DESCRIPTION (provided by applicant): Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is an inborn error of fatty acid metabolism. The overall frequency of the disease is ~1:12,000 in Caucasians of mostly Northern European ancestry. MCADD patients are normal at birth but are at risk for episodes of acute, life threatening metabolic decompensation. These usually occur between three and twenty four months of age but can occur at any age in association with physiologic stress such as fasting or infection. The mortality rate during an acute crisis in previously undiagnosed patients can be as high as 20%. With the introduction of expanded newborn screening via tandem mass spectrometry, MCADD can now be identified pre-symptomatically, nearly eliminating mortality due to this disease. However, treatment requires lifelong dietary monitoring, and significant morbidity still occurs due to hospitalizations for IV glucose therapy in the face of reduced oral intake. A single mutation in the MCAD gene (a G985A point mutation) has been identified in 90% of the alleles in the MCAD gene in deficient patients. This mutation substitutes a glutamate for a lysine at position 304 of the mature enzyme (K304E), introducing four abnormal negative charges, destabilizing the quaternary structure of the enzyme. As a result, the mutant protein is rapidly degraded. In vitro studies have shown that the mutant protein is catalytically active when it can be stabilized. Importantly, published in vivo and in vitro data suggest that restoration of only a few percent of normal MCAD activity will restore near normal metabolic balance in patients. The long range objective of this study is to develop molecular strategies for treatment of deficiencies of MCAD and other structurally similar acyl-CoA dehydrogenases (ACDs). The specific objective of this application is to establish the drugability of the MCAD most common mutant by identifying small molecule lead compounds that can stabilize the MCAD K304E mutant protein in vitro. There are two specific aims. Specific Aim 1 is focused on the identification of chemical chaperonins using two random in vitro chemical library screening techniques, an HCS (high content-image based screening) immunoassay and HTS (high throughput screening) enzymatic assay, and to identify candidate compounds using in silico small fragments library screening. The crystal structure of MCAD K304E mutant the investigators obtained recently will be used to design compounds that are predicted to bind to the abnormal K304E tetramer and stabilize it. Specific Aim 2 is focused on the in vitro testing of candidate compounds. Efficacy of potential drugs will be measured by the ability to rescue the MCAD deficient phenotype. This provides a much stronger indication of the drugability of the mutant enzyme while providing a higher bar for initial determination of safety of candidate compounds. Synthesized candidate compounds will be examined for their ability to improve stability of the K304E mutant enzyme using two in vitro model systems. PROJECT NARRATIVE: Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is an inborn error of fatty acid metabolism with patients found to be normal at birth but are at risk for episodes of acute, life threatening metabolic decompensation. A single mutation in the MCAD gene, which causes the replacement of a lysine with a glutamate destabilizing the enzyme and resulting in its rapid degradation, has been identified in 90% of the MCAD gene in deficient patients. The long term objective of this project is to develop a chemical chaperonin for stabilizing the MCAD enzyme, using in vitro and in silico approaches, and hence reduce patients' risk for life threatening episodes of decompensation and hospitalization, and improve their overall quality of life.

rjc artve Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is an inborn error of fatty acid metabolism with patients found to be normal at birth but are at risk for episodes of acute, life threatening metabolic decompensa- tion. A single mutation in the MCAD gene, which causes the replacement of a lysine with a glutamate destabiliz- ing the enzyme and resulting in its rapid degradation, has been identified in 90% of the MCAD gene in deficient patients. The long term objective of this project is to develop a chemical chaperonin for stabilizing the MCAD enzyme, using n irno n iioapproaches, and hence reduce patients' risk for life threatening episodes of decompensation and hospitalization, and improve their overall quality of life.