An exploration of the effects of L- and D-tetrahydroisoquinoline-3-carboxylic acid substitutions at positions 2,3 and 7 in cyclic and linear antagonists of vasopressin and oxytocin and at position 3 in arginine vasopressin

We have investigated the effects of mono-substitutions with the conformationally restricted amino acid, 1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid (Tic) at position 3 in arginine vasopressin (AVP), at positions 2, 3 and 7 in potent non-selective cyclic AVP V₂/V_1a antagonists, in potent and selective cyclic and linear AVP V_1a antagonists, in a potent and selective oxytocin antagonist and in a new potent linear oxytocin antagonist Phaa-D -Tyr(Me)-Ile-Val-Asn-Orn-Pro-Orn-NH₂ (10). We report here the solid-phase synthesis of peptide 10 together with the following Tic-substituted peptides: 1, [Tic³]AVP; 2, d(CH₂)₅[D -Tic²]VAVP; 3, d(CH₂)₅[D -Tyr(Et)²Tic³]VAVP; 4, d(CH₂)₅[Tic²Ala-NH₂⁹]AVP; 5, d(CH₂)₅[Tyr(Me)², Tic³, Ala-NH₂⁹]AVP; 6, d(CH₂)₅ [Tyr(Me)², Tic⁷]AVP; 7, Phaa-D -Tyr(Me)-Phe-Gln-Asn-Lys-Tic-Arg-NH₂; 8, desGly-NH₂,d(CH₂)₅[Tic²,Thr⁴]OVT; 9, desGly-NH₂d(CH₂)₅[Tyr(Me)²Thr⁴, Tic⁷]OVT; 11, Phaa-D-Tic-Ile-Val-Asn-Orn-Pro-Orn-NH₂, using previously described methods. The protected precursors were synthesized by the solid-phase method, cleaved, purified and deblocked with sodium in liquid ammonia to give the free peptides 1–11 which were purified by methods previously described. Peptides 1–11 were examined for agonistic and antagonistic potency in oxytocic (in vitro, without Mg²⁺) and AVP antidiuretic (V₂-receptor) and vasopressor (V_1a-receptor) assays. Tic³ substitution in AVP led to drastic losses of V₂, V_1a and oxytocic agonistc activities in peptide 1.L - and D -Tic² substitutions led to drastic losses of anti-V₂/anti-V_1a and anti-oxytocic potencies in peptides 2, 4, 8 and 11 (peptide 2 retained substantial anti-oxytocic potency; pA₂ = 7.25 ± 0.25). Whereas Tic³ substitution in the selective V_1a antagonist d(CH₂)₅[Tyr(Me)², Ala-NH₂⁹]APV(C) led to a drastic reduction in anti-V_1a potency (from anti-V_1a pA₂) 8.75 to 6.37 for peptide 5, remarkably, Tic³ substitution in the V₂/V_1a antagonist d(CH₂)₅[D -Tyr(Et)²]VAVP(B) led to full retention of anti-V₂ potency and a 95% reduction in anti-V_1a potency. With an anti-V₂ pA₂ = 7.69 ± 0.05 and anti-V_1a pA₂ = 6.95 ± 0.03, d(CH₂)₅[D -Tyr(Et)²,Tic³]VAVP exhibits a 13-fold gain in anti-V₂/anti-V_1a selectivity compared to (B). Tic⁷ substitutions are very well tolerated in peptides 6, 7 and 9 with excellent retention of the characteristic potencies of the parent peptides. The findings on the effects of Tic³ substitutions reported here may provide promising leads to the design of more selective and possibly orally active V₂ antagonists for use as pharmacological tools and as therapeutic clinical agents for the treatment of the syndrome of the inappropriate secretion of antidiuretic hormone (SIADH).     

