Erratum to Machine learning prediction of cyanobacterial toxin (microcystin) toxicodynamics in humans.

In this manuscript, which appeared in ALTEX (2020), 37(1), 24-36, doi:10.14573/altex.1904031 , there were errors in Tables 1 and 3.

Machine learning prediction of cyanobacterial toxin (microcystin) toxicodynamics in humans.

Microcystins (MC) represent a family of cyclic peptides with approx. 250 congeners presumed harmful to human health due to their ability to inhibit ser/thr-proteinphosphatases (PPP), albeit all hazard and risk assessments (RA) are based on data of one MC-congener (MC-LR) only. MC congener structural diversity is a challenge for the risk assessment of these toxins, especially as several different PPPs have to be included in the RA. Consequently, the inhibition of PPP1, PPP2A and PPP5 was determined with 18 structurally different MC and demonstrated MC congener dependent inhibition activity and a lower susceptibility of PPP5 to inhibition than PPP1 and PPP2A. The latter data were employed to train a machine learning algorithm that should allow prediction of PPP inhibition (toxicity) based on MCs 2D chemical structure. IC50 values were classified in toxicity classes and three machine learning models were used to predict the toxicity class, resulting in 80-90% correct predictions.

Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards.

Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuterated MC standards. For proof of concept, tissue and plasma samples from mice i.p. injected with MC-LR and MC-LF were analysed. While MC-LF was detected in all tissue samples of both sexes, detection of MC-LR was restricted to liver samples of male mice, suggesting different toxicokinetics in males, e.g., transport, conjugation or protein binding. Thus, deconjugation/-proteinisation steps should be employed to improve detection of bound MC.

Total Synthesis of Microcystin-LF and Derivatives Thereof.

Microcystins (MCs) are highly toxic natural products which are produced by cyanobacteria. They can be released to the water during harmful algal blooms and are a serious threat to animals and humans. Described is the total synthesis of the cyanotoxin microcystin-LF (MC-LF, 1a) and two derivatives thereof. Deuterated derivative 1b is of interest as an internal standard during MC quantification in biological samples by mass spectrometry and alkyne-labeled 1c can be employed for toxin derivatization by click chemistry with an azide-containing reporter molecule or as an activity-based probe to identify interaction partners. Application of tert-butyl ester protecting groups for erythro-ß-d-methylaspartic acid and γ-d-glutamic acid were key for an isomerization-free synthesis. The analytical data of synthetic MC-LF were identical to those of an authentic sample of the natural product. All derivatives 1a-c were determined to be potent inhibitors of protein phosphatase-1 with similar activity.

Synthesis of Highly Selective Submicromolar Microcystin-Based Inhibitors of Protein Phosphatase (PP)2A over PP1.

Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure-activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid- and solution-phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues.

Regioselective Cleavage of Thioether Linkages in Microcystin Conjugates.

Microcystins are cyanobacterial toxins that can be found in fresh and coastal waters during algal blooms. Microcystin contamination of water can cause severe poisoning of animals and humans. Quantification of these toxins in biological samples is complicated because a major proportion of microcystins is covalently linked to proteins through thioether bonds formed through a Michael-type addition of cysteine residues of proteins to an N-methyldehydroalanine residue in the microcystins. We investigated chemical methods that can be used to cleave such thioether bonds by means of an elimination reaction that leaves the microcystin backbone intact for subsequent analysis. The known reagent O-mesitylenesulfonylhydroxylamine (MSH) led to regioselective thioether cleavage, but a large excess of reagent was needed, thus making purification challenging. An unexpected side reaction observed during the investigation of the base-induced elimination inspired us to develop a new thioether-cleavage methodology based on the addition of propargylamine as a nucleophile that can trap the elimination product. This methodology could be successfully applied to the quantitative cleavage of a microcystin-LF-glutathione conjugate. The alkyne moiety introduced by this procedure offers the possibility for further reactions with azides by using click chemistry, which might be useful for the derivatization or isolation of microcystins.