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Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is an inborn error of fatty acid metabolism, rivaling phenylalanine hydroxylase deficiency (PKU) as the most common biochemical genetic disorder. Patients with MCAD deficiency are asymptomatic at birth but are at risk for episodes of acute, life threatening metabolic decompensation. These usually first occur between 3 and 24 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 percent
With the introduction of expanded newborn screening via tandem mass spectrometry, MCAD deficiency can now be identified pre-symptomatically, nearly eliminating mortality from this disease. However, treatment requires lifelong dietary monitoring, and significant morbidity still occurs due to hospitalizations for intravenous 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 percent 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). According to the crystal structures of recombinant human MCAD, the K304 side chain extends at the edge of a unique cavity in the inner core of the tetramer, which is about 14 angstroms in diameter comprised by the four monomers, where its side chain amino group hydrogen bonds with the side chain amide oxygen of Q342. The K304E mutation introduces instead four abnormal negative charges into the core, 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-term objective of this research program is to develop molecular strategies for treatment of ACAD deficiencies taking advantage of structural similarities of ACADs. While this is hypothetically feasible, the specific objective of this current research is to establish the drugability of the MCAD K304E mutant by identifying lead compounds that can stabilize the mutant protein using in vitro and in silico approaches.
Experimental results have led to two different approaches. One approach is the use of sodium phenylbutyrate to help stabilize the protein in vivo. Results show that its CoA ester form is a substrate for MCAD and that its presence in vitro with MCAD forms a ternary complex that has higher thermal stability. A clinical study has started to evaluate the effect of the drug Buphenol on MCAD K304E patients. The second approach was to identify ligands that can bind to known sites on the MCAD tetramer that has reasonable pharmacophore qualities. These sites could be used for structure- or fragment-based drug design. We have identified the electron transfer flavoprotein docking site as a target on MCAD and other ACADs. A mutant 12-mer peptide designed to bind to the docking site increased thermal stability by 2–2.5°C as shown by enzyme activity and circular dichroism spectral studies. This proof-of-concept finding is critical in pursuing this site for drug design. In the near future, this peptide and/or critical components in its structure will be used as scaffold for fragment-based drug design and screening of in silico libraries of chemicals that already exist.
Another important and potential breakthrough is related to screening of chemical libraries for chaperone therapy in a high throughput setting. An article has reported the adaptation of real-time polymerase chain reaction technology using 96-well plates to follow the melting of flavoproteins; therefore, thermal stability could easily be monitored and quantitated. This potential breakthrough will be tested shortly and would be the high-throughput assay for screen chemical libraries in a 384-well plates setting
Al-Walid A. Mohsen, PhD
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UPMC Children’s Hospital of Pittsburgh
One Children’s Hospital Way
4401 Penn Ave.
Pittsburgh, PA 15224
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