RESUMEN
Protein tyrosine phosphatase type IVA member 3 (PTP4A3 or PRL-3) is a nonreceptor, oncogenic, dual-specificity phosphatase that is highly expressed in many human tumors, including ovarian cancer, and is associated with a poor patient prognosis. Recent studies suggest that PTP4A3 directly dephosphorylates SHP-2 phosphatase as part of a STAT3-PTP4A3 feedforward loop and directly dephosphorylates p38 kinase. The goal of the current study was to examine the effect of a PTP4A phosphatase inhibitor, 7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053), on ovarian cancer STAT3, SHP-2, and p38 kinase phosphorylation. JMS-053 caused a concentration- and time-dependent decrease in the activated form of STAT3, Y705 phospho-STAT3, in ovarian cancer cells treated in vitro. In contrast, the phosphorylation status of two previously described direct PTP4A3 substrates, SHP-2 phosphatase and p38 kinase, were rapidly increased with JMS-053 treatment. We generated A2780 and OVCAR4 ovarian cancer cells resistant to JMS-053, and the resulting cells were not crossresistant to paclitaxel, cisplatin, or teniposide. JMS-053-resistant A2780 and OVCAR4 cells exhibited a 95% and 50% decrease in basal Y705 phospho-STAT3, respectively. JMS-053-resistant OVCAR4 cells had an attenuated phosphorylation and migratory response to acute exposure to JMS-053. These results support a regulatory role for PTP4A phosphatase in ovarian cancer cell STAT3 and p38 signaling circuits. SIGNIFICANCE STATEMENT: This study demonstrates that chemical inhibition of PTP4A phosphatase activity with JMS-053 decreases STAT3 activation and increases SHP-2 phosphatase and p38 kinase phosphorylation activation in ovarian cancer cells. The newly developed JMS-053-resistant ovarian cancer cells should provide useful tools to further probe the role of PTP4A phosphatase in ovarian cancer cell survival and cell signaling.
Asunto(s)
Neoplasias Ováricas , Femenino , Humanos , Línea Celular Tumoral , Fosforilación , Transducción de Señal , Factor de Transcripción STAT3/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 14 Activada por MitógenosRESUMEN
We developed JMS-053, a potent inhibitor of the dual specificity phosphatase PTP4A3 that is potentially suitable for cancer therapy. Due to the emerging role of the unfolded protein response (UPR) in cancer pathology, we sought to identify derivatives that combine PTP4A3 inhibition with induction of endoplasmatic reticulum (ER) stress, with the goal to generate more potent anticancer agents. We have now generated bifunctional analogs that link the JMS-053 pharmacophore to an adamantyl moiety and act in concert with the phosphatase inhibitor to induce ER stress and cell death. The most potent compound in this series, 7a, demonstrated a ca. 5-fold increase in cytotoxicity in a breast cancer cell line and strong activation of UPR and ER stress response genes in spite of a ca. 13-fold decrease in PTP4A3 inhibition. These results demonstrate that the combination of phosphatase inhibition with UPR/ER-stress upregulation potentiates efficacy.
Asunto(s)
Antineoplásicos/farmacología , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Iminas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Piridinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Femenino , Humanos , Iminas/síntesis química , Iminas/química , Estructura Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
Protein tyrosine phosphatase (PTP) 4A3 is frequently overexpressed in human solid tumors and hematologic malignancies and is associated with tumor cell invasion, metastasis, and a poor patient prognosis. Several potent, selective, and allosteric small molecule inhibitors of PTP4A3 were recently identified. A lead compound in the series, JMS-053 (7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione), has a long plasma half-life (â¼ 24 hours) in mice, suggesting possible binding to serum components. We confirmed by isothermal titration calorimetry that JMS-053 binds to human serum albumin. A single JMS-053 binding site was identified by X-ray crystallography in human serum albumin at drug site 3, which is also known as subdomain IB. The binding of JMS-053 to human serum albumin, however, did not markedly alter the overall albumin structure. In the presence of serum albumin, the potency of JMS-053 as an in vitro inhibitor of PTP4A3 and human A2780 ovarian cancer cell growth was reduced. The reversible binding of JMS-053 to serum albumin may serve to increase JMS-053's plasma half-life and thus extend the delivery of the compound to tumors. SIGNIFICANCE STATEMENT: X-ray crystallography revealed that a potent, reversible, first-in-class small molecule inhibitor of the oncogenic phosphatase protein tyrosine phosphatase 4A3 binds to at least one site on human serum albumin, which is likely to extend the compound's plasma half-life and thus assist in drug delivery into tumors.
