Identification of molecules that correct structural and functional defects of naturally-occurring pathogenic apoA-I mutants

Background and Aims : Naturally-occurring mutations in human apolipoprotein A-I (apoA-I) have been shown to disturb protein conformation and induce functional defects that impair the levels and atheroprotective properties of HDL. One such apoA-I mutation, L178P, induces major defects in the structural integrity and functions of the protein that may underlie the reduced HDL-cholesterol and increased cardiovascular risk observed in carriers of the mutation. Here, a library of marketed drugs (∼1000 compounds) was screened against apoA-I[L178P] to identify molecules that can prevent mutant apoA-I from adopting its pathological conformation.Methods: To evaluate the effect of the drugs on the thermodynamic stability and structure of the mutant protein, we utilized the thermal stability shift assay in the presence of the fluorescent dye SYPRO Orange. As an orthogonal assay, we used the change of fluorescence intensity of 1-anilinonaphthalene-8-sulfonic acid (ANS) upon its binding on hydrophobic sites on apoA-I as a reporter of protein structural integrity.Results: Thermal stability shift analysis identified twenty-one compounds that induce the mutant protein to undergo a melting transition similar to that of WT apoA-I. Subsequent analysis of the capacity of the twenty-one drugs to induce apoA-I[L178P] to have a similar binding capacity of ANS as WT apoA-I, narrowed the potential structure correctors to six. Functional analyses have so far identified one compound that restores the defective capacity of apoA-I[L178P] to induce ABCA1-mediated cholesterol efflux.Conclusions: Our findings indicate that small molecules can correct defective apoA-I structure and function and may lead to novel therapeutic approaches for apoA-I-related dyslipidemias and increased cardiovascular risk. Background and Aims : Naturally-occurring mutations in human apolipoprotein A-I (apoA-I) have been shown to disturb protein conformation and induce functional defects that impair the levels and atheroprotective properties of HDL. One such apoA-I mutation, L178P, induces major defects in the structural integrity and functions of the protein that may underlie the reduced HDL-cholesterol and increased cardiovascular risk observed in carriers of the mutation. Here, a library of marketed drugs (∼1000 compounds) was screened against apoA-I[L178P] to identify molecules that can prevent mutant apoA-I from adopting its pathological conformation. Methods: To evaluate the effect of the drugs on the thermodynamic stability and structure of the mutant protein, we utilized the thermal stability shift assay in the presence of the fluorescent dye SYPRO Orange. As an orthogonal assay, we used the change of fluorescence intensity of 1-anilinonaphthalene-8-sulfonic acid (ANS) upon its binding on hydrophobic sites on apoA-I as a reporter of protein structural integrity. Results: Thermal stability shift analysis identified twenty-one compounds that induce the mutant protein to undergo a melting transition similar to that of WT apoA-I. Subsequent analysis of the capacity of the twenty-one drugs to induce apoA-I[L178P] to have a similar binding capacity of ANS as WT apoA-I, narrowed the potential structure correctors to six. Functional analyses have so far identified one compound that restores the defective capacity of apoA-I[L178P] to induce ABCA1-mediated cholesterol efflux. Conclusions: Our findings indicate that small molecules can correct defective apoA-I structure and function and may lead to novel therapeutic approaches for apoA-I-related dyslipidemias and increased cardiovascular risk.