RESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted adenosine monophosphate-activated serine/threonine protein kinase (AMPK) activation and downregulation of mammalian target of rapamycin (mTOR) signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL patient-derived xenografts (PDXs) led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47-driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease.
Assuntos
Antineoplásicos/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Desacopladores/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Mitocôndrias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Desacopladores/farmacologiaRESUMO
p53 is a Zn(2+)-dependent tumor suppressor inactivated in >50% of human cancers. The most common mutation, R175H, inactivates p53 by reducing its affinity for the essential zinc ion, leaving the mutant protein unable to bind the metal in the low [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) can repopulate the defective binding site, but how it accomplishes this in the context of living cells and organisms is unclear. In this study, we demonstrated that ZMC1 increases intracellular [Zn(2+)]free by functioning as a Zn(2+) ionophore, binding Zn(2+) in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn(2+)]free in cancer (TOV112D) and noncancer human embryonic kidney cell line 293 to 15.8 and 18.1 nM, respectively, with half-times of 2-3 minutes. These [Zn(2+)]free levels are predicted to result in â¼90% saturation of p53-R175H, thus accounting for its observed reactivation. This mechanism is supported by the X-ray crystal structure of the [Zn(ZMC1)2] complex, which demonstrates structural and chemical features consistent with those of known metal ionophores. These findings provide a physical mechanism linking zinc metallochaperone-1 in both in vitro and in vivo activities and define the remaining critical parameter necessary for developing synthetic metallochaperones for clinical use.
Assuntos
Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Ionóforos/metabolismo , Metalochaperonas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Zinco/metabolismo , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Mutação/genética , Conformação Proteica , Proteína Supressora de Tumor p53/genéticaAssuntos
Proteínas Reguladoras de Apoptose/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Citosol/metabolismo , Regulação para Baixo , Neoplasias Pulmonares/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Células A549 , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proteínas Mitocondriais/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidoresRESUMO
Controlling malaria requires new drugs against Plasmodium falciparum. The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the parasite's life cycle. We defined the structure-activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.
Assuntos
Antimaláricos , Malária Falciparum , Animais , Humanos , Antimaláricos/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum , Animais Geneticamente Modificados , Relação Estrutura-AtividadeRESUMO
Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.
Assuntos
Antineoplásicos/uso terapêutico , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/classificação , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/química , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Citocromos c/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Linfoma/tratamento farmacológico , Linfoma/patologia , Espectroscopia de Ressonância Magnética , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Nitrofenóis , Paclitaxel/farmacologia , Piperazinas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Relação Estrutura-Atividade , Sulfonamidas , Taxa de SobrevidaRESUMO
Pancreatic ductal adenocarcinoma (PDA) is aggressive cancer characterized by rapid progression, metastatic recurrence, and highly resistant to treatment. PDA cells exhibit aerobic glycolysis, or the Warburg effect, which reduces the flux of pyruvate into mitochondria. As a result, more glycolytic metabolites are shunted to pathways for the production of building blocks (e.g., ribose) and reducing agents (e.g., NADPH) for biosynthesis that are necessary for cell proliferation. In addition, PDA cells are highly addicted to glutamine for both maintaining biosynthetic pathways and achieving redox balance. Mitochondrial uncoupling facilitates proton influx across the mitochondrial inner membrane without generating ATP, leading to a futile cycle that consumes glucose metabolites and glutamine. We synthesized a new mitochondrial uncoupler MB1-47 and tested its effect on cancer cell metabolism and the anticancer activity in pancreatic cancer cell models and murine tumor transplantation models. MB1-47 uncouples mitochondria in the pancreatic cancer cells, resulting in: (1) the acceleration of pyruvate oxidation and TCA turnover; (2) increases in AMP/ATP and ADP/AMP ratios; and (3) a decrease in the synthesis rate of nucleotides and sugar nucleotides. Moreover, MB1-47 arrests cell cycle at G0-G1 phase, reduces clonogenicity, and inhibits cell growth of murine and human pancreatic cancer cells. In vivo studies showed that MB1-47 inhibits tumor growth in murine tumor transplantation models, and inhibits the hepatic metastasis when tumor cells were transplanted intrasplenically. Our results provide proof of concept for a potentially new strategy of treating PDA, and a novel prototype experimental drug for future studies and development.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Ciclo do Ácido Cítrico/genética , Neoplasias Hepáticas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Difosfato de Adenosina/genética , Monofosfato de Adenosina/genética , Trifosfato de Adenosina/genética , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Glucose/metabolismo , Glicólise/genética , Xenoenxertos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismoRESUMO
Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved inhibitors against ricin. We used a new strategy to disrupt RTA-ribosome interactions by fragment screening using surface plasmon resonance. Here, using a structure-guided approach, we improved the affinity and inhibitory activity of small-molecular-weight lead compounds and obtained improved compounds with over an order of magnitude higher efficiency. Four advanced compounds were characterized by X-ray crystallography. They bind at the RTA-ribosome binding site as the original compound but in a distinctive manner. These inhibitors bind remotely from the catalytic site and cause local conformational changes with no alteration of the catalytic site geometry. Yet they inhibit depurination by ricin holotoxin and inhibit the cytotoxicity of ricin in mammalian cells. They are the first agents that protect against ricin holotoxin by acting directly on RTA.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Ribossomos/efeitos dos fármacos , Ricina/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Chlorocebus aethiops , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Ricina/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Células VeroRESUMO
We identified a set of thiosemicarbazone (TSC) metal ion chelators that reactivate specific zinc-deficient p53 mutants using a mechanism called zinc metallochaperones (ZMCs) that restore zinc binding by shuttling zinc into cells. We defined biophysical and cellular assays necessary for structure-activity relationship studies using this mechanism. We investigated an alternative class of zinc scaffolds that differ from TSCs by substitution of the thiocarbamoyl moiety with benzothiazolyl, benzoxazolyl, and benzimidazolyl hydrazones. Members of this series bound zinc with similar affinity and functioned to reactivate mutant p53 comparable to the TSCs. Acute toxicity and efficacy assays in rodents demonstrated C1 to be significantly less toxic than the TSCs while demonstrating equivalent growth inhibition. We identified C85 as a ZMC with diminished copper binding that functions as a chemotherapy and radiation sensitizer. We conclude that the benzothiazolyl, benzoxazolyl, and benzimidazolyl hydrazones can function as ZMCs to reactivate mutant p53 in vitro and in vivo.
Assuntos
Benzotiazóis/uso terapêutico , Benzoxazóis/uso terapêutico , Quelantes/uso terapêutico , Hidrazonas/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Zinco/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzotiazóis/síntese química , Benzotiazóis/farmacologia , Benzoxazóis/síntese química , Benzoxazóis/farmacologia , Linhagem Celular Tumoral , Quelantes/síntese química , Quelantes/farmacologia , Humanos , Hidrazonas/síntese química , Hidrazonas/farmacologia , Camundongos Nus , Estrutura Molecular , Neoplasias/tratamento farmacológico , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of potent piperidine-linked cytosine derivatives were prepared as inhibitors of deoxycytidine kinase (dCK). Compound 9h was discovered to be a potent inhibitor of dCK and shows a good combination of cellular potency and pharmacokinetic parameters. Compound 9h blocks the incorporation of radiolabeled cytosine into mouse T-cells in vitro, as well as in vivo in mice following a T-cell challenge.
Assuntos
Desoxicitidina Quinase/antagonistas & inibidores , Flucitosina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Desenho de Fármacos , Flucitosina/síntese química , Flucitosina/química , Humanos , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of deoxycytidine kinase inhibitors was simultaneously optimized for potency and PK properties. A co-crystal structure then allowed merging this series with a high throughput screening hit to afford a highly potent, selective and orally bioavailable inhibitor, compound 10. This compound showed dose dependent inhibition of deoxycytidine kinase in vivo.
Assuntos
Desoxicitidina Quinase/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Desoxicitidina/síntese química , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
BMP receptor inhibitors induce death of cancer cells through the downregulation of antiapoptotic proteins XIAP, pTAK1, and Id1-Id3. However, the current most potent BMP receptor inhibitor, DMH2, does not downregulate BMP signaling in vivo because of metabolic instability and poor pharmacokinetics. Here we identified the site of metabolic instability of DMH2 and designed a novel BMP receptor inhibitor, JL5. We show that JL5 has a greater volume of distribution and suppresses the expression of Id1 and pTak1 in tumor xenografts. Moreover, we demonstrate JL5-induced tumor cell death and tumor regression in xenograft mouse models without immune cells and humanized with adoptively transferred human immune cells. In humanized mice, JL5 additionally induces the infiltration of immune cells within the tumor microenvironment. Our studies show that the BMP signaling pathway is targetable in vivo and BMP receptor inhibitors can be developed as a therapeutic to treat cancer patients.
Assuntos
Antineoplásicos/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinolonas/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Células A549 , Transferência Adotiva , Animais , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteína 1 Inibidora de Diferenciação/biossíntese , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirazóis/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Quinolonas/química , Quinolonas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The synthesis and structure-activity relationships of novel dipeptidyl peptidase IV inhibitors replacing the classical cyanopyrrolidine P1 group with other small nitrogen heterocycles are described. A unique potency enhancement was achieved with beta-branched natural and unnatural amino acids, particularly adamantylglycines, linked to a (2S,3R)-2,3-methanopyrrolidine based scaffold.
