StaN is a cytochrome P450 (P450) involved in the biosynthesis of the antibiotic and anticancer indolocarbazole staurosporine. It was identified in 2005 once the whole biosynthetic cluster of staurosporine in Streptomyces sp TP-A0274 was sequenced. It can be classified as a P450 given its high sequence homology with other members of this superfamily and the presence of several typical molecular signatures of P450s, e.g. the universally conserved cysteine residue as the thiolate ligand to the heme. Nevertheless, StaN is supposed to catalyze an uncommon reaction for a P450 because it involves the formation of the glycosidic C-N bond between the staurosporine aglycone and the deoxyaminosugar, allowing for the formation of the intermediate O-demethyl-N-demethyl-staurosporine. To date, StaN represents the very first example of a P450 involved in C-N bond formation and its mechanism of action is unknown. My goal is to determine the high-resolution tridimensional structure of StaN by using x-ray crystallography in order to identify the molecular features critical for its activity and to understand the molecular mechanism behind its unusual reaction. I will also carry on a functional characterization of this system by means of substrate and inhibitors binding experiments. This also implies the production of site-specific mutants directed on critical residues, that will be identified by the structure, in order to better understand their specific role in catalysis and with the further aim to manipulate and redirect StaN activity against alternative substrate molecules. The long-term aim of this project is to produce new indolocarbazole derivatives able to bypass the non-selectivity that until now has precluded the use of staurosporine in anticancer therapy.
Among over 9000 individual enzymes belonging to this superfamily, StaN is the only cytochrome P450 able to catalyze the formation of a glycosidic C-N bond. Furthermore, its mechanism of action is unique since the substrate is elaborated through an oxidative chemistry, not shared with any other enzyme involved in C-N bond formation up to date.
The determination of StaN structure through x-ray crystallography is the best way to clarify its reaction mechanism, which is still a matter of debate. I aim at unveiling the atomic details of the conformational changes endowed with StaN function, thus providing a "mise au point" about the catalytic pathway promoted by this enzyme. Moreover, StaN plays a key role in the biological process of staurosporine production, which has been recently discovered to possess anticancer properties, which makes this enzyme even more attractive. The whole indolocarbazole family of natural products constitutes a new class of antitumor drugs divided into two major groups depending on their mechanisms of action and structural features [1]. One group includes inhibitors of DNA topoisomerase I, such as rebeccamycin. Many members of this group contain a sugar moiety attached by a beta-glycosidic linkage to one of the indole nitrogen atoms of the aglycone. The second group includes protein kinase inhibitors, such as staurosporine. Usually, they contain a sugar moiety linked to both indole nitrogen atoms of the indolocarbazole core. Staurosporine is a very potent inhibitor of protein kinase C [2], but lacks the selectivity required for pharmaceutical applications involving the very specific inhibition of individual protein kinases [3]. Its discovery stimulated the development of medicinal chemistry programs aimed at generating novel derivatives with higher inhibitory specificity for protein kinase C. Some of these novel compounds (UCN-01, CGP 41251, CEP-751) have entered clinical trials for their use as antitumor agents [4]. The methodology I propose for
discovering new staurosporine-derived compounds is based on a structural approach: once I will clarify the molecular mechanism behind the reaction of StaN, I plan to modify its specificity through rational site-directed mutagenesis. Producing StaN mutants will provide a deeper understanding on essential residues involved in substrate recognition and catalysis and it will open the perspective of creating novel drugs for cancer therapy. Since nature requires thousand years to develop the exquisitely fine tool of enzyme catalysis, modifying its
specificity to produce artificial compounds for pharmaceutical applications is a very arduous challenge. Once achieved, this would provide a powerful tool for drug design and discovery, with a very wide potential.
[1] Gribble G.W. (1993) Studies in Natural Products Chemistry
[2] Tamaoki T. (1986) Biochemical Biophysical Research Communications
[3] Gescher A. (2003) Critical Reviews in Oncology/Hematology
[4] Akinaga S. (2000) Anticancer Drug Design