RESUMO
Despite advances in treatment, a significant proportion of patients with chronic lymphocytic leukemia (CLL) will relapse with drug-resistant disease. The imipridones, ONC-201 and ONC-212, are effective against a range of different cancers, including acute myeloid leukemia (AML) and tumors of the brain, breast, and prostate. These drugs induce cell death through activation of the mitochondrial protease, caseinolytic protease (CIpP), and the unfolded protein response (UPR). Here we demonstrate that the novel imipridone analog, TR-57, has efficacy as a single agent and synergises with venetoclax against CLL cells under in vitro conditions that mimic the tumor microenvironment. Changes in protein expression suggest TR-57 activates the UPR, inhibits the AKT and ERK1/2 pathways and induces pro-apoptotic changes in the expression of proteins of the BCL-2 family. The study suggests that TR-57, as a single agent and in combination with venetoclax, may represent an effective treatment option for CLL.
Assuntos
Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes , Sinergismo Farmacológico , Leucemia Linfocítica Crônica de Células B , Sulfonamidas , Humanos , Sulfonamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified â¼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying â¼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation.
RESUMO
The mitochondrial ClpP protease is responsible for mitochondrial protein quality control through specific degradation of proteins involved in several metabolic processes. ClpP overexpression is also required in many cancer cells to eliminate reactive oxygen species (ROS)-damaged proteins and to sustain oncogenesis. Targeting ClpP to dysregulate its function using small-molecule agonists is a recent strategy in cancer therapy. Here, we synthesized imipridone-derived compounds and related chemicals, which we characterized using biochemical, biophysical, and cellular studies. Using X-ray crystallography, we found that these compounds have enhanced binding affinities due to their greater shape and charge complementarity with the surface hydrophobic pockets of ClpP. N-terminome profiling of cancer cells upon treatment with one of these compounds revealed the global proteomic changes that arise and identified the structural motifs preferred for protein cleavage by compound-activated ClpP. Together, our studies provide the structural and molecular basis by which dysregulated ClpP affects cancer cell viability and proliferation.
Assuntos
Mitocôndrias , Proteômica , Mitocôndrias/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , ProteóliseRESUMO
We recently described the identification of a new class of small-molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small-molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. We selected one compound (TR-107) with excellent potency, specificity, and drug-like properties for further evaluation. TR-107 showed ClpP-dependent growth inhibition in the low nanomolar range that was equipotent to paclitaxel in triple-negative breast cancer (TNBC) cell models. TR-107 also reduced specific mitochondrial proteins, including OXPHOS and TCA cycle components, in a time-, dose-, and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed the inactivation of OXPHOS and increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared with other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. Using human TNBC MDA-MB-231 xenografts, the antitumor response to TR-107 was investigated. Oral administration of TR-107 resulted in a reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.
Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Endopeptidase Clp/química , Endopeptidase Clp/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Peptídeo Hidrolases/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Despite advances in therapy, a significant proportion of patients with chronic lymphocytic leukemia (CLL) relapse with drug resistant disease. Novel treatment approaches are required, particularly for high risk disease. The imipridones represent a new class of cancer therapy that has been investigated in pre-clinical and clinical trials against a range of different cancers. We investigated the effects of the imipridone, ONC-212, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment and a TP53ko CLL cell line (OSU-CLL-TP53ko). ONC-212 induced dose-dependent apoptosis, cell cycle arrest and reduced the migration of CLL cells in vitro, including cells from patients with TP53 lesions and OSU-CLL-TP53ko cells. The effects of ONC-212 were associated with protein changes consistent with activation of the mitochondrial protease, CIpP, and the integrated stress response. We also observed inhibition of pathways downstream of the B-cell receptor (BCR) (AKT and MAPK-ERK1/2) and a pro-apoptotic shift in the balance of proteins of the BCL2 family of proteins (BCL2, MCL1, BCLxL, BAX and NOXA). In conclusion, the study suggests ONC-212 may represent an effective treatment for high risk CLL disease by inhibiting multiple facets of the BCR signaling pathway and the pro-survival effects of the BCL2-family proteins.
RESUMO
ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be â¼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with â¼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a â¼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.
Assuntos
Antineoplásicos/farmacologia , Endopeptidase Clp/antagonistas & inibidores , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia de Afinidade , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Ativação Enzimática , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Imidazóis , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Piridinas , PirimidinasRESUMO
Inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanosine nucleotides, catalyzes the irreversible nicotinamide-adenine dinucleotide dependent oxidation of inosine-5'-monophosphate to xanthosine-5'-monophosphate. Mycophenolate Mofetil (MMF), a prodrug of mycophenolic acid, has clinical utility for the treatment of transplant rejection based on its inhibition of IMPDH. The overall clinical benefit of MMF is limited by what is generally believed to be compound-based, dose-limiting gastrointestinal (GI) toxicity that is related to its specific pharmacokinetic characteristics. Thus, development of an IMPDH inhibitor with a novel structure and a different pharmacokinetic profile may reduce the likelihood of GI toxicity and allow for increased efficacy. This article will detail the discovery and SAR leading to a novel and potent acridone-based IMPDH inhibitor 4m and its efficacy and GI tolerability when administered orally in a rat adjuvant arthritis model.
