cantharidin and tautomycin

Topics: Alkenes; Antibiotics, Antineoplastic; Antifungal Agents; Cantharidin; Crystallography, X-Ray; Cyclosporine; Enzyme Inhibitors; Humans; Microcystins; Models, Molecular; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Spiro Compounds; Structure-Activity Relationship

Topics: Alkenes; Animals; Antifungal Agents; Cantharidin; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Spiro Compounds

Protein phosphatase 1 occurs in all tissues and regulates many pathways, ranging from cell-cycle progression to carbohydrate metabolism. Many naturally occurring, molecular toxins modulate PP1 activity, though the exact mechanism of this differential regulation is not understood. A detailed elucidation of these interactions is crucial for understanding the cellular basis of phosphatase function and signaling pathways but, more importantly, they can serve as the basis for highly specific therapeutics, e.g. against cancer. We report the crystal structures of PP1 in complex with nodularin-R at 1.63 A and tautomycin at 1.70 A resolution. The PP1:nodularin-R complex was used to demonstrate the utility of our improved PP1 production technique, which produces highly active, soluble PP1. Tautomycin is one of the few toxins that reportedly preferentially binds PP1>PP2A. Therefore, the PP1:tautomycin structure is the first complex structure with a toxin with preferred PP1 specificity. Furthermore, since tautomycin is a linear non-peptide-based toxin, our reported structure will aid the design of lead compounds for novel PP1-specific pharmaceuticals.

Topics: Binding Sites; Cantharidin; Crystallography, X-Ray; Drug Design; Enzyme Inhibitors; Humans; Hydrogen Bonding; Models, Molecular; Peptides, Cyclic; Protein Phosphatase 1; Protein Structure, Secondary; Pyrans; Spiro Compounds; Structure-Activity Relationship

Natural product extracts have proven to be a rich source of small molecules that potently inhibit the catalytic activity of certain PPP-family ser/thr protein phosphatases. To date, the list of inhibitors includes okadaic acid (produced by marine dinoflagelates, Prorocentrum sp. and Dinophysis sp.), calyculin A, dragmacidins (isolated from marine sponges), microcystins, nodularins (cyanobacteria, Microcystis sp. and Nodularia sp.), tautomycin, tautomycetin, cytostatins, phospholine, leustroducsins, phoslactomycins, fostriecin (soil bacteria, Streptomyces sp.), and cantharidin (blister beetles, approx 1500 species). Many of these compounds share structural similarities, and several have become readily available for research purposes. Here we will review the specificity of available inhibitors and present methods for their use in studying sensitive phosphatases. Common mistakes in the employment of these compounds will also be addressed briefly, notably the widespread misconception that they only inhibit the activity of PP1 and PP2A. Inhibitors of PP2B (calcineurin) will only be mentioned in passing, except to state that, in our hands, cypermethrin, deltamethrin, and fenvalerate, which are sold as potent inhibitors of PP2B, do not inhibit the catalytic activity of PP2B.

Topics: Cantharidin; Enzyme Inhibitors; Furans; Indole Alkaloids; Lactones; Lipids; Marine Toxins; Microcystins; Okadaic Acid; Organophosphates; Organophosphorus Compounds; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Pyrans; Pyrones; Spiro Compounds

Stimulation of insulin secretion is accompanied by changes in the phosphorylation state of several islet polypeptides. Protein (de)phosphorylation is mediated by the action of protein kinases and phosphoprotein phosphatases. In this study we have investigated expression of phospho-serine/threonine phosphatases (PPs) in rat islets of Langerhans and studied the role of these enzymes in the regulation of insulin secretion. PP1, PP2A and PP2B were identified in rat islets and high levels of PP1/2A activities were detected. Inhibition of PP1/2A markedly inhibited glucose-stimulated insulin secretion, whilst glucose increased islet PP1/2A activities in situ. Insulin secretion at basal glucose was unaffected by inhibitors of PP1/2A. Inhibition of PP2B had no effect on either basal or glucose stimulated insulin secretion. These results suggest that PP1/2A are stimulated by glucose in rat islets and the presence of active PP1/2A is required for stimulation of insulin secretion by glucose.

Topics: Animals; Antifungal Agents; Cantharidin; Cyclosporine; Glucose; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Male; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds

Although the role of protein kinases and phosphorylation in steroidogenesis has received much attention, very little is known about the activities of phosphoprotein phosphatases (PP) and dephosphorylation in steroidogenic tissues. The aims of the present study were therefore to identify which of those serine/threonine PPs more commonly involved in intracellular signalling are expressed in rat luteal cells; to quantify, in vitro, the effects of inhibitors on PP activity extracted from purified rat luteal cells; and to measure the effects of PP inhibitors on the phosphorylation of endogenous luteal cell proteins. Polyclonal antibodies raised against the catalytic subunits of PP types 1 and 2A, and a monoclonal antibody raised against the Ca(2+)-binding subunit of PP2B, were used to identify immunoreactive proteins that migrated on SDS-PAGE with approximate molecular masses of 37, 34 and 16 kDa, corresponding well with the reported molecular mass of PP1, PP2A and PP2B respectively. Five selective inhibitors of PP1/PP2A: okadaic acid, calyculin A, cantharidin, tautomycin and microcystin-RR, each caused a dose-dependent decrease in the activity of PPs in luteal cell homogenates, and also enhanced 32P incorporation into numerous luteal cell proteins; most notably, proteins with approximate molecular masses of 20 and 22 kDa. The results of this study suggest that PPs may play an important role in the regulation of rat luteal cell functions.

Topics: Animals; Antifungal Agents; Calcineurin; Calmodulin-Binding Proteins; Cantharidin; Cells, Cultured; Corpus Luteum; Dose-Response Relationship, Drug; Female; Marine Toxins; Microcystins; Molecular Weight; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Pyrans; Rats; Rats, Sprague-Dawley; Signal Transduction; Spiro Compounds