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

I型谷氨酸尿症是赖氨酸代谢中戊二酰辅酶A脱氢酶（GCDH）的常染色体隐性缺陷。由于酶缺陷，神经毒性代谢物戊二酰辅酶A、谷氨酸和3-羟基谷氨酸蓄积。未经治疗的患者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出现神经系统表型。2-氨基-/2-氧代己二酸尿症是赖氨酸代谢的另一种遗传缺陷。最近，我们已经确定了致病突变的蛋白质DHTKD 1（脱氢酶E1和转酮醇酶域包含1）。一半的患者保持无症状，而另一半则表现出轻度的神经系统表型。所有受影响的个体的特征在于2-氨基己二酸和2-氧代己二酸的积累。DHTKD 1是另一种2-氧代戊二酸脱氢酶复合物（OGDHc）的亚基，催化2-氧代己二酸形成戊二酰辅酶A，因此是GCDH之前的一个酶促步骤。受2-氨基- / 2-氧代己二酸酸尿症影响的患者的缺乏或轻度表型表明，DHTKD 1的药理学抑制是I型谷氨酸尿症的一种有前途的新治疗方法。为了更好地了解DHTKD 1在细胞代谢中的作用，并评估上述治疗的潜在风险，我们使用转录激活因子样效应核酸酶技术创建了DHTKD 1-/-小鼠。对这些小鼠进行表征后，将其与Gcdh-/-小鼠杂交。双基因敲除小鼠将用于研究新治疗方法的功效，即降低谷氨酸和3-羟基谷氨酸水平以及降低对富含赖氨酸的饮食的敏感性。同时，我们将纯化和表征OGDHc样复合物含有DHTKD 1从原核和真核表达系统。纯化的蛋白质将用于建立抑制剂研究的酶测定系统。使用高通量药物筛选，我们将鉴定抑制DHTKD 1的小分子/抗代谢物。将使用纯化的蛋白质在体外和在I型谷氨酸尿症患者的成纤维细胞中以及在Gcdh-/-小鼠体内研究这些化合物的抑制功效。

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.