Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

Abstract

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In "proof of concept" experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 muM IC(50) and having no effect on human ACC1 at 100 muM.

Fig. 1.

An overview of the domain structure of the multidomain ACC and the structure of various chimeric genes complementing the ACC1 null mutation in yeast. The apicoplast targeting signal of Toxoplasma ACC was deleted in the construct shown (7). Two additional human ACC2 genes were constructed in which the N-terminal mitochondrial targeting signal was deleted (29- and 148-amino acid deletion, respectively). Only the coding parts of the synthetic genes in yeast shuttle vectors pRS423 carrying a GAL10 promoter and the 5′-UTR and 3′-UTR from the yeast ACC1 gene (28) are shown. Numbers indicate amino acid positions in native ACCs. Positions of mutations and other features described in the text and Table S1 are also shown. Names of the corresponding yeast gene-replacement strains (Table S1) are shown on the left, underlined.

Fig. 2.

Specificity of the best hits. Growth inhibition of yeast strain ACC2m1 versus strains WT and ACC1 (A and B), and correlation between growth inhibition of strain ACC2m1 versus inhibition of ACC2 enzymatic activity (C) by the best hits found by screening chemical libraries described in the text. Arrow points to data points for compound CD-017-0191. Chemotypes: green, thieno[2,3-d]pyrimidine; purple, 5H-pyrrolo[1,2-a]quinoxalin-4-one; orange, oxazolo[4,5-b]pyridines; blue, 2-piperidin-1-yl-1H-benzoimidazole; red, 1H-pyrazolo[3,4-b]pyridine. Inhibition data are shown in Table S3.

Fig. 3.

Structure of CD-017-0191, 2-[[[5-methyl-2-[4-(1-methylethoxy)phenyl]-4-oxazolyl]methyl]thio]-oxazolo[4,5-b]pyridine), and CD-017-0213, 4-(2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-1-ethoxycarbonylpiperazine.

Fig. 4.

(A). Growth inhibition of yeast gene-replacement strains ACC2m1 and ACC1 (Fig. 1, Table S1) and WT yeast by compound CD-017-0191. (B). Inhibition of ACC1 and ACC2 enzymatic activity in vitro by compound CD-017-0191. Squares, growth of strain ACC2m1 and enzymatic activity of ACC2; circles, growth of strain ACC1 and enzymatic activity of ACC1; triangles, growth of strain WT.

Fig. 5.

Growth inhibition of a collection of yeast gene-replacement strains by CD-017-0191. ACC composition in different yeast strains is shown in Fig. 1. Inhibitor concentration: lighter gray bar, 10 μM; darker gray bar, 100 μM. Culture density in the absence of inhibitor = 100%.