Neocarzinostatin chromophore

The natural complex Neocarzinostatin comprises a labile chromophore noncovalently bound to an 11.2 kDa protein. We present the first high-resolution structure of a novel complex derived from the recombinant apoprotein bound to a non-natural synthetic chromophore. Fluorescence and nuclear magnetic resonance spectroscopy were used to probe the strength and location of binding. Binding occurred in a location similar to that observed for the chromophore in the natural Neocarzinostatin complex, but with a distinct orientation. These results provide structural evidence that the apoprotein can readily accommodate small druglike entities, other than the natural chromophore within its binding cleft. The clinical use of the natural complex described by others, together with the results reported here, suggests potential applications for small molecule binding by apo-Neocarzinostatin.

A 2-naphthol derivative 2 corresponding to the aromatic ring moiety of neocarzinostatin chromophore was found to degrade proteins under photo-irradiation with long-wavelength UV light without any additives under neutral conditions. Structure-activity relationship studies of the derivative revealed that methylation of the hydroxyl group at the C2 position of 2 significantly suppressed its photodegradation ability. Furthermore, a purpose-designed synthetic tumor-related biomarker, a H2 O2 -activatable photosensitizer 8 possessing a H2 O2 -responsive arylboronic ester moiety conjugated to the hydroxyl group at the C2 position of 2, showed significantly lower photodegradation ability compared to 2. However, release of the 2 from 8 by reaction with H2 O2 regenerated the photodegradation ability. Compound 8 exhibited selective photo-cytotoxicity against high H2 O2 -expressing cancer cells upon irradiation with long-wavelength UV light.

To determine the role of the epoxide moiety of the nonprotein chromophore of the antitumor antibiotic neocarzinostatin in its ability to damage DNA, the diol monomethyl ether derivative was prepared, and its stability and biological properties were studied. This analogue was found to be more stable than the epoxide (about 9-fold), but to be much less active in nicking supercoiled DNA and in forming covalent adducts with poly(dA-dT). However it is able to bind noncovalently to DNA and to the neocarzinostatin apoprotein. Another analogue, the chlorohydrin derivative, is about half as active as the epoxide in the DNA scission reaction and appears to produce the same covalent adducts with poly(dA-dT) as does the epoxide, suggesting that both compounds undergo similar types of activation by thiol. These results indicate that the epoxide moiety of the neocarzinostatin chromophore is an important part of the highly unsaturated, strained bicyclo[7.3.0]dodecadiendiyne in the thiol-dependent, DNA-damaging reaction. It may be involved in the activation of the drug to its active species and/or may be the site of new bond formation in its reaction with DNA.