RESUMEN
Nonalcoholic steatohepatitis (NASH) is an inflammatory and fibrotic liver disease that has reached epidemic proportions and has no approved pharmacologic therapies. Research and drug development efforts are hampered by inadequate preclinical models. This research describes a three-dimensional bioprinted liver tissue model of NASH built using primary human hepatocytes and nonparenchymal liver cells (hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) from either healthy or NASH donors. Three-dimensional tissues bioprinted with cells sourced from diseased patients showed a NASH phenotype, including fibrosis. More importantly, this NASH phenotype occurred without the addition of disease-inducing agents. Bioprinted tissues composed entirely of healthy cells exhibited significantly less evidence of disease. The role of individual cell types in driving the NASH phenotype was examined by producing chimeric bioprinted tissues composed of healthy cells together with the addition of one or more diseased nonparenchymal cell types. These experiments reveal a role for both hepatic stellate and liver sinusoidal endothelial cells in the disease process. This model represents a fully human system with potential to detect clinically active targets and eventually therapies.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Células Endoteliales/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Macrófagos del Hígado/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/patologíaRESUMEN
Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. Here, we characterize verinurad (RDEA3170), which is currently under evaluation for gout therapy. Verinurad specifically inhibits URAT1 with a potency of 25 nM. High affinity inhibition of uric acid transport requires URAT1 residues Cys-32, Ser-35, Phe-365 and Ile-481. Unlike other available uricosuric agents, the requirement for Cys-32 is unique to verinurad. Two of these residues, Ser-35 and Phe-365, are also important for urate transport kinetics. A URAT1 binding assay using radiolabeled verinurad revealed that distinct URAT1 inhibitors benzbromarone, sulfinpyrazone and probenecid all inhibit verinurad binding via a competitive mechanism. However, mutations made within the predicted transporter substrate channel differentially altered the potency for individual URAT1 inhibitors. Overall, our results suggest that URAT1 inhibitors bind to a common site in the core of the transporter and sterically hinder the transit of uric acid through the substrate channel, albeit with vastly different potencies and with differential interactions with specific URAT1 amino acids.
Asunto(s)
Gota/tratamiento farmacológico , Gota/metabolismo , Hiperuricemia/tratamiento farmacológico , Hiperuricemia/metabolismo , Transportadores de Anión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Uricosúricos/uso terapéutico , Adulto , Animales , Benzbromarona/farmacología , Benzbromarona/uso terapéutico , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Concentración 50 Inhibidora , Cinética , Masculino , Terapia Molecular Dirigida , Mutación , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Unión Proteica , Ratas , Ácido Úrico/sangre , Ácido Úrico/metabolismo , Uricosúricos/farmacología , Adulto JovenRESUMEN
Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions.
Asunto(s)
Transportadores de Anión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Sustitución de Aminoácidos , Animales , Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/genética , Sitios de Unión/genética , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteína 1 de Transporte de Anión Orgánico/química , Transportadores de Anión Orgánico/química , Transportadores de Anión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/química , Proteínas de Transporte de Catión Orgánico/genética , Dominios y Motivos de Interacción de Proteínas , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ácido Úrico/metabolismoRESUMEN
BACKGROUND: Excess body burden of uric acid promotes gout. Diminished renal clearance of uric acid causes hyperuricemia in most patients with gout, and the renal urate transporter (URAT)1 is important for regulation of serum uric acid (sUA) levels. The URAT1 inhibitors probenecid and benzbromarone are used as gout therapies; however, their use is limited by drug-drug interactions and off-target toxicity, respectively. Here, we define the mechanism of action of lesinurad (Zurampic®; RDEA594), a novel URAT1 inhibitor, recently approved in the USA and Europe for treatment of chronic gout. METHODS: sUA levels, fractional excretion of uric acid (FEUA), lesinurad plasma levels, and urinary excretion of lesinurad were measured in healthy volunteers treated with lesinurad. In addition, lesinurad, probenecid, and benzbromarone were compared in vitro for effects on urate transporters and the organic anion transporters (OAT)1 and OAT3, changes in mitochondrial membrane potential, and human peroxisome proliferator-activated receptor gamma (PPARγ) activity. RESULTS: After 6 hours, a single 200-mg dose of lesinurad elevated FEUA 3.6-fold (p < 0.001) and reduced sUA levels by 33 % (p < 0.001). At concentrations achieved in the clinic, lesinurad inhibited activity of URAT1 and OAT4 in vitro, did not inhibit GLUT9, and had no effect on ABCG2. Lesinurad also showed a low risk for mitochondrial toxicity and PPARγ induction compared to benzbromarone. Unlike probenecid, lesinurad did not inhibit OAT1 or OAT3 in the clinical setting. CONCLUSION: The pharmacodynamic effects and in vitro activity of lesinurad are consistent with inhibition of URAT1 and OAT4, major apical transporters for uric acid. Lesinurad also has a favorable selectivity and safety profile, consistent with an important role in sUA-lowering therapy for patients with gout.
