Soluble epoxide hydrolase inhibitors and their potential for treatment of multiple pathologic conditions.

Epoxyeicosanoids, including the epoxyeicosatrienoic acids are signaling molecules which appear to help ameliorate the effects of a wide variety of pathological conditions. The enzyme soluble epoxide hydrolase (sEH) metabolizes these molecules by converting them to their corresponding vicinal diols. Inhibition of sEH either by knockout or chemical inhibitors increases epoxyeicosanoid levels in vivo and provides significant organ protection in models of brain, cardiac, and renal injury. sEH also appears to be involved in modulating inflammation, pain pathways, pulmonary function, hypertension, and diabetes. Potent sEH inhibitors have been developed in academic, pharmaceutical, and biotech laboratories and described in the patent and scientific literature. Most of the inhibitor scaffolds employ a urea or amide which functions as an active-site transition state mimic. Arête Therapeutics compound AR9281 successfully completed phase Ia and 1b studies. A phase IIa proof of concept trial for treatment of impaired glucose tolerance has been completed, but the results are not yet reported.

The use of a modular rotating hinge component in salvage revision total knee arthroplasty.

Revision total knee arthroplasty (TKA) using a second-generation modular rotating hinge design was performed on 16 knees in 15 patients over a 5-year period. Follow-up of 2 to 6 years (mean, 51 months) was obtained in 14 knees in 13 patients. Indications for revision were aseptic loosening of a hinged prosthesis (8 knees), loosening and bone loss associated with chronic extensor mechanism disruption (2 knees), component instability with chronic medial collateral ligament disruption (3 knees), and comminuted distal femur fracture (1 knee). Clinical and radiographic results were reviewed and compared with 87 patients who underwent revision TKA using a standard condylar revision design during the same period. Early results showed comparable postoperative knee scores and range of motion between the 2 groups despite the use of the rotating hinge component in more complex revision cases. No patient has exhibited radiographic evidence of definite component loosening. Alignment of 5 degrees to 10 degrees of valgus in the frontal plane and within 2 degrees of neutral in the sagittal plane was achieved consistently. Short-term clinical and radiographic results are encouraging and suggest that a second-generation modular rotating hinge component can be used successfully in selected salvage revision cases.

High-affinity interactions of tumor necrosis factor receptor-associated factors (TRAFs) and CD40 require TRAF trimerization and CD40 multimerization.

Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TRAFs) that interact with receptor cytoplasmic domains following ligand-induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that directly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domains of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-terminal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences necessary for trimer formation and indicated that the coiled coil TRAF-N region is required for trimerization. Consistent with the idea that TRAF trimerization is required for high-affinity interactions with CD40, monomeric TRAF-C domains bound to CD40 significantly weaker than trimeric TRAFs. In surface plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2 > TRAF3 >> TRAF1 and TRAF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-kappaB and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary for maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomerized receptors as well as different requirements for degree of receptor multimerization appear to contribute to the selectivity of TRAF recruitment to receptor cytoplasmic domains.

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Revision knee arthroplasty with patella replacement versus bony shell.

A study was undertaken to assess the clinical results of revision total knee arthroplasty in which an unresurfaced bony shell was left after removing a patellar component versus those in which a patellar component was implanted. Followup was obtained in 123 of 130 consecutive revision total knee replacements (94%) from three centers. In 21 knees a shell of patellar bone was left and 92 knees had a patellar component in place. Ten patients had a patellectomy and were excluded from consideration. The group with the bony shell had a lower postoperative knee score but the preoperative Knee Society clinical score was significantly lower as well in this group of patients. Compared with the group of patients with the patellar component in place, the group of patients with knees left with a bony shell had a significantly higher percentage of patients who had difficulty using stairs, a higher percentage of patients who were not satisfied with their surgery, and a higher percentage of patients who rated their surgery as unsuccessful in returning them to normal daily activities. When a patellar component was not able to be implanted in revision total knee arthroplasty, a lower quality result was observed.

Interaction between the SH2 domains of ZAP-70 and the tyrosine-based activation motif 1 sequence of the zeta subunit of the T-cell receptor.

One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR zeta ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon zeta ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of zeta chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein-phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein-phosphopeptide complex relative to the protein alone.

Determination of affinities for lck SH2 binding peptides using a sensitive fluorescence assay: comparison between the pYEEIP and pYQPQP consensus sequences reveals context-dependent binding specificity.

