RESUMO
Dihydroorotate dehydrogenase (DHODH) catalyzes a key step in pyrimidine biosynthesis and has recently been validated as a therapeutic target for malaria through clinical studies on the triazolopyrimidine-based Plasmodium DHODH inhibitor DSM265. Selective toxicity towards Plasmodium species could be achieved because malaria parasites lack pyrimidine salvage pathways, and DSM265 selectively inhibits Plasmodium DHODH over the human enzyme. However, while DSM265 does not inhibit human DHODH, it inhibits DHODH from several preclinical species, including mice, suggesting that toxicity could result from on-target DHODH inhibition in those species. We describe here the use of dihydroorotate (DHO) as a biomarker of DHODH inhibition. Treatment of mammalian cells with DSM265 or the mammalian DHODH inhibitor teriflunomide led to increases in DHO where the extent of biomarker buildup correlated with both dose and inhibitor potency on DHODH. Treatment of mice with leflunomide (teriflunomide prodrug) caused a large dose-dependent buildup of DHO in blood (up to 16-fold) and urine (up to 5,400-fold) that was not observed for mice treated with DSM265. Unbound plasma teriflunomide levels reached 20-85-fold above the mouse DHODH IC50, while free DSM265 levels were only 1.6-4.2-fold above, barely achieving â¼ IC90 concentrations, suggesting that unbound DSM265 plasma levels are not sufficient to block the pathway in vivo. Thus, any toxicity associated with DSM265 treatment in mice is likely caused by off-target mechanisms. The identification of a robust biomarker for mammalian DHODH inhibition represents an important advance to generally monitor for on-target effects in preclinical and clinical applications of DHODH inhibitors used to treat human disease.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Pró-Fármacos , Animais , Biomarcadores , Crotonatos , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Hidroxibutiratos , Leflunomida/farmacologia , Leflunomida/uso terapêutico , Mamíferos/metabolismo , Camundongos , Nitrilas , Plasmodium falciparum/metabolismo , Pró-Fármacos/farmacologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , ToluidinasRESUMO
OBJECTIVES: To investigate the efficacy and potential molecular mechanism of Huangkui capsule in combination with leflunomide (HKL) for the treatment of immunoglobulin A nephropathy (IgAN). METHODS: IgAN rat models were constructed by treating rats with bovine serum albumin, lipopolysaccharide, and tetrachloromethane. Th22 cells were isolated from the blood samples of patients with IgAN using a CD4+ T cell isolation kit. The expression levels of the components of the TGF-ß1/Smad3 signaling pathway, namely, TGF-ß1, Smad2, Smad3, Smad4, and Smad7, were detected using quantitative reverse transcription polymerase chain reaction. Cell proliferation was determined using the MTT assay, cell viability was determined using the WST 1 method, and the chemotaxis of Th22 cells was observed using the wound healing assay. Changes in the histology of the kidney tissues were analyzed using hematoxylin and eosin staining. RESULTS: Compared with IgAN rats, the rats subjected to HKL treatment showed good improvement in kidney injuries, and the combined drug treatment performed much better than the single-drug treatment. In addition, following HKL treatment, the viability, proliferation, and chemotaxis of Th22 cells dramatically decreased (*p<0.05, **p<0.01, and ***p<0.001). In addition, CCL20, CCL22, and CCL27 levels decreased and the expression of the key components of the TGF-ß1/Smad3 signaling pathway was downregulated in IgAN rats and Th22 cells (*p<0.05, ***p<0.001). CONCLUSIONS: By targeting the TGF-ß1/Smad3 signaling pathway, HKL treatment can improve kidney injury in IgAN rats as well as the excessive proliferation and metastasis of Th22 cells.
Assuntos
Medicamentos de Ervas Chinesas , Glomerulonefrite por IGA , Leflunomida , Proteína Smad3 , Fator de Crescimento Transformador beta1 , Animais , Medicamentos de Ervas Chinesas/farmacologia , Glomerulonefrite por IGA/tratamento farmacológico , Glomerulonefrite por IGA/metabolismo , Humanos , Rim/metabolismo , Leflunomida/farmacologia , Ratos , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
OBJECTIVES: To investigate the efficacy and potential molecular mechanism of Huangkui capsule in combination with leflunomide (HKL) for the treatment of immunoglobulin A nephropathy (IgAN) METHODS: IgAN rat models were constructed by treating rats with bovine serum albumin, lipopolysaccharide, and tetrachloromethane. Th22 cells were isolated from the blood samples of patients with IgAN using a CD4+ T cell isolation kit. The expression levels of the components of the TGF-β1/Smad3 signaling pathway, namely, TGF-β1, Smad2, Smad3, Smad4, and Smad7, were detected using quantitative reverse transcription polymerase chain reaction. Cell proliferation was determined using the MTT assay, cell viability was determined using the WST 1 method, and the chemotaxis of Th22 cells was observed using the wound healing assay. Changes in the histology of the kidney tissues were analyzed using hematoxylin and eosin staining. RESULTS: Compared with IgAN rats, the rats subjected to HKL treatment showed good improvement in kidney injuries, and the combined drug treatment performed much better than the single-drug treatment. In addition, following HKL treatment, the viability, proliferation, and chemotaxis of Th22 cells dramatically decreased (*p<0.05, **p<0.01, and ***p<0.001). In addition, CCL20, CCL22, and CCL27 levels decreased and the expression of the key components of the TGF-β1/Smad3 signaling pathway was downregulated in IgAN rats and Th22 cells (*p<0.05, ***p<0.001). CONCLUSIONS: By targeting the TGF-β1/Smad3 signaling pathway, HKL treatment can improve kidney injury in IgAN rats as well as the excessive proliferation and metastasis of Th22 cells.
Assuntos
Humanos , Animais , Ratos , Medicamentos de Ervas Chinesas/farmacologia , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Leflunomida/farmacologia , Glomerulonefrite por IGA/metabolismo , Glomerulonefrite por IGA/tratamento farmacológico , Transdução de Sinais , Rim/metabolismoRESUMO
Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.
Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Oxirredutases/antagonistas & inibidores , Pandemias , Pneumonia Viral/tratamento farmacológico , Vírus de RNA/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Sítios de Ligação/efeitos dos fármacos , COVID-19 , Linhagem Celular , Infecções por Coronavirus/virologia , Crotonatos/farmacologia , Síndrome da Liberação de Citocina/tratamento farmacológico , Di-Hidro-Orotato Desidrogenase , Avaliação Pré-Clínica de Medicamentos , Técnicas de Inativação de Genes , Humanos , Hidroxibutiratos , Vírus da Influenza A/efeitos dos fármacos , Leflunomida/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Nitrilas , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/uso terapêutico , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Pirimidinas/biossíntese , Vírus de RNA/fisiologia , SARS-CoV-2 , Relação Estrutura-Atividade , Tiazóis/uso terapêutico , Toluidinas/farmacologia , Ubiquinona/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.