Isolation and partial characterization of an opioid-like 88 kDa hibernation-related protein

Previous studies show that infusion of hibernating woodchuck albumin (HWA) induces hibernation in summer-active ground squirrels and results in profound behavioral and physiological depression in primates. These effects are reversed by the administration of opiate antagonists, suggesting that the putative hibernation induction trigger (HIT) may act through opioid receptors. We have demonstrated that both HIT-containing plasma and the synthetic α opioid -Ala²- -Leu⁵-enkephalin (DADLE), which mimics the activity of HIT in hibernators, extend tissue survival time of a multi-organ autoperfusion system by 3-fold. In this study we present the first data showing biological activity with a much more highly purified plasma fraction from hibernating woodchucks, identified as the hibernation-related factor (HRF). Both the HRF and DADLE show opiate-like contractile inhibition in the mouse vas deferens (Mvd) bioassay. We also have preliminary evidence in an isolated rabbit heart preparation indicating that the HRF and DADLE act similarly to restore left ventricular function following global myocardial ischemia. Furthermore, we have partially sequenced an α 1-glycoprotein-like 88 kDa hibernation-related protein (p88 HRP) present in this fraction, which may prove to be the blood-borne HIT molecule.     

Modelling of H2 antagonists and tridimensional modelling of H2 receptor interactions. Part 2

The interactions between the H2 antagonists cimetidine, ranitidine and famotidine with a basic molecular model for the histamine H2 receptor have been analyzed. The calculated potential energies of the antagonist-H2 receptor complexes follow an order consistent with the published binding data, indicating that famotidine is the best H2 receptor ligand. Comparison with the interactions found for histamine and this H2 receptor model suggests that the protonated imiddazole moiety of cimetidine, the dimethylammonio moiety of protonated ranitidine and the protonated guanidinyl moiety of famotidine are bioisosteric with the protonated aliphatic amine group of histamine. Asp 98 in helix 3 appears to be the main residue for antagonist recognition, but some residues in helix 5 may be involved, apparently by serving to guide the antagonist into the binding pocket.     

Positive and negative allosteric modulation of GluK2 kainate receptors by BPAM344 and antiepileptic perampanel

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Recombinant FimH Adhesin Demonstrates How the Allosteric Catch Bond Mechanism Can Support Fast and Strong Bacterial Attachment in the Absence of Shear

The FimH protein of Escherichia coli is a model two-domain adhesin that is able to mediate an allosteric catch bond mechanism of bacterial cell attachment, where the mannose-binding lectin domain switches from an ‘inactive’ conformation with fast binding to mannose to an ‘active’ conformation with slow detachment from mannose. Because mechanical tensile force favors separation of the domains and, thus, FimH activation, it has been thought that the catch bonds can only be manifested in a fluidic shear-dependent mode of adhesion. Here, we used recombinant FimH variants with a weakened inter-domain interaction and show that a fast and sustained allosteric activation of FimH can also occur under static, non-shear conditions. Moreover, it appears that lectin domain conformational activation happens intrinsically at a constant rate, independently from its ability to interact with the pilin domain or mannose. However, the latter two factors control the rate of FimH deactivation. Thus, the allosteric catch bond mechanism can be a much broader phenomenon involved in both fast and strong cell-pathogen attachments under a broad range of hydrodynamic conditions. This concept that allostery can enable more effective receptor-ligand interactions is fundamentally different from the conventional wisdom that allostery provides a mechanism to turn binding off under specific conditions.     

Large-scale production of chlamydospores of Gliocladium virens strain GL-21 in submerged culture

SoilGard™ is a commercial microbial fungicide containing chlamydospores of Gliocladium virens strain GL-21. The formulation is registrated with the US Environmental Protection Agency (EPA) by Thermo Trilogy Corporation, Columbia, Maryland. The biocontrol agent was developed in cooperation with the Biocontrol of Plant Diseases Laboratory (BPDL), US Department of Agriculture (USDA), and is targeted for controlling damping-off diseases caused by Rhizoctonia solani and Pythium ultimum in vegetable and ornamental greenhouses. Formulation requires production of chlamydospore biomass in liquid fermentation which was carried out in 20-L, 1500-L and 4000-L submerged liquid fermentors. Key economic process parameters, fermentation kinetics and the reproducibility of the fermentation at this scale were evaluated and analyzed. Starter culture quality and maturity of chlamydospores were identified as key issues for obtaining successful fermentations. Received 07 February 1997/ Accepted in revised form 27 May 1997