The development of a sensitive fluorescence binding assay for evaluating the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides containing the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the acetamido-anilino-naphthyl sulfonic acid (AANS), acrylodan, or dansyl groups as fluorophores. The spectral features of these probes were characterized in the presence and absence of the lck SH2 domain. The binding affinities (Kd) for the fluorescent peptides studied ranged from 40 to 500 nM. The fluorescent peptide containing the sequence FTATEC(AANS)QpYEEIP exhibited the highest binding affinity (Kd = 3.98 x 10(-8) M) and largest change in emission intensity (approximately 8.7-fold) upon binding the SH2 domain. This probe was subsequently used in competitive binding assays to study the interaction of the lck SH2 domain with a series of phosphopeptides related to the pYEEIP and pYQPQP (the pY505 C-terminal) consensus sequences. The effects of peptide length and substitutions of residues within the pYEEIP sequence are discussed in terms of binding affinities. Comparison between the two peptide series revealed that the contributions of individual substitutions to binding affinity are context-dependent. The data also led to the conclusion that the presence of P at +2 results in a functional "truncation" of the binding sequence; i.e., residues at positions higher than +2 do not participate significantly in binding. This implicit truncation may actually be a desired property for the autoregulatory nature of the pYQPQP sequence, since it retains specificity for the SH2 domain while adjusting the Kd to a value appropriate for maintaining the delicate balance of receptor-ligand interactions that are involved in signal transduction events.

Binding affinities of the SH2 domains of ZAP-70, p56lck and Shc to the zeta chain ITAMs of the T-cell receptor determined by surface plasmon resonance.

The zeta chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the zeta chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated. The phosphotyrosine residues, along with flanking amino acids, form an activation motif (and are shared by signaling subunits in the TCR, B cell receptor, and FcgammaRI) termed tyrosine-based activation motifs (ITAMs). ITAMs serve as binding sites for SH2 domain-containing proteins. Recent evidence suggests that the zeta chains provide docking space for several key signal transduction molecules such as ZAP-70, p56lck, and Shc. To determine if ZAP-70, p56lck, and Shc bind to particular zeta chain ITAM sequences, quantitative free-solution measurements of binding affinities (Kd) were obtained by use of surface plasmon resonance technology. The results indicate that binding affinities of distinct SH2 domains to individual and paired phosphorylation sites greatly differ, and may dictate the sequence of signal transduction events.

Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides.

Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.

Regulation of L-selectin expression by membrane proximal proteolysis.

L-selectin is a lectin cell adhesion molecule expressed on the cell surfaces of lymphocytes, monocytes and granulocytes. Upon leukocyte activation or L-selectin cross-linking the transmembrane-bound L-selectin is rapidly shed from the cell surface. Based on these observations, it has been proposed that L-selectin is proteolytically cleaved from the cell surface. However a panel of common protease inhibitors have no effect on L-selectin proteolysis. To further define the mechanism of L-selectin down-regulation we have produced reagents to study proteolytic fragments of L-selectin. We have developed a trapping ELISA for the detection of soluble L-selectin. In addition we have produced a high affinity polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled PHA lymphoblasts and peripheral blood neutrophils. We review here our progress in defining a 6 kD L-selectin transmembrane peptide (L-STMP) from PMA activated lymphoblasts and fMLP-activated neutrophils. Radiochemical sequencing data indicate that the cleavage site occurs between Lys321 and Ser322 in a short membrane-proximal region of the extracellular domain.

High-performance liquid chromatography and photoaffinity crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human immunodeficiency virus.

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.

Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin.

Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti-cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L-selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif.

Amino acid substitutions in HIV-1 reverse transcriptase with corresponding residues from HIV-2. Effect on kinetic constants and inhibition by non-nucleoside analogs.

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.

Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: preparation of heterodimeric enzyme devoid of unprocessed gene product.

A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.

Proteolytic processing of the cardioviral P2 region: primary 2A/2B cleavage in clone-derived precursors.

The primary 2A/2B cleavage within cardiovirus polyprotein was examined by construction of cDNA plasmids which linked fragments from the P2 region of encephalomyocarditis virus (EMCV) and Mengovirus genomes to the EMCV 5' nontranslated region. When RNA transcripts from these clones were tested in reticulocyte extracts, the synthesized proteins were cotranslationally processed at the 2A/2B site. No viral segments outside of the P2 region were required for this activity. Engineered deletions which removed the amino-terminal two-thirds of protein 2A or the carboxyl half of protein 2B had no effect on this scission, nor did insertions into a Ser-Ala-Phe sequence (SAF) within 2B, which is conserved in most cardio- and aphthoviruses. In contrast, mutations which disrupted a conserved Asn-Pro-Gly-Pro (NPGP) sequence abolished primary scission. Precursors thus inactivated were unable to serve as substrate when simultaneously expressed with active (wild-type) 2AB sequences. Microsequencing placed the EMCV primary cleavage site between the Gly/Pro pair within the NPGP sequence. It was also determined that endogenous viral protease 3C is the previously unidentified agent responsible for cardiovirus 1D/2A scission, a cleavage that is part of the primary processing reaction in poliovirus.

