RESUMEN
Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by a mutation in the X-linked CDKL5 gene. CDKL5 is a serine/threonine kinase that is critical for axon outgrowth and dendritic morphogenesis as well as synapse formation, maturation, and maintenance. This disorder is characterized by early-onset epilepsy, hypotonia, and failure to reach cognitive and motor developmental milestones. Because the disease is monogenic, delivery of the CDKL5 gene to the brain of patients should provide clinical benefit. To this end, we designed a gene therapy vector, adeno-associated virus (AAV)9.Syn.hCDKL5, in which human CDKL5 gene expression is driven by the synapsin promoter. In biodistribution studies conducted in mice, intracerebroventricular (i.c.v.) injection resulted in broader, more optimal biodistribution than did intra-cisterna magna (i.c.m.) delivery. AAV9.Syn.hCDKL5 treatment increased phosphorylation of EB2, a bona fide CDKL5 substrate, demonstrating biological activity in vivo. Our data provide proof of concept that i.c.v. delivery of AAV9.Syn.hCDKL5 to neonatal male Cdkl5 knockout mice reduces pathology and reduces aberrant behavior. Functional improvements were seen at doses of 3e11 to 5e11 vector genomes/g brain, which resulted in transfection of ≥50% of the neurons. Functional improvements were not seen at lower doses, suggesting a requirement for broad distribution for efficacy.
RESUMEN
Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.
Asunto(s)
Atrofia Muscular Espinal , Empalme Alternativo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Exones , Femenino , Humanos , Masculino , Ratones , Atrofia Muscular Espinal/metabolismo , Empalme del ARN , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína 2 para la Supervivencia de la Neurona MotoraRESUMEN
Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.
Asunto(s)
Bencimidazoles/farmacología , Síndromes Mielodisplásicos/tratamiento farmacológico , Pirazinas/farmacología , Moduladores de Tubulina/farmacología , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Bencimidazoles/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Decitabina/farmacología , Fase G2/efectos de los fármacos , Células HL-60 , Xenoinjertos , Humanos , Ratones , Síndromes Mielodisplásicos/genética , Paclitaxel/farmacología , Pirazinas/uso terapéutico , Análisis de Secuencia de ARN/métodos , Tubulina (Proteína)/efectos de los fármacos , Moduladores de Tubulina/uso terapéutico , Vincristina/farmacologíaRESUMEN
Nonsense mutations resulting in a premature stop codon in an open reading frame occur in critical tumor suppressor genes in a large number of the most common forms of cancers and are known to cause or contribute to the progression of disease. Low molecular weight compounds that induce readthrough of nonsense mutations offer a new means of treating patients with genetic disorders or cancers resulting from nonsense mutations. We have identified the nucleoside analog clitocine as a potent and efficacious suppressor of nonsense mutations. We determined that incorporation of clitocine into RNA during transcription is a prerequisite for its readthrough activity; the presence of clitocine in the third position of a premature stop codon directly induces readthrough. We demonstrate that clitocine can induce the production of p53 protein in cells harboring p53 nonsense-mutated alleles. In these cells, clitocine restored production of full-length and functional p53 as evidenced by induced transcriptional activation of downstream p53 target genes, progression of cells into apoptosis, and impeded growth of nonsense-containing human ovarian cancer tumors in xenograft tumor models. Thus, clitocine induces readthrough of nonsense mutations by a previously undescribed mechanism and represents a novel therapeutic modality to treat cancers and genetic diseases caused by nonsense mutations.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Materiales Biomiméticos/farmacología , Codón sin Sentido/efectos de los fármacos , Furanos/farmacología , Nucleósidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Nucleósidos de Pirimidina/farmacología , Proteína p53 Supresora de Tumor/agonistas , Animales , Antimetabolitos Antineoplásicos/síntesis química , Antimetabolitos Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/metabolismo , Línea Celular Tumoral , Femenino , Furanos/síntesis química , Furanos/metabolismo , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Desnudos , Nucleósidos/síntesis química , Nucleósidos/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Biosíntesis de Proteínas , Nucleósidos de Pirimidina/síntesis química , Nucleósidos de Pirimidina/metabolismo , Transducción de Señal , Activación Transcripcional , Carga Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a â¼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment.
