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Biomedical subjects

P C Weber

Publications and source records attributed to P C Weber.

At least 19 recordsLinked to original sources
PubMed1999

Tricyclic farnesyl protein transferase inhibitors: crystallographic and calorimetric studies of structure-activity relationships.

Crystallographic and thermodynamic studies of farnesyl protein transferase (FPT) complexed with novel tricyclic inhibitors provide insights into the observed SAR for this unique class of nonpeptidic FPT inhibitors. The crystallographic structures reveal a binding pattern conserved across the mono-, di-, and trihalogen series. In the complexes, the tricycle spans the FPT active site cavity and interacts with both protein atoms and the isoprenoid portion of bound farnesyl diphosphate. An amide carbonyl, common to the tricyclic compounds described here, participates in a water-mediated hydrogen bond to the protein backbone. Ten high-resolution crystal structures of inhibitors complexed with FPT are reported. Included are crystallographic data for FPT complexed with SCH 66336, a compound currently undergoing clinical trials as an anticancer agent (SCH 66336, 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl]-1-piperidinecarbo xamide ). Thermodynamic binding parameters show favorable enthalpies of complex formation and small net entropic contributions as observed for 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-ylidene)-1-piperidinyl]-2-oxoethyl]pyridine N-oxide where DeltaH degrees bind = -12.5 kcal/mol and TDeltaS degrees bind = -1.5 kcal/mol.

Alkyl and Aryl Transferases↗
PubMed1999

Stable ubiquitination of the ICP0R protein of herpes simplex virus type 1 during productive infection.

ICP0R is the polypeptide product of an alternatively spliced transcript of the gene encoding the transactivator protein ICP0 of herpes simplex virus type 1 (HSV-1). Although it has been shown to act as a transrepressor of gene expression in transfection assays, overexpression of the ICP0R protein in the recombinant virus HSV-KST was previously found to have no detectable effect on virus replication, so that the role it plays in HSV-1 infection remains unclear. Analysis of HSV-KST-infected cell lysates by Western blotting revealed the presence of not only the 41-kDa ICP0R polypeptide but also a 64-kDa processed form of the protein. This processing event was the result of ubiquination of the ICP0R protein, as demonstrated by the reactivity of the 64-kDa species with antibody specific for the influenza virus hemagglutinin (HA) protein epitope in experiments where the gene encoding ICP0R was coexpressed with a gene encoding HA-tagged ubiquitin. Surprisingly, the 64-kDa form of ICP0R was found to be remarkably stable and persisted in infected cells for many hours after processing, despite the fact that ubiquitination normally functions as a means of tagging proteins for rapid degradation. Analyses of mutant polypeptides containing arginine substitutions at each of the lysine residues of ICP0R, which represent potential ubiquitin conjugation sites, revealed that a single lysine residue at codon 248 was both necessary and sufficient for the appearance of the 64-kDa processed form. However, a number of ICP0R mutants that retained the ubiquitination site at lysine 248 but contained disruptions of sequences at distant sites also lacked detectable 64-kDa protein, indicating that the integrity of the overall structure of ICP0R was an additional determinant for ubiquitination.

Amino Acid Sequence↗
PubMed1999

Differential immobilization and hierarchical involvement of chemokines in monocyte arrest and transmigration on inflamed endothelium in shear flow.

Monocyte extravasation into areas of inflammation involves sequential interactions of multiple adhesion molecules. However, differential contribution of chemokines produced by cytokine-stimulated endothelium and their receptors to leukocyte attachment and transmigration under flow conditions remains to be elucidated. The activation of endothelial cells with TNF-alpha up-regulated mRNA and protein expression of the CXC chemokine GRO-alpha and the CC chemokine monocyte chemotactic protein (MCP)-1, which act through the receptors CXCR2 and CCR2 expressed on monocytes, respectively. Whereas GRO-alpha was immobilized to endothelial cells via heparan sulfate proteoglycans, MCP-1 was secreted in a soluble form. Consequently, inhibition experiments with blocking peptide analogues and monoclonal antibodies revealed that GRO-alpha and its receptor CXCR2 but not MCP-1 and its receptors substantially contributed to conversion of rolling into firm, shear-resistant arrest of monocytes to TNF-alpha-stimulated endothelium in physiological flow. In contrast, MCP-1 and CCR2 but not GRO-alpha and CXCR2 mediated spreading, shape change and subsequent transendothelial migration, which was evident in flow but rarely in stasis and may thus require the establishment of a diffusible MCP-1 gradient. Differential patterns of presentation may determine a functional specialization and hierarchy of chemokines and their receptors in sequential steps of monocyte emigration on inflamed endothelium and shear flow.

