Characterization, structure and inhibition of the human succinyl-CoA:glutarate-CoA transferase, a genetic modifier of glutaric aciduria type 1

Abstract

Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or non-toxic metabolites. Here, we report a novel target, SUGCT, which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA. We report the structure of SUGCT, the first eukaryotic structure of a type III CoA transferase, develop a high-throughput enzyme assay and a cell-based assay, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme validating the screening approach. These results may form the basis for future development of new pharmacological intervention to treat GA1.

Figure 1.. Characterization of SUGCT overexpressing and parental Flp-In 293 cells.

(A) Immunoblot showing SUGCT overexpression in 2 representative clones (+ SUGCT). (B) SUGCT enzyme activity. (C) C5DC levels in cell pellets after 24 hours of exposure to different concentration of glutarate in the media. (D) C5DC levels in cell pellets upon GCDH KO using CRISPR-Cas9 genome editing. Three pellets from three independent clones were evaluated. A Mann Whitney test was significant at P < 0.0001.

Figure 2.. A novel SUGCT enzyme assay.

(A) Schema of the SUGCT reaction coupled to HMGCR. (B) IC₅₀ curves for malonate, succinate, glutarate, adipate and suberate. HMG concentration is 4 mM.

Figure 3.. High throughput screen of FDA-approved drugs on SUGCT activity.

(A) Z-score of 853 screening wells. Z-scores were calculated for each 384-well plate separately using the average and SD of all screening wells after removal of outliers due to compound fluorescence or quenching. Negative control (NC, DMSO) and positive control (PC, no enzyme) not included in the calculation of the average and SD. Compounds selected for repurchase are indicated in blue. (B) Repurchase of 15 FDA approved drugs. Each compound was measured in duplicate at 100 μM and 10 μM. The hit LCZ969 is a fixed-dose combination medication containing Sacubitril (AHU377) and valsartan. Rosuvastatin was selected as positive control. Therefore 5/13 hits (38%) confirmed at the 100 μM screening dose. (C) Effect of 10 different Angiotensin II Receptor Blockers (ARBs) on SUGCT activity. Each ARB was measured in duplicate at 10 μM. Telmisartan displayed some fluorescence, which may explain the lower RFU/min. (D) IC₅₀ curve for valsartan. (E) IC₅₀ curve for losartan carboxylic acid.

Figure 4.. Overall structure of SUGCT.

(A) Dimeric structure with catalytic residue (Asp212) shown. (B) Surface and cartoon view of SUGCT dimer, indicating dimeric assembly of polypeptide chains.

Figure 5.. Structure of SUGCT:Losartan carboxylic acid complex.

(A) Overall structure of the complex with Losartan carboxylic shown in purple in the left active site. (B) Close-up view of Losartan bound in the pocket, colored by chain. (C) Comparison of apo (yellow) and Losartan (cyan) active sites. Key residues are shown in stick form. (D) Close-up view of the losartan carboxylic acid interaction with the catalytic site. Ammonium (blue and white) and sulfate (red and orange) ions are shown in sphere form, with potential interactions shown with dashed lines.