Asunto(s)
Isocumarinas/administración & dosificación , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/genética , Piperazinas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/farmacocinética , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Exones/genética , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Ratones Transgénicos , Atrofia Muscular Espinal/sangre , Atrofia Muscular Espinal/patología , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genética , Piel/metabolismo , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Proteína 2 para la Supervivencia de la Neurona Motora/sangreRESUMEN
The continued emergence of bacteria resistant to current standard of care antibiotics presents a rapidly growing threat to public health. New chemical entities (NCEs) to treat these serious infections are desperately needed. Herein we report the discovery, synthesis, SAR and in vivo efficacy of a novel series of 4-hydroxy-2-pyridones exhibiting activity against Gram-negative pathogens. Compound 1c, derived from the N-debenzylation of 1b, preferentially inhibits bacterial DNA synthesis as determined by standard macromolecular synthesis assays. The structural features of the 4-hydroxy-2-pyridone scaffold required for antibacterial activity were explored and compound 6q, identified through further optimization of the series, had an MIC90 value of 8⯵g/mL against a panel of highly resistant strains of E. coli. In a murine septicemia model, compound 6q exhibited a PD50 of 8â¯mg/kg in mice infected with a lethal dose of E. coli. This novel series of 4-hydroxy-2-pyridones serves as an excellent starting point for the identification of NCEs treating Gram-negative infections.
Asunto(s)
Antibacterianos/metabolismo , Compuestos de Azabiciclo/química , ADN/metabolismo , Niacina/análogos & derivados , Piridinas/química , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Compuestos de Azabiciclo/metabolismo , Compuestos de Azabiciclo/farmacología , Compuestos de Azabiciclo/uso terapéutico , ADN/química , Evaluación Preclínica de Medicamentos , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/veterinaria , Semivida , Ratones , Pruebas de Sensibilidad Microbiana , Niacina/metabolismo , Niacina/farmacología , Niacina/uso terapéutico , Piridinas/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Relación Estructura-ActividadRESUMEN
Dihydroorotate dehydrogenase (DHODH) is the enzyme that catalyzes a rate-determining step during the de novo synthesis of uridine, an important source of cellular pyrimidine nucleotides. Ability to modulate the activity of this enzyme may be used to control diseases associated with rapid, out-of-control cell growth in oncology, immunology, and virology. Emvododstat (PTC299) is a tetrahydro-ß-carboline DHODH inhibitor discovered through the GEMS technology (Gene Expression Modulation by Small-Molecules). Described in this paper is the lead optimization campaign that culminated in the discovery of this highly potent DHODH inhibitor.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/farmacología , CarbamatosRESUMEN
Blocking the pyrimidine nucleotide de novo synthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH) results in the cell cycle arrest and/or differentiation of rapidly proliferating cells including activated lymphocytes, cancer cells, or virally infected cells. Emvododstat (PTC299) is an orally bioavailable small molecule that inhibits DHODH. We evaluated the potential for emvododstat to inhibit the progression of acute myeloid leukemia (AML) using several in vitro and in vivo models of the disease. Broad potent activity was demonstrated against multiple AML cell lines, AML blasts cultured ex vivo from patient blood samples, and AML tumor models including patient-derived xenograft models. Emvododstat induced differentiation, cytotoxicity, or both in primary AML patient blasts cultured ex vivo with 8 of 10 samples showing sensitivity. AML cells with diverse driver mutations were sensitive, suggesting the potential of emvododstat for broad therapeutic application. AML cell lines that are not sensitive to emvododstat are likely to be more reliant on the salvage pathway than on de novo synthesis of pyrimidine nucleotides. Pharmacokinetic experiments in rhesus monkeys demonstrated that emvododstat levels rose rapidly after oral administration, peaking about 2 hours post-dosing. This was associated with an increase in the levels of dihydroorotate (DHO), the substrate for DHODH, within 2 hours of dosing indicating that DHODH inhibition is rapid. DHO levels declined as drug levels declined, consistent with the reversibility of DHODH inhibition by emvododstat. These preclinical findings provide a rationale for clinical evaluation of emvododstat in an ongoing Phase 1 study of patients with relapsed/refractory acute leukemias.
RESUMEN
Growing evidence has demonstrated that clonogenic cancer stem (initiating) cells are responsible for tumor regrowth and disease relapse. Bmi-1 plays a critical role in the self-renewal of adult stem cells. The Bmi-1 protein is elevated in many types of cancers, and experimental reduction of Bmi-1 protein levels by small interfering RNA (siRNA) causes apoptosis and/or senescence in tumor cells in vitro and increases susceptibility to cytotoxic agents. The Bmi-1 protein has no known enzymatic activity, but serves as the key regulatory component of the PRC1 complex (polycomb repressive complex-1). This complex influences chromatin structure and regulates transcriptional activity of a number of important loci including the Ink4a locus which encodes the tumor suppressor proteins p16(Ink4a) and p14(Arf) . In this prospective study, we will discuss the implication of BMI1 in cancers, the biology of BMI1, and the regulatory control of BMI1 expression. The target validation and the future prospects of targeting BMI1 in cancer therapy are also discussed.
