Design and Synthesis of Fluorescent Acyclic Nucleoside Phosphonates as Potent Inhibitors of Bacterial Adenylate Cyclases

Abstract

Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with submicromolar potency (IC50 values 11 – 622 nM). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase of direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly CaM-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC50 = 12 µM.

Synthesis of α‐Branched Acyclic Nucleoside Phosphonates as Potential Inhibitors of Bacterial Adenylate Cyclases

“…For this reason, and to increase the flexibility of the ribose moiety of natural NTPs, novel fluorescent 5‐chloroanthraniloyl‐substituted ANPs were recently synthesized either as diphosphates, that is, Cl‐ANT‐ANPpp analogues (e.g., compound V , Figure ) or as bisamidate prodrugs to improve their bioavailability . These compounds were shown to be potent competitive ACT and EF inhibitors with sub‐micromolar potency (IC 50 values: 11–622 n m ) …”

“… Structures of bis‐Cl‐ANT‐ATP ( IV ), Cl‐ANT‐ANPpp derivative ( V ), and new target molecules VI (the hemiaminal linkage is highlighted in red).…”

“…Fluorescent (M)ANT‐NTPs were also shown to be suitable candidates for detailed studies of their binding interactions with ACT using direct fluorescence and/or fluorescence resonance energy transfer, but clearly were not therapeutically applicable due to their rapid degradation in vivo. For this reason, and to increase the flexibility of the ribose moiety of natural NTPs, novel fluorescent 5‐chloroanthraniloyl‐substituted ANPs were recently synthesized either as diphosphates, that is, Cl‐ANT‐ANPpp analogues (e.g., compound V , Figure ) or as bisamidate prodrugs to improve their bioavailability . These compounds were shown to be potent competitive ACT and EF inhibitors with sub‐micromolar potency (IC 50 values: 11–622 n m ) …”

Synthesis and evaluation of symmetric acyclic nucleoside bisphosphonates as inhibitors of the Plasmodium falciparum, Plasmodium vivax and human 6-oxopurine phosphoribosyltransferases and the antimalarial activity of their prodrugs

“…12 In general, ANPs have ability to target various metabolic pathways where they exhibit a wide range of biological activities, e.g. antibacterial, immunomodulatory and antineoplastic 13,14,15,16,17 and can serve as monomers for oligonucleotide synthesis. 18 Figure 2.…”

Novel nucleotide analogues bearing (1 H -1,2,3-triazol-4-yl)phosphonic acid moiety as inhibitors of Plasmodium and human 6-oxopurine phosphoribosyltransferases

“…Several different chemical types of ANPs, including modified PMEA analogues, have also been studied as potent inhibitors of bacterial adenylate cyclases, namely adenylate cyclase toxin from Bordetella pertussis and edema factor from Bacillus anthracis. [12][13][14] Such analogues may have potential for treatment or prevention of toxaemia caused by the invasion of these bacteria into the human host. Inhibition of plasmodial hypoxanthine-guanine-(xanthine) phosphoribosyltransferases (HG(X)PRTs) by the ANPs is well-correlated with their antimalarial activity.…”