pubs.acs.org/Biochemistry Published on Web 07/27/2010 r 2010 American Chemical Society 7722 Biochemistry 2010, 49, 7722–7732 DOI: 10.1021/bi100735v Lipid Bilayer-Assisted Release of an Enediyne Antibiotic from Neocarzinostatin Chromoprotein † Parameswaran Hariharan, ‡,§ Christopher Gunasekaran Sudhahar, ‡ Shan-Ho Chou, § and Der-Hang Chin* ,‡ ‡ Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC, and § Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC Received May 10, 2010; Revised Manuscript Received June 30, 2010 ABSTRACT: The nine-membered enediyne class has drawn extensive interest because of extremely high antitumor potency and intricate interactions with its carrier protein. While the drug-induced DNA cleavage reactions have been mostly elucidated, the critical release-transport process of the labile enediyne molecule in cellular environment remained obscure. Using neocarzinostatin chromoprotein as a model, we demonstrated a lipid bilayer-assisted release mechanism. The in vitro enediyne release rate under aqueous conditions was found to be too slow to account for its efficient DNA cleavage action. Via the presence of lipid bilayers, chaotropic agents, or organic solvents, we found the release was substantially enhanced. The increased rate was linearly dependent on the lipid bilayer concentration and the dielectric value of the binary organic solvent mixtures. While lipid bilayers provided a low surrounding dielectricity to assist in drug release, there were no major conformational changes in the apo and holo forms of the carrier protein. In addition, the lifespan of the released enediyne chromophore was markedly extended through partitioning of the chromophore in the hydrophobic bilayer phase, and the lipid bilayer-stabilized enediyne chromophore significantly enhanced DNA cleavage in vitro. Collectively, we depicted how a lipid bilayer membrane efficiently enhanced dissociation of the enediyne chromophore through a hydrophobic sensing release mechanism and then acted as a protector of the released enediyne molecule until its delivery to the target DNA. The proposed membrane- assisted antibiotic release-transport model might signify a new dimension to our understanding of the modus operandi of the antitumor enediyne drugs. The release and transport of bioactive small molecules that are naturally bound to carrier proteins are interesting topics in exploring fundamental and applied biosciences. The recen- tly discovered nine-membered enediyne antibiotic chromo- proteins (1, 2), which belong to one of the most potent antitumor categories, are particularly attractive. While the drug-induced DNA cleavage reactions have been mostly elucidated, the intri- cate mode of action in the release-transport process remained unclear, even though such a process could critically affect drug efficacy. Among the members of the enediyne family, the first member, neocarzinostatin (NCS) 1 (3), is a very useful role model for studying small molecule-protein interactions (4). The NCS chromoprotein complex must first release its very tightly bound enediyne chromophore (NCS-C) (5), which is otherwise highly labile in a naked form (6, 7). In the cellular environment, how the antibiotic is released and securely transported to the target DNA without losing its activity must be an ingeniously designed and adaptively evolved process. NCS, isolated from Streptomyces carzinostaticus (3), is a 1:1 noncovalent complex (holoNCS) of NCS-C (8) and a protective apoprotein (apoNCS) (Figure 1) (7). The unique architecture of the nine-membered bicyclo[7,3,0]dodecadiyne ring (Figure 1A) makes NCS-C highly labile (7). A thiol-activated rearrangement of the enediyne core generates an active radical species, which abstracts hydrogen and leads to DNA lesions (7, 9). Crystallo- graphy and solution NMR studies revealed that both holoNCS and apoNCS share a similar protein structure (10, 11). apoNCS is an all-β-sheet protein (11 kDa) folded into a β-sandwich with overall dimensions of ∼20 A ˚ Â 25 A ˚ Â 40 A ˚ (10, 12). The chro- mophore is bound in a deep cavity and is tightly secured by the two loops on the exterior of the binding cleft (Figure 1B). Scientific interest in NCS is overwhelming because of its therapeutic usage as a potent antitumor drug (13, 14). apoNCS carries the potent enediyne antibiotic and possesses attractive features as a prototype for the development of versatile cargo- delivery vehicles. Its small size and strong tolerance of denatu- rants (15) are favorable for an ideal drug carrier. With an immu- noglobulin-like topology and attractive ligand binding scaffold, apoNSC provides a structural framework for the development of new recognition sites and binding specificities for nonenediyne molecules through protein engineering (4, 16, 17). At this † This work was supported by Grant NHRI-EX90-8807BL from National Health Research Institutes and Grants 95-2311-B-005-012- MY3 and 95-2113-M-005-007-MY3 from the National Science Council, Taiwan, Republic of China, to D.-H.C. This work was also supported in part by the Ministry of Education, Taiwan, Republic of China, under the ATU plan to National Chung Hsing University. *To whom correspondence should be addressed: Department of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 40227, Taiwan, Republic of China. Telephone: þ886-4- 22840411, ext. 304. Fax: þ886-4-22862547. E-mail: chdhchin@dragon. nchu.edu.tw. 1 Abbreviations: NCS, neocarzinostatin; NCS-C, neocarzinostatin chromophore; apoNCS, apoprotein component of neocarzinostatin; holoNCS, chromoprotein complex of neocarzinostatin; PC, L-R-phos- phatidylcholine; PS, L-R-phosphatidylserine; PE, L-R-phosphatidyl- ethanolamine; LUV, large unilamellar vesicle; TBS, Tris-HCl-buffered saline; GSH, glutathione; MPS, 3-mercapto-1-propanesulfonic acid; CD, circular dichroism; HPLC, high-performance liquid chromatography.