ABSTRACT
The mechanistic target of rapamycin (mTOR) was identified in rapamycin resistant mutants of S. cerevisiae. The mTOR consists of two structurally and functionally distinct complexes, TORC1 and TORC2. As an inhibitor of mTORC1, rapamycin extends the life spans of yeast, flies, and mice. As a side effect, chronic administration of rapamycin impairs glucose tolerance and insulin action. It was shown that rapamycin disrupted mTORC2 in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis (Lamming et al., 2012). A decrease in mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis. Thus, rapamycin-induced insulin resistance is due to mTORC2 loss and is uncoupled from longevity effects.Rapamycin has anticancer properties. Rapamycin analogs temsirolimus, everolimus, and ridaforolimus inhibit mTORC1 and have improved pharmaceutical properties for treatment of cancer (Gibbons et al., 2009). Clinical trials of these analogs validated the importance of mTOR inhibition as an attractive anti-tumor target. Temsirolimus and enverolimus with the clinical names Torisel and Afinitor, respectively, have been approved as intravenous drugs for advanced renal cell carcinoma other cancer treatments (Table 25.1).Owing to its anti-proliferation property of halting cells at G1 phase, rapamycin and its analogs have been used in drug-eluting stents to prevent restenosis as summarized in Table 25.1. The bare metal stents were originally used to reduce the angiographic and clinical restenosis rates of balloon angioplasty. In-stent restenosis occurred after 6 months in about 20% of cases. Drug-eluting stents improved the bare metal stents by delivering drugs locally to inhibit neointimal hyperplasia (Doostzadeh et al., 2010). The sirolimuseluting stent Cypher was the first drug-eluting stent approved by the FDA. The second generation of drug-eluting stents are made of rapamycin analogs: the zotarolimus-eluting stent Endeavor® from
Medtronic, and the everolimus-eluting stent Xience V® from Abbott Vascular (Puranik et al., 2013). These analogs are more efficacious and selective with lower systemic toxicity in stent applications. 25.2.2 Biosynthesis of RapamycinRapamycin is a polyketide and its biosynthesis is carried out by polyketide synthases (PKSs), in a manner similar to fatty acid biosynthesis (Kwan and Schulz, 2011). The polyketide chain assembly is performed through the successive condensation of activated carboxylic acid-derived units. The starter unit for the rapamycin is the dihydrocyclohexane carboxylic acid (DHCHC) which is derived from the shikimate. The first committed step is the hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid (DCDC; Andexer et al., 2011). This chorismatase activity is encoded by the rapK gene in the rapamycin gene cluster of Streptomyces hygroscopicus. The extender units for rapamycin biosynthesis are malonyl-CoA and methylmalonyl-CoA. In Streptomyces hygroscopicus, three polyketide synthases, RapA, RapB, and RapC, are responsible for pre-rapamycin biosynthesis using DHCHC. Each enzyme has multiple modules and there are a total of 14 homologous modules (Schwecke et al., 1995). Each module catalyzes a specific round of chain elongation. The pipecolate-incorporating enzyme RapL then incorporates the L-pipecolate unit to complete the macrocycle (Gatto et al., 2006). Further modifications of the pre-rapamycin are performed by RapI, RapJ, RapM, RapN, and RapQ (Gregory et al., 2006). RapI is the O-methyltransferase, which methylates the oxygen at C39. RapM is another methylase that adds a methyl group to the oxygen group at C16. The carbonyl group at C9 is formed by RapJ, a P-450 monooxygenases. Finally, RapN and RapQ are responsible for the formation of the O-methyl group at C27. 25.2.3 Improvement of Rapamycin Production by
Traditional Mutagenesis and Precursor Metabolic EngineeringClassical strain improvement often involves mutagenesis carried out by chemicals, UV light, and radiation. Other methods such as protoplast fusion and mating are also employed (Chen et al., 2009).
