Enzyme inhibitors from algae

Abstract

This paper reports the results of a screen for inhibitors of acetylcholinesterase and cAMP phosphodiesterase. Acetylcholinesterase inhibitors are clinically important in the treatment of myasthena gravisll], post-operative atony of smooth muscle [2] and potentially in the treatment of chronic pain [3]. In addition they also constitute two economically important groups of insecticides; organophosphorus and carbamate insecticides. The positive inotropic and relaxant effects of cAMP phosphodiesterase inhibitors have made these compounds potentially important in the treatment of congestive heart failure [4].As part of an on-going project to isolate compounds of potential clinical value from algae and cyanobacteria we have screened aqueous culture supernatants and methanolic cell extracts of 80 randomly selected freshwater and marine algae for these inhibitors. 100ml cultures of each of the algae tested were grown for 28 days in marine [5], blue-green [6] or M4[7] media as appropriate. During this time the cultures were shaken at 11 Orev./min and illuminated at 27001~. using flourescent strips for l6h/day. The algae were harvested by centrifugation (4000xg, 15min) and the culture supernatant retained. The algal pellet was extracted with methanol (6ml, HPLC grade) by shaking at 1lOrevJmin for 30 min at 25°C. Following centrifugation of the algal extract (4000xg, 15 min) the methanolic supernatant was retained and the algal pellet discarded. The methanolic cell extract and the aqueous culture supernatant were then screened for the presence of acetylcholinesterase inhibitors and cAMP phosphodiesterase inhibitors.A microtitre plate based method was employed for the detection of acetylcholinesterase inhibitors. 100~1 of aqueous test solution or 30y1 of methanolic cell extract with 70~~1 of water was added to each well and 25pl of 0.5M citrate buffer pH 7.7 and 100~1 of enzyme solution (Sigma, Electeric eel cholinesterase, 5 units/ml in 0.1 M citrate buffer) added. The plate was incubated at 37°C for 30 min and then 5Op1 of 2mg/ml 59dithiobis-(2-nitrobenzoic acid) and 25p1 of 1 mg/ml butrylthiocholine iodide both prepared in 0.1 M citrate buffer pH 7.7 added. The absorbance of the wells was read at 410 nm and the plate incubated at 37°C for a further hour after which time the absorbance was again determined. The presence of acetylcholinesterase inhibitors was indicated by i n h i b i t i o n o f c o l o u r d e v e l o p m e n t . Acetylcholinesterase inhibitors were detected in the culture supernatant of the cyanobacterium Audouinella parvula and the methanolic extract of the chlorophyte Cladophora globulina; and in both the culture supernatant and methanolic cell extract of the chrysophyte Ochromonas villosa. Cultures of these algae have been grown on at least 3 Occasions and been found positive on each occasion. Further purification of the active compounds from the rnethanolic cell extract of Ochromonas villosa is currently underway in our laboratory.A modified version of the method of Thompson and Appleman [8] has been ...

Biogeography and biodiscovery hotspots of macroalgal marine natural products

“…Macroalgae have been historically an important group of organisms for marine drug development. While the pharmacological properties of MNPs from macroalgae were already well known from traditional and folk medicine prior to the 1950s, 14 the rst comprehensive publications were only published in the 1970s and covered MNPs discovered since the 1960s. 15,16 It was in the 1970s that MNP research began with great promise, followed by a period of rapid discovery of novel molecules.…”

Marine natural products

“…23 In one of the most important and controversial papers of the year, it was reported that the phenol (6), incorrectly named 3,5-dibromo-2-(3', 5'-dibromo-2'-methoxyphenoxy)phenol, 24 which had previously been regarded as a sponge metabolite, was produced by a bacterium of the genus Vibrio that had been isolated from the sponge Dysidea The controversial aspect of this paper concerns the very low yield of phenol (6) detected in the bacterial culture versus the high yields typically obtained from the sponge. 26 Four benzothiazoles (7)- ( 10) have been obtained from cultures of a Micrococcus species that was isolated from tissues of the sponge Tedania ignis.2' The structures of benzothiazoles ( 7)-(lo), all of which were known compounds, were elucidated by analysis of spectral data. A structure-activity study revealed that the antimicrobial activity of pentabromopseudilin (1 l), which is produced by Alteromonas luteoviolaceus,28g 29 is absent in isopentabromopseudilin (1 2) and some related synthetic analogues.3o…”