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A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.

The allosteric inhibition of the lymphocyte function associated antigen-1/intercellullar adhesion molecule (LFA-1/ICAM-1) interaction, by a class of small molecules, is characterized by a battery of mass spectrometric techniques. Binding of hydantoins to the I domain of LFA-1 is observed by size exclusion chromatography/mass spectrometry (SEC/MS) and by direct electrospray ionization mass spectrometry (ESI/MS). A photoactive hydantoin analog specifically labels an amino acid residue of LFA-1 I domain. Competition with this photoaffinity labeling by a panel of inhibitors is correlated with their Kd’s for inhibition of the LFA-1/ICAM interaction. Alterations to the tertiary structure of LFA-1 I domain, upon compound binding, are inferred from perturbation in the ESI mass spectrum of the polypeptide’s charge state distribution and by an altered level of nonspecific multimer formation. The results demonstrate specific, stoichiometric, reversible binding of the hydantoins to LFA-1. They further show correlation of this binding with activity and indicate alterations in the polypeptide’s tertiary structure, on hydantoin binding, consistent with the proposed mechanism for inhibition of the protein–protein interaction.

The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.

Unlike its thiocyanate analogues, which involve the formation of the geminite [Pd{(C 6H 5) 2P(CH 2) 2-P(C 6H 5) 2}(SCN)(NCS)] and the di-N-bound complex [Pd{(C 6H 5) 2P(CH 2) 3P(C 6H 5) 2}(NCS) 2], the corresponding selenocyanate complexes contain only Sebound groups, as determined by the results of single crystal X-ray diffraction studies. Se-bonding is also maintained in the [Pd{(C 6H 5) 2PCH 2 o C 6H 4-CH 2P-(C 6H 5) 2}(SeCN) 2] complex, based on the results of IR studies. Only in the case of the (C 6H 5) 2PCH 2P(C 6H 5) 2 palladium(II) complexes, do both pseudohalides exhibit analogous bonding via their chalcogen atoms. The selenocyanate's bonding pattern is thus dominated by the soft/soft interaction afforded by Pd-SeCN bonding, remaining impervious to increasing steric demands of the diphosphine ligands.

The reaction, at 25 °C in methanol, between [Pd(SeCN)4]2− and bis(diphenylphosphino)methane (dpm) has been found to produce cyano(selenocyanato) [diphenyl(diphenylphosphinomethyl) phosphine selenide]palladium(II), [Pd(dpmSe)(CN)(SeCN)], wherein the cyanide group is trans to an Se atom which has been inserted into one PdP bond, and the selenocyanate group is trans to the unchanged diphenylphosphino group. The structure has been confirmed by the results of a single crystal X-ray diffraction study. The structural isomer, [Pd(dpm)- (SeCN)2], of the foregoing complex has also been prepared by the reaction of Pd(C2H3O2)2 with dpm, followed by reaction with KSeCN. Heating the [Pd(dpm)(SeCN)2] isomer converts it into [Pd- (dpmSe)(CN)(SeCN)]. A mechanism is proposed for the isomerization which involves an intramolecular selenium atom insertion.

Carboxylate and guanidinium groups of amino acids have been studied by ultraviolet resonance Raman (UVRR) spectroscopy. On near-resonant excitation (192 nm) into the allowed π–π* transition of the carboxylate chromophore, the vibrational spectrum of aspartate displays only one band originating from the COO− symmetric stretching mode, consistent with an A-term enhancement mechanism. The UVRR spectra of the guanidinium ion reveals a complex vibrational pattern, and the bands have been assigned with the aid of a normal coordinate analysis. UV enhancement is observed for both asymmetric (E′) and totally symmetric (A1′) modes, and also for the overtone of the out-of-plane CN3 bending mode, Γ(A2″). The E′ enhancement is suggested to reflect Jahn—Teller splitting in the first allowed electronic transition A2″ E″, whereas the 2Γ(A2″) enhancement requires a lowering of the force constant for out-of-plane bending in the excited state, consistent with its expected tendency toward pyramidalization. Similar enhancement patterns are seen in the UVRR spectra of the guanidinium chromophore in methylguanidinium and arginine, which show, in addition, splittings and intensity redistribution due to the substituents. The excitation profiles of guanidinium and arginine reveal a complex structure that may arise from an excited-state Jahn—Teller effect. The Raman enhancements are too low to permit detection of guanidinium or carboxylate in the UVRR spectra of proteins, even at wavelengths as low as 192 nm.