Reversal of acid-induced and inflammatory pain by the selective ASIC3 inhibitor, APETx2.

Background And Purpose: Inflammatory pain is triggered by activation of pathways leading to the release of mediators such as bradykinin, prostaglandins, interleukins, ATP, growth factors and protons that sensitize peripheral nociceptors. The activation of acid-sensitive ion channels (ASICs) may have particular relevance in the development and maintenance of inflammatory pain. ASIC3 is of particular interest due to its restricted tissue distribution in the nociceptive primary afferent fibres and its high sensitivity to protons.

Characterizations of four monoclonal antibodies against M2 protein ectodomain of influenza A virus.

M2 protein of influenza A virus has been implicated as a target for vaccines with broad cross-strain coverage. Studies in small animal models have shown that antibody responses induced by 23-mer M2 peptide vaccines can provide protection against influenza A virus challenge. To study antiviral mechanisms of Merck M2-OMPC conjugate vaccine, we generated and characterized four M2 peptide-specific monoclonal antibodies (mAbs).

Blockers of the delayed-rectifier potassium current in pancreatic beta-cells enhance glucose-dependent insulin secretion.

Delayed-rectifier K+ currents (I(DR)) in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltage-gated K+ channel, K(V)2.1, is expressed in beta-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I(DR) in rodent beta-cells.

Pharmaceutical and immunological evaluation of human papillomavirus viruslike particle as an antigen carrier.

We report the preparation and the immunogenicity of a conjugate vaccine obtained by chemically conjugating a variant of the extracellular peptide fragment of influenza type A M2 protein to the human papillomavirus (HPV) viruslike particle (VLP). Conjugates comprised of approximately 4,000 copies of the antigenic peptide per VLP are obtained as the result of the reaction between a C-terminal cysteine residue on the peptide and the maleimide-activated HPV VLP. The resulting conjugates have an average particle size slightly larger than the carrier and present enhanced overall stability against chemical and thermal-induced denaturation.

Novel mutations introduced at the beta-site of amyloid beta protein precursor enhance the production of amyloid beta peptide by BACE1 in vitro and in cells.

Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEV" (APP_NFEV) in vitro and in cells.

Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys.

A universal influenza virus vaccine that does not require frequent updates and/or annual immunizations will offer significant advantages over current seasonal flu vaccines. The highly conserved influenza virus A M2 membrane protein has been previously suggested as a potential antigen target for such a vaccine. Here, we report systematic evaluation of M2 peptide conjugate vaccines (synthetic peptides of M2 extracellular domain conjugated to keyhole limpet hemocyanin (KLH) or Neisseria meningitidis outer membrane protein complex (OMPC)) in mice, ferrets, and rhesus monkeys.

Progress towards the development of a HIV-1 gp41-directed vaccine.

The HIV-1 gp41 envelope glycoprotein mediates fusion of the viral and cellular membranes. The core of the gp41 ectodomain undergoes a receptor-triggered conformational transition forming a trimeric, alpha-helical coiled-coil structure. This trimer-of-hairpins species facilitates insertion of the viral envelope protein into the host cell membrane promoting viral entry.

Rational design and synthesis of selective BACE-1 inhibitors.

An effective approach for enhancing the selectivity of beta-site amyloid precursor protein cleaving enzyme (BACE 1) inhibitors is developed based on the unique features of the S1' pocket of the enzyme. A series of low molecular weight (<600) compounds were synthesized with different moieties at the P1' position. The selectivity of BACE 1 inhibitors versus cathepsin D and renin was enhanced 120-fold by replacing the hydrophobic propyl group with a hydrophilic propionic acid group.

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb.

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins.

A prostate-specific antigen (PSA)-activated vinblastine prodrug selectively kills PSA-secreting cells in vivo.

Currently, there is no therapy for men with androgen-refractory prostate cancer that substantially extends survival. This report characterizes by in vitro and in vivo techniques a new chemotherapeutic that is composed of desacetyl-vinblastine covalently linked to a peptide that contains a peptide bond that can be hydrolyzed by prostate-specific antigen (PSA). This compound (referred to as vinblastine-conjugate) is minimally toxic to cells in culture which do not express PSA.

Two tarantula peptides inhibit activation of multiple sodium channels.

Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels.

Design and synthesis of a pro-drug of vinblastine targeted at treatment of prostate cancer with enhanced efficacy and reduced systemic toxicity.

Chemotherapy of prostate cancer with antimitotic agents such as vinblastine and doxorubicin is only marginally effective, due to dose-limiting systemic toxicity. Herein we report the development of peptidyl conjugate 5 of the cytotoxic agent vinblastine (1), along with the results of its in vitro and in vivo evaluation as a pro-drug targeted at prostate cancer cells. Prostate-derived tumors are known to produce significant amounts of prostate specific antigen (PSA), a serine protease with chymotrypsin-like properties.

Enhancement of alpha -helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro. Implications for vaccine design.

The synthetic peptide DP178, derived from the carboxyl-terminal heptad repeat region of human immunodeficiency virus type 1 GP41 protein is a potent inhibitor of viral-mediated fusion and contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody 2F5. Efforts at eliciting a 2F5-like immune response by immunization with peptides or fusion proteins containing this sequence have not met with success, possibly because of incorrect structural presentation of the epitope. Although the structure of the carboxyl-terminal heptad repeat on the virion is not known, several recent reports have suggested a propensity for alpha-helical conformation.