Asunto(s)
Iminas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Piridinas/farmacología , Albúmina Sérica Humana/metabolismo , Sitios de Unión , Calorimetría , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Pruebas de Enzimas , Semivida , Humanos , Iminas/química , Iminas/uso terapéutico , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas Tirosina Fosfatasas/metabolismo , Piridinas/química , Piridinas/uso terapéutico , Albúmina Sérica Humana/ultraestructuraRESUMEN
Oncogenic protein tyrosine phosphatases (PTPs) are overexpressed in numerous human cancers but they have been challenging pharmacological targets. The emblematic oncogenic PTP4A tyrosine phosphatase family regulates many fundamental malignant processes. 7-Imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053) is a novel, potent, and selective PTP4A inhibitor but its mechanism of action has not been fully elucidated, nor has the chemotype been fully investigated. Because tyrosine phosphatases are notoriously susceptible to oxidation, we interrogated JMS-053 and three newly synthesized analogs with specific attention on the role of oxidation. JMS-053 and its three analogs were potent in vitro PTP4A3 inhibitors, but 7-imino-5-methyl-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (NRT-870-59) appeared unique among the thienopyridinediones with respect to its inhibitory specificity for PTP4A3 versus both a PTP4A3 A111S mutant and an oncogenic dual specificity tyrosine phosphatase, CDC25B. Like JMS-053, NRT-870-59 was a reversible PTP4A3 inhibitor. All of the thienopyridinediones retained cytotoxicity against human ovarian and breast cancer cells grown as pathologically relevant three-dimensional spheroids. Inhibition of cancer cell colony formation by NRT-870-59, like JMS-053, required PTP4A3 expression. JMS-053 failed to generate significant detectable reactive oxygen species in vitro or in cancer cells. Mass spectrometry results indicated no disulfide bond formation or oxidation of the catalytic Cys104 after in vitro incubation of PTP4A3 with JMS-053 or NRT-870-59. Gene expression profiling of cancer cells exposed to JMS-053 phenocopied many of the changes seen with the loss of PTP4A3 and did not indicate oxidative stress. These data demonstrate that PTP4A phosphatases can be selectively targeted with small molecules that lack prominent reactive oxygen species generation and encourage further studies of this chemotype. SIGNIFICANCE STATEMENT: Protein tyrosine phosphatases are emerging as important contributors to human cancers. We report on a new class of reversible protein phosphatase small molecule inhibitors that are cytotoxic to human ovarian and breast cancer cells, do not generate significant reactive oxygen species in vitro and in cells, and could be valuable lead molecules for future studies of PTP4A phosphatases.
Asunto(s)
Antineoplásicos/farmacología , Iminas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Piridinas/farmacología , Piridonas/farmacología , Línea Celular Tumoral , Humanos , Mutación , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatasas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
he mechanistic details of the aldol addition of N-amino cyclic carbamate (ACC) hydrazones is provided herein from both an experimental and computational perspective. When the transformation is carried out at room temperature the anti-aldol product is formed exclusively. Under these conditions the anti- and syn-aldolate intermediates are in equilibrium and the transformation is under thermodynamic control. The anti-aldolate that leads to the anti-aldol product was calculated to be 3.7â kcal mol-1 lower in energy at room temperature than that leading to the syn-aldol product, which sufficiently accounts for the exclusive formation of the anti-aldol product. When the reaction is conducted at -78 °C it is under kinetic control and favors formation of the syn-aldol addition product. In this case, it was found that a solvent separated aza-enolate anion and aldehyde form a σ-intermediate in which the lithium cation is coordinated to the aldehyde. The σ-intermediate collapses with a very small activation barrier to form the ß-alkoxy hydrazone intermediate. The chiral nonracemic lithium aza-enolate discriminates between the two diastereotopic faces of the pro-chiral aldehyde, and there is no rapid direct pathway that interconverts the two diastereomeric intermediates. Consequently, the reaction does not follow the Curtin-Hammett principle and the stereochemical outcome at low temperature instead depends on the relative energies of the two σ-intermediates.
