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1.
Bioorg Chem ; 144: 107137, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38245951

RESUMO

Based on the mimicry of microbial metabolites, functionalized indoles were demonstrated as the ligands and agonists of the pregnane X receptor (PXR). The lead indole, FKK6, displayed PXR-dependent protective effects in DSS-induced colitis in mice and in vitro cytokine-treated intestinal organoid cultures. Here, we report on the initial in vitro pharmacological profiling of FKK6. FKK6-PXR interactions were characterized by hydrogen-deuterium exchange mass spectrometry. Screening FKK6 against potential cellular off-targets (G protein-coupled receptors, steroid and nuclear receptors, ion channels, and xenobiotic membrane transporters) revealed high PXR selectivity. FKK6 has poor aqueous solubility but was highly soluble in simulated gastric and intestinal fluids. A large fraction of FKK6 was bound to plasma proteins and chemically stable in plasma. The partition coefficient of FKK6 was 2.70, and FKK6 moderately partitioned into red blood cells. In Caco2 cells, FKK6 displayed high permeability (A-B: 22.8 × 10-6 cm.s-1) and no active efflux. These data are indicative of essentially complete in vivo absorption of FKK6. The data from human liver microsomes indicated that FKK6 is rapidly metabolized by cytochromes P450 (t1/2 5 min), notably by CYP3A4. Two oxidized FKK6 derivatives, including DC73 (N6-oxide) and DC97 (C19-phenol), were detected, and these metabolites had 5-7 × lower potency as PXR agonists than FKK6. This implies that despite high intestinal absorption, FKK6 is rapidly eliminated by the liver, and its PXR effects are predicted to be predominantly in the intestines. In conclusion, the PXR ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development.


Assuntos
Colite , Humanos , Animais , Camundongos , Receptor de Pregnano X/agonistas , Células CACO-2 , Colite/induzido quimicamente , Receptores Citoplasmáticos e Nucleares , Anti-Inflamatórios/uso terapêutico
2.
Int J Mol Sci ; 23(18)2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36142735

RESUMO

Aryl hydrocarbon receptor (AHR) plays pivotal roles in intestinal physiology and pathophysiology. Intestinal AHR is activated by numerous dietary, endogenous, and microbial ligands. Whereas the effects of individual compounds on AHR are mostly known, the effects of real physiological mixtures occurring in the intestine have not been studied. Using reporter gene assays and RT-PCR, we evaluated the combinatorial effects (3520 combinations) of 11 microbial catabolites of tryptophan (MICTs) on AHR. We robustly (n = 30) determined the potencies and relative efficacies of single MICTs. Synergistic effects of MICT binary mixtures were observed between low- or medium-efficacy agonists, in particular for combinations of indole-3-propionate and indole-3-lactate. Combinations comprising highly efficacious agonists such as indole-3-pyruvate displayed rather antagonist effects, caused by saturation of the assay response. These synergistic effects were confirmed by RT-PCR as CYP1A1 mRNA expression. We also tested mimic multicomponent and binary mixtures of MICTs, prepared based on the metabolomic analyses of human feces and colonoscopy aspirates, respectively. In this case, AHR responsiveness did not correlate with type of diet or health status, and the indole concentrations in the mixtures were determinative of gross AHR activity. Future systematic research on the synergistic activation of AHR by microbial metabolites and other ligands is needed.


Assuntos
Receptores de Hidrocarboneto Arílico , Triptofano , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Humanos , Indóis/metabolismo , Indóis/farmacologia , Intestinos , Ligantes , Propionatos , Piruvatos , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Triptofano/metabolismo , Triptofano/farmacologia
3.
J Med Chem ; 65(9): 6859-6868, 2022 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-35416668