Asunto(s)
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genéticaRESUMEN
PTC596 is an investigational small-molecule tubulin-binding agent. Unlike other tubulin-binding agents, PTC596 is orally bioavailable and is not a P-glycoprotein substrate. So as to characterize PTC596 to position the molecule for optimal clinical development, the interactions of PTC596 with tubulin using crystallography, its spectrum of preclinical in vitro anticancer activity, and its pharmacokinetic-pharmacodynamic relationship were investigated for efficacy in multiple preclinical mouse models of leiomyosarcomas and glioblastoma. Using X-ray crystallography, it was determined that PTC596 binds to the colchicine site of tubulin with unique key interactions. PTC596 exhibited broad-spectrum anticancer activity. PTC596 showed efficacy as monotherapy and additive or synergistic efficacy in combinations in mouse models of leiomyosarcomas and glioblastoma. PTC596 demonstrated efficacy in an orthotopic model of glioblastoma under conditions where temozolomide was inactive. In a first-in-human phase I clinical trial in patients with cancer, PTC596 monotherapy drug exposures were compared with those predicted to be efficacious based on mouse models. PTC596 is currently being tested in combination with dacarbazine in a clinical trial in adults with leiomyosarcoma and in combination with radiation in a clinical trial in children with diffuse intrinsic pontine glioma.
Asunto(s)
Bencimidazoles/farmacología , Glioblastoma/tratamiento farmacológico , Leiomiosarcoma/tratamiento farmacológico , Pirazinas/farmacología , Moduladores de Tubulina/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Bencimidazoles/farmacocinética , Proliferación Celular , Femenino , Glioblastoma/patología , Humanos , Leiomiosarcoma/patología , Masculino , Dosis Máxima Tolerada , Ratones , Ratones Desnudos , Persona de Mediana Edad , Pronóstico , Pirazinas/farmacocinética , Distribución Tisular , Moduladores de Tubulina/farmacocinética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.
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Bencimidazoles/farmacología , Proliferación Celular/efectos de los fármacos , Mieloma Múltiple/patología , Inhibidores de Proteasoma/farmacología , Pirazinas/farmacología , Tubulina (Proteína)/metabolismo , Animales , Bencimidazoles/administración & dosificación , Bencimidazoles/metabolismo , Bortezomib/administración & dosificación , Bortezomib/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Quimioterapia Combinada , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Polimerizacion , Inhibidores de Proteasoma/administración & dosificación , Pirazinas/administración & dosificación , Pirazinas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.
Asunto(s)
Síndromes Mielodisplásicos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Animales , ADN , Decitabina/farmacología , Dihidroorotato Deshidrogenasa , Ratones , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genéticaRESUMEN
G418 is currently the most potent and active aminoglycoside to promote readthrough of eukaryotic nonsense mutations. However, owing to its toxicity G418 cannot be used in vivo to study readthrough activity A robust and scalable method for selective derivatization of G418 was developed to study the biological activity and toxicity of a series of analogs. Despite our synthetic efforts, an improvement in readthrough potency was not achieved. We discovered several analogs that demonstrated reduced zebra fish hair cell toxicity (a surrogate for ototoxicity), but this reduction in cellular toxicity did not translate to reduced in vivo toxicity in rats.
Asunto(s)
Aminoglicósidos/farmacología , Gentamicinas/farmacología , Cabello/efectos de los fármacos , Aminoglicósidos/síntesis química , Aminoglicósidos/química , Animales , Gentamicinas/química , Conformación Molecular , Ratas , Pez CebraRESUMEN
PURPOSE: Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer that is broadly chemoresistant, due in part to biophysical properties of tumor stroma, which serves as a barrier to drug delivery for most classical chemotherapeutic drugs. The goal of this work is to evaluate the preclinical efficacy and mechanisms of PTC596, a novel agent with potent anticancer properties in vitro and desirable pharmacologic properties in vivo.Experimental Design: We assessed the pharmacology, mechanism, and preclinical efficacy of PTC596 in combination with standards of care, using multiple preclinical models of PDA. RESULTS: We found that PTC596 has pharmacologic properties that overcome the barrier to drug delivery in PDA, including a long circulating half-life, lack of P-glycoprotein substrate activity, and high systemic tolerability. We also found that PTC596 combined synergistically with standard clinical regimens to improve efficacy in multiple model systems, including the chemoresistant genetically engineered "KPC" model of PDA. Through mechanistic studies, we learned that PTC596 functions as a direct microtubule polymerization inhibitor, yet a prior clinical trial found that it lacks peripheral neurotoxicity, in contrast to other such agents. Strikingly, we found that PTC596 synergized with the standard clinical backbone regimen gemcitabine/nab-paclitaxel, yielding potent, durable regressions in a PDX model. Moreover, similar efficacy was achieved in combination with nab-paclitaxel alone, highlighting a specific synergistic interaction between two different microtubule-targeted agents in the setting of pancreatic ductal adenocarcinoma. CONCLUSIONS: These data demonstrate clear rationale for the development of PTC596 in combination with standard-of-care chemotherapy for PDA.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/metabolismo , Microtúbulos/metabolismo , Neoplasias Pancreáticas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Moduladores de Tubulina/farmacología , Albúminas/farmacología , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/mortalidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Inmunohistoquímica , Ratones , Microtúbulos/química , Paclitaxel/farmacología , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/mortalidad , Moduladores de Tubulina/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematologic cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability, and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies.
