I. Comparison of Translesion Bypass of Guanine–N2 Monoadducts of Mitomycin C and Guanine-N7 Monoadducts of 2,7-diaminomitosene by T7 exo-, Klenow exo-, eta and Klenow exo+ DNA Polymerases. II. Structure-based Design, Synthesis, Structure-conformation and Structure-activity Relationships Studies of D-Phe-Pro-D-Arg-P1’-CONH2 Tetrapeptides with Inhibitory Activity for Thrombin.

Abstract

The guanine (G)-N2 DNA monoadduct of mitomycin C (MC), a cytotoxic anticancer drug, inhibits translesion bypass by DNA polymerases. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2\u27-deoxyguanosine (2,7-DAM-dG) and is noncytotoxic. In part I of this study we tested a potential correlation between the lack of cytotoxicity of 2,7-DAM and the relative ease of bypass of this adduct as compared with the MC adduct. 24-mer and 27-mer templates, adducted at a single guanine either with MC or 2,7-DAM were synthesized and submitted to extension of primers by T7 exo-, Klenow exo-, Klenow exo+, and eta DNA polymerases. The G-N7-2,7-DAM adduct was bypassed by all four polymerases, resulting in the production of a fully extended primer. In sharp contrast, the G-N2-MC monoadduct was not bypassed beyond the adduct position under the same conditions by any of the four polymerases. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5\u2732P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. These results showed for the first time that the dG-N7-2, 7-DAM lesion is non-mutagenic in cell-free systems. In part II of this research structure-based design and molecular docking were employed to design in silico libraries of peptides as potential reversible inhibitors of thrombin. The candidate inhibitors were selected from two original classes of amino acids sequences (1)-D-Phe-Pro-Arg (P1)-D-Pro(P1\u27)-P2\u27-P3\u27-CONH2 and (2)-D-Phe-Pro-D-Arg(P1)-P1\u27-P2\u27-P3\u27-CONH2. For the first time in the field of peptides inhibitors for thrombin we showed that the presence of D-Pro at P1\u27 Position and the use of D-Arg instead of L-Arg at P1 Position is responsible for inhibiting hydrolysis of these of peptides by thrombin, causing these sequences to be inhibitors. In vitro kinetics of thrombin inhibition showed a specific structure-activity relationship at P1\u27 position in the peptide sequence space (2)-D-Phe-Pro-D-Arg(P1)-P1\u27-CONH2. The lead peptides (D-Phe-Pro-D-Arg-D-Ala-CONH2, D-Phe-Pro-D-Arg-D-Thr-CONH2, D-Phe-Pro-D-Arg-D-Cys-CONH2, D-Phe-Pro-D-Arg-D-Ser-CONH2) had competitive or mixed inhibition with respect to thrombin and are characterized by inhibitory constant in the 20-0.8 micromolar range