Evaluation of phosphoramidon and three synthetic phosphonates for inhibition of botulinum neurotoxin B catalytic activity

Abstract

Three putative metalloprotease inhibitors were synthesized and tested for their ability to inhibit the catalytic activity of botulinum neurotoxin B light chain (BoNT/B LC). The compounds were designed to emulate the naturally occurring metalloprotease inhibitor phosphoramidon, which has been reported to be a weak antagonist of BoNT/B action. All three analogs contained the dipeptide Phe‐Glu in place of Leu‐Trp of phosphoramidon and possessed a phenyl, ethyl or methyl group in place of the rhamnose sugar of the parent compound. The inhibitors were evaluated in a cell‐free assay based on the detection of a fluorescent product following cleavage of a 50‐mer synaptobrevin peptide ([Pya88] S 39–88) by BoNT/B LC. This peptide corresponds to the hydrophilic core of synaptobrevin‐2 and contains a fluorescent analog L‐pyrenylalanine (Pya) in place of Tyr88. Cleavage of [Pya88] S 39–88 by BoNT/B LC gives rise to fragments of 38 and 12 amino acid residues. Quantification of BoNT/B‐mediated substrate cleavage was achieved by separating the 12‐mer fragment (FETSAAKLKRK‐Pya) that contains the C‐terminal fluorophore and measuring fluorescence at 377 nm. The results indicate that the phenyl‐substituted synthetic compound ICD 2821 was slightly more active than phosphoramidon, but analogs with methyl or ethyl substitutions were relatively inactive. These findings suggest that phosphonate monoesters may be useful for providing insights into the structural requirement of BoNT/B protease inhibitors.

A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)

“…Nevertheless, the discovery of highly selective small molecule protease inhibitors across a wide variety of protease is still achievable. For example, neprilysin (neutral endopeptidase 24.11; NEP) inhibitor, phosphoramidon, obtained by analogs with methyl or ethyl substitutions, was relatively not potent [32]. The docking simulation of SC44463 revealed that this compound blocked the direction of the peptide chain in contact with the active site is in the reverse direction of that seen in complex of stromelysin with synthetic inhibitors [33].…”

Development of Potent Inhibitors of Botulinum Neurotoxin Type B

“…were shown to be inactive. 5,[36][37][38] Other inhibitors have also been tested: 7-N-phenylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (elastase inhibitor) with an inhibitory potency of 27.6 µM, 35,36 phosphonates and phenylmethylsulfonyl fluoride (serine protease inhibitor) which were not really effective, 35,39 BABIM (bis(5-amidino-2-benzimidazolyl)methane) or keto-BABIM with inhibitory potencies of 1.6 and 0.8 µM, 40 and buforin-1 with an inhibitory potency of 1 µM were the first synthetic substances with a relatively good affinity for BoNT/B. 41 However, their potency was too low for a possible clinical use.…”

“…Zinc chelating agents are effective blockers but are devoid of selectivity and have important adverse effects and thus cannot be used in therapy. − Small peptides derived from the substrate of BoNT/B are neither substrates nor inhibitors. , Classical inhibitors of zinc metalloproteases (ACE, NEP, etc.) were shown to be inactive. ,− Other inhibitors have also been tested: 7- N -phenylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (elastase inhibitor) with an inhibitory potency of 27.6 μM, , phosphonates and phenylmethylsulfonyl fluoride (serine protease inhibitor) which were not really effective, , BABIM (bis(5-amidino-2-benzimidazolyl)methane) or keto-BABIM with inhibitory potencies of 1.6 and 0.8 μM, and buforin-1 with an inhibitory potency of 1 μM were the first synthetic substances with a relatively good affinity for BoNT/B . However, their potency was too low for a possible clinical use.…”

Targeting Metalloenzymes for Therapeutic Intervention

“…644 It is worth noting that phosphoramidon was found to be more effective at protecting against BoNT in the tissue model 645 than what would be expected from the poor activity reported in vitro enzymatic assay. 644 This discrepancy between the in vitro enzymatic assay and cell culture and tissue models has been noted elsewhere, and suggests that there are relevant differences between the truncated form of BoNT used in the in vitro assays and the intact form of BoNT used in the in vivo assays that are not yet fully understood with respect to drug development. 657 The most prolifically explored class of inhibitor against BoNT uses the hydroxamic acid MBP (Figure 70).…”

“…Subsequent work to replace the synthetically liable rhamnose moiety resulted in the discovery of compounds BoNTi-2, BoNTi-3, and BoNTi-4, of which the phenyl-substituted BoNTi-4 was found to be the most effective with a poor IC 50 value of 8.1 mM against BoNTB . It is worth noting that phosphoramidon was found to be more effective at protecting against BoNT in the tissue model than what would be expected from the poor activity reported in vitro enzymatic assay . This discrepancy between the in vitro enzymatic assay and cell culture and tissue models has been noted elsewhere, and suggests that there are relevant differences between the truncated form of BoNT used in the in vitro assays and the intact form of BoNT used in the in vivo assays that are not yet fully understood with respect to drug development …”