RESUMEN
Protein tyrosine phosphatases (PTPs) are emerging new targets for drug discovery. PTPs and protein tyrosine kinases (PTKs) maintain cellular homeostasis through opposing roles: tyrosine O-dephosphorylation and -phosphorylation, respectively. An imbalance in the phosphorylation equilibrium results in aberrant protein signaling and pathophysiological conditions. PTPs have historically been considered 'undruggable', in part due to a lack of evidence defining their relationship to disease causality and a focus on purely competitive inhibitors. However, a better understanding of protein-protein interfaces and shallow active sites has recently renewed interest in the pursuit of allosteric and orthosteric modulators of targets outside the major druggable protein families. While their biological mechanism of action still remains to be clarified, PTP4A1-3 (also referred to as PRL1-3) are validated oncology targets and play an important role in cell proliferation, metastasis, and tumor angiogenesis. In this Digest, recent syntheses and structure-activity relationships (SAR) of small molecule inhibitors (SMIs) of PTP4A1-3 are summarized, and enzyme docking studies of the most potent chemotype are highlighted. In particular, the thienopyridone scaffold has emerged as a potent lead structure to interrogate the function and druggability of this dual-specificity PTP.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Animales , Dominio Catalítico , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Piridonas/síntesis química , Piridonas/metabolismo , Piridonas/farmacología , Piridonas/uso terapéutico , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/metabolismo , Tiofenos/farmacología , Tiofenos/uso terapéuticoRESUMEN
A continuous flow photooxygenation of 7-aminothieno[3,2-c]pyridin-4(5H)-ones to produce 7-iminothieno[3,2-c]pyridine-4,6(5H,7H)-diones has been developed, utilizing ambient air as the sole reactant. N-H Imines are formed as the major products, and excellent functional group tolerance and conversion on gram-scale without the need for chromatographic purification allow for facile late-stage diversification of the aminothienopyridinone scaffold. Several analogs exhibit potent in vitro inhibition of the cancer-associated protein tyrosine phosphatase PTP4A3, and the SAR supports an exploratory docking model.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Piridonas/química , Piridonas/farmacología , Tienopiridinas/química , Tienopiridinas/farmacología , Aminación , Humanos , Luz , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Oxidación-Reducción , Proteínas Tirosina Fosfatasas/metabolismo , Relación Estructura-ActividadRESUMEN
The asymmetric (er > 99:1) total synthesis of (+)-anti- and (-)-syn-mefloquine hydrochloride from a common intermediate is described. The Sharpless asymmetric dihydroxylation is the key asymmetric transformation used in the synthesis of this intermediate. It is carried out on an olefin that is accessed in three steps from commercially available materials, making the overall synthetic sequence very concise. The common diol intermediate derived from the Sharpless asymmetric dihydroxylation is converted into either a trans- or cis-epoxide, and these are subsequently converted to (+)-anti- and (-)-syn-mefloquine, respectively. X-ray crystallographic analysis of derivatives of (+)-anti- and (-)-syn-mefloquine is used to lay to rest a 40 year argument regarding the absolute stereochemistry of the mefloquines. A formal asymmetric (er > 99:1) synthesis of (+)-anti-mefloquine hydrochloride is also presented that uses a Sharpless asymmetric epoxidation as a key step.
RESUMEN
A concise asymmetric (>99:1 e.r.) total synthesis of (+)-anti- and (-)-syn-mefloquine hydrochloride from a common intermediate is described. The key asymmetric transformation is a Sharpless dihydroxylation of an olefin that is accessed in three steps from commercially available materials. The Sharpless-derived diol is converted into either a trans or cis epoxide, and these are subsequently converted into (+)-anti- and (-)-syn-mefloquine, respectively. The synthetic (+)-anti- and (-)-syn-mefloquine samples were derivatized with (S)-(+)-mandelic acid tert-butyldimethylsilyl ether, and a crystal structure of each derivative was obtained. These are the first X-ray structures for mefloquine derivatives that were obtained by coupling to a known chiral, nonracemic compound, and provide definitive confirmation of the absolute stereochemistry of (+)-anti- as well as (-)-syn-mefloquine.
Asunto(s)
Mefloquina/química , Mefloquina/síntesis química , Cristalografía por Rayos X , Modelos Moleculares , Conformación Molecular , EstereoisomerismoRESUMEN
High grade serous ovarian cancer (OvCa) frequently becomes drug resistant and often recurs. Consequently, new drug targets and therapies are needed. Bioinformatics-based studies uncovered a relationship between high Protein Tyrosine Phosphatase of Regenerating Liver-3 (PRL3 also known as PTP4A3) expression and poor patient survival in both early and late stage OvCa. PTP4A3 mRNA levels were 5-20 fold higher in drug resistant or high grade serous OvCa cell lines compared to nonmalignant cells. JMS-053 is a potent allosteric small molecule PTP4A3 inhibitor and to explore further the role of PTP4A3 in OvCa, we synthesized and interrogated a series of JMS-053-based analogs in OvCa cell line-based phenotypic assays. While the JMS-053 analogs inhibit in vitro PTP4A3 enzyme activity, none were superior to JMS-053 in reducing high grade serous OvCa cell survival. Because PTP4A3 controls cell migration, we interrogated the effect of JMS-053 on this cancer-relevant process. Both JMS-053 and CRISPR/Cas9 PTP4A3 depletion blocked cell migration. The inhibition caused by JMS-053 required the presence of PTP4A3. JMS-053 caused additive or synergistic in vitro cytotoxicity when combined with paclitaxel and reduced in vivo OvCa dissemination. These results indicate the importance of PTP4A3 in OvCa and support further investigations of the lead inhibitor, JMS-053.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Bases de Datos Genéticas , Femenino , Humanos , Iminas/química , Iminas/farmacología , Proteínas de Neoplasias/fisiología , Neoplasias Ováricas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Piridinas/química , Piridinas/farmacologíaRESUMEN
Nonsense mutations constitute ~10% of TP53 mutations in cancer. They introduce a premature termination codon that gives rise to truncated p53 protein with impaired function. The aminoglycoside G418 can induce TP53 premature termination codon readthrough and thus increase cellular levels of full-length protein. Small molecule phthalimide derivatives that can enhance the readthrough activity of G418 have also been described. To determine whether readthrough enhancers exist among drugs that are already approved for use in humans, we tested seven antimalarial drugs for readthrough of the common R213X TP53 nonsense mutation in HDQ-P1 breast cancer cells. Mefloquine induced no TP53 readthrough activity as a single agent but it strongly potentiated readthrough by G418. The two enantiomers composing pharmaceutical mefloquine potentiated readthrough to similar levels in HDQ-P1 cells and also in SW900, NCI-H1688 and HCC1937 cancer cells with different TP53 nonsense mutations. Exposure to G418 and mefloquine increased p53 phosphorylation at Ser15 and P21 transcript levels following DNA damage, indicating p53 produced via readthrough was functional. Mefloquine does not appear to enhance readthrough via lysosomotropic effects as it did not significantly affect lysosomal pH, the cellular levels of G418 or its distribution in organellar or cytosolic fractions. The availability of a readthrough enhancer that is already approved for use in humans should facilitate study of the therapeutic potential of TP53 readthrough in preclinical cancer models.
Asunto(s)
Antimaláricos/farmacología , Codón sin Sentido , Codón de Terminación , Gentamicinas/farmacología , Mefloquina/farmacología , Terminación de la Cadena Péptídica Traduccional , Proteína p53 Supresora de Tumor , Células HCT116 , Humanos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Terminación de la Cadena Péptídica Traduccional/genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
A new strategy for the rhodium-catalyzed enantioselective hydroacylation is described. This has been achieved through the merger of iminium ion catalysis and transition-metal catalysis such that asymmetric induction derives from a readily accessible, inexpensive chiral nonracemic secondary amine catalyst rather than a chiral nonracemic phosphine as is typical of conventional asymmetric hydroacylation methods.