BW-A4C

A combinatorial pool of hydroxamic acid fragments as potential metalloprotein drug leads was generated from the enzymatic hydrolysis of the natural product desferrioxamine B (DFOB). DFOB is a metabolite produced by Streptomyces pilosus for iron acquisition, and can be selectively catabolised by Niveispirillum irakense to access carbon for growth. The supernatant of a DFOB‐supplemented culture of N. irakense was analysed by LC‐MS at intervals over 168 h. This identified a mixture of endo‐hydroxamic acid fragments that contained reactive terminal groups. The supernatants from two cultures (at 48 h and 168 h) were reacted with 1,8‐naphthalic anhydride in a microwave synthesiser to generate pools of scriptaid analogues, which were screened against ZnII‐containing histone deacetylases (HDACs) and FeIII‐containing 5‐lipoxygenase (5‐LO). Compound S2 showed relative potency against 5‐LO (IC50=59 μm; BWA4C, 17 μm); it was 28‐fold more selective towards 5‐LO than HDAC1. Compound S1 inhibited HDAC1 but not 5‐LO. Enzyme‐mediated reverse biosynthesis could yield new benefits from structurally complex natural products in drug design.

Recently, we published that nitro-fatty acids (NFA) are potent electrophilic molecules which inhibit 5-lipoxygenase (5-LO) by interacting catalytically with cysteine residues next to a substrate entry channel. The electrophilicity is derived from an intramolecular Michael acceptor moiety consisting of an electron-withdrawing group in close proximity to a double bond. The potential of the Michael acceptor moiety to interact with functionally relevant cysteines of proteins potentially renders them effective and sustained enzyme activity modulators. We screened a large library of naturally derived and synthetic electrophilic compounds to investigate whether other types of Michael acceptor containing drugs suppress 5-LO enzyme activity. The activity was measured by assessing the effect on the 5-LO product formation of intact human polymorphonuclear leukocytes. We demonstrated that a number of structurally different compounds were suppressive in the activity assays and showed that Michael acceptors of the quinone and nitro-alkene group produced the strongest inhibition of 5-LO product formation. Reactivity with the catalytically relevant cysteines 416 and 418 was confirmed using mutated recombinant 5-LO and mass spectrometric analysis (MALDI-MS). In the present study, we show for the first time that a number of well-recognized naturally occurring or synthetic anti-inflammatory compounds carrying a Michael acceptor, such as thymoquinone (TQ), the paracetamol metabolite NAPQI, the 5-LO inhibitor AA-861, and bardoxolone methyl (also known as RTA 402 or CDDO-methyl ester) are direct covalent 5-LO enzyme inhibitors that target the catalytically relevant cysteines 416 and 418.