RESUMO

Targeting the aryl hydrocarbon receptor (AhR) is an emerging therapeutic strategy for multiple diseases (e.g., inflammatory bowel disease). Thermosporothrix hazakensis microbial metabolite 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a putative AhR endogenous ligand. To improve the chemical stability, we synthesized a series of ITE chemical mimics. Using a series of in vitro assays, we identified 2-(1H-indole-3-carbonyl)-N-methyl thiazole-4-carboxamide (ITE-CONHCH3) as a highly potent (EC50 = 1.6 nM) AhR agonist with high affinity (Ki = 88 nM). ITE-CONHCH3 triggered AhR nuclear translocation and dimerization of AhR-ARNT, enhanced AhR binding in the CYP1A1 promoter, and induced AhR-regulated genes in an AhR-dependent manner. The metabolic stability of ITE-CONHCH3 in a cell culture was 10 times higher than that of ITE. Finally, we observed protective effects of ITE-CONHCH3 in mice with DSS-induced colitis. Overall, we demonstrate and validate a concept of microbial metabolite mimicry in the therapeutic targeting of AhR.


Assuntos
Colite , Receptores de Hidrocarboneto Arílico , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , Citocromo P-450 CYP1A1 , Indóis/farmacologia , Indóis/uso terapêutico , Camundongos , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Tiazóis/farmacologia
4.
Bioorg Chem ; 109: 104661, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33636438

RESUMO

Microbial metabolite mimicry is a new concept that promises to deliver compounds that have minimal liabilities and enhanced therapeutic effects in a host. In a previous publication, we have shown that microbial metabolites of L-tryptophan, indoles, when chemically altered, yielded potent anti-inflammatory pregnane X Receptor (PXR)-targeting lead compounds, FKK5 and FKK6, targeting intestinal inflammation. Our aim in this study was to further define structure-activity relationships between indole analogs and PXR, we removed the phenyl-sulfonyl group or replaced the pyridyl residue with imidazolopyridyl of FKK6. Our results showed that while removal of the phenyl-sulfonyl group from FKK6 (now called CVK003) shifts agonist activity away from PXR towards the aryl hydrocarbon receptor (AhR), the imidazolopyridyl addition preserves PXR activity in vitro. However, when these compounds are administered to mice, that unlike the parent molecule, FKK6, they exhibit poor induction of PXR target genes in the intestines and the liver. These data suggest that modifications of FKK6 specifically in the pyridyl moiety can result in compounds with weak PXR activity in vivo. These observations are a significant step forward for understanding the structure-activity relationships (SAR) between indole mimics and receptors, PXR and AhR.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Indóis/química , Indóis/farmacologia , Receptor de Pregnano X/metabolismo , Adenocarcinoma , Animais , Linhagem Celular Tumoral , Neoplasias do Colo , Desenho de Fármacos , Feminino , Hepatócitos , Humanos , Intestinos , Fígado , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Receptor de Pregnano X/química , Conformação Proteica , Relação Estrutura-Atividade
5.
Mol Biol Evol ; 38(4): 1225-1240, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33247726

RESUMO

Although gene duplications provide genetic backup and allow genomic changes under relaxed selection, they may potentially limit gene flow. When different copies of a duplicated gene are pseudofunctionalized in different genotypes, genetic incompatibilities can arise in their hybrid offspring. Although such cases have been reported after manual crosses, it remains unclear whether they occur in nature and how they affect natural populations. Here, we identified four duplicated-gene based incompatibilities including one previously not reported within an artificial Arabidopsis intercross population. Unexpectedly, however, for each of the genetic incompatibilities we also identified the incompatible alleles in natural populations based on the genomes of 1,135 Arabidopsis accessions published by the 1001 Genomes Project. Using the presence of incompatible allele combinations as phenotypes for GWAS, we mapped genomic regions that included additional gene copies which likely rescue the genetic incompatibility. Reconstructing the geographic origins and evolutionary trajectories of the individual alleles suggested that incompatible alleles frequently coexist, even in geographically closed regions, and that their effects can be overcome by additional gene copies collectively shaping the evolutionary dynamics of duplicated genes during population history.