Asunto(s)
Gota , Transportadores de Anión Orgánico/efectos de los fármacos , Proteínas de Transporte de Catión Orgánico/efectos de los fármacos , Tioglicolatos/farmacocinética , Triazoles/farmacocinética , Ácido Úrico/sangre , Uricosúricos/farmacocinética , Línea Celular , Humanos , Riñón/efectos de los fármacos , MasculinoRESUMEN
Uric acid is the highly insoluble end-product of purine metabolism in humans. Serum levels exceeding the solubility threshold can trigger formation of urate crystals resulting in gouty arthritis. Uric acid is primarily excreted through the kidneys with 90% reabsorbed back into the bloodstream through the uric acid transporter URAT1. This reabsorption process is essential for the high serum uric acid levels found in humans. We discovered that URAT1 proteins from humans and baboons have higher affinity for uric acid compared with transporters from rats and mice. This difference in transport kinetics of URAT1 orthologs, along with inability of modern apes to oxidize uric acid due to loss of the uricase enzyme, prompted us to ask whether these events occurred concomitantly during primate evolution. Ancestral URAT1 sequences were computationally inferred and ancient transporters were resurrected and assayed, revealing that affinity for uric acid was increased during the evolution of primates. This molecular fine-tuning occurred between the origins of simians and their diversification into New- and Old-World monkey and ape lineages. Remarkably, it was driven in large-part by only a few amino acid replacements within the transporter. This alteration in primate URAT1 coincided with changes in uricase that greatly diminished the enzymatic activity and took place 27-77 Ma. These results suggest that the modifications to URAT1 transporters were potentially adaptive and that maintaining more constant, high levels of serum uric acid may have provided an advantage to our primate ancestors.
Asunto(s)
Gota/sangre , Gota/genética , Transportadores de Anión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/genética , Primates/genética , Urato Oxidasa/genética , Ácido Úrico/sangre , Animales , Evolución Biológica , Simulación por Computador , Evolución Molecular , Gota/metabolismo , Células HEK293 , Homeostasis , Hominidae , Humanos , Hiperuricemia/sangre , Hiperuricemia/genética , Ratones , Modelos Genéticos , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Filogenia , Ratas , Análisis de Secuencia de ADN , Urato Oxidasa/metabolismo , Ácido Úrico/metabolismoRESUMEN
A limited number of whole-cell assays allow monitoring of receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. We present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase (RTK) superfamily. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTKs and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for nine RTKs derived from four subfamilies [erythroblastic leukemia viral (v-erb-b) oncogene homolog (ErbB), platelet-derived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLCgamma1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in the carboxyl terminus. RTK BRET assays are highly sensitive for quantifying ligand-independent (constitutive), agonist-induced, or antagonist-inhibited RTK activity levels. We studied the signaling properties of the PDGF receptor, alpha polypeptide (PDGFRA) isoforms (V561D; D842V and delta842-845) carrying activating mutations identified in gastrointestinal stromal tumors (GIST). All three PDGFRA isoforms are fully constitutively activated, insensitive to the growth factor PDGF-BB, but show differential sensitivity of their constitutive activity to be inhibited by the inhibitor imatinib (Gleevec). Epidermal growth factor receptor (EGFR) BRET structure-function studies identify the tyrosine residues 1068, 1114, and 1148 as the main residues mediating the interaction of EGFR with the adapter protein Grb2. The BRET technology provides an assay platform to study signaling pathway-specific RTK structure-function and will facilitate drug discovery efforts for the identification of novel RTK modulators.