Pyrrocidine A, a metabolite of endophytic fungi, has a potent apoptosis-inducing activity against HL60 cells through caspase activation via the Michael addition

Abstract

Pyrrocidine A is a known antimicrobial compound produced by endophytic fungi and has a unique 13-membered macrocyclic alkaloid structure with an α,β-unsaturated carbonyl group. We have previously reported that pyrrocidine A shows potent cytotoxicity against human acute promyelocytic leukemia HL60 cells, and the activity is 70-fold higher than that of pyrrocidine B which is the analog lacking the α,β-unsaturated carbonyl group. Pyrrocidine A induced nuclear condensation, DNA fragmentation and caspase activation in HL60 cells. Since the DNA fragmentation was suppressed by pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-fmk), caspase-mediated apoptosis contributes to pyrrocidine A-induced cytotoxicity. JFCR39 human cancer cells panel indicated that the mechanism of action of pyrrocidine A is different from other clinical anticancer drugs, and this compound broadly inhibited the growth of various cancer cell lines. The apoptosis induction by pyrrocidine A was suppressed by both N-acetyl-l-cysteine (NAC) and glutathione, both of which are thiol-containing antioxidants. Furthermore, pyrrocidine A directly bound to N-acetyl-l-cysteine methyl ester (NACM) through the Michael-type addition at the α,β-unsaturated carbonyl group and was detected by HPLC and liquid chromatography-ESI-tandem MS (LC-ESI-MS/MS) analyses. This indicates that this moiety is crucial for the potent apoptosis-inducing activity of pyrrocidine A.

Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions

“…44 Lactam 73 reacts rapidly with N -acetyl-cysteine methyl ester in less than an hour in a 70% MeOH/water solution at 37 °C. The reaction was monitored by HPLC and the adduct confirmed by LC-MS.…”

Pyrrocidine, a molecular off switch for fumonisin biosynthesis

“…Pyrrocidine A differs from pyrrocidine B by presence of a double bond in its lactam ring, resulting, by an unknown mechanism, in a higher toxicity of pyrrocidine A than B against a number of fungal and bacterial species [8,9]. The crucial role of this double bond in determining apoptosis inducing cytotoxicity against human acute promyelocytic leukemia HL60 cells [10]. Pyrrocidine A and B can both be detected in S. zeae-infested kernels through liquid chromatography-mass spectrometry (LC-MS), but the concentration of pyrrocidines during natural occurrence in maize seeds has not been described [8,9].…”

Asymmetric Primaquine and Halogenaniline Fumardiamides as Novel Biologically Active Michael Acceptors

“…Vernolepin and helenalin are sesquiterpenes with exomethylene lactones responsible for irreversible DNA polymerase inhibition. Pyrrocidine A is a 13-membered macrocyclic alkaloid produced by endophytic fungi, which directly binds to N -acetyl- l -cysteine methyl ester through the Michael-type addition and exerts both antimicrobial and cytostatic effect on leukemia HL60 cells [ 12 ]. Angelic acid ester ingenol mebutate has been identified as the most active component of Euphorbia peplus L. latex sap, effective against human nonmelanoma skin cancer [ 13 ] and actinic keratosis [ 14 ].…”