Paxilline

Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism.[Pubmed:25348413]

J Gen Physiol. 2014 Nov;144(5):415-40.

Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca(2+)- and voltage-activated K(+) (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of Paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by Paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that Paxilline increases occupancy of closed states, effectively reducing the closed-open equilibrium constant, L(0). Gating current measurements exclude an effect of Paxilline on voltage sensors. Steady-state inhibition by multiple Paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC50 for Paxilline shifted from around 10 nM when channels were largely closed to near 10 microM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single Paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 microM Paxilline, with a slope of 2 x 10(6) M(-1)s(-1). Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, Paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that Paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller molecules to this cavity.

A fungal prenyltransferase catalyzes the regular di-prenylation at positions 20 and 21 of paxilline.[Pubmed:25036831]

Biosci Biotechnol Biochem. 2014;78(3):448-54.

A putative indole diterpene biosynthetic gene cluster composed of eight genes was identified in a genome database of Phomopsis amygdali, and from it, biosynthetic genes of fusicoccin A were cloned and characterized. The six genes showed significant similarities to pax genes, which are essential to Paxilline biosynthesis in Penicillium paxilli. Recombinants of the three putative prenyltransferase genes in the cluster were overexpressed in Escherichia coli and characterized by means of in vitro experiments. AmyG is perhaps a GGDP synthase. AmyC and AmyD were confirmed to be prenyltransferases catalyzing the transfer of GGDP to IGP and a regular di-prenylation at positions 20 and 21 of Paxilline, respectively. AmyD is the first know example of an enzyme with this function. The Km values for AmyD were calculated to be 7.6 +/- 0.5 muM for Paxilline and 17.9 +/- 1.7 muM for DMAPP at a kcat of 0.12 +/- 0.003/s.

A Concise Approach to Paxilline Indole Diterpenes.[Pubmed:26593869]

J Am Chem Soc. 2015 Dec 16;137(49):15410-3.

A synthetic approach to Paxilline indole diterpenes is described. The route to the pentacyclic core relies on a new regioselective alkenylation of ketones and a tandem radical addition-aldol reaction sequence to access vicinal quaternary stereocenters. Emindole SB, the simplest member of the family, is synthesized in 11 steps from commercially available material to demonstrate the application of this approach.

Development and Evaluation of Monoclonal Antibodies for Paxilline.[Pubmed:26426046]

Toxins (Basel). 2015 Sep 25;7(10):3903-15.

Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC50s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 microg/kg and the limit of quantification was 90 microg/kg. Recovery from samples spiked over the range of 100 to 1000 microg/kg averaged 106% +/- 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage.

The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline.[Pubmed:12234490]

Arch Biochem Biophys. 2002 Oct 1;406(1):55-64.

Paxilline, an indole alkaloid mycotoxin from Penicillium paxilli, is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). Paxilline inhibited differing isoforms of SERCA with IC50s between 5 and 50 microM. It inhibited more potently the purified Ca2+ ATPase activity from skeletal muscle with an IC50 of 5 microM. Detailed effects of this inhibitor on the Ca2+ and ATP dependence upon activity indicate that it affects the high-affinity Ca2+-binding (E1) form of the ATPase. In addition, Paxilline is a "competitive" inhibitor with respect to high concentrations of ATP, increasing the regulatory binding site K(m), without affecting the catalytic binding site K(m). At higher concentrations, Paxilline inhibits phosphoenzyme formation from ATP and inorganic phosphate, without affecting nucleotide binding. We therefore suggest that Paxilline has two effects on the Ca2+ ATPase. At lower concentrations (5-10 microM), Paxilline inhibits the ATP-dependent acceleration of Ca2+ release from the phosphoenzyme and/or phosphoenzyme decay. At higher concentrations, Paxilline inhibits phosphoenzyme formation.

Paxilline inhibition of the alpha-subunit of the high-conductance calcium-activated potassium channel.[Pubmed:8938726]

Neuropharmacology. 1996;35(7):963-8.

High conductance calcium-activated (maxi-K) channels are potently blocked by a family of indole diterpenes that includes Paxilline. Paxilline stimulates binding of charybdotoxin (ChTX) to maxi-K channels in vascular smooth muscle and blocks these channels in electrophysiological experiments (Knaus et al., 1994b). These results suggested that Paxilline blocked maxi-K channels at a site distance from the ChTX binding site located near the external entrance to the pore. Here we have examined block of the cloned alpha subunit (slo) of the maxi-K channel in excised membrane patches after internal application of Paxilline. Paxilline caused a reversible inhibition of channel currents with slow washout kinetics. In the presence of 10 muM intracellular calcium, Paxilline blocked currents elicited by brief voltage pulses with a Ki of 1.9 nM and a Hill coefficient near one. Changing the internal calcium by the fold caused a two to three fold change in the Ki for Paxilline block, with less block occurring at high calcium concentrations. Paxilline reduced the maximum of the conductance-voltage relation in a calcium-sensitive manner with less block occurring at high calcium concentrations, and caused a 20 mV depolarizing shift in the midpoint for channel opening. The time-course of relief of Paxilline block by elevated calcium was more rapid than washout of Paxilline suggesting an allosteric interaction between calcium and Paxilline.

Tremorgenic indole alkaloids potently inhibit smooth muscle high-conductance calcium-activated potassium channels.[Pubmed:7514038]

Biochemistry. 1994 May 17;33(19):5819-28.

Tremorgenic indole alkaloids produce neurological disorders (e.g., staggers syndromes) in ruminants. The mode of action of these fungal mycotoxins is not understood but may be related to their known effects on neurotransmitter release. To determine whether these effects could be due to inhibition of K+ channels, the interaction of various indole diterpenes with high-conductance Ca(2+)-activated K+ (maxi-K) channels was examined. Paspalitrem A, paspalitrem C, aflatrem, penitrem A, and paspalinine inhibit binding of [125I]charybdotoxin (ChTX) to maxi-K channels in bovine aortic smooth muscle sarcolemmal membranes. In contrast, three structurally related compounds, Paxilline, verruculogen, and paspalicine, enhanced toxin binding. As predicted from the binding studies, covalent incorporation of [125I]ChTX into the 31-kDa subunit of the maxi-K channel was blocked by compounds that inhibit [125I]ChTX binding and enhanced by compounds that stimulate [125I]ChTX binding. Modulation of [125I]ChTX binding was due to allosteric mechanisms. Despite their different effects on binding of [125I]ChTX to maxi-K channels, all compounds potently inhibited maxi-K channels in electrophysiological experiments. Other types of voltage-dependent or Ca(2+)-activated K+ channels examined were not affected. Chemical modifications of Paxilline indicate a defined structure-activity relationship for channel inhibition. Paspalicine, a deshydroxy analog of paspalinine lacking tremorgenic activity, also potently blocked maxi-K channels. Taken together, these data suggest that indole diterpenes are the most potent nonpeptidyl inhibitors of maxi-K channels identified to date. Some of their pharmacological properties could be explained by inhibition of maxi-K channels, although tremorgenicity may be unrelated to channel block.

Paxilline is an indole alkaloid mycotoxin from Penicillium paxilli, acts as a potent BK channels inhibitor by an almost exclusively closed-channel block mechanism. Paxilline also inhibits the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) with IC50s between 5 μM and 50 μM for differing isoforms. Paxilline possesses significant anticonvulsant activity.

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