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Major histocompatibility complex (MHC) class II genes are regulated at the transcriptional level by coordinate action of a limited number of transcription factors that include regulatory factor X (RFX), class II transcriptional activator (CIITA), nuclear factor Y (NF-Y), and cyclic AMP-response element binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB were expressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical and biophysical methods. Analytical ultracentrifugation (AUC) has demonstrated that RFX is a heterotrimer. A heterodimer of RFX5 and RFX-AP was also observed. A high-affinity interaction (K(d) = 25 nM) between RFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction between RFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that the interaction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescence anisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits and complexes binding to duplex DNA. The RFX5 subunit was found to drive recognition of the promoter, while the auxiliary RFX-AP and RFX-ANK subunits were shown to contribute to the specificity of binding for the overall complex. AUC experiments demonstrate that in the absence of additional subunits, monomeric RFX5 binds to X-box DNA with a 1:1 stoichiometry. Interactions between CREB, CIITA, and RFX in the absence of DNA were demonstrated using bead-based immunoprecipitation assays, confirming that preassociation with DNA is not required for forming the macromolecular assemblies that drive MHC class II gene expression.

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.

Herpes simplex virus infections are the cause of significant morbidity, and currently used therapeutics are largely based on modified nucleoside analogs that inhibit viral DNA polymerase function. To target this disease in a new way, we have identified and optimized selective thiazolylphenyl-containing inhibitors of the herpes simplex virus (HSV) helicase-primase enzyme. The most potent compounds inhibited the helicase, the primase and the DNA-dependent ATPase activities of the enzyme with IC50 (50% inhibitory concentration) values less than 100 nM. Inhibition of the enzymatic activities was through stabilization of the interaction between the helicase-primase and DNA substrates, preventing the progression through helicase or primase catalytic cycles. Helicase-primase inhibitors also prevented viral replication as demonstrated in viral growth assays. One compound, BILS 179 BS, displayed an EC50 (effective concentration inhibiting viral growth by 50%) of 27 nM against viral growth with a selectivity index greater than 2,000. Antiviral activity was also demonstrated for multiple strains of HSV, including strains resistant to nucleoside-based therapies. Most importantly, BILS 179 BS was orally active against HSV infections in murine models of HSV-1 and HSV-2 disease and more effective than acyclovir when the treatment frequency per day was reduced or when initiation of treatment was delayed up to 65 hours after infection. These studies validate the use of helicase-primase inhibitors for the treatment of acute herpesvirus infections and provide new lead compounds for optimization and design of superior anti-HSV agents.

Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.

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 specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.

Ultraviolet resonance Raman spectra are reported for cytochrome c (cyt c) in FeII and FeIII oxidation states at low (0.005 M) and high (0.9-1.5 M) ionic strength. With 200-nm excitation the amide band intensities are shown to remain constant, establishing that redox state and ionic strength have no influence on the alpha-helical content. The tyrosine 830/850-cm-1 doublet, however, shows a loss in 830-cm-1 intensity at I = 0.005 M for the FeIII protein, suggesting a weakening or a loss of H-bonding from an internal tyrosine, probably Tyr-48, which is H-bonded to a heme propionate group in cyt c crystals. Excitation profiles of tryptophan peak at approximately 229 nm for both FeII and FeIII forms of cyt c, but at approximately 218 nm for aqueous tryptophan. The approximately 2200-cm-1 red shift of the resonant electronic transition is attributed to the Trp-59 residue being buried and H-bonded. Consistent with this Trp environment, the H-bond-sensitive 877-cm-1 Trp band is strong and sharp, and the 1357/1341-cm-1 doublet has a large intensity ratio, approximately 1.5, for both FeII and FeIII cyt c. The 877-cm-1-band frequency shifts to 860 cm-1 when the Trp indole proton is replaced by a deuteron. This band was used to show that Trp H/D exchange in D2O is much faster for FeIII than FeII cyt c. The half-time for exchange at room temperature is estimated to be approximately 30 and approximately 5 h, respectively, for FeII and FeIII when examined at I = 0.005.(ABSTRACT TRUNCATED AT 250 WORDS)