Discovery of Highly Potent Solute Carrier 13 Member 5 (SLC13A5) Inhibitors for the Treatment of Hyperlipidemia.

In the face of escalating metabolic disease prevalence, largely driven by modern lifestyle factors, this study addresses the critical need for novel therapeutic approaches. We have identified the sodium-coupled citrate transporter (NaCT or SLC13A5) as a target for intervention. Utilizing rational drug design, we developed a new class of SLC13A5 inhibitors, anchored by the hydroxysuccinic acid scaffold, refining the structure of PF-06649298. Among these, LBA-3 emerged as a standout compound, exhibiting remarkable potency with an IC50 value of 67 nM, significantly improving upon PF-06649298. In vitro assays demonstrated LBA-3's efficacy in reducing triglyceride levels in OPA-induced HepG2 cells. Moreover, LBA-3 displayed superior pharmacokinetic properties and effectively lowered triglyceride and total cholesterol levels in diverse mouse models (PCN-stimulated and starvation-induced), without detectable toxicity. These findings not only spotlight LBA-3 as a promising candidate for hyperlipidemia treatment but also exemplify the potential of targeted molecular design in advancing metabolic disorder therapeutics.

Bempedoic Acid Unveils Therapeutic Potential in Non-Alcoholic Fatty Liver Disease: Suppression of the Hepatic PXR-SLC13A5/ACLY Signaling Axis.

The hepatic SLC13A5/SLC25A1-ATP-dependent citrate lyase (ACLY) signaling pathway, responsible for maintaining the citrate homeostasis, plays a crucial role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Bempedoic acid (BA), an ACLY inhibitor commonly used for managing hypercholesterolemia, has shown promising results in addressing hepatic steatosis. This study aimed to elucidate the intricate relationships in processes of hepatic lipogenesis among SLC13A5, SLC25A1, and ACLY and to examine the therapeutic potential of BA in NAFLD, providing insights into its underlying mechanism. In murine primary hepatocytes and HepG2 cells, the silencing or pharmacological inhibition of SLC25A1/ACLY resulted in significant upregulation of SLC13A5 transcription and activity. This increase in SLC13A5 activity subsequently led to enhanced lipogenesis, indicating a compensatory role of SLC13A5 when the SLC25A1/ACLY pathway was inhibited. However, BA effectively counteracted this upregulation, reduced lipid accumulation, and ameliorated various biomarkers of NAFLD. The disease-modifying effects of BA were further confirmed in NAFLD mice. Mechanistic investigations revealed that BA could reverse the elevated transcription levels of SLC13A5 and ACLY, and the subsequent lipogenesis induced by PXR activation in vitro and in vivo. Importantly, this effect was diminished when PXR was knocked down, suggesting the involvement of the hepatic PXR-SLC13A5/ACLY signaling axis in the mechanism of BA action. In conclusion, SLC13A5-mediated extracellular citrate influx emerges as an alternative pathway to SLC25A1/ACLY in the regulation of lipogenesis in hepatocytes, BA exhibits therapeutic potential in NAFLD by suppressing the hepatic PXR-SLC13A5/ACLY signaling axis, while PXR, a key regulator in drug metabolism may be involved in the pathogenesis of NAFLD. SIGNIFICANCE STATEMENT: This work describes that bempedoic acid, an ATP-dependent citrate lyase (ACLY) inhibitor, ameliorates hepatic lipid accumulation and various hallmarks of non-alcoholic fatty liver disease. Suppression of hepatic SLC25A1-ACLY pathway upregulates SLC13A5 transcription, which in turn activates extracellular citrate influx and the subsequent DNL. Whereas in hepatocytes or the liver tissue challenged with high energy intake, bempedoic acid reverses compensatory activation of SLC13A5 via modulating the hepatic PXR-SLC13A5/ACLY axis, thereby simultaneously downregulating SLC13A5 and ACLY.