Assuntos
Arabidopsis/genética , Duplicação Gênica , Isolamento Reprodutivo , Alelos , Filogeografia
6.
Toxicol Lett ; 334: 87-93, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002526

RESUMO

The interplays between the metabolic products of intestinal microbiota and the host signaling through xenobiotic receptors, including pregnane X receptor (PXR), are of growing interest, in the context of intestinal health and disease. A distinct class of microbial catabolites is formed from dietary tryptophan, having the indole scaffold in their core structure, which is a biologically active entity. In the current study, we examined a series of ten tryptophan microbial catabolites for their interactions with PXR signaling. Utilizing a reporter gene assay, we identified indole (IND) and indole-3-acetamide (IAD) as PXR agonists. IND and IAD induced PXR-regulated genes CYP3A4 and MDR1 in human intestinal cancer cells. Using time-resolved fluorescence resonance energy transfer, we show that IND (IC50 292 µM) and IAD (IC50 10 µM) are orthosteric ligands of PXR. Binding of PXR in its DNA response elements was enhanced by IND and IAD, as revealed by chromatin immunoprecipitation assay. We demonstrate that tryptophan microbial intestinal metabolites IND and IAD are ligands and agonists of human PXR. These findings are of particular importance in understanding the roles of microbial catabolites in human physiology and pathophysiology. Furthermore, these results are seminal in expanding potential drug repertoire through microbial metabolic mimicry.


Assuntos
Microbioma Gastrointestinal , Ácidos Indolacéticos/metabolismo , Indóis/metabolismo , Mucosa Intestinal , Receptor de Pregnano X/agonistas , Triptofano/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Genes Reporter , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Ligantes , Masculino , Receptor de Pregnano X/genética , Ligação Proteica , Transfecção
7.
Int J Mol Sci ; 21(8)2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32316498

RESUMO

The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citocromo P-450 CYP1A1/genética , Hepatócitos/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Sulfonamidas/farmacologia , Triptaminas/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Células Cultivadas , Reposicionamento de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Especificidade de Órgãos , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/química , Regulação para Cima
8.
Int J Mol Sci ; 21(7)2020 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-32283770

RESUMO

We examined the effects of gut microbial catabolites of tryptophan on the aryl hydrocarbon receptor (AhR). Using a reporter gene assay, we show that all studied catabolites are low-potency agonists of human AhR. The efficacy of catabolites differed substantially, comprising agonists with no or low (i3-propionate, i3-acetate, i3-lactate, i3-aldehyde), medium (i3-ethanol, i3-acrylate, skatole, tryptamine), and high (indole, i3-acetamide, i3-pyruvate) efficacies. We displayed ligand-selective antagonist activities by i3-pyruvate, i3-aldehyde, indole, skatole, and tryptamine. Ligand binding assay identified low affinity (skatole, i3-pyruvate, and i3-acetamide) and very low affinity (i3-acrylate, i3-ethanol, indole) ligands of the murine AhR. Indole, skatole, tryptamine, i3-pyruvate, i3-acrylate, and i3-acetamide induced CYP1A1 mRNA in intestinal LS180 and HT-29 cells, but not in the AhR-knockout HT-29 variant. We observed a similar CYP1A1 induction pattern in primary human hepatocytes. The most AhR-active catabolites (indole, skatole, tryptamine, i3-pyruvate, i3-acrylate, i3-acetamide) elicited nuclear translocation of the AhR, followed by a formation of AhR-ARNT heterodimer and enhanced binding of the AhR to the CYP1A1 gene promoter. Collectively, we comprehensively characterized the interactions of gut microbial tryptophan catabolites with the AhR, which may expand the current understanding of their potential roles in intestinal health and disease.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Microbioma Gastrointestinal , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/metabolismo , Triptofano/metabolismo , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Expressão Gênica , Genes Reporter , Humanos , Indóis , Ligantes , Redes e Vias Metabólicas , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica
9.
Plant Cell ; 31(7): 1579-1597, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036599

RESUMO

The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Subunidades Proteicas/metabolismo , Sementes/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Óvulo Vegetal/genética , Pólen/genética , Ligação Proteica , Sementes/genética , Transcriptoma/genética , Regulação para Cima/genética
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