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Exploring therapeutic approaches for treatment of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency

Kang, Heejung (2014) Exploring therapeutic approaches for treatment of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is a common biochemical genetic disorder in the US. Nearly 90% of alleles from MCADD patients contain a common mutation in the ACADM (c.985A>G). The change replaces a lysine with a glutamate (K304E), causing improper folding. The K304E protein can fold to a mature form and is then stable and active when expressed in a prokaryotic system with molecular chaperonins. The goal of this project was to identify chemical chaperones capable of stabilizing the K304E MCAD protein. Since even a small amount of MCAD activity restores metabolic flux, inducing intra-mitochondrial folding of K304E MCAD has the potential to be protective for patients. To demonstrate proof of principle, dimethylsulfoxide, glycerol, betaine, trimethylamine N-oxide (TMAO), and L-proline were tested for the ability to increase MCAD activity in lymphoblasts having c.985G>A alleles. TMAO and glycerol significantly increased MCAD activity in these cells. Phenylbutyrate is converted to its CoA ester form and metabolized to phenylacetyl-CoA through β-oxidation initiated by MCAD. As a substrate analogue for MCAD, phenylbutyryl-CoA is expected to improve protein stability. Experiments in HEK293 cells containing inducible wild type or K304E MCAD alleles, phenylbutyrate increased wild type MCAD activity by 30% and K304E MCAD activity by 154%. A clinical trial testing the efficacy of phenylbutyrate in MCAD patients is underway. Drug targeting sites were also investigated using molecular modeling. The docking site for electron transfer flavoprotein (ETF) was hypothesized to be a viable site. Twelve amino acid peptides with variable sequences were synthesized based on ETF βArg191-βLys202. One of the peptides significantly increased thermal stability of K304E MCAD. Circular dichroism spectroscopy confirmed binding of the synthetic peptide, inducing a shift in the Tm of the enzymes. The ETF docking peptide analogue also protected K304E MCAD protein against limited proteolysis by Staphylococcus aureus V8. These results confirm that ETF docking site is a viable target for MCADD.
PUBLIC HEALTH SIGNIFICANCE: Even though newborn screening has reduced the mortality of the MCADD, patients still require frequent hospital visits during metabolic decompensation. New treatments for MCADD will significantly reduce the burden of disease on these patients.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Kang, Heejunghek25@pitt.eduHEK25
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairFerrell, Robertrferrell@pitt.eduRFERRELL
Thesis AdvisorVockley, Jerryvockleyg@upmc.eduGEV1
Committee MemberMohsen, Al-WalidAl-Walid.Mohsen@chp.eduAAM27
Committee MemberUrban, Zsolturbanz@pitt.eduURBANZ
Committee MemberFinegold,
Date: 27 June 2014
Date Type: Publication
Defense Date: 15 April 2014
Approval Date: 27 June 2014
Submission Date: 7 April 2014
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 135
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Human Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: English
Date Deposited: 27 Jun 2014 20:12
Last Modified: 01 May 2019 05:15


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