正在研究SLC6A19抑制剂作为苯丙酮尿症的治疗剂。在这项工作中,有效的SLC6A19抑制剂(RA836)使大鼠肾脏尿毒症毒素硫酸吲哚酚(IDS)水平升高,强度(任意单位)为13.7{正负}7.7,而溶媒0.3{正负}0.1(P=0.01),通过组织质谱成像(tMSI)分析确定。我们假设IDS的血浆和肾脏水平升高可能是由于Slc6a19和负责将IDS排泄到尿液中的肾脏IDS转运蛋白同时受到抑制所致。为了测试这个,我们首先通过给大鼠喂食无Trp饮食来证实通过色氨酸代谢形成IDS。用RA836抑制Slc6a19导致这些大鼠的IDS增加。接下来,体外评价RA836及其关键代谢产物对肾转运体OAT1、OAT3和BCRP的抑制作用。RA836抑制BCRP,IC50为0.045µM,但对OAT1或OAT3无明显抑制作用。最后,合成了对SLC6A19和/或BCRP具有有效抑制或不具有抑制的RA836类似物,并将其施用给饲喂正常饮食的大鼠。收集血浆和肾脏样品以使用LC-MS定量IDS。SLC6A19无活性但有效的BCRP抑制剂或SLC6A19有活性但较弱的BCRP抑制剂均未升高IDS水平,而抑制两种转运蛋白的化合物在大鼠血浆和肾脏中引起IDS积累,支持大鼠Bcrp有助于IDS排泄的假设。总之,我们发现抑制Slc6a19会增加IDS的形成,同时抑制Bcrp导致肾脏和血浆中的IDS积累。意义陈述这是破译通过抑制Slc6a19和Bcrp在大鼠中积累IDS(尿毒症毒素)的机制的第一份出版物。具体来说,在胃肠道中抑制Slc6a19会增加IDS的形成,并在肾脏中抑制Bcrp会阻断IDS的排泄。因此,我们应该避免同时抑制SLC6A19和BCRP,以防止IDS的积累,一个已知的心血管疾病的危险因素,精神焦虑,慢性肾脏病患者的死亡率。
SLC6A19 inhibitors are being studied as therapeutic agents for Phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7{plus minus}7.7 compared to vehicle 0.3{plus minus}0.1 (P=0.01) as determined by tissue mass spectrometry imaging (tMSI) analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney
transporters OAT1, OAT3 and BCRP. RA836 inhibits BCRP with an IC50 of 0.045 µM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using LC-MS. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, while compounds inhibiting both
transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma. Significance Statement This is the first publication to decipher the mechanism for accumulation of IDS (a uremic toxin) in rats via inhibition of both Slc6a19 and Bcrp. Specifically, inhibition of Slc6a19 in the GI track increases IDS formation and inhibition of Bcrp in the kidney blocks IDS excretion. Therefore, we should avoid inhibiting both SLC6A19 and BCRP simultaneously in humans to prevent accumulation of IDS, a known risk factor for cardiovascular disease, psychic anxiety, and mortality in chronic kidney disease patients.