; molecular weight, 540.2) (Fig. ), also known as FR901228 or depsipeptide and registered as NSC 630176 or romidepsin, is a natural product discovered in the fermentation broth of Chromobacterium violaceum
no. 968 in a screening program for agents that reverse the malignant phenotype of a Ha-ras
oncogene-transformed NIH 3T3 cell line (51
). It exhibited outstanding anticancer activities against an array of tumor cell lines, including many members of a standard panel of 60 cell lines from the U.S. National Cancer Institute (18
). FK228 has entered extensive clinical trials and has shown promising properties as a new type of anticancer drug (5
). A multinational pivotal trial of FK228 for the treatment of cutaneous T-cell lymphoma has been launched by Gloucester Pharmaceuticals, Inc., and the company plans to file for U.S. Food and Drug Administration approval in late 2007.
FIG. 1. FK228 structure and mode of action (modified from reference 17 with permission of the publisher). Hydrophobic FK228 can diffuse across the cell membrane. Inside cells, FK228 is activated by cellular reduction, and a freed sulfhydryl group chelates Zn (more ...)
Structurally, FK228 is a bicyclic depsipeptide that features a 16-membered macrolactone ring containing an ester linkage and a 17-membered ring containing the same ester linkage and a disulfide bond, the latter of which endows FK228 with an unprecedented molecular scaffold (Fig. ). Its structure was determined by spectroscopic and X-ray crystallographic analyses (45
) and was confirmed by total synthesis (27
). A close examination of the FK228 structure identified building blocks of three amino acids (d
-valine, and l
-valine), an amino acid derivative (2,3-dehydro-2-aminobutanoic acid; also called 2,3-dehydrothreonine), and a complex l
)-3-hydroxy-7-mercaptohept-4-enoic acid moiety that is likely built from one Cys and two C2
units derived from malonyl coenzyme A (MCoA). These observations suggest a hybrid nonribosomal peptide (NRP)-polyketide (PK)-NRP nature for FK228.
Mechanistically, FK228 was originally discovered as an anti-ras
); later, it was found to interfere with mitogen-induced signaling pathways (38
), and more recently it has been identified as a potent histone deacetylase (HDAC) inhibitor (17
). Histone acetylation catalyzed by histone acetyltransferases is an important component of chromatin remodeling and gene expression regulation; histone hypoacetylation mediated by HDACs is often associated with the onset and progression of cancer (24
). HDAC inhibitors are a diverse group of molecules that can induce growth arrest, differentiation, apoptosis, and autophagocytic cell death of cancer cells (10
). Interestingly, FK228 has an intramolecular disulfide bond, which makes it structurally distinct from other known HDAC inhibitors, such as hydroxamic acids, apicidin, and trapoxin. This disulfide bond has been postulated to mediate a novel mechanism of cytotoxic action of FK228 (Fig. ). Furumai and coworkers showed that FK228 serves as a stable prodrug and is activated by intracellular reduction of the disulfide bond after uptake into cells or organisms. The freed sulfhydryl group on the longer aliphatic tail of reduced FK228 fits inside the catalytic pocket of preferred class I HDACs, chelating Zn2+
, and thus inhibits the enzyme activities (17
). Xiao and coworkers also independently detected more active metabolites in rat plasma and human plasma following their incubation with FK228 in the presence of glutathione (56
). The 50% inhibitory concentration of FK228 was found to be nanomolar for inducing apoptosis in cells from patients with chronic lymphocytic leukemia (6
). Research on FK228 has been expanding rapidly in recent years.
HDAC inhibitors are prime agents for the development of novel anticancer drugs (1
). One HDAC inhibitor, Zolinza (vorinostat or suberoylanilide hydroxamic acid), was approved by the U.S. Food and Drug Administration in October 2006, and at least nine other HDAC inhibitors, including FK228, are in various stages of clinical trials as monotherapies or in combinations with other agents (18
). Due to its outstanding anticancer activities and novel structural characteristics, as well as certain levels of undesirable cardiac toxicity, FK228 may serve as an excellent molecular scaffold for the generation of structural analogs, from which compounds with improved anticancer properties may be identified. However, chemical synthesis of FK228 has been difficult (27
), and derivatization of FK228 by chemical synthesis has not been reported.
We set out to take an alternative approach to making FK228 analogs by means of pathway engineering, combinatorial biosynthesis, or chemoenzymatic synthesis. As the first essential step towards this goal, we identified, cloned, and partially characterized a biosynthetic gene cluster (designated dep
for depsipeptide) responsible for FK228 biosynthesis. Here we report the identification of candidate biosynthetic genes by a genome-scanning approach, the adaptation of a Pseudomonas aeruginosa
gene replacement system to create targeted gene- inactivated mutant strains, and the subsequent cloning and characterization of an unusual hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS)-NRPS pathway for FK228 biosynthesis in C. violaceum
no. 968. Acquisition of the dep
gene cluster and acclimation of an efficient genetic system should provide a solid foundation for the generation of FK228 analogs by engineered biosynthesis strategies (for recent comprehensive reviews, see references 20