Evaluating two different evaporative cooling management systems for dairy cows in a hot, dry climate.

Milk production, rectal temperature, live weight gain, reproductive performance, and weather data were obtained on 150 Holstein cows managed under two cooling systems on a large dairy farm in Saudi Arabia during the summer months. Cows were paired at the onset of the trial according to days postpartum, lactation number, and current milk production. Females were then allocated either to a system that forced air, precooled by evaporative cooling, over the cows or to a system that alternately showered a fine mist onto the surface of the cows and then forced air at ambient temperature over them. The cows receiving evaporative cooling and those with spray and fan cooling were on sand and on slatted concrete floor, respectively, during the periods of cooling. The onset of estrus was observed during the night when the cows preferred the unshaded corral. For the 120-d trial period, 84% (62 of 75) of the cows receiving evaporative cooling and 60% (44 of 75) of the cows receiving spray and fan cooling became pregnant. In the evaporative cooling system, the pregnancy rate per insemination was 35.2% (179 inseminations) versus 23.2% (194 inseminations) for spray and fan cooling. The mean postpartum interval to pregnancy was 117.6 d for the evaporative cooling cows and 146.7 d for spray and fan cooling cows. The evaporative cooling system, with its open shades and sand bedding, enhanced reproductive performance and milk production compared with that of cows cooled with a spray and fan system with slatted flooring in this hot climate.

Nonnucleoside inhibitors of HIV-1 reverse transcriptase: nevirapine as a prototype drug.

Nevirapine, a dipyridodiazepinone, is a highly specific inhibitor of HIV-1 reverse transcriptase (RT) which exhibits an IC50 = 84nM in enzyme assays and IC50 = 40nM against HIV-1 replication in cell culture. This nonnucleoside inhibitor acts noncompetitively with respect to nucleoside triphosphates, template and primer suggesting that nevirapine does not bind to the active site of RT. Studies employing an azido analogue of nevirapine as a photoaffinity probe indicated that one molecule of inhibitor is sufficient to inactivate one molecule of heterodimeric enzyme and demonstrated that only the p66 subunit of p66/p51 heterodimeric RT is covalently labeled by this probe. When subjected to trypic mapping, Tyr 181 and Tyr 188 were labeled with probe and consequently these aromatic residues are apparently near or actually within the RT binding site for nevirapine. The extent to which Tyr 181 and Tyr 188 participate/contribute to nevirapine binding was determined by making amino acid substitutions at these positions using the corresponding residues from HIV-2 RT which is not sensitive to nevirapine. A change at either position dramatically decreased the enzymes' sensitivity to nevirapine, as well as to TIBO derivative and Merck L-693,593, indicating that both Tyr 181 and 188 are crucial for inhibitor-enzyme interaction. Cell culture selection in the continued presence of nevirapine results in the appearance of resistant HIV-1, Tyr 181 to Cys, raising the concern that combination drug therapy will be required in the clinic.

The phospholipase A2 of human spermatozoa; purification and partial sequence.

In view of its proposed key role in the acrosome reaction, phospholipase A2 has been isolated and purified from human spermatozoa. Following SDS-PAGE, a single major band was obtained with an estimated molecular mass of 16.7 kDa. Sequence analysis of the N-terminal portion of the molecule revealed the identity of the first 19 amino acids to be YNYQFGLMIVITKGHFAMV. From this partial analysis it is evident that the phospholipase A2 of human spermatozoa represents a new sequence. Of interest is the location of glutamine-4, phenylalanine-5, methionine-8 and isoleucine-9; this sequence appears to be highly conserved throughout evolution.

Characterization of the binding site for nevirapine (BI-RG-587), a nonnucleoside inhibitor of human immunodeficiency virus type-1 reverse transcriptase.

Nevirapine (BI-RG-587) is a potent and specific non-nucleoside inhibitor of human immunodeficiency virus type-1 reverse transcriptase. The compound is non-competitive with respect to template, primer, and nucleoside triphosphates indicating that BI-RG-587 does not act directly at the catalytic site. The binding site for this inhibitor was investigated by employing an azido photoaffinity analogue, BI-RJ-70, to covalently label the enzyme. The resulting photoadduct was subjected to enzymatic digestion by trypsin and endoproteinase lys-C and a single, highly labeled peptide was identified as residues 174-199. Sequencing of this peptide identified Tyr-181 and Tyr-188 as labeled residues.