RESUMO
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.
Assuntos
Morte Celular Imunogênica , Proteólise , Proteína Serina-Treonina Quinases de Interação com Receptores , Transdução de Sinais , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Humanos , Animais , Camundongos , Proteólise/efeitos dos fármacos , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Morte Celular Imunogênica/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Necroptose/imunologia , Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Camundongos Endogâmicos C57BL , Antineoplásicos/farmacologia , Imunoterapia/métodosRESUMO
B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.
Assuntos
Linfoma Difuso de Grandes Células B , Quinolonas , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-6/química , Fatores de TranscriçãoRESUMO
By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.
Assuntos
Linfoma Difuso de Grandes Células B , Humanos , Cristalografia por Raios X , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Desenho de Fármacos , LigantesRESUMO
The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.
Assuntos
Carcinogênese , Regulação Neoplásica da Expressão Gênica , Animais , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6/metabolismoRESUMO
To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.
Assuntos
Domínio BTB-POZ , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6RESUMO
We describe the optimization of modestly active starting points to potent inhibitors of BCL6 by growing into a subpocket, which was occupied by a network of five stably bound water molecules. Identifying potent inhibitors required not only forming new interactions in the subpocket but also perturbing the water network in a productive, potency-increasing fashion while controlling the physicochemical properties. We achieved this goal in a sequential manner by systematically probing the pocket and the water network, ultimately achieving a 100-fold improvement of activity. The most potent compounds displaced three of the five initial water molecules and formed hydrogen bonds with the remaining two. Compound 25 showed a promising profile for a lead compound with submicromolar inhibition of BCL6 in cells and satisfactory pharmacokinetic (PK) properties. Our work highlights the importance of finding productive ways to perturb existing water networks when growing into solvent-filled protein pockets.
Assuntos
Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Antineoplásicos/química , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Solubilidade , Relação Estrutura-AtividadeRESUMO
Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.
Assuntos
Benzimidazóis/administração & dosagem , Benzimidazóis/química , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Descoberta de Drogas , Microssomos Hepáticos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , Triazóis/química , Triazóis/farmacologia , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Ensaios Clínicos Fase I como Assunto , Feminino , Humanos , Masculino , Metilação , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Distribuição Tecidual , Triazóis/farmacocinéticaRESUMO
Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.
Assuntos
4-Aminopiridina/análogos & derivados , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , 4-Aminopiridina/síntese química , 4-Aminopiridina/química , 4-Aminopiridina/farmacologia , Quinase 1 do Ponto de Checagem/metabolismo , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazinas/síntese química , Pirazinas/química , Relação Estrutura-AtividadeRESUMO
Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells.
Assuntos
Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Compostos de Anilina/farmacologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Células HCT116 , Células HEK293 , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/metabolismo , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacologiaRESUMO
The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.
Assuntos
Compostos de Anilina/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Desenho de Fármacos , Compostos Heterocíclicos com 2 Anéis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Administração Oral , Compostos de Anilina/administração & dosagem , Compostos de Anilina/química , Disponibilidade Biológica , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 2 Anéis/administração & dosagem , Compostos Heterocíclicos com 2 Anéis/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
We report herein a series of Nek2 inhibitors based on an aminopyridine scaffold. These compounds have been designed by combining key elements of two previously discovered chemical series. Structure based design led to aminopyridine (R)-21, a potent and selective inhibitor able to modulate Nek2 activity in cells.
Assuntos
Aminopiridinas/síntese química , Antineoplásicos/síntese química , Benzamidas/síntese química , Mitose , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Alcenos/síntese química , Alcenos/química , Alcenos/farmacologia , Aminopiridinas/química , Aminopiridinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Derivados de Benzeno/síntese química , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Estrutura Molecular , Quinases Relacionadas a NIMA , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química , Tiofenos/farmacologiaRESUMO
Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.
Assuntos
Antineoplásicos/síntese química , Isoquinolinas/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/metabolismo , Pirazinas/síntese química , Piridinas/síntese química , Trifosfato de Adenosina/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Disponibilidade Biológica , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Camundongos , Camundongos Nus , Conformação Molecular , Transplante de Neoplasias , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/química , Pirazinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
We report herein the first systematic exploration of inhibitors of the mitotic kinase Nek2. Starting from HTS hit aminopyrazine 2, compounds with improved activity were identified using structure-based design. Our structural biology investigations reveal two notable observations. First, 2 and related compounds bind to an unusual, inactive conformation of the kinase which to the best of our knowledge has not been reported for other types of kinase inhibitors. Second, a phenylalanine residue at the center of the ATP pocket strongly affects the ability of the inhibitor to bind to the protein. The implications of these observations are discussed, and the work described here defines key features for potent and selective Nek2 inhibition, which will aid the identification of more advanced inhibitors of Nek2.
Assuntos
Modelos Moleculares , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazinas/síntese química , Cristalografia por Raios X , Humanos , Quinases Relacionadas a NIMA , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Pirazinas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Avaliação Pré-Clínica de Medicamentos , Células HT29 , Humanos , Ligação de Hidrogênio , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%.
Assuntos
Antineoplásicos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/síntese química , Resorcinóis/síntese química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Ligação Competitiva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Polarização de Fluorescência , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Isoxazóis/farmacocinética , Isoxazóis/farmacologia , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Resorcinóis/farmacocinética , Resorcinóis/farmacologia , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/farmacologia , Pirazóis/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Feminino , Células HCT116 , Proteínas de Choque Térmico HSP90/química , Células HT29 , Humanos , Isoxazóis/química , Isoxazóis/farmacocinética , Camundongos , Camundongos Nus , NAD(P)H Desidrogenase (Quinona)/metabolismo , Ligação Proteica/efeitos dos fármacos , Pirazóis/química , Pirazóis/farmacocinética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/farmacologia , Pirazóis/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Compostos Heterocíclicos com 2 Anéis/química , Humanos , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Pirazóis/química , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties. Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90.