Pseudoxazolones as hepatitis A virus 3C proteinase inhibitors and bacterially derived antimicrobial peptides

A number of imino analogues of amino acids were synthesized and tested as inhibitors of the 3C proteinase enzyme of the Hepatitis A Virus (HAV 3C). The 3C proteinase is essential for viral replication and the conservation of its active site among a wide range of serotypes makes the enzyme an attractive target for chemotherapy. The most effective compounds tested were the monophenyl pseudoxazolones E-(21a) and Z-( 21b)-2-benzylidene-2H-oxazol-5-one with IC50 values of 6 and 4 muM, respectively.; Antibiotic peptides derived from bacterial sources were also studied. Mattacin (52) was originally identified as a peptide antibiotic isolated from Paenibacillus kobensis M with potent antimicrobial activity towards a variety of Gram-negative organisms. After performing a series of multidimensional NMR experiments and stereochemical analysis of the component amino acids, a full structural characterization of mattacin (52) was achieved, showing it to be a member of the polymyxin family of peptide antibiotics. Conformational analyses were undertaken using 2D NMR techniques (TNROESY and transferred NOE) to investigate the solution structure of mattacin, both in isolation and in the presence of lipid A ( 51). Lipid A is the endotoxic principle of the outer membrane of Gram-negative bacteria and is the putative receptor for polymyxin peptides. Isothermal titration calorimetry was used to compare the binding energy of mattacin and polymyxin B (50) (a common therapeutic agent) with lipid A. Results from these experiments indicate that mattacin behaves in a manner similar to polymyxin B, both in its preferred solution conformation and affinity for lipid A.; New methods for the characterization of lantibiotic peptides were also developed. Isolated from bacterial sources, lantibiotics exhibit antibacterial activity towards Gram-positive organisms, often several orders of magnitude more potent than traditional antibiotics such as penicillin. Lantibiotics contain a number of unique structural features including dehydro and lanthionine (thioether) residues. Introduced after ribosomal translation of the parent peptide, these moieties render conventional means of peptide analysis ineffective. A new analytical approach, successfully applied to the most common lantibiotic nisin (53), involved the use of nickel boride (Ni2B) in the presence of D2, to reduce dehydro side chains and cleave lanthionine bridges with concomitant deuterium incorporation at the site of reduction or desulfurization. Using this method it was possible to identify and distinguish the original locations of dehydro side chains and lanthionine bridges by use of peptide sequencing (Edman degradation) coupled with mass spectrometry. This methodology was also successfully applied to a new two-component lantibiotic, lacticin 3147, produced by Lactococcus lactis subsp. lactis DPC3147. After acquiring a lactococcal strain with amplified production levels of lacticin 3147, and optimizing the isolation protocol, quantities of material suitable for NMR analysis were obtained. The primary structures, including the lanthionine bridging patterns of both peptides were determined by using multidimensional NMR. These results indicate that lacticin A1 (68) has a specific lanthionine bridging pattern similar to the known lantibiotic mersacidin (66) and that the A2 peptide 69 bears a resemblance to lactosin S (67).