Assuntos
Acridinas/síntese química , IMP Desidrogenase/antagonistas & inibidores , Piperazinas/síntese química , Acridinas/farmacologia , Acridinas/toxicidade , Administração Oral , Animais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Disponibilidade Biológica , Linhagem Celular , Proliferação de Células , Trato Gastrointestinal/efeitos dos fármacos , Meia-Vida , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Macaca fascicularis , Masculino , Piperazinas/farmacologia , Piperazinas/toxicidade , Ratos , Ratos Endogâmicos Lew , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
LFA-1 (leukocyte function-associated antigen-1), is a member of the beta2-integrin family and is expressed on all leukocytes. This letter describes the discovery and preliminary SAR of spirocyclic hydantoin based LFA-1 antagonists that culminated in the identification of analog 8 as a clinical candidate. We also report the first example of the efficacy of a small molecule LFA-1 antagonist in combination with CTLA-4Ig in an animal model of transplant rejection.
Assuntos
Antígeno-1 Associado à Função Linfocitária/metabolismo , Compostos de Espiro/síntese química , Tiofenos/síntese química , Animais , Adesão Celular/efeitos dos fármacos , Cristalografia por Raios X , Cães , Rejeição de Enxerto/prevenção & controle , Humanos , Antígeno-1 Associado à Função Linfocitária/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Pneumonia/tratamento farmacológico , Pneumonia/imunologia , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/farmacocinética , Tiofenos/farmacologia , Transplante HomólogoRESUMO
LFA-1 (leukocyte function-associated antigen-1), is a member of the beta(2)-integrin family and is expressed on all leukocytes. The LFA-1/ICAM interaction promotes tight adhesion between activated leukocytes and the endothelium, as well as between T cells and antigen-presenting cells. Evidence from both animal models and clinical trials provides support for LFA-1 as a target in several different inflammatory diseases. This paper describes the de novo design, synthesis and in vitro activity of LFA-1 antagonists based on a bicyclic[5.5]hydantoin scaffold.
Assuntos
Hidantoínas/síntese química , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Desenho de Fármacos , Células HeLa , Humanos , Hidantoínas/farmacologia , Inflamação/tratamento farmacológico , Concentração Inibidora 50 , Molécula 1 de Adesão Intercelular/química , Antígeno-1 Associado à Função Linfocitária/química , Conformação Molecular , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
A series of novel anilino 5-azaimidazoquinoxaline analogues possessing potent in vitro activity against p56Lck and T cell proliferation have been discovered. Subsequent SAR studies led to the identification of compound 4 (BMS-279700) as an orally active lead candidate that blocks the production of proinflammatory cytokines (IL-2 and TNFalpha) in vivo. In addition, an expanded set of imidazoquinoxalines provided several descriptive QSAR models highlighting the influence of significant steric and electronic features. The H-bonding (Met319) contribution to observed binding affinities within a tightly congeneric series was found to be significant.
Assuntos
Anti-Inflamatórios/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Quinoxalinas/química , Quinoxalinas/farmacocinética , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Disponibilidade Biológica , Citocinas/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Feminino , Ligação de Hidrogênio , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Pirazinas/química , Pirazinas/farmacologia , Quinoxalinas/farmacologia , Quinases da Família src/antagonistas & inibidoresRESUMO
A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.
Assuntos
Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Indóis/farmacologia , Catálise , Cinética , Relação Estrutura-AtividadeRESUMO
The first reported structure-activity relationships (SARs) about the N-[3-methoxy-4-(5-oxazolyl)phenyl moiety for a series of recently disclosed inosine monophosphate dehydrogenase (IMPDH) inhibitors are described. The syntheses and in vitro inhibitory values for IMPDH II, and T-cell proliferation (for select analogues) are given.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Oxazóis/química , Oxazóis/farmacologia , Animais , Carbamatos/química , Carbamatos/farmacologia , Linhagem Celular , Cricetinae , Cricetulus , Concentração Inibidora 50 , Ativação Linfocitária/efeitos dos fármacos , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacologia , Oxazóis/síntese química , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologiaRESUMO
The development of a series of novel indole-based inhibitors of 5'-inosine monophosphate dehydrogenase (IMPDH) is described. Various hydrogen bond acceptors at C-3 of the indole were explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are outlined.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Indóis/química , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Glucuronídeos/metabolismo , Humanos , Ligação de Hidrogênio , Sistema Imunitário/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Mitógenos/farmacologia , Quinolonas/síntese química , Quinolonas/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Screening of our in-house compound collection led to the discovery of 5-bromo-6-amino-2-isoquinoline 1 as a weak inhibitor of IMPDH. Subsequent optimization of 1 afforded a series of novel 2-isoquinolinoaminooxazole-based inhibitors, represented by 17, with single-digit nanomolar potency against the enzyme.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Oxazóis/síntese química , Oxazóis/farmacologia , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/enzimologia , Humanos , NAD/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of novel quinolone-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Quinolonas/síntese química , Quinolonas/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fenômenos Químicos , Físico-Química , Cristalografia por Raios X , Humanos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologiaRESUMO
The synthesis and the structure-activity relationships (SAR) of analogues derived from the introduction of basic residues on ring D of quinolone-based inhibitors of IMPDH are described. This led to the identification of compound 27 as a potent inhibitor of IMPDH with significantly improved aqueous solubility over the lead compound 1.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Quinolonas/síntese química , Quinolonas/farmacologia , Humanos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Humanos , Técnicas In Vitro , Indicadores e Reagentes , Ligantes , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of anilino(imidazoquinoxaline) analogues bearing solubilizing side chains at the 6- and 7-positions of the fused phenyl ring has been prepared and evaluated for inhibition against Lck enzyme and of T-cell proliferation. Significant improvement of the cellular activity was achieved over the initial lead, compound 2.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Quinoxalinas/síntese química , Quinoxalinas/farmacologia , Divisão Celular/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacosRESUMO
A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.