RESUMEN
Malignant gliomas are the most common primary central nervous system tumor in adults. Despite current therapeutics, these tumors are associated with poor prognosis and a median survival of 16 to 19 months. This highlights the need for innovative treatments for this incurable disease. Rac1 has long been associated with tumor progression and plays a key role in glioma's infiltrative and invasive nature. The aim of this study is to evaluate the 1A-116 molecule, a Rac1 inhibitor, as targeted therapy for this aggressive disease. We found that targeting Rac1 inhibits cell proliferation and cell cycle progression using different in vitro human glioblastoma models. Additionally, we evaluated 1A-116 in vivo, showing a favorable toxicological profile. Using in silico tools, 1A-116 is also predicted to penetrate the blood-brain barrier and present a favorable metabolic fate. In line with these results, 1A-116 i.p daily treatment resulted in a dose-dependent antitumor effect in an orthotopic IDH-wt glioma model. Altogether, our study provides a strong potential for clinical translation of 1A-116 as a signal transduction-based precision therapy for glioma and also increases the evidence of Rac1 as a key molecular target.
RESUMEN
Tumor cells have unlimited replicative potential, principally due to telomerase activity, which requires assembly of components such as dyskerin (hDKC1), human telomerase reverse transcriptase and human telomerase RNA (hTR). The present study aimed to develop novel inhibitors of telomerase to target the interaction between hTR and hDKC1. Based on dockingbased virtual screening, the candidates R1D210 and R1D215, which exert an in vitro inhibitory effect on telomerase activity, were selected. Human mammary adenocarcinoma MDAMB 231 cell line was selected to evaluate the treatment with the aforementioned compounds; the effect on telomere length was evaluated by qPCR, where both compounds caused telomere shortening. Furthermore, expression of genes related to apoptosis and senescence process, as well SA ß galactosidase staining and caspase 3 activity. We determine that only compound R1D210 showed and effect on the induction of these cellular processes. To identify a lead compound from R1D210, 100 analogs were designed by LigDream server and then analyzed by AutoDock Vina and ProteinLigand Interaction Profile to calculate their docking energy and target interaction. Those with the best values and specific residue interactions were selected for in silico prediction of absorption, distribution, metabolism, excretion (ADME), offtarget interaction, toxicity and chemical diversity. A total of nine chemically different analogs was identified with higher docking affinity to the target, suitable ADME properties and not offtarget interaction and side effects. These results indicated R1D210 and its analogs may serve as potential novel inhibitors of telomerase and antitumoral drugs in clinical use.
Asunto(s)
Neoplasias de la Mama , Telomerasa , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Células MCF-7 , Proteínas de Unión al ARN/metabolismo , Telomerasa/metabolismoRESUMEN
The Ras homologous family of small guanosine triphosphate-binding enzymes (GTPases) is critical for cell migration and proliferation. The novel drug 1A-116 blocks the interaction site of the Ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase with some of its guanine exchange factors (GEFs), such as T-cell lymphoma invasion and metastasis 1 (TIAM1), inhibiting cell motility and proliferation. Knowledge of circadian regulation of targets can improve chemotherapy in glioblastoma. Thus, circadian regulation in the efficacy of 1A-116 was studied in LN229 human glioblastoma cells and tumor-bearing nude mice. METHODS: Wild-type LN229 and BMAL1-deficient (i.e., lacking a functional circadian clock) LN229E1 cells were assessed for rhythms in TIAM1, BMAL1, and period circadian protein homolog 1 (PER1), as well as Tiam1, Bmal1, and Rac1 mRNA levels. The effects of 1A-116 on proliferation, apoptosis, and migration were then assessed upon applying the drug at different circadian times. Finally, 1A-116 was administered to tumor-bearing mice at two different circadian times. RESULTS: In LN229 cells, circadian oscillations were found for BMAL1, PER1, and TIAM1 (mRNA and protein), and for the effects of 1A-116 on proliferation, apoptosis, and migration, which were abolished in LN229E1 cells. Increased survival time was observed in tumor-bearing mice when treated with 1A-116 at the end of the light period (zeitgeber time 12, ZT12) compared either to animals treated at the beginning (ZT3) or with vehicle. CONCLUSIONS: These results unveil the circadian modulation in the efficacy of 1A-116, likely through RAC1 pathway rhythmicity, suggesting that a chronopharmacological approach is a feasible strategy to improve glioblastoma treatment.