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Neoplasias Hematológicas/tratamiento farmacológico , Imidazoles/administración & dosificación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Tiazoles/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dihidroorotato Deshidrogenasa , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/enzimología , Humanos , Imidazoles/farmacología , Células K562 , Ratones , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/sangre , Tiazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.
Asunto(s)
Neoplasias Cerebelosas/tratamiento farmacológico , Meduloblastoma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Complejo Represivo Polycomb 1/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Niño , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Ratones , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 1/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nonsense mutations, resulting in a premature stop codon in the open reading frame of mRNAs are responsible for thousands of inherited diseases. Readthrough of premature stop codons by small molecule drugs has emerged as a promising therapeutic approach to treat disorders resulting from premature termination of translation. The aminoglycoside antibiotics are a class of molecule known to promote readthrough at premature termination codons. Gentamicin consists of a mixture of major and minor aminoglycoside components. Here, we investigated the readthrough activities of the individual components and show that each of the four major gentamicin complex components representing 92-99% of the complex each had similar potency and activity to that of the complex itself. In contrast, a minor component (gentamicin X2) was found to be the most potent and active readthrough component in the gentamicin complex. The known oto- and nephrotoxicity associated with aminoglycosides preclude long-term use as readthrough agents. Thus, we evaluated the components of the gentamicin complex as well as the so-called "designer" aminoglycoside, NB124, for in vitro and in vivo safety. In cells, we observed that gentamicin X2 had a safety/readthrough ratio (cytotoxicity/readthrough potency) superior to that of gentamicin, G418 or NB124. In rodents, we observed that gentamicin X2 showed a safety profile that was superior to G418 overall including reduced nephrotoxicity. These results support further investigation of gentamicin X2 as a therapeutic readthrough agent.
Asunto(s)
Codón sin Sentido/síntesis química , Enfermedades Genéticas Congénitas/tratamiento farmacológico , Gentamicinas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Aminoglicósidos/farmacología , Aminoglicósidos/uso terapéutico , Animales , Antibióticos Antineoplásicos/farmacología , Células Cultivadas , Codón de Terminación/síntesis química , Embrión no Mamífero , Gentamicinas/química , Gentamicinas/uso terapéutico , Humanos , Enfermedades Renales/inducido químicamente , Enfermedades Renales/patología , Masculino , Sistemas de Lectura Abierta/efectos de los fármacos , Sistemas de Lectura Abierta/genética , Inhibidores de la Síntesis de la Proteína/uso terapéutico , Ratas , Ratas Sprague-Dawley , Pez Cebra/embriologíaRESUMEN
There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC90 values against Escherichia coli (0.5-1 µg/mL) and Acinetobacter baumannii (8-16 µg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.
Asunto(s)
Antibacterianos/farmacología , ADN-Topoisomerasas de Tipo II/química , Descubrimiento de Drogas , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Sepsis/tratamiento farmacológico , Inhibidores de Topoisomerasa II/farmacología , Animales , Antibacterianos/química , Femenino , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Piridinas/química , Sepsis/microbiología , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/químicaRESUMEN
Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal "homeostatic" state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5' and 3' untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Antineoplásicos/administración & dosificación , Ensayos Analíticos de Alto Rendimiento/métodos , Neoplasias/tratamiento farmacológico , Regiones no Traducidas/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genética , Administración Oral , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/farmacología , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Ratones , Neoplasias/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The underlying cause of spinal muscular atrophy (SMA) is a deficiency of the survival motor neuron (SMN) protein. Starting from hits identified in a high-throughput screening campaign and through structure-activity relationship investigations, we have developed small molecules that potently shift the alternative splicing of the SMN2 exon 7, resulting in increased production of the full-length SMN mRNA and protein. Three novel chemical series, represented by compounds 9, 14, and 20, have been optimized to increase the level of SMN protein by >50% in SMA patient-derived fibroblasts at concentrations of <160 nM. Daily administration of these compounds to severe SMA Δ7 mice results in an increased production of SMN protein in disease-relevant tissues and a significant increase in median survival time in a dose-dependent manner. Our work supports the development of an orally administered small molecule for the treatment of patients with SMA.