Role of the chloride channel blocker in the formation of filtering tract scars after glaucoma surgery in rats.

Filtration surgery is commonly performed for glaucoma treatment to reduce intraocular pressure (IOP); however, scarring of the filtering bleb is the main cause of failure. In this study, we evaluated the effects of the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) on scar formation in filtering blebs. A glaucoma filtering surgery model was generated using Sprague-Dawley rats, divided into the control and NPPB groups receiving injections of different NPPB concentrations. The IOP of all rats decreased 1-day post-surgery and gradually increased afterward. However, IOP in rats from the NPPB groups recovered more slowly than that of the control group rats. In addition, the area and survival times of filtering blebs in rats from the NPPB groups were substantially larger and longer than those in the control group. Twenty-eight days after surgery, the protein and mRNA expression of collagen I, fibronectin and α-smooth muscle actin in the filtering area of rats from the NPPB groups were significantly lower than that in the control group rats. Collectively, our study demonstrates that NPPB inhibits filtering bleb scar formation, maintains filtering bleb morphology and prolongs filtering bleb survival time by inhibiting the differentiation of conjunctival fibroblasts and extracellular matrix synthesis.

Contributions of intestine and liver to the absorption and disposition of FZJ-003, a selective JAK1 inhibitor with structure modification of filgotinib.

FZJ-003 is a selective Janus kinase 1 (JAK1) inhibitor with structural modification of filgotinib for rheumatoid arthritis (RA) treatment. In this study, a series of in vivo and in vitro experiments were conducted to investigate the specific contribution of the intestine and liver to the disposition of FZJ-003 compared with filgotinib. Results showed that FZJ-003 exhibited over 2-fold higher systemic exposure and lower clearance than those of filgotinib, after intravenous or intragastric administration at the equivalent mole dose level to conscious rats. In anesthetized rats treated with different dosing routes, FZJ-003 exhibited higher intestinal bioavailability (Fa·Fg, 98.47 vs 34.54%) but lower hepatic bioavailability (Fh, 61.45 vs 92.07%). Permeability test in Caco-2 cells indicated that FZJ-003 was probably transported by passive diffusion (efflux ratio 1.37 < 2, indicating the approximately equivalent Papp values in two directions) with a little higher permeability (Papp,AP-to-BL, 1.42 × 10-6vs 1.01 × 10-6 cm·s-1, FZJ-003 vs filgotinib). Metabolic studies in pre-systemic incubation systems showed that FZJ-003 experienced more NADPH-dependent metabolism, especially in hepatic microsomes fractions. Unlike filgotinib, there was no obvious amide-hydrolyzed metabolite of FZJ-003 detected throughout the pre-systemic metabolic sites. Collectively, these data suggest that the higher systemic exposure of FZJ-003 than filgotinib is mainly attributed to the higher intestinal bioavailability including bypassing the amide hydrolysis and possible efflux by intestinal epithelial cells, which strongly support the structural design purpose in terms of pharmacokinetics.

Regulation on Citrate Influx and Metabolism through Inhibiting SLC13A5 and ACLY: A Novel Mechanism Mediating the Therapeutic Effects of Curcumin on NAFLD.

Upregulated de novo lipogenesis (DNL) plays a pivotal role in the progress of the nonalcoholic fatty liver disease (NAFLD). Cytoplasmic citrate flux, mediated by plasma membrane citrate transporter (SLC13A5), mitochondrial citrate carrier (SLC25A1), and ATP-dependent citrate lyase (ACLY), determines the central carbon source for acetyl-CoA required in DNL. Curcumin, a widely accepted dietary polyphenol, can attenuate lipid accumulation in NAFLD. Here, we first investigated the lipid-lowering effect of curcumin against NAFLD in oleic and palmitic acid (OPA)-induced primary mouse hepatocytes and high-fat plus high-fructose diet (HFHFD)-induced mice. Curcumin profoundly attenuated OPA- or HFHFD-induced hyperlipidemia and aberrant hepatic lipid deposition via modulating the expression and function of SLC13A5 and ACLY. The possible mechanism of curcumin on the citrate pathway was investigated using HepG2 cells, HEK293T cells transfected with human SLC13A5, and recombinant human ACLY. In OPA-stimulated HepG2 cells, curcumin rectified the dysregulated expression of SLC13A5/ACLY possibly via the AMPK-mTOR signaling pathway. Besides, curcumin also functionally inhibited both citrate transport and metabolism mediated by SLC13A5 and ACLY, respectively. These findings confirm that curcumin improves the lipid accumulation in the liver by blocking citrate disposition and hence may be used to prevent NAFLD.