Assuntos
Dipeptídeos/química , Inibidores da Dipeptidil Peptidase IV , Inibidores da Dipeptidil Peptidase IV/química , Dipeptídeos/farmacologia , Inibidores da Dipeptidil Peptidase IV/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Nitrilas/química , Nitrilas/farmacologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) represents one of the most lethal cancers worldwide due to therapy resistance and disease recurrence. Tumor relapse following treatment could be driven by the persistence of liver cancer stem-like cells (CSCs). The protein BMI1 is a member of the polycomb epigenetic factors governing cellular self-renewal, proliferation, and stemness maintenance. BMI1 expression also correlates with poor patient survival in various cancer types. OBJECTIVE: We aimed to elucidate the extent to which BMI1 can be used as a potential therapeutic target for CSC eradication in HCC. METHODS: We have recently participated in characterizing the first known pharmacological small molecule inhibitor of BMI1. Here, we synthesized a panel of novel BMI1 inhibitors and examined their ability to alter cellular growth and eliminate cancer progenitor/stem-like cells in HCC with different p53 backgrounds. RESULTS: Among various molecules examined, RU-A1 particularly downregulated BMI1 expression, impaired cell viability, reduced cell migration, and sensitized HCC cells to 5-fluorouracil (5-FU) in vitro. Notably, long-term analysis of HCC survival showed that, unlike chemotherapy, RU-A1 effectively reduced CSC content, even as monotherapy. BMI1 inhibition with RU-A1 diminished the number of stem-like cells in vitro more efficiently than the model compound C-209, as demonstrated by clonogenic assays and impairment of CSC marker expression. Furthermore, xenograft assays in zebrafish showed that RU-A1 abrogated tumor growth in vivo. CONCLUSIONS: This study demonstrates the ability to identify agents with the propensity for targeting CSCs in HCC that could be explored as novel treatments in the clinical setting.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Complexo Repressor Polycomb 1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/química , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células HEK293 , Células Hep G2 , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Complexo Repressor Polycomb 1/biossíntese , Complexo Repressor Polycomb 1/genética , Bibliotecas de Moléculas Pequenas/química , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
The antiapoptotic proteins Bcl-x(L) and Bcl-2 play key roles in the maintenance of normal cellular homeostasis. However, their overexpression can lead to oncogenic transformation and is responsible for drug resistance in certain types of cancer. This makes Bcl-x(L) and Bcl-2 attractive targets for the development of potential anticancer agents. Here we describe the structure-based discovery of a potent Bcl-x(L) inhibitor directed at a hydrophobic groove on the surface of the protein. This groove represents the binding site for BH3 peptides from proapoptotic Bcl-2 family members such as Bak and Bad. Application of NMR-based screening yielded an initial biaryl acid with an affinity (K(d)) of approximately 300 microM for the protein. Following the classical "SAR by NMR" approach, a second-site ligand was identified that bound proximal to the first-site ligand in the hydrophobic groove. From NMR-based structural studies and parallel synthesis, a potent ligand was obtained, which binds to Bcl-x(L) with an inhibition constant (K(i)) of 36 +/- 2 nM.
Assuntos
Compostos de Anilina/síntese química , Modelos Moleculares , Sulfonamidas/síntese química , Proteína bcl-X/antagonistas & inibidores , Compostos de Anilina/química , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Espectroscopia de Ressonância Magnética , Ligação Proteica , Solubilidade , Relação Estrutura-Atividade , Sulfonamidas/química , Proteína bcl-X/químicaRESUMO
Efforts to further elucidate structure-activity relationships (SAR) within our previously disclosed series of beta-quaternary amino acid linked l-cis-4,5-methanoprolinenitrile dipeptidyl peptidase IV (DPP-IV) inhibitors led to the investigation of vinyl substitution at the beta-position of alpha-cycloalkyl-substituted glycines. Despite poor systemic exposure, vinyl-substituted compounds showed extended duration of action in acute rat ex vivo plasma DPP-IV inhibition models. Oxygenated putative metabolites were prepared and were shown to exhibit the potency and extended duration of action of their precursors in efficacy models measuring glucose clearance in Zucker(fa/fa) rats. Extension of this approach to adamantylglycine-derived inhibitors led to the discovery of highly potent inhibitors, including hydroxyadamantyl compound BMS-477118 (saxagliptin), a highly efficacious, stable, and long-acting DPP-IV inhibitor, which is currently undergoing clinical trials for treatment of type 2 diabetes.
