Design and Synthesis of a Nitrogen Mustard Derivative Stabilized by Apo-neocarzinostatin

Abstract

Neocarzinostatin (NCS) is an antitumor antibiotic comprising a 1:1 protein-chromophore complex and exhibits cytotoxic action through DNA cleavage via H-abstraction. Cytotoxic activity resides with the chromophore 1 alone, while the protein (apoNCS) protects and transports labile 1. The naphthoate portion (2) of NCS chromophore (1) is important for binding to apoNCS and DNA intercalation. In this paper we describe our attempts to use apoNCS to improve the hydrolytic stability of novel bifunctional DNA alkylating agents. The nitrogen mustards, melphalan and chlorambucil, were both conjugated to 2, and the biological activities of these conjugates were assessed. Chlorambucil did not benefit from conjugation. The melphalan conjugate (6) formed covalent DNA adducts at guanine bases and exhibited greater in vitro cytotoxic activity than unmodified melphalan. Fluorescence and NMR spectroscopy showed that 6 binds to apoNCS. Binding to apoNCS-protected 6 reduced the extent of hydrolysis of the conjugate. This novel approach demonstrates for the first time that an enediyne apo-protein can be used to improve the stability of substances that are of potential interest in cancer chemotherapy.

Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

“…Because of a technical problem, we lost the enriched sample after the structure determination and thus measured the relaxation data with a native protein at high concentration as described later. We confirmed that there is no concentration dependence for 13 C and 1 H chemical shift in the range of 2.5-8 mM by 13 C and 1 H-HSQC NMR spectra.…”

“…However, there was no clarification between conformational ambiguity and the internal motion, which should be investigated by relaxation data in NMR. 13 C NMR relaxation studies were achieved for apo-NCS by Izadi-Pruneyre et al (24) and Mispelter et al (25), and yet there was no clear relation between the internal motion and the apo-NCS structural convergence (12,26). Here, we performed holo-NCS structural analysis with a combination of 13 C NMR methine relaxation experiments at natural abundance to investigate the internal motion of the loop and the chromophore-releasing mechanism.…”

“…N, 13 C Uniform Label and Purification of Holo-NCS-To obtain stable isotope-labeled holo-NCS, S. carzinostaticus was grown in Waxman medium (0.05% meat extract, 0.05% peptone, 0.03% NaCl, and 0.1% D-glucose) containing algae 15 N-and 15 N/ 13 C-labeled amino acid mixtures (Chlorella Industry Co., Ltd). After a 72-h incubation, the cell-free culture medium was collected using filter paper.…”

“…NMR Measurements and Resonance Assignments-NMR experiments were carried out at 30°C on Bruker AMX 600 and Varian Unity 750 spectrometers equipped with shielded gradient triple resonance probes. Sequence-specific 1 H, 15 N, and 13 C NMR assignments for holo-NCS were obtained with heteronuclear three-dimensional HNCA, 15 Nedited three-dimensional NOESY-HMQC and 13 C-edited three-dimensional HMQC spectra following the standard strategy for proteins (27)(28)(29)(30). The mixing times employed for the NOESY spectra were 150 ms. All of the 1 H and 15 N resonances, with the exception of N ⑀ H 2 protons of Gln 27 and Gln 94 and N ␦ H 2 of Asn 48 , were unambiguously assigned.…”

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

“…There has been recent interest in exploring the possibility of docking small synthetic molecules such as EtBr [32], naphthoate ester derivatives [33][34][35], and flavone-based ligand [36] in place of natural chromophore. Among the synthetic ligands, EtBr exhibits a relatively high binding constant (8.57 × 10 −7 M) [32] and has been frequently used to examine the binding properties of NCS mutants [37,38].…”