Characterization and pharmacological Modulation of DHTKD1: new therapeutic approach for glutaric aciduria type I

戊二酸尿I型是赖氨酸代谢中戊二醛辅酶A脱氢酶(GCDH)的常染色体隐性缺陷。由于酶的缺陷，神经毒性代谢物戊二酰辅酶A、戊二酸和3-羟基戊二酸积累。未经治疗的患者会出现双侧纹状体病变。Gcdh缺陷(Gcdh-/-)小鼠表现出与患者相同的生化表型，并在富含赖氨酸的饮食下形成神经表型。2-氨基/2-氧代己二酸尿是赖氨酸代谢的另一种遗传性缺陷。最近，我们发现了DHTKD1蛋白(脱氢酶E1和转酮醇酶结构域包含1)的致病突变。一半的患者仍然没有症状，而另一半则表现出轻微的神经表型。所有受影响的人的特征是2-氨基己二酸酯和2-氧代己二酸酯的积累。DHTKD1是另一种2-氧代戊二酸-脱氢酶复合体(OGDHc)的一个亚基，它催化2-氧代己二酸生成戊二酰辅酶A，从而比GCDH早一步。2-氨基/2-氧代己二酸尿症患者的缺乏或轻度表型表明，DHTKD1的药理抑制是治疗I型戊二酸尿症的一种很有前途的新方法。为了更好地了解DHTKD1在细胞代谢中的作用并评估上述治疗的潜在风险，我们使用转录激活物样效应核酸酶技术创建了Dhtkd1-/-小鼠。在对这些小鼠进行鉴定后，它们将与Gcdh-/-小鼠杂交。双基因敲除小鼠将被用来研究新治疗方法的有效性，即降低戊二酸和3-羟基戊二酸水平，以及降低对富含赖氨酸的饮食的敏感性。同时，我们将从原核和真核表达系统中提纯和鉴定含有DHTKD1的OGDHc-like复合体。纯化的蛋白将用于建立用于抑制剂研究的酶检测系统。利用高通量药物筛选，我们将鉴定抑制DHTKD1的小分子/抗代谢产物。这些化合物的抑制效果将在体外使用纯化的蛋白和在I型戊二酸尿症患者的成纤维细胞中进行研究，以及在Gcdh-/-小鼠体内进行研究。

Glutaric aciduria type I is an autosomal-recessive defect of the enzyme glutaryl-CoA dehydrogenase (GCDH) in lysine metabolism. Due to the enzymatic defect the neurotoxic metabolites glutaryl-CoA, glutaric acid, and 3-hydroxyglutaric acid accumulate. Untreated patients develop bilateral striatal lesions. Gcdh-deficient (Gcdh-/-) mice show the same biochemical phenotype as patients and develop a neurological phenotype under lysine-enriched diet. 2-Amino-/2-oxoadipic aciduria is another inherited defect in lysine metabolism. Recently, we have identified disease-causing mutations in the protein DHTKD1 (dehydrogenase E1 and transketolase domain containing 1). Half of the patients remains asymptomatic, whereas the other half displays a mild neurological phenotype. All affected individuals are characterized by accumulation of 2-aminoadipate and 2-oxoadipate. DHTKD1 is a subunit of an alternative 2-oxoglutarate-dehydrogenase complex (OGDHc) catalyzing the formation of glutaryl-CoA from 2-oxoadipate and, thereby, being one enzymatic step before GCDH. The lacking or mild phenotype of patients affected with 2-amino- / 2-oxoadipate aciduria suggests that pharmacologic inhibition of DHTKD1 is a promising new therapeutic approach for glutaric aciduria type I. To gain a better understanding of the role of DHTKD1 in cellular metabolism and to evaluate potential risks of the aforementioned therapy, we have created Dhtkd1-/- mice using transcription activator-like effector nucleases technique. After characterization of these mice, they will be crossbred with Gcdh-/- mice. The double knock out-mice will be used to investigate the efficacy of the new therapeutic approach, i.e. reduction of glutaric and 3-hydroxyglutaric acid levels as well as a reduced susceptibility to a lysine-enriched diet. In parallel we will purify and characterize the OGDHc-like complex containing DHTKD1 from prokaryotic and eukaryotic expression systems. The purified protein will be used to establish an enzymatic assay system for inhibitor studies. Using High-Throughput-Drug screen we will identify small molecules / antimetabolites to inhibit DHTKD1. Inhibitory efficacy of these compounds will be studied in vitro using the purified protein and in fibroblasts of glutaric aciduria type I patients as well as in vivo in Gcdh-/- mice.