Assuntos
Adamantano/análogos & derivados , Adamantano/síntese química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptídeos/síntese química , Dipeptidil Peptidase 4/metabolismo , Glicina/análogos & derivados , Glicina/síntese química , Hipoglicemiantes/síntese química , Inibidores de Proteases/síntese química , Adamantano/farmacologia , Animais , Disponibilidade Biológica , Glicemia/análise , Diabetes Mellitus Tipo 2/fisiopatologia , Dipeptídeos/farmacologia , Teste de Tolerância a Glucose , Glicina/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Insulina/sangue , Masculino , Camundongos , Camundongos Obesos , Microssomos Hepáticos/metabolismo , Nitrilas/síntese química , Nitrilas/farmacologia , Prolina/análogos & derivados , Prolina/síntese química , Prolina/farmacologia , Inibidores de Proteases/farmacologia , Ratos , Ratos Zucker , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The NMR-based discovery of biaryl hydroxamate inhibitors of the matrix metalloproteinase stromelysin (MMP-3) has been previously described (Hajduk et al. J. Am. Chem. Soc. 1997, 119, 5818-5827). While potent in vitro, these inhibitors exhibited no in vivo activity due, at least in part, to the poor pharmacokinetic properties of the alkylhydroxamate moiety. To circumvent this liability, NMR-based screening was implemented to identify alternative zinc-chelating groups. Using this technique, 1-naphthyl hydroxamate was found to bind tightly to the protein (K(D) = 50 microM) and was identified as a candidate for incorporation into the lead series. On the basis of NMR-derived structural information, the naphthyl hydroxamate and biaryl fragments were linked together to yield inhibitors of this enzyme that exhibited improved bioavailability. These studies demonstrate that the NMR-based screening of fragments can be effectively applied to improve the physicochemical or pharmacokinetic profile of lead compounds.
Assuntos
Ácidos Hidroxâmicos/síntese química , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/síntese química , Animais , Disponibilidade Biológica , Domínio Catalítico , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Espectroscopia de Ressonância Magnética , Metaloproteinase 3 da Matriz/química , Modelos Moleculares , Naftalenos/síntese química , Naftalenos/química , Naftalenos/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
A series of methanoprolinenitrile-containing dipeptide mimetics were synthesized and assayed as inhibitors of the N-terminal sequence-specific serine protease dipeptidyl peptidase IV (DPP-IV). The catalytic action of DPP-IV is the principle means of degradation of glucagon-like peptide-1, a key mediator of glucose-stimulated insulin secretion, and DPP-IV inhibition shows clinical benefit as a novel mechanism for treatment of type 2 diabetes. However, many of the reversible inhibitors to date suffer from chemical instability stemming from an amine to nitrile intramolecular cyclization. Installation of a cyclopropyl moiety at either the 3,4- or 4,5-position of traditional 2-cyanopyrrolidide proline mimetics led to compounds with potent inhibitory activity against the enzyme. Additionally, cis-4,5-methanoprolinenitriles with beta-branching in the N-terminal amino acid provided enhanced chemical stability and high inhibitory potency. This class of inhibitors also exhibited the ability to suppress prandial glucose elevations after an oral glucose challenge in male Zucker rats.
Assuntos
Ciclopropanos/síntese química , Dipeptidil Peptidase 4/metabolismo , Inibidores Enzimáticos/síntese química , Nitrilas/síntese química , Prolina/análogos & derivados , Prolina/síntese química , Animais , Simulação por Computador , Cristalografia por Raios X , Ciclopropanos/química , Ciclopropanos/farmacologia , Dipeptídeos/química , Dipeptidil Peptidase 4/química , Estabilidade de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Masculino , Modelos Moleculares , Conformação Molecular , Mimetismo Molecular , Nitrilas/química , Nitrilas/farmacologia , Prolina/química , Prolina/farmacologia , Ratos , Ratos Zucker , SoluçõesRESUMO
UNLABELLED: NSC319726 (ZMC1) is a small molecule that reactivates mutant p53 by restoration of WT structure/function to the most common p53 missense mutant (p53-R175H). We investigated the mechanism by which ZMC1 reactivates p53-R175H and provide evidence that ZMC1: 1) restores WT structure by functioning as a zinc-metallochaperone, providing an optimal concentration of zinc to facilitate proper folding; and 2) increases cellular reactive oxygen species that transactivate the newly conformed p53-R175H (via post-translational modifications), inducing an apoptotic program. We not only demonstrate that this zinc metallochaperone function is possessed by other zinc-binding small molecules, but that it can reactivate other p53 mutants with impaired zinc binding. This represents a novel mechanism for an anti-cancer drug and a new pathway to drug mutant p53. SIGNIFICANCE: We have elucidated a novel mechanism to restore wild-type structure/function to mutant p53 using small molecules functioning as zinc-metallochaperones. The pharmacologic delivery of a metal ion to restore proper folding of a mutant protein is unique to medicinal chemistry and represents a new pathway to drug mutant p53.
Assuntos
Antineoplásicos/farmacologia , Metalochaperonas/farmacologia , Piridinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Células HCT116 , Humanos , Células MCF-7 , Metalochaperonas/metabolismo , Mutação , Ligação Proteica , Dobramento de Proteína/efeitos dos fármacos , Piridinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Zinco/metabolismoRESUMO
Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.