Antigen Presentation↗
PubMed1999

Lipids in vascular function.

Physiological and pathological vascular responses depend on the action of numerous intercellular mediators, ranging from hormones to gases like nitric oxide, proteins, and lipids. The last group consists not only of the different types of lipoproteins, but also includes a broad array of other lipophilic signaling molecules such as fatty acids, eicosanoids, phospholipids and their derivatives, sphingolipids and isoprenoids. Due to space limitations, it is impossible to discuss all the vascular effects of lipophilic mediators or compounds. Therefore, we will focus on one of the most important lipid-mediated diseases, atherosclerosis. Lipoproteins and especially their native or oxidized lipid compounds affect vascular function in many different ways, and these effects do not only modulate atherogenesis but are of paramount physiological and pathophysiological importance in other diseases, such as inflammation, tumor metastasis, or normal wound healing.

Animals↗
PubMed1999

Hearing loss.

Hearing loss may be caused by either a conductive problem, such that the sound waves do not reach the tympanic membrane, ossicles or inner ear, or a sensorineural problem, in which the cochlea and/or auditory nerve are somehow damaged. A combination of the above may also occur. The various causes of hearing loss and the options in treatment will be presented.

Cochlear Nerve↗
PubMed1999

Hearing aids.

The first step in the hearing aid fitting and selection process is to determine the patient's hearing disability, motivation, lifestyle, finances, and cosmetic concerns. Most hearing impairments can be helped with a modern hearing aid. With a symmetric hearing loss, a binaural fitting provides the most benefit and should be attempted initially. The binaural fitting may not be successful in cases with large asymmetries in auditory function. The advances in electronic technology, such as the digital hearing aid, are moving faster than the research to determine which is the most effective signal processing method. The near future should yield more significant improvements in hearing aids that will provide a better quality of life of the hearing-impaired population.

Attitude to Health↗
PubMed1999

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation.

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 A resolution) and iNOS (2.25 A resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.

Amino Acid Sequence↗
PubMed1999

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase beta-subunit C-terminal mutants.

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimeric enzyme that catalyzes post-translational farnesylation of many cellular regulatory proteins including p21 Ras. To facilitate the construction of site-directed mutants, a novel translationally coupled, two-cistron Escherichia coli expression system for rat FPT has been developed. This expression system enabled yields of >5 mg of purified protein per liter of E.coli culture to be obtained. The E.coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was used to construct three beta-subunit C-terminal truncation mutants, Delta5, Delta10 and Delta14, which were designed to eliminate a lattice interaction between the beta-subunit C-terminus of one molecule and the active site of a symmetry-related molecule. Steady-state kinetic analyses of these mutants showed that deletion up to 14 residues at the C-terminus did not reduce the value of kcat; however, Km values for both peptide and FPP increased 2-3-fold. A new crystalline form of FPT was obtained for the Delta10 C-terminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val-Ile-Met-COOH peptide and alpha-hydroxyfarnesylphosphonic acid. The crystals diffract to beyond 2.0 A resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.

Alkyl and Aryl Transferases↗
PubMed1999

Lovastatin decreases the receptor-mediated degradation of acetylated and oxidized LDLs in human blood monocytes during the early stage of differentiation into macrophages.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors are used therapeutically to upregulate the LDL receptor-mediated removal of plasma cholesterol by the liver. Several lines of evidence indicate that these drugs also exert direct effects on the metabolism of native and modified LDL in extrahepatic cells. We studied the effects of lovastatin (LOV) on the degradation of native, acetylated, and oxidized LDL, and on levels of mRNA encoding for the LDL, types I and II class A macrophage scavenger, and CD36 receptors in human blood monocytes at different stages of their maturation into adherent macrophages. LOV (10 micromol/L) reduced the degradation of acetylated LDL when added to freshly isolated cells cultured for 2 (81+/-4% of control, P<0.05) and 5 (76+/-6%, of control, P<0.05) days. The degradation of oxidized LDL was also reduced in cells treated with LOV for 2 days after seeding (51+/-3% of control, P<0. 001) but not in 5-day-old cells. LOV had no significant effect on the degradation of either acetylated or oxidized LDL when added to fully matured macrophages allowed to differentiate under control conditions for 7 days before incubations with 10 micromol/L LOV for an additional 2 days. In contrast, LOV increased the degradation of native LDL in these cells at all 3 stages of cell differentiation. LOV also reduced class A types I and II macrophage scavenger receptor and CD36 mRNA levels in 2- and 5-day-old cells but not in the more mature macrophages. These data suggest that 3-hydroxy-3-methylglutaryl-coenzyme A inhibitors may reduce the expression and function of the class A types I and II macrophage scavenger receptor and CD36 in monocytes, during the early stages of their differentiation into adherent macrophages. These effects, if operative in vivo, may slow down the development of the atherosclerotic plaque and thus contribute to the beneficial effects of these drugs.

