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 μM IC 50 and having no effect on human ACC1 at 100 μM.fatty acid metabolism | human health

Analysis of Arabidopsis Accessions Hypersensitive to a Loss of Chloroplast Translation

“…Human ACCase is controlled by allosteric regulation, protein-protein interactions, reversible phosphorylation, and oligomerization (Brownsey et al, 2006;Tong, 2013). Chemical inhibitors have been developed that interfere with dimerization of one or both isoforms, modify fatty acid metabolism, and represent promising treatments for diabetes, obesity, and other human diseases (Marjanovic et al, 2010;Harriman et al, 2016).…”

The utility of yeast as a tool for cell-based, target-directed high-throughput screening

“…Importantly, as outlined above, all yeast-based HTS must employ panels of counter-screens and secondary assays to rule out offtarget effects. In particular, those platforms relying on growth inhibition read-outs are prone to identify off-target hits and the Molecular Mode of Action (MMoA) must be corroborated (Marjanovic et al 2010).…”

“…Until very recently this was the only published yeast-based HTS for anti-parasitics, although the potential of these platforms for this purpose has been recognized with respect to the protozoal acetyl-CoA carboxylase (Marjanovic et al 2010). However, drug sensitivity yeast mutants have been used to identify the primary targets and possible toxic effects of the anti-malarials quinine (Khozoie et al 2009), St. John's Wort (McCue and Phang, 2008) and artemisinin (Li et al 2005).…”

“…This approach has been widely used in the study of parasite (Klein et al 1997;Sibley et al 1997;Bilsland et al 2011) and mammalian proteins (Munder and Hinnen, 1999) through the constitution of phenotypic screening assays. One recent example involved the development of an HTS assay to identify inhibitors of the human acetyl-CoA carboxylase 2 (AAC2) with a view to discovering potential agents for the treatment of obesity (Marjanovic et al 2010). In this example, the S. cerevisiae AAC was replaced using homologous recombination by orthologous human AAC 1 and 2, as well as by the corresponding wheat and protozoan (Toxoplasma gondii) AACs.…”

Identification of an Aminothiazole with Antifungal Activity against Intracellular Histoplasma capsulatum

“…Yeast cells were grown in 96-well microtiter plates by modification of protocols reported previously for Saccharomyces growth in microtiter plates (25). OSU76 yeast cells at the exponential phase of growth were enumerated by using a hemacytometer and added to microtiter plates at 2.5 ϫ 10 5 yeast cells per well (for library screening) or 1 ϫ 10 5 yeast cells per well (for follow-up screening) (2.5 ϫ 10 6 yeast cells/ml and 1 ϫ 10 6 yeast cells/ml, respectively) in HMM supplemented with 10 g/ml tetracycline and 10 g/ml gentamicin.…”