The Ras/Raf/MEK/ERK signal transduction, an oncogenic pathway implicated in a variety of human cancers, is a key target in anticancer drug design. A novel series of pyrimidylpyrrole ERK inhibitors has been identified. Discovery of a conformational change for lead compound 2, when bound to ERK2 relative to antitarget GSK3, enabled structure-guided selectivity optimization, which led to the discovery of 11e, a potent, selective, and orally bioavailable inhibitor of ERK.

Compound 3 is a potent aminobenzimidazole urea with broad-spectrum Gram-positive antibacterial activity resulting from dual inhibition of bacterial gyrase (GyrB) and topoisomerase IV (ParE), and it demonstrates efficacy in rodent models of bacterial infection. Preclinical in vitro and in vivo studies showed that compound 3 covalently labels liver proteins, presumably via formation of a reactive metabolite, and hence presented a potential safety liability. The urea moiety in compound 3 was identified as being potentially responsible for reactive metabolite formation, but its replacement resulted in loss of antibacterial activity and/or oral exposure due to poor physicochemical parameters. To identify second-generation aminobenzimidazole ureas devoid of reactive metabolite formation potential, we implemented a metabolic shift strategy, which focused on shifting metabolism away from the urea moiety by introducing metabolic soft spots elsewhere in the molecule. Aminobenzimidazole urea 34, identified through this strategy, exhibits similar antibacterial activity as that of 3 and did not label liver proteins in vivo, indicating reduced/no potential for reactive metabolite formation.

Collision-induced dissociation (CID) was performed on multiply deprotonated ions from three commercial peptides: hirudin (54-65), fibrinopeptide B, and oxidized insulin chain A. Ions were produced by electrospray ionization in a Fourier transform ion cyclotron resonance mass spectrometer. Each of these peptides contains multiple acidic residues, which makes them very difficult to ionize in the positive mode. However, the peptides deprotonate readily making negative ion studies a viable alternative. The CID spectra indicated that the likely deprotonation sites are acidic residues (aspartic, glutamic, and cysteic acids) and the C-terminus. The spectra are rife with c, y, and internal ions, although some a, b, x, and z ions form. Many of the fragment ions were formed from cleavage adjacent to acidic residues, both N- and C-terminal to the acidic site. In addition, neutral loss (e.g., NH3, CH3, H2O, and CO2) was prevalent from both the parent ions and from fragment ions. These neutral eliminations were often indicative of specific amino acid residues. The fragmentation patterns from several charge states of the parent ions, when combined, provide significant primary sequence information. These results suggest that negative mode CID of multiply deprotonated ions provides useful structural information and can be worthwhile for highly acidic peptides that do not form positive ions in abundance.

Apparent gas-phase basicities (GB(app)s) for [M + H]+ of bradykinin, des-Arg1-bradykinin and des-Arg9-bradykinin have been assigned by deprotonation reactions of [M + 2H]2+ in a Fourier transform ion cyclotron resonance mass spectrometer. With a GB(app) of 225.8 +/- 4.2 kcal x mol(-1), bradykinin [M + H]+ is the most basic of the ions studied. Ions from des-Arg1-bradykinin and des-Arg9-bradykinin have GB(app) values of 222.8 +/- 4.3 kcal x mol(-1) and 214.9 +/- 2.3 kcal x mol(-1), respectively. One purpose of this work was to determine a suitable reaction efficiency 'break point' for assigning GB(app) values to peptide ions using the bracketing method. An efficiency value of 0.1 (i.e. approximately 10% of all collisions resulting in a deprotonation reaction) was used to assign GB(app)s. Support for this criterion is provided by the fact that our GB(app) values for des-Arg1-bradykinin and des-Arg9-bradykinin are identical, within experimental error, to literature values obtained using a modified kinetic method. However, the GB(app)s for bradykinin ions from the two studies differ by 10.3 kcal x mol(-1). The reason for this is not clear, but may involve conformation differences produced by experimental conditions. The results may be influenced by salt-bridge conformers and/or by conformational changes caused by the use of a proton-bound heterodimer in the kinetic method. Factors affecting the basicities of these peptide ions are also discussed, and molecular modeling is used to provide information on protonation sites and conformations. The presence of two highly basic arginine residues on bradykinin results in its high GB(app), while the basicity of des-Arg1-bradykinin ions is increased by the presence of two proline residues at the N-terminus. The proline residue in the second position folds the peptide chain in a manner that increases intramolecular hydrogen bonding to the protonated N-terminal amino group of the proline at the first position.

The gas-phase basicity (GB) of proline, which is the only imino acid and is reported to play a significant role in protein folding, was determined by deprotonation reactions in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Protonated peptide ions were generated by fast atom bombardment in an external ion source. The GB of proline was found to be 213.3 kcal/mol. Among the dipeptides studied, the GB of glycylproline (GlyPro) was determined to be 214.8 kcal/mol, while prolyglycine (ProGly) was 4.2 kcal/mol more basic (GB = 219.0 kcal/mol). The basicity of the tripeptide prolylglycylglycine (ProGlyGly, GB = 219.0 kcal/mol) is 2 kcal/mol higher than the basicities of glycylglycylproline (GlyGlyPro) and glycylprolylglycine (GlyProGly), both of which have GB = 217.0 kcal/mol. The enhanced basicity of ProGlyGly is consistent with the protonation site being the terminal amino nitrogen with enhanced stabilization of the charge by the nearby bulkier residue. Interestingly, prolyproline (ProPro), which has a GB of 223.3 kcal/mol, is more basic than the other di- and tripeptides studied. Consequently, semi-empirical AM1 calculations were employed to probe the structural characteristics and intramolecular hydrogen bonding interactions for ProPro, GlyPro, ProGly and glycylglycine (GlyGly). ProPro was found to have a unique structure with two potential hydrogen bonds between amino hydrogens and both carbonyl oxygens.