RESUMEN
BACKGROUND: Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2) is activated downstream of p38 MAPK and regulates stability of mRNAs encoding inflammatory cytokines. CC-99677 is a novel, irreversible, covalent MK2 inhibitor under development for the treatment of ankylosing spondylitis (AS) and other inflammatory diseases. As part of a phase I clinical trial to assess safety and tolerability, we evaluated target engagement, pharmacokinetics, and pharmacodynamics of CC-99677. METHODS: The MK2 inhibitor CC-99677 was evaluated for its effect on cytokine expression in vitro in peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with a definitive AS diagnosis. A novel in vitro model was developed to compare the potential for tachyphylaxis of CC-99677 and p38 inhibitors in THP-1 cells. The effect of CC-99677 on tristetraprolin (TTP) and cytokine mRNA was assessed in stimulated human monocyte-derived macrophages. In a first-in-human study, thirty-seven healthy volunteers were randomly assigned to daily oral doses of CC-99677 or placebo, and blood was collected at pre-specified time points before and after dosing. CC-99677 concentrations were assessed in the plasma, and CC-99677 binding to MK2 was evaluated in PBMCs. Ex vivo stimulation of the whole blood was conducted from participants in the first-in-human study to assess the pharmacodynamic effects. RESULTS: In vitro, CC-99677 inhibited tumor necrosis factor (TNF), interleukin (IL)-6, and IL-17 protein production in samples of monocytes and macrophages from AS patients and healthy volunteers via an mRNA-destabilization mechanism. In the in vitro model of tachyphylaxis, CC-99677 showed a differentiated pattern of sustained TNF protein inhibition compared with p38 inhibitors. CC-99677 reduced TTP phosphorylation and accelerated the decay of inflammatory cytokine mRNA in lipopolysaccharide-stimulated macrophages. Administration of CC-99677 to healthy volunteers was safe and well-tolerated, with linear pharmacokinetics and sustained reduction of ex vivo whole blood TNF, IL-6, and chemokine synthesis. CONCLUSIONS: CC-99677 inhibition of MK2 is a promising approach for the treatment of inflammatory diseases and may overcome the limitations of p38 MAPK inhibition. TRIAL REGISTRATION: ClinicalTrials.gov NCT03554993 .
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas , Citocinas/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , ARN Mensajero , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
As an anti-inflammatory strategy, MAPK-activated protein kinase-2 (MK2) inhibition can potentially avoid the clinical failures seen for direct p38 inhibitors, especially tachyphylaxis. CC-99677, a selective targeted covalent MK2 inhibitor, employs a rare chloropyrimidine that bonds to the sulfur of cysteine 140 in the ATP binding site via a nucleophilic aromatic substitutions (SNAr) mechanism. This irreversible mechanism translates biochemical potency to cells shown by potent inhibition of heat shock protein 27 (HSP27) phosphorylation in LPS-activated monocytic THP-1 cells. The cytokine inhibitory profile of CC-99677 differentiates it from known p38 inhibitors, potentially suppressing a p38 pathway inflammatory response while avoiding tachyphylaxis. Dosed orally, CC-99677 is efficacious in a rat model of ankylosing spondylitis. Single doses, 3 to 400 mg, in healthy human volunteers show linear pharmacokinetics and apparent sustained tumor necrosis factor-α inhibition, with a favorable safety profile. These results support further development of CC-99677 for autoimmune diseases like ankylosing spondylitis.
Asunto(s)
Enfermedades Autoinmunes , Espondilitis Anquilosante , Adenosina Trifosfato , Animales , Antiinflamatorios , Enfermedades Autoinmunes/tratamiento farmacológico , Cisteína , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos , Proteínas Serina-Treonina Quinasas , Ratas , Azufre , Factor de Necrosis Tumoral alfa , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Quinolinate phosphoribosyltransferase was examined for susceptibility to different chemical modification reagents. Loss of enzyme activity with trinitrobenzenesulfonate (TNBS) occurred when 1.1 lysines per subunit were modified. Tryptic digestion of the modified enzyme followed by HPLC-MS analysis of the peptides showed Lys70 reacts with TNBS. Based on x-ray studies, this amino acid participates in a conformational change distant from the active site.
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Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Lisina/química , Pentosiltransferasa/antagonistas & inhibidores , Pentosiltransferasa/metabolismo , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Secuencia Conservada , Inhibidores Enzimáticos/metabolismo , Lisina/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Molecular , Pentosiltransferasa/química , Alineación de SecuenciaRESUMEN
Recent studies of human cancers and mutant mouse models have implicated the Nkx3.1 homeobox gene as having a key role in prostate carcinogenesis. Consistent with such a role, here we show that Nkx3.1 displays growth-suppressing activities in cell culture, and that aged Nkx3.1 mutant mice display histopathological defects resembling prostatic intraepithelial neoplasia (PIN), the presumed precursor of human prostate cancer. Using a tissue recombination approach, we found that PIN-like lesions from Nkx3.1 mutants can undergo progressively severe histopathological alterations after serial transplantation in nude mice. Our findings indicate that Nkx3.1 loss-of-function is a critical event in prostate cancer initiation, and that Nkx3.1 mutant mice accurately model early stages of prostate carcinogenesis. More generally, our tissue recombination assay provides an empirical test to examine the relationship of PIN to prostate carcinoma.
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Proteínas de Homeodominio/genética , Neoplasia Intraepitelial Prostática/genética , Neoplasias de la Próstata/genética , Factores de Transcripción/genética , Animales , División Celular/genética , División Celular/fisiología , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genes Supresores de Tumor , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/fisiología , Endogamia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Desnudos , Mutación , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/patología , Factores de Transcripción/fisiologíaRESUMEN
UNLABELLED: Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. IMPLICATIONS: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.
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Carcinoma Hepatocelular/metabolismo , Proliferación Celular/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Xenoinjertos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/química , Invasividad Neoplásica , Oligorribonucleótidos Antisentido/uso terapéuticoRESUMEN
Teriflunomide is an oral disease-modifying therapy recently approved in several locations for relapsing-remitting multiple sclerosis. To gain insight into the effects of teriflunomide, immunocyte population changes were measured during progression of experimental autoimmune encephalomyelitis in Dark Agouti rats. Treatment with teriflunomide attenuated levels of spinal cord-infiltrating T cells, natural killer cells, macrophages, and neutrophils. Teriflunomide also mitigated the disease-induced changes in immune cell populations in the blood and spleen suggesting an inhibitory effect on pathogenic immune responses.