Acetyl CoA carboxylase (ACC) is the rate-determining enzyme of fatty acid biosynthesis in plants and animals. ACC is a biotin containing enzyme which catalyzes the carboxylation of acetyl CoA to form malonyl CoA in a two-step reaction (Beaty & Lane, (1982). J. Biol. Chem. 257:924–929). The first step is the ATP-dependent carboxylation of biotin covalently linked to the enzyme. In the second step, a carboxyltransferase step, the carboxyl group is transferred to the substrate, acetyl CoA, to form malonyl CoA. Citrate is a potent allosteric activator of ACC. Malonyl CoA is the C2 donor for de novo synthesis of long chain fatty acids.
In mammals, there are two subtypes of ACC, ACC1 and ACC2. ACC1 is mainly localized in lipogenic tissues such as adipose tissue and liver, where fatty acids are synthesized. ACC2 is found primarily in non-lipogenic tissues such as skeletal muscle and heart muscle, although some is also found in liver. Malonyl CoA allosterically inhibits carnitine palmitoyl transferase 1 (CPT1), which is a critical enzyme to transfer the long chain fatty acid into the mitochondria for β-oxidation. Because ACC2 is co-localized with CPT-1, the primary role of malonyl CoA that is synthesized by ACC2 has been suggested to regulate the rate of β-oxidation.
ACC is a potential target in metabolic diseases for the treatment of metabolic syndrome including obesity, insulin resistance and dyslipidemia. Increased rates of muscle fatty acid oxidation, a reduced fat content and a reduction in total body fat were observed in ACC-2 knock-out mice (Abu-Elheiga et al., (2001) Science 291:2613–2616; Abu-Elheiga et al., (2003) Proc. Natl. Acad. Sci. USA. 100:10207–10212). Harwood et al. reported that ACC inhibitors caused reduction in fatty acid synthesis, increase in fatty acid oxidation, and reduction of respiratory quotient in rats (Harwood et al., (2003) J. Biol. Chem. 278:37099–37111). Chronic dosing of these compounds resulted in the reduction of whole body fat mass and improvement of insulin sensitivity (Harwood et al., (2003) J. Biol. Chem. 278:37099–37111). These observations further validated the enzyme as a drug target.
Several human ACC2 and rat ACC2 nucleotide and amino acid sequences have been published (see, e.g., Human ACC2: GenBank Accession No. NM—001093 (SEQ ID NOs:1 and 2) and GenBank Accession No. AC007637 (SEQ ID NOs:3 and 4); Rat ACC2: GenBank Accession No. NM—053922 (SEQ ID NOs:7 and 8) and GenBank Accession No. AB004329 (SEQ ID NOs:9 and 10)). It was found, however, that for each species, each of the published amino acid and/or nucleotide sequences was different from one another by one or more residues. More specifically, it was found that the nucleotide sequences of human ACC2 and rat ACC2 contain non-silent mutations that introduce substitutions into several of the published encoded amino acid sequences of these enzymes.
In order to identify the most effective modulators of human ACC2 and rat ACC2, accurate nucleotide and amino acid sequences are required. Therefore, what is needed to advance research on human and rat ACC2 is an accurate amino acid sequence for these enzymes, as well as the encoding nucleotide sequences. The present invention solves this and other problems.
USPTO Backgrounds
USPTO Backgrounds