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Acyl-CoA synthetase 4 (ACSL4) is an isoenzyme of the fatty acid ligase-coenzyme-A family taking part in arachidonic acid metabolism and steroidogenesis. ACSL4 is involved in the development of tumor aggressiveness in breast and prostate tumors through the regulation of various signal transduction pathways. Here, a bioinformatics analysis shows that the ACSL4 gene expression and proteomic signatures obtained using a cell model was also observed in tumor samples from breast and cancer patients. A well-validated ACSL4 inhibitor, however, has not been reported hindering the full exploration of this promising target and its therapeutic application on cancer and steroidogenesis inhibition. In this study, ACSL4 inhibitor PRGL493 was identified using a homology model for ACSL4 and docking based virtual screening. PRGL493 was then chemically characterized through nuclear magnetic resonance and mass spectroscopy. The inhibitory activity was demonstrated through the inhibition of arachidonic acid transformation into arachidonoyl-CoA using the recombinant enzyme and cellular models. The compound blocked cell proliferation and tumor growth in both breast and prostate cellular and animal models and sensitized tumor cells to chemotherapeutic and hormonal treatment. Moreover, PGRL493 inhibited de novo steroid synthesis in testis and adrenal cells, in a mouse model and in prostate tumor cells. This work provides proof of concept for the potential application of PGRL493 in clinical practice. Also, these findings may prove key to therapies aiming at the control of tumor growth and drug resistance in tumors which express ACSL4 and depend on steroid synthesis.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Coenzima A Ligasas/metabolismo , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Animales , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Coenzima A Ligasas/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , Próstata/citología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Esteroides/sangre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the last years, the development of new drugs in oncology has evolved notably. In particular, drug development has shifted from empirical screening of active cytotoxic compounds to molecularly targeted drugs blocking specific biologic pathways that drive cancer progression and metastasis. Using a rational design approach, our group has developed 1A-116 as a promising Rac1 inhibitor, with antitumoral and antimetastatic effects in several types of cancer. Rac1 is over activated in a wide range of tumor types and and it is one of the most studied proteins of the Rho GTPase family. Its role in actin cytoskeleton reorganization has effects on endocytosis, vesicular trafficking, cell cycle progression and cellular migration. In this context, the regulatory activity of Rac1 affects several key processes in the course of the cancer including invasion and metastasis. The purpose of this preclinical study was to focus on the mode of action of 1A-116, conducting an interdisciplinary approach with in silico bioinformatics tools and in vitro assays. Here, we demonstrate that the tryptophan 56 residue is necessary for the inhibitory effects of 1A-116 since this compound interferes with protein-protein interactions (PPI) of Rac1GTPase involving several GEF activators. 1A-116 is also able to inhibit the oncogenic Rac1P29S mutant protein, one of the oncogenic drivers found in sun-exposed melanoma. It also inhibits numerous Rac1-regulated cellular processes such as membrane ruffling and lamellipodia formation. These results deepen our knowledge of 1A-116 inhibition of Rac1 and its biological impact on cancer progression. They also represent a good example of how in silico analyses represent a valuable approach for drug development.
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AIMS: G protein-coupled receptor (GPCR) kinases (GRKs) are mainly involved in the desensitization of GPCRs. Among them, GRK2 has been described to be upregulated in many pathological conditions and its crucial role in cardiac hypertrophy, hypertension, and heart failure promoted the search for pharmacological inhibitors of its activity. There have been several reports of potent and selective inhibitors of GRK2, most of them directed to the kinase domain of the protein. However, the homologous to the regulator of G protein signaling (RH) domain of GRK2 has also been shown to regulate GPCRs signaling. Herein, we searched for potential inhibitors of receptor desensitization mediated by RH domain of GRK2. MATERIALS AND METHODS: We performed a docking-based virtual screening utilizing the crystal structure of GRK2 to search for potential inhibitors of the interaction between GRK2 and Gαq protein. To evaluate the biological activity of compounds we measured, calcium response of histamine H1 receptor (H1R) using Fura-2AM dye and H1R internalization by saturation binding experiments in A549â¯cells. GRK2(45-178)GFP translocation was determined in HeLa cells through confocal fluorescence imaging. KEY FINDINGS: We identified inhibitors of GRK2 able to reduce the RH mediated desensitization of the histamine H1 receptor and GRK2 translocation to plasma membrane. Also candidates presented adequate lipophilia and cytotoxicity profile. SIGNIFICANCE: We obtained compounds with the ability of reducing RH mediated actions of GRK2 that can be useful as a starting point in the development of novel drug candidates aimed to treat pathologies were GRK2 plays a key role.
