reveals that all three classes behave as simple reversible inhibitors of the enzyme and that they are noncompetitive with respect to both substrates, Ub-Ubc4 and p53. Studies of the effects of combinations of two inhibitory molecules on hdm2 activity indicate that the three types of compounds bind in a mutually exclusive fashion, suggesting a common binding site on hdm2 for all of these inhibitors. These compounds establish the feasibility of selectively blocking hdm2-mediated ubiquitination of p53 by small molecule inhibitors. Selective inhibitors of hdm2 E3 ligase activity could provide a novel mechanism for the development of new chemotherapeutics for the treatment of human cancers.