Discovery of therapeutic agents targeting PKLR for NAFLD using drug repositioning

Background & Aims Non-alcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of liver pathologies. However, not medical treatment has been approved for the disease so far. In our previous study, we found PKLR could be a potential target for treatment of NALFD. Here the aim is to investigate the effect of PKLR in in vivo model and reposition a drug which could be used for treatment of NAFLD. Results The Pklr KO reversed the increased hepatic triglyceride level in mice fed with high sucrose diet and partly recovered the transcriptomic changes in liver as well as other three tissues. Both liver and white adipose tissues exhibited dysregulated circadian transcriptomic profiles, and these dysregulations were reversed by hepatic knockout of Pklr. In addition, 10 small molecule drugs were identified as potential inhibitor of PKLR by the drug repositioning pipeline, and two of them significantly inhibited both the PKLR expression and triglyceride level in in vitro model. Finally, the two selected small molecule drugs were evaluated in in vivo rat models and it was demonstrated that these drugs attenuated hepatic steatosis without side effect on other tissues. Conclusion: In conclusion, our study provided biological insights about the critical role of PKLR in NAFLD progression and proposed a treatment strategy for NAFLD patients, which could be validated in further clinical trials. Eight male C57BL/6J mice wild-type mice and eight Pklr-/- mutant mice were fed a standard mouse chow diet and housed in a 12-h light-dark cycle. From the age of 8 weeks, both the wild-type and mutant mice were then divided into two groups of 4 mice fed with chow diet, high-sucrose diet for another 8 weeks, respectively. At the age of 16 weeks, all mice are sacrificed and necropsies from liver, muscle, WAT and heart tissue were taken for RNA sequencing and lipid quantification.研究背景与研究目的 非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）涵盖广泛的肝脏病理谱系，但截至目前，该疾病尚无获批的临床治疗方案。本团队前期研究发现，PKLR可作为治疗非酒精性脂肪性肝病的潜在靶点。本研究旨在探究PKLR在体内模型中的作用，并筛选可用于治疗NAFLD的药物重定位候选化合物。 研究结果 Pklr基因敲除可逆转高蔗糖饮食喂养小鼠的肝脏甘油三酯水平升高，并部分恢复肝脏及其他三种组织的转录组表达异常。肝脏与白色脂肪组织（White Adipose Tissue, WAT）均出现节律性转录组表达紊乱，而Pklr肝脏特异性敲除可纠正上述转录组异常。此外，通过药物重定位流程，本研究筛选出10种可靶向抑制PKLR的小分子化合物，其中2种可在体外模型中显著下调PKLR表达并降低甘油三酯水平。最后，本研究在大鼠体内模型中对上述2种筛选得到的小分子化合物进行了验证，结果显示其可减轻肝脏脂肪变性，且未对其他组织产生不良反应。 研究结论 综上，本研究阐明了PKLR在NAFLD进展中的关键调控作用，为NAFLD患者提供了潜在治疗策略，该方案有待后续临床试验进一步验证。 实验方法 本研究选用8只雄性野生型C57BL/6J小鼠与8只Pklr-/-突变小鼠，所有小鼠均喂食标准小鼠饲料，并饲养于12小时光照-12小时黑暗的循环环境中。自8周龄起，将野生型与突变型小鼠各分为2组，每组4只，分别继续喂食标准饲料与高蔗糖饲料，持续8周。至16周龄时，处死所有小鼠并采集肝脏、肌肉、WAT及心脏组织样本，用于RNA测序（RNA sequencing）与脂质定量检测。