Pharmacological gating modulation of small- and intermediate-conductance Ca2+-activated K+ channels (KCa2.x and KCa3.1)

This short review discusses pharmacological modulation of the opening/closing properties (gating) of small- and intermediate-conductance Ca²⁺-activated K⁺ channels (K_Ca2 and K_Ca3.1) with special focus on mechanisms-of-action, selectivity, binding sites, and therapeutic potentials. Despite K_Ca channel gating-modulation being a relatively novel field in drug discovery, efforts in this area have already revealed a surprising plethora of pharmacological sites-of-actions and channel subtype selectivity exerted by different chemical classes. The currently published positive modulators show that such molecules are potentially useful for the treatment of various neurodegenerative disorders such as ataxia, alcohol dependence, and epilepsy as well as hypertension. The negative K_Ca2 modulators are very effective agents for atrial fibrillation. The prediction is that further unraveling of the molecular details of gating pharmacology will allow for the design of even more potent and subtype selective K_Ca modulators entering into drug development for these indications.     

Synthesis and evaluation of 4,5-dihydro-5-methylisoxazolin-5-carboxamide derivatives as VLA-4 antagonists

A novel set of compounds containing a 4,5-dihydro-5-methylisoxazoline have been successfully designed as VLA-4 receptor antagonists. Compound (14p) had a high receptor binding affinity of 4 nM and also found to be metabolically stable in vitro.     

Discovery of a potent and selective small molecule hGPR91 antagonist

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC₅₀: 0.8 μM)—originally synthesized in Merck for Bradykinin B₁ Receptor (BK₁R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5g (IC₅₀: 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC₅₀: 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.     

The cAMP response to isoprenaline in mononuclear cells is markedly increased in the presence of platelets

We have previously shown that the addition of platelets to mononuclear cells (MNC) increases cAMP in MNC. This response may be of interest because the physical interaction between platelets and MNC plays an important role in the inflammatory process. We have now demonstrated that the addition of both isoprenaline and platelets to MNC resulted in a marked amplification of the cAMP response. Prostaglandins, ATP and adenosine and the P-selectin ligand PSGL-1 could not account for the response. No substance was found in the supernatant that could increase cAMP in MNC. W7, a Ca(2+)-calmodulin inhibitor and the addition of EDTA reduced the response to both platelets and isoprenaline. Furthermore, we have demonstrated that mRNA for type I adenylyl cyclase, which is sensitive to Ca(2+), is present in MNC. No increase in Ca(2+) in the cytoplasma in MNC was recorded, however, by quantitative fluorescence microscopy after addition of platelets to MNC. It is possible that there are small increments in Ca(2+) at the binding sites, which we were unable to detect by our technique. Alternatively the binding of platelets to MNC may induce intermolecular interactions in the cell membrane which may facilitate the synthesis of cAMP.     

2-substituted pi system derivatives of adenosine that are coronary vasodilators acting via the A2A adenosine receptor

Compound 20 (CVT-3146--a 2-[(N-1-(4-N-methylcarboxamidopyrazolyl)] adenosine derivative) and compound 31 (CVT-3033--a 2-[(4-(1-N-pentylpyrazolyl)] adenosine derivative), were found to be short acting functionally selective coronary vasodilators (CV t0.5 = 5.2 +/- 0.2 and 3.4 +/- 0.5 min, respectively--rat isolated heart 50% reversal time) with good potency (EC50S = 6.4 +/- 1.2 nM and 67.9 +/- 16.7 nM, respectively), but they possess low affinity for the ADO A2A receptor (Ki = 1122 +/- 323 nM and 2138 +/- 952 nM, respectively; pig striatum).     

Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption

Cell cultures of Plumbago rosea were immobilized in calcium alginate and cultured in Murashige and Skoog's basal medium containing 10 mM CaCl(2) for the production of plumbagin, an important medicinal compound. Studies were carried to find out the impact of immobilization on the increased accumulation of this secondary metabolite. Immobilization in calcium alginate enhanced the production of plumbagin by three, two and one folds compared to that of control, un-crosslinked alginate and CaCl(2) treated cells respectively. Cell loading at a level of 20% to the polymer volume (Na-alginate) was optimal and maximum plumbagin was obtained. At higher cell loading (40-50%), lower plumbagin accumulation was noticed. Addition of 200 mg l(-1) chitosan as an elicitor to the immobilized cells resulted in eight and two folds higher accumulation of plumbagin over control and immobilized cells. Also, more than 70% of the plumbagin was released into the medium, which is highly desirable for easy recovery of the product. Sucrose utilization rate of the cells was higher when cells were subjected to in situ product removal using Amberlite XAD-7. This may indicate that the toxicity of plumbagin was reduced on cells when it was removed from the medium. Cells subjected to combined treatments of chitosan, immobilization and in situ extraction showed a synergistic effect and yielded 92.13 mg g(-1) DCW of plumbagin which is 21, 5.7, 2.5 times higher than control, immobilized, immobilized and elicited cells respectively.