The synthesis of a prodrug of doxorubicin designed to provide reduced systemic toxicity and greater target efficacy.

Doxorubicin (Dox) can provide some stabilization in prostate cancer; however, its use is limited because of systemic toxicities, primarily cardiotoxicity and immunosuppression. The administration of a prodrug of doxorubicin, designed to permit selective activation by the tumor, would reduce general systemic exposure to the active drug and would thereby increase the therapeutic index. Prostate specific antigen (PSA) is a serine protease with chymotrypsin-like activity that is a member of the kallikrein gene family.

Thermodynamics of peptide inhibitor binding to HIV-1 gp41.

The gp41 subunit of the human immunodeficiency virus type 1 envelope glycoprotein mediates fusion of the cellular and viral membranes. The gp41 ectodomain is a trimer of alpha-helical hairpins, where N-terminal helices form a parallel three-stranded coiled-coil core and C-terminal helices pack around the core. A deep hydrophobic pocket on the N-terminal core represents an attractive target for antiviral therapeutics.

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression.

PSA-specific and non-PSA-specific conversion of a PSA-targeted peptide conjugate of doxorubicin to its active metabolites.

Tumor-selective delivery of doxorubicin by a prostate-specific antigen (PSA)-targeted peptide conjugate prodrug of doxorubicin was demonstrated in a nude mouse xenograft model of human prostate cancer. The prodrug (referred to as doxorubicin conjugate) contains doxorubicin linked to a seven-amino acid peptide conjugate that was designed to increase delivery of doxorubicin to tumor sites through the hydrolytic properties of PSA, which prostate tumors express in high amounts. Following i.

A peptide-doxorubicin 'prodrug' activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo.

We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen.

The significance of monoisotopic and carbon-13 isobars for the identification of a 19-component dodecapeptide library by positive ion electrospray Fourier transform ion cyclotron resonance mass spectrometry.

Harnessing the ultra high resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and positive ion electrospray, we have demonstrated the significance and utility of cumulative mass defect high resolution mass separation stable isotope distribution, exact mass measurement and elemental formula as a means of simultaneously identifying 19 components of the dodecapeptide library Ac-ANKISYQS[X]STE-NH(2). With an instrument resolution of 275 000 (average), isobaric multiplets attributed to monoisotopic and carbon-13 components of peptides: Ac approximately SLS approximately NH(2); Ac approximately SNS approximately NH(2); Ac approximately SOS approximately NH(2); Ac approximately SDS approximately NH(2); within the mass window of 1380-1385 Da, and Ac approximately SQS approximately NH(2); Ac approximately SKS approximately NH(2); Ac approximately SES approximately NH(2); Ac approximately SMS approximately NH(2), within the mass window 1395-1400 Da, were mass resolved, accurately mass measured and identified from the computed molecular formulas. This experimental procedure enabled the separation of monoisotopic and carbon-13 isobars yielding enhanced selectivity and specificity and serves to illustrate the significance of monoisotopic and carbon-13 isobars in final product analysis.

Structural studies on phospholamban and implications for regulation of the Ca(2+)-ATPase.

The cardiac sarcoplasmic reticulum (SR) protein phospholamban (PLB) is an endogenous inhibitor of the SR Ca(2+)-ATPase. Phosphorylation of PLB relieves this inhibition and up-regulates calcium transport. PLB has proved remarkably difficult to study by conventional solution-state nuclear magnetic resonance (NMR) methods, due primarily to the extreme hydrophobic nature of the protein and its propensity to form pentamers.

Conformation of a novel tetrapeptide inhibitor NH2-D-Trp-D-Met-Phe(pCl)-Gla-NH2 bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers.

Selective inhibition of factor Xa in the prothrombinase complex by the carboxyl-terminal domain of antistasin.

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112.

Mutational anatomy of an HIV-1 protease variant conferring cross-resistance to protease inhibitors in clinical trials. Compensatory modulations of binding and activity.

Site-specific substitutions of as few as four amino acids (M46I/L63P/V82T/I84V) of the human immunodeficiency virus type 1 (HIV-1) protease engenders cross-resistance to a panel of protease inhibitors that are either in clinical trials or have recently been approved for HIV therapy (Condra, J. H., Schleif, W.

Site-directed mutagenesis of residues in a conserved region of bovine aspartyl (asparaginyl) beta-hydroxylase: evidence that histidine 675 has a role in binding Fe2+.

The roles in catalysis of several residues in bovine aspartyl (asparaginyl) beta-hydroxylase that are located in a region of homology among alpha-ketoglutarate-dependent dioxygenases were investigated using site-directed mutagenesis. Previous studies have shown that when histidine 675, an invariant residue located in this highly conserved region, was mutated to an alanine residue, no enzymatic activity was detected. A more extensive site-directed mutagenesis study at position 675 has been undertaken to define the catalytic role of this essential residue.

Biochemical and biophysical comparison of native and chemically synthesized phospholamban and a monomeric phospholamban analog.

Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplasmic reticulum using immunoaffinity chromatography and prepared by solid phase peptide synthesis. The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentameric oligomeric states. They are similarly detected on Western blots, are phosphorylation substrates, have identical amino acid compositions that directly reflect their predicted values, yield the same internal amino acid sequences upon CNBr digestion, and have molecular mass values agreeing with the expected value (approximately 6123 Da).