Adult↗
PubMed1999

Effects of oxidized low density lipoprotein, lipid mediators and statins on vascular cell interactions.

The integrin heterodimer CDllb/CD18 (alphaMbeta2, Mac-1, CR3) expressed on monocytes or polymorphonuclear leukocytes (PMN) is a receptor for iC3b, fibrinogen, heparin, and for intercellular adhesion molecule (ICAM)-1 on endothelium, crucially contributing to vascular cell interactions in inflammation and atherosclerosis. In this report, we summarize our findings on the effects of lipid mediators and lipid-lowering drugs. Exposure of endothelial cells to oxidized low density lipoprotein (oxLDL) induces upregulation of ICAM-1 and increases adhesion of monocytic cells expressing Mac-1. Inhibition experiments show that monocytes use distinct ligands, i.e. ICAM-1 and heparan sulfate proteoglycans for adhesion to oxLDL-treated endothelium. An albumin-transferable oxLDL activity is inhibited by the antioxidant pyrrolidine dithiocarbamate (PDTC), while 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) or lysophosphatidylcholine had no effect, implicating yet unidentified radicals. Sequential adhesive and signaling events lead to the firm adhesion of rolling PMN on activated and adherent platelets, which may occupy areas of endothelial denudation. Shear-resistant arrest of PMN on thrombin-stimulated platelets in flow conditions requires distinct regions of Mac-1, involving its interactions with fibrinogen bound to platelet alphallbbeta3, and with other platelet ligands. Both arrest and adhesion strengthening under flow are stimulated by platelet-activating factor and leukotriene B4, but not by the chemokine receptor CXCR2. We tested whether Mac-1-dependent monocyte adhesiveness is affected by inhibitors of hydroxy-methylglutaryl-Coenzyme A reductase (statins) which improve morbidity and survival of patients with coronary heart disease. As compared to controls, adhesion of isolated monocytes to endothelium ex vivo was increased in patients with hypercholesterolemia. Treatment with statins decreased total and low density lipoprotein (LDL) cholesterol plasma levels, surface expression of Mac-1, and resulted in a dramatic reduction of Mac-1-mediated monocyte adhesion to endothelium. The inhibition of monocyte adhesion was reversed by mevalonate but not LDL in vitro, indicating that isoprenoid precursors are crucial for adhesiveness of Mac-1. Such effects may crucially contribute to the clinical benefit of statins, independent of cholesterol-lowering, and may represent a paradigm for novel, anti-inflammatory mechanisms of action by this class of drugs.

Anticholesteremic Agents↗
PubMed1998

Crystal structure of farnesyl protein transferase complexed with a CaaX peptide and farnesyl diphosphate analogue.

The crystallographic structure of acetyl-Cys-Val-Ile-selenoMet-COOH and alpha-hydroxyfarnesylphosphonic acid (alphaHFP) complexed with rat farnesyl protein transferase (FPT) (space group P61, a = b = 174. 13 A, c = 69.71 A, alpha = beta = 90 degrees, gamma = 120 degrees, Rfactor = 21.8%, Rfree = 29.2%, 2.5 A resolution) is reported. In the ternary complex, the bound substrates are within van der Waals contact of each other and the FPT enzyme. alphaHFP binds in an extended conformation in the active-site cavity where positively charged side chains and solvent molecules interact with the phosphate moiety and aromatic side chains pack adjacent to the isoprenoid chain. The backbone of the bound CaaX peptide adopts an extended conformation, and the side chains interact with both FPT and alphaHFP. The cysteine sulfur of the bound peptide coordinates the active-site zinc. Overall, peptide binding and recognition appear to be dominated by side-chain interactions. Comparison of the structures of the ternary complex and unliganded FPT [Park, H., Boduluri, S., Moomaw, J., Casey, P., and Beese, L. (1997) Science 275, 1800-1804] shows that major rearrangements of several active site side chains occur upon substrate binding.

