RESUMO
Toxicology in the 21st Century (Tox21) is a federal collaboration employing a high-throughput robotic screening system to test 10,000 environmental chemicals. One of the primary goals of the program is prioritizing toxicity evaluations through in vitro high-throughput screening (HTS) assays for large numbers of chemicals already in commercial use for which little or no toxicity data is available. Within the Tox21 screening program, disruption in nuclear receptor (NR) signaling represents a particular area of interest. Given the role of NR's in modulating a wide range of biological processes, alterations of their activity can have profound biological impacts. Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that has demonstrated importance in bile acid homeostasis, glucose metabolism, lipid homeostasis and hepatic regeneration. In this study, we re-evaluated 24 FXR agonists and antagonists identified through Tox21 using select orthogonal assays. In transient transactivation assays, 7/8 putative agonists and 4/4 putative inactive compounds were confirmed. Likewise, we confirmed 9/12 antagonists tested. Using a mammalian two hybrid approach we demonstrate that both FXR agonists and antagonists facilitate FXRα-coregulator interactions suggesting that differential coregulator recruitment may mediate activation/repression of FXRα mediated transcription. Additionally, we tested the ability of select FXR agonists and antagonists to facilitate hepatic transcription of FXR gene targets Shp and Bsep in a teleost (Medaka) model. Through application of in vitro cell-based assays, in silico modeling and in vivo gene expressions, we demonstrated the molecular complexity of FXR:ligand interactions and confirmed the ability of diverse ligands to modulate FXRα, facilitate differential coregulator recruitment and activate/repress receptor-mediated transcription. Overall, we suggest a multiplicative approach to assessment of nuclear receptor function may facilitate a greater understanding of the biological and mechanistic complexities of nuclear receptor activities and further our ability to interpret broad HTS outcomes.
RESUMO
We have previously described the first Bayesian machine learning models from FDA-approved drug screens, for identifying compounds active against the Ebola virus (EBOV). These models led to the identification of three active molecules in vitro: tilorone, pyronaridine, and quinacrine. A follow-up study demonstrated that one of these compounds, tilorone, has 100% in vivo efficacy in mice infected with mouse-adapted EBOV at 30 mg/kg/day intraperitoneal. This suggested that we can learn from the published data on EBOV inhibition and use it to select new compounds for testing that are active in vivo. We used these previously built Bayesian machine learning EBOV models alongside our chemical insights for the selection of 12 molecules, absent from the training set, to test for in vitro EBOV inhibition. Nine molecules were directly selected using the model, and eight of these molecules possessed a promising in vitro activity (EC50 < 15 µM). Three further compounds were selected for an in vitro evaluation because they were antimalarials, and compounds of this class like pyronaridine and quinacrine have previously been shown to inhibit EBOV. We identified the antimalarial drug arterolane (IC50 = 4.53 µM) and the anticancer clinical candidate lucanthone (IC50 = 3.27 µM) as novel compounds that have EBOV inhibitory activity in HeLa cells and generally lack cytotoxicity. This work provides further validation for using machine learning and medicinal chemistry expertize to prioritize compounds for testing in vitro prior to more costly in vivo tests. These studies provide further corroboration of this strategy and suggest that it can likely be applied to other pathogens in the future.
RESUMO
Drug resistant tuberculosis (TB) is a major worldwide health problem. In addition to the bacterial mechanisms, human drug transporters limiting the cellular accumulation and the pharmacological disposition of drugs also influence the efficacy of treatment. Mycobacterium tuberculosis topoisomerase-I (MtTopo-I) is a promising target for antimicrobial treatment. In our previous work we have identified several hit compounds targeting the MtTopo-I by in silico docking. Here we expand the scope of the compounds around three scaffolds associated with potent MtTopo-I inhibition. In addition to measuring the effect of newly generated compounds on MtTopo-I activity, we characterized the compounds' antimicrobial activity, toxicity in human cells, and interactions with human multidrug transporters. Some of the newly developed MtTopo-I inhibitors have strong antimicrobial activity and do not harm mammalian cells. Moreover, our studies revealed significant human ABC drug transporter interactions for several MtTopo-I compounds that may modify their ADME-Tox parameters and cellular effects. Promising new drug candidates may be selected based on these studies for further anti-TB drug development.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Mycobacterium tuberculosis/enzimologia , Inibidores da Topoisomerase I/metabolismo , Inibidores da Topoisomerase I/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Animais , Linhagem Celular , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Inibidores da Topoisomerase I/toxicidadeRESUMO
Tilorone dihydrochloride (tilorone) is a small-molecule, orally bioavailable drug that is used clinically as an antiviral outside the United States. A machine-learning model trained on anti-Ebola virus (EBOV) screening data previously identified tilorone as a potent in vitro EBOV inhibitor, making it a candidate for the treatment of Ebola virus disease (EVD). In the present study, a series of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays demonstrated the drug has excellent solubility, high Caco-2 permeability, was not a P-glycoprotein substrate, and had no inhibitory activity against five human CYP450 enzymes (3A4, 2D6, 2C19, 2C9, and 1A2). Tilorone was shown to have 52% human plasma protein binding with excellent plasma stability and a mouse liver microsome half-life of 48 min. Dose range-finding studies in mice demonstrated a maximum tolerated single dose of 100 mg/kg of body weight. A pharmacokinetics study in mice at 2- and 10-mg/kg dose levels showed that the drug is rapidly absorbed, has dose-dependent increases in maximum concentration of unbound drug in plasma and areas under the concentration-time curve, and has a half-life of approximately 18 h in both males and females, although the exposure was â¼2.5-fold higher in male mice. Tilorone doses of 25 and 50 mg/kg proved efficacious in protecting 90% of mice from a lethal challenge with mouse-adapted with once-daily intraperitoneal (i.p.) dosing for 8 days. A subsequent study showed that 30 mg/kg/day of tilorone given i.p. starting 2 or 24 h postchallenge and continuing through day 7 postinfection was fully protective, indicating promising activity for the treatment of EVD.
Assuntos
Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Tilorona/farmacologia , Animais , Antivirais/farmacologia , Células CACO-2 , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/efeitos dos fármacosRESUMO
GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.
Assuntos
Lisofosfolipídeos/química , Piperazinas/química , Pirrolidinas/química , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes de Fusão/química , Elemento de Resposta Sérica , Motivos de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Expressão Gênica , Células HEK293 , Humanos , Cinética , Ligantes , Lisofosfolipídeos/farmacologia , Simulação de Acoplamento Molecular , Mutação , Piperazinas/farmacologia , Ligação Proteica , Pirrolidinas/farmacologia , Receptores de Canabinoides , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides delta/química , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Resposta Sérica/química , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Glycine max , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
A series of 1,3,4-oxadiazol-2-ones was synthesized and tested for activity as antagonists at GPR55 in cellular beta-arrestin redistribution assays. The synthesis was designed to be modular in nature so that a sufficient number of analogues could be rapidly accessed to explore initial structure-activity relationships. The design of analogues was guided by the docking of potential compounds into a model of the inactive form of GPR55. The results of the assays were used to learn more about the binding pocket of GPR55. With this oxadiazolone scaffold, it was determined that modification of the aryl group adjacent to the oxadiazolone ring was often detrimental and that the distal cyclopropane was beneficial for activity. These results will guide further exploration of this receptor.