Discovery of imidazopyrrolopyridines derivatives as novel and selective inhibitors of JAK2.

Herein, we describe the design, synthesis, and structure-activity relationships of a series of imidazopyrrolopyridines derivatives that selectively inhibit Janus kinase 2 (JAK2). These screening cascades revealed that 6k was a preferred compound, with IC50 values of 10 nM for JAK2. Moreover, 6k was a selective JAK2 inhibitor with 19-fold, >30-fold and >30-fold selectivity over JAK1, JAK3 and TYK2 respectively. In cytokine-stimulated cell-based assays, 6k exhibited a higher JAK2 selectivity over JAK1 isoforms. Indeed, at a dose of 20 mg/kg compound 6k, pSTAT3 and pSTAT5 expression was reduced to levels comparable to those of control animals untreated with GM-CSF. Additionally, 6k showed a relatively good bioavailability (F = 38%), a suitable half-life time (T1/2 = 1.9 h), a satisfactory metabolic stability, suggesting that 6k might be a promising inhibitor of JAK2 for further development research for the treatment of MPNs.

Tanshinone IIA prevents LPS-induced inflammatory responses in mice via inactivation of succinate dehydrogenase in macrophages.

Metabolic reprogramming is associated with NLRP3 inflammasome activation in activated macrophages, contributing to inflammatory responses. Tanshinone IIA (Tan-IIA) is a major constituent from Salvia miltiorrhiza Bunge, which exhibits anti-inflammatory activity. In this study, we investigated the effects of Tan-IIA on inflammation in macrophages in focus on its regulation of metabolism and redox state. In lipopolysaccharides (LPS)-stimulated mouse bone marrow-derived macrophages (BMDMs), Tan-IIA (10 µM) significantly decreased succinate-boosted IL-1ß and IL-6 production, accompanied by upregulation of IL-1RA and IL-10 release via inhibiting succinate dehydrogenase (SDH). Tan-IIA concentration dependently inhibited SDH activity with an estimated IC50 of 4.47 µM in LPS-activated BMDMs. Tan-IIA decreased succinate accumulation, suppressed mitochondrial reactive oxygen species production, thus preventing hypoxia-inducible factor-1α (HIF-1α) induction. Consequently, Tan-IIA reduced glycolysis and protected the activity of Sirtuin2 (Sirt2), an NAD+-dependent protein deacetylase, by raising the ratio of NAD+/NADH in activated macrophages. The acetylation of α-tubulin was required for the assembly of NLRP3 inflammasome; Tan-IIA increased the binding of Sirt2 to α-tubulin, and thus reduced the acetylation of α-tubulin, thus impairing this process. Sirt2 knockdown or application of Sirt2 inhibitor AGK-2 (10 µM) neutralized the effects of Tan-IIA, suggesting that Tan-IIA inactivated NLRP3 inflammasome in a manner dependent on Sirt2 regulation. The anti-inflammatory effects of Tan-IIA were observed in mice subjected to LPS challenge: pre-administration of Tan-IIA (20 mg/kg, ip) significantly attenuated LPS-induced acute inflammatory responses, characterized by elevated IL-1ß but reduced IL-10 levels in serum. The peritoneal macrophages isolated from the mice displayed similar metabolic regulation. In conclusion, Tan-IIA reduces HIF-1α induction via SDH inactivation, and preserves Sirt2 activity via downregulation of glycolysis, contributing to suppression of NLRP3 inflammasome activation. This study provides a new insight into the anti-inflammatory action of Tan-IIA from the respect of metabolic and redox regulation.