Alkyl and Aryl Transferases↗
PubMed1998

Pasteurella multocida toxin increases endothelial permeability via Rho kinase and myosin light chain phosphatase.

Pasteurella multocida toxin (PMT) has been shown to induce actin reorganization through activation of the GTPase Rho. Here we investigated the involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial permeability and the underlying actin reorganization of endothelial cells. Stimulation of endothelial layers with PMT enhanced transendothelial permeability > 10-fold, and this was abolished by pretreatment with the specific Rho inactivator C3 transferase from Clostridium botulinum. The PMT-induced increase in endothelial permeability was associated with 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphorylation, and 3) endothelial cell retraction and actin stress fiber formation. PMT-stimulated actin reorganization could be prevented by 1) pretreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase. Together, these results suggest that PMT activates Rho/Rho kinase, which inactivates MLC phosphatase. The resulting increase in MLC phosphorylation causes endothelial cell retraction and a rise in endothelial permeability.

Bacterial Proteins↗
PubMed1998

Biochemical and crystallographic characterization of homologous non-peptidic thrombin inhibitors having alternate binding modes.

The X-ray crystallographic structure of [N-(3-phenylpropionyl)-N-(phenethyl)]-Gly-boroLys-OH (HPBK, Ki = 0. 42 nM, crystallographic R factor to 1.8 A resolution, 19.6%) complexed with human alpha-thrombin shows that the boron adopts a tetrahedral geometry and is covalently bonded to the active serine, Ser195. The HPBK phenethyl aromatic ring forms an edge-to-face interaction with the indole side chain of Trp215. Four HPBK analogs containing either electron-withdrawing or electron-donating substitutents at the 3' position of the phenethyl ring were synthesized in an attempt to modulate ligand affinity by inductive stabilization of the edge-to-face interaction. Refined crystallographic structures of the trifluoromethyl (Ki = 0.37 nM, crystallographic R factor to 2.0 A resolution = 18.7%), fluoro (Ki = 0.60; R factor to 2.3 A resolution = 18.4%), methoxy (Ki = 0.91 nM, R factor to 2.2 A resolution = 19.8%) and methyl (Ki = 0.20 nM, R factor to 2.5 A resolution = 16.9%) HPBK analogs complexed with thrombin revealed two binding modes for the closely related compounds. A less than 1.5-fold variation in affinity was observed for analogs (trifluoromethyl-HPBK and fluoro-HPBK) binding with the edge-to-face interaction. The slight inductive modulation is consistent with the overall weak nature of the edge-to-face interaction. Owing to an unexpected rotation of the phenethyl aromatic ring, the 3' substituent of two analogs, methoxy-HPBK and methyl-HPBK, made direct contact with the Trp215 indole side chain. Increased affinity of the 3' methyl analog is attributed to favorable interactions between the methyl group and the Trp215 indole ring. Differences in inhibitor, thrombin and solvent structure are discussed in detail. These results demonstrate the subtle interplay of weak forces that determine the equilibrium binding orientation of inhibitor, solvent and protein.

Anticoagulants↗
PubMed1998

Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells.

The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.

ADP Ribose Transferases↗
PubMed1998

Molecular recognition of cyclic urea HIV-1 protease inhibitors.

As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.

Anti-HIV Agents↗
PubMed1998

Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex.

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein into its constituent nonstructural proteins. The NS3/4A complex is thus an attractive target for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during expression, producing a noncovalent complex between HisNS3 and NS4A with a subnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatography. We removed the His tag by thrombin cleavage and then further purified the complex by gel filtration. The purified NS3/4A complex is active in a protease assay using a synthetic peptide substrate derived from the NS5A-NS5B junction, with kcat/K(m) of 3700 (+/- 600) M-1 s-1, an order of magnitude above those previously reported for NS3 expressed by other strategies. This high protease activity implies that the full-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activity is dependent on the aggregation state of the NS3/4A complex. The monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.

Adenosine Triphosphatases↗