Targeting non-alcoholic fatty liver disease: Design, X-ray co-crystal structure and synthesis of 'first-in-kind' inhibitors of serine/threonine kinase25.

We describe the synthesis of a series of 3-t-butyl 5-aminopyrazole p-substituted arylamides as inhibitors of serine-threonine25 (STK25), an enzyme implicated in the progression of non-alcoholic fatty liver disease (NAFLD). Appending a p-N-pyrrolidinosulphonamide group to the arylamide group led to a 'first-in kind' inhibitor with IC50 = 228 nM. A co-crystal structure with STK 25 revealed productive interactions which were also reproduced using molecular docking. A new series of triazolo dihydro oxazine carboxamides of 3-t-butyl 5-aminopyrazole was not active against STK25.

A molecule inducing androgen receptor degradation and selectively targeting prostate cancer cells.

Aberrant androgen signaling drives prostate cancer and is targeted by drugs that diminish androgen production or impede androgen-androgen receptor (AR) interaction. Clinical resistance arises from AR overexpression or ligand-independent constitutive activation, suggesting that complete AR elimination could be a novel therapeutic strategy in prostate cancers. IRC117539 is a new molecule that targets AR for proteasomal degradation. Exposure to IRC117539 promotes AR sumoylation and ubiquitination, reminiscent of therapy-induced PML/RARA degradation in acute promyelocytic leukemia. Critically, ex vivo, IRC117539-mediated AR degradation induces prostate cancer cell viability loss by inhibiting AR signaling, even in androgen-insensitive cells. This approach may be beneficial for castration-resistant prostate cancer, which remains a clinical issue. In xenograft models, IRC117539 is as potent as enzalutamide in impeding growth, albeit less efficient than expected from ex vivo studies. Unexpectedly, IRC117539 also behaves as a weak proteasome inhibitor, likely explaining its suboptimal efficacy in vivo. Our studies highlight the feasibility of AR targeting for degradation and off-target effects' importance in modulating drug activity in vivo.

Transcriptional activation of apolipoprotein CIII expression by glucose may contribute to diabetic dyslipidemia.

OBJECTIVE: Hypertriglyceridemia and fatty liver are common in patients with type 2 diabetes, but the factors connecting alterations in glucose metabolism with plasma and liver lipid metabolism remain unclear. Apolipoprotein CIII (apoCIII), a regulator of hepatic and plasma triglyceride metabolism, is elevated in type 2 diabetes. In this study, we analyzed whether apoCIII is affected by altered glucose metabolism. METHODS AND RESULTS: Liver-specific insulin receptor-deficient mice display lower hepatic apoCIII mRNA levels than controls, suggesting that factors other than insulin regulate apoCIII in vivo. Glucose induces apoCIII transcription in primary rat hepatocytes and immortalized human hepatocytes via a mechanism involving the transcription factors carbohydrate response element-binding protein and hepatocyte nuclear factor-4α. ApoCIII induction by glucose is blunted by treatment with agonists of farnesoid X receptor and peroxisome proliferator-activated receptor-α but not liver X receptor, ie, nuclear receptors controlling triglyceride metabolism. Moreover, in obese humans, plasma apoCIII protein correlates more closely with plasma fasting glucose and glucose excursion after oral glucose load than with insulin. CONCLUSIONS: Glucose induces apoCIII transcription, which may represent a mechanism linking hyperglycemia, hypertriglyceridemia, and cardiovascular disease in type 2 diabetes.

BN82451 attenuates L-dopa-induced dyskinesia in 6-OHDA-lesioned rat model of Parkinson's disease.

The development of L-dopa-induced dyskinesia (LID) remains a major problem in the long-term treatment of Parkinson's disease (PD). This study aimed to assess the effect of the multitargeting molecule BN82451 on LID and to measure striatal mRNA expression of several genes in a rat model of PD. Rats were administered two unilateral injections of 6-OHDA in the striatum. After four weeks, the animals started a chronic daily treatment with increasing doses of L-dopa over a further four-week period. Over the course of L-dopa treatment, the rats developed abnormal involuntary movements (AIMs) classified as locomotive, axial, orolingual and forelimb dyskinesia. In animals rendered dyskinetic by L-dopa, administration of BN82451 at doses ranging from 1 to 10 mg/kg p.o. attenuated the severity of fully-established AIMs in a dose-related manner. This anti-dyskinetic effect could be achieved with lower doses of BN82451 administered sub chronically vs. acute single treatment. The improvement of AIMs is not due to a reduction in the general motor activity of dyskinetic rats. BN82451 treatment significantly reversed the overexpression of c-Fos, FosB and Arc mRNA associated with the dyskinesiogenic action of L-dopa. A significant correlation between the degree of overexpression of c-Fos, FosB and Arc mRNA and the dyskinesiogenic action of L-dopa was observed. The data demonstrate that BN82451 effectively attenuates LID and the associated molecular alterations in an animal model of PD and may represent a treatment option for managing dyskinesia.

Glucose regulates the expression of the farnesoid X receptor in liver.

An increased prevalence of hypertriglyceridemia and gallbladder disease occurs in patients with diabetes or insulin resistance. Hypertriglyceridemia is positively associated to gall bladder disease risk. The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that plays a key role in bile acid and triglyceride homeostasis. The mechanisms controlling FXR gene expression are poorly understood. This study evaluated whether FXR gene expression is regulated by alterations in glucose homeostasis. FXR expression was decreased in livers of streptozotocin-induced diabetic rats and normalized upon insulin supplementation. Concomitantly with diabetes progression, FXR expression also decreased in aging diabetic Zucker rats. In primary rat hepatocytes, D-glucose increased FXR mRNA in a dose- and time-dependent manner, whereas insulin counteracted this effect. Addition of xylitol, a precursor of xylulose-5-phosphate, to primary rat hepatocytes increased FXR expression to a comparable level as D-glucose. Finally, expression of the FXR target genes, SHP and apolipoprotein C-III, were additively regulated by D-glucose and FXR ligands. This study demonstrates that FXR is decreased in animal models of diabetes. In addition, FXR is regulated by glucose likely via the pentose phosphate pathway. Dysregulation of FXR expression may contribute to alterations in lipid and bile acid metabolism in patients with diabetes or insulin resistance.

Transcriptional regulation of human Rev-erbalpha gene expression by the orphan nuclear receptor retinoic acid-related orphan receptor alpha.

The Rev-erb and retinoic acid-related orphan receptors (ROR) are two related families of orphan nuclear receptors that recognize similar response elements but have opposite effects on transcription. Recently, the Rev-erbalpha gene promoter has been characterized and shown to harbor a functional Rev-erbalpha-binding site known as Rev-DR2, responsible for negative feedback down-regulation of promoter activity by Rev-erbalpha itself. The present study aimed to investigate whether Rev-erbalpha gene expression is regulated by RORalpha. Gel shift analysis demonstrated that in vitro translated hRORalpha1 protein binds to the Rev-DR2 site, both as monomer and dimer. Chromatin immunoprecipitation assays demonstrated that binding of RORalpha to this site also occurred in vivo in human hepatoma HepG2 cells. The Rev-DR2 site was further shown to be functional as it conferred hRORalpha1 responsiveness to a heterologous promoter and to the natural human Rev-erbalpha gene promoter in these cells. Mutation of this site in the context of the natural Rev-erbalpha gene promoter abolished its activation by RORalpha, indicating that this site plays a key role in hRORalpha1 action. Finally, adenoviral overexpression of hRORalpha1 in HepG2 cells led to enhanced hRev-erbalpha mRNA accumulation, further confirming the physiological importance of RORalpha1 in the regulation of Rev-erbalpha expression.

ATP-dependent mobilization of the glucocorticoid receptor during chromatin remodeling.

Chromatin remodeling by the glucocorticoid receptor (GR) is associated with activation of transcription at the mouse mammary tumor virus (MMTV) promoter. We reconstituted this nucleoprotein transition with chromatin assembled on MMTV DNA. The remodeling event was ATP dependent and required either a nuclear extract from HeLa cells or purified human Swi/Snf. Through the use of a direct interaction assay (magnetic bead pull-down), we demonstrated recruitment of human Swi/Snf to MMTV chromatin by GR. Unexpectedly, we found that GR is actively displaced from the chromatin template during the remodeling process. ATP-dependent GR displacement was reversed by the addition of apyrase and was specific to chromatin templates. The disengagement reaction could also be induced with purified human Swi/Snf. Although GR apparently dissociated during chromatin remodeling by Swi/Snf, it participated in binding of the secondary transcription factor, nuclear factor 1. These results are paralleled by a recent discovery that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment in living cells. Both the in vitro and in vivo results are consistent with a dynamic model (hit and run) in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, facilitates transcription factor binding, and is simultaneously lost from the template.