RESUMO
C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Polipose Intestinal/tratamento farmacológico , Triptofano/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Animais , Antineoplásicos/química , Domínio Catalítico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Hidroxilaminas/química , Hidroxilaminas/farmacologia , Polipose Intestinal/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The design and development of irreversible kinase inhibitors is an expanding frontier of kinase drug discovery. The current approach to develop these inhibitors utilizes ATP-competitive inhibitor scaffolds to target non-catalytic cysteines in the kinase ATP-binding site. However, this approach is limited as not all kinases have a cysteine in the ATP-binding site that can be targeted. In this work, we report a complementary approach to developing irreversible kinase inhibitors that utilizes the substrate-binding site. Using the catalytic subunit of cAMP-dependent protein kinase (PKACα) as a model system, we have designed and synthesized an irreversible inhibitor based on the substrate-competitive inhibitor scaffold PKI(14-22) that covalently modifies non-catalytic Cys199 in the PKACα substrate-binding site. The new compound inhibits PKACα (IC50 = 11.8 ± 1.1 nM), is â¼100-fold selective for PKACα in a kinase panel, and covalently labels the kinase as demonstrated by fluorescence, mass spectrometry, and kinetics experiments. This study demonstrates the feasibility of utilizing this new approach to develop irreversible inhibitors for any of the eighty-nine kinases that possess a similar non-catalytic cysteine in their substrate-binding sites.
Assuntos
Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/química , Desenho de Fármacos , Corantes Fluorescentes/química , Cetonas/síntese química , Cetonas/farmacologia , Sequência de Aminoácidos , Técnicas de Química Sintética , Cetonas/química , Modelos MolecularesRESUMO
C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24µM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18µM) and 3-chloro- (IC50=0.17µM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Oximas/química , Oximas/farmacologia , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Oxirredutases do Álcool/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Desenho de Fármacos , Halogenação , Humanos , Metionina/análogos & derivados , Metionina/metabolismo , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Oximas/síntese química , Fenilpropionatos/síntese química , Relação Estrutura-AtividadeRESUMO
The pyranonaphthoquinone (PNQ) lactone natural products, including 7-deoxykalafungin, have been reported to be potent and selective covalent inhibitors of AKT kinase. In this work we seek to identify structural features of the natural product scaffold that are essential for potency and selectivity. Using a deconstruction approach, we designed and prepared simplified analogues of 7-deoxykalafungin. Testing of the compounds for their ability to inhibit AKT and the closely related kinase PKA revealed that the 3,6-dihydro-2H-pyran ring of the PNQ lactones is required for potent and selective inhibition of AKT. We have also unexpectedly identified a new submicromolar inhibitor of PKA.
Assuntos
Naftoquinonas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Relação Dose-Resposta a Droga , Estrutura Molecular , Naftoquinonas/síntese química , Naftoquinonas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-AtividadeRESUMO
Chelation-directed C-H activation/C-C bond forming reactions utilizing homogeneous palladium(ii) and the Pd(ii)/Pd(iv) catalytic cycle have been previously reported. Here we report the first use of a solid-supported Pd(ii) catalyst [Pd(ii) nanoparticles on multiwalled carbon nanotubes, Pd(ii)/MWCNT] to carry out C-H activation/C-C bond forming reactions. The results presented demonstrate that the solid-supported Pd(ii)/MWCNT catalyst can effectively catalyze these arylation reactions using the Pd(ii)/Pd(iv) catalytic cycle. We also show that the solid-supported catalyst is recyclable, has turnover frequencies up to 2.9-fold higher than the homogeneous catalyst, and results in low levels of residual palladium contamination in the products.
RESUMO
N-Chelation-directed C-H activation reactions that utilize the Pd(II)/Pd(IV) catalytic cycle have been previously reported. To date, these reactions employ only homogeneous palladium catalysts. The first use of a solid-supported Pd(II) catalyst [Pd(II) nanoparticles on multiwalled carbon nanotubes, Pd(II)/MWCNT] to carry out N-chelation-directed C-H to C-O, C-Cl, and C-Br transformations is reported. The results presented demonstrate that the solid-supported Pd(II)/MWCNT catalyst can effectively catalyze C-H activation reactions using the Pd(II)/Pd(IV) catalytic cycle.