Changes in GABAergic markers accompany degradation of neuronal function in the primary visual cortex of senescent rats.

Numerous studies have reported age-dependent degradation of neuronal function in the visual cortex and have attributed this functional decline to weakened intracortical inhibition, especially GABAergic inhibition. However, whether this type of functional decline is linked to compromised GABAergic inhibition has not been fully confirmed. Here, we compared the neuronal response properties and markers of GABAergic inhibition in the primary visual cortex (V1) of young adult and senescent rats. Compared with those of young adult rats, old rats' V1 neurons exhibited significantly increased visually evoked responses and spontaneous activity, a decreased signal-to-noise ratio and reduced response selectivity for the stimulus orientation and motion direction. Additionally, the ratio of GABA-positive neurons to total cortical neurons in old rats was significantly decreased compared with that in young rats. Expression of the key GABA-synthesizing enzyme GAD67 was significantly lower in old rats than in young rats, although GAD65 expression showed a marginal difference between the two age groups. Further, expression of an important GABAA receptor subunit, GABAAR α1, was significantly attenuated in old rats relative to young ones. These results demonstrate that ageing may result in decreased GABAergic inhibition in the visual cortex and that this decrease in GABAergic inhibition accompanies neuronal function degradation.

Design, synthesis and molecular docking of amide and urea derivatives as Escherichia coli PDHc-E1 inhibitors.

By targeting the ThDP binding site of Escherichia coli PDHc-E1, two new 'open-chain' classes of E. coli PDHc-E1 inhibitors, amide and urea derivatives, were designed, synthesized, and evaluated. The amide derivatives of compound 6d, with 4-NO2 in the benzene ring, showed the most potent inhibition of E. coli PDHc-E1. The urea derivatives displayed more potent inhibitory activity than the corresponding amide derivatives with the same substituent. Molecular docking studies confirmed that the urea derivatives have more potency due to the two hydrogen bonds formed by two NH of urea with Glu522. The docking results also indicate it might help us to design more efficient PDHc-E1 inhibitors that could interact with Glu522.

Design, synthesis and biological evaluation of novel 2-methylpyrimidine-4-ylamine derivatives as inhibitors of Escherichia coli pyruvate dehydrogenase complex E1.

As potential inhibitors of Escherichia coli pyruvate dehydrogenase complex E1 (PDHc E1), a series of novel 2-methylpyrimidine-4-ylamine derivatives were designed based on the structure of the active site of PDHc E1 and synthesized using 'click chemistry'. Their inhibitory activity in vitro against PDHc E1 and fungicidal activity were examined. Some of these compounds such as 3g, 3l, 3n, 3o, and 5b demonstrated to be effective inhibitors of PDHc E1 from E. coli and exhibited antifungal activity. SAR analysis indicated that both, the inhibitory potency against E. coli PDHc E1 and the antifungal activity of title compounds, could be increased greatly by optimizing substituent groups in the compounds. The structures of substituent group in 5-position on the 1,2,3-triazole and 4-position on the benzene ring in title compounds were found to play a pivotal role in both above-mentioned biological activities. Amongst all the compounds, compound 5b with iodine in the 5-position of 1,2,3-triazole and with nitryl group in the 4-position of benzene ring acted as the best inhibitor against PDHc E1 from E. coli. It was also found to be the most effective compound with higher antifungal activity against Rhizoctonia solani and Botrytis cinerea at the dosage of 100 µg mL(-1). Therefore, in this study, compound 5b was used as a lead compound for further optimization.