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Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Células A549 , Simulación por Computador , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Células HeLa , Humanos , Tamizaje Masivo , Simulación del Acoplamiento Molecular/métodos , Fosforilación , Unión Proteica , Dominios Proteicos , Inhibidores de Proteínas Quinasas/química , Receptores Histamínicos H1/metabolismo , Transducción de SeñalRESUMEN
Immortality is one of the main features of cancer cells. Tumor cells have an unlimited replicative potential, principally due to the holoenzyme telomerase. Telomerase is composed mainly by dyskerin (DKC1), a catalytic retrotranscriptase (hTERT) and an RNA template (hTR). The aim of this work is to develop new inhibitors of telomerase, selecting the interaction between hTRâ»DKC1 as a target. We designed two models of the human protein DKC1: homology and ab initio. These models were evaluated by different procedures, revealing that the homology model parameters were the most accurate. We selected two hydrophobic pockets contained in the PUA (pseudouridine synthase and archaeosine transglycosylase) domain, using structural and stability analysis. We carried out a docking-based virtual screen on these pockets, using the reported mutation K314 as the center of the docking. The hDKC1 model was tested against a library of 450,000 drug-like molecules. We selected the first 10 molecules that showed the highest affinity values to test their inhibitory activity on the cell line MDA MB 231 (Monroe Dunaway Anderson Metastasis Breast cancer 231), obtaining three compounds that showed inhibitory effect. These results allowed us to validate our design and set the basis to continue with the study of telomerase inhibitors for cancer treatment.
Asunto(s)
Antineoplásicos/química , Proteínas de Ciclo Celular/química , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Nucleares/química , Telomerasa/química , Aminoácidos , Antineoplásicos/farmacología , Proteínas de Ciclo Celular/metabolismo , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Conformación Molecular , Mutación , Proteínas Nucleares/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Telomerasa/antagonistas & inhibidores , Telomerasa/genéticaRESUMEN
Rho GTPases are a key protein family controlling the transduction of external signals to cytoplasmatic and nuclear effectors. In the last few years, the development of genetic and pharmacological tools has allowed a more precise definition of the specific roles of Rho GTPases. The aim of this review is to describe the cellular functions regulated by these proteins with focus on the molecular mechanism involved. We also address the role of Rho GTPases in the development of different human diseases such as cancer. Finally, we describe different experimental therapeutic strategies with Rho GTPases as molecular targets.
Asunto(s)
Neoplasias/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Proteínas de Unión al GTP rho/uso terapéutico , Antineoplásicos/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Humanos , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
Las Rho GTPasas son una familia de proteínas clave en la transmisión de señales provenientes del exterior celular hacia efectores intracelulares tanto citoplasmáticos como nucleares. En los últimos año ha habido un desarrollo vertiginoso de múltiples herramientas genéticas y farmacológicas, lo que ha permitido establecer de manera mucho más precisa las funciones específicas de estas proteínas. El objetivo de la presente revisión es hacer foco en las múltiples funciones celulares reguladas por las Rho GTPasas, describiendo en detalle el mecanismo molecular involucrado. Se discute además la participación de estas proteínas en diversas enfermedades humanas haciendo énfasis en su vinculación con el cáncer. Por último, se hace una actualización detallada sobre las estrategias terapéuticas en experimentación que tienen a las Rho GTPasas como blancos moleculares.
Rho GTPases are a key protein family controlling the transduction of external signals to cytoplasmatic and nuclear effectors. In the last few years, the development of genetic and pharmacological tools has allowed a more precise definition of the specific roles of Rho GTPases. The aim of this review is to describe the cellular functions regulated by these proteins with focus on the molecular mechanism involved. We also address the role of Rho GTPases in the development of different human diseases such as cancer. Finally, we describe different experimental therapeutic strategies with Rho GTPases as molecular targets.