Structure–Activity Relationship of Semicarbazone EGA Furnishes Photoaffinity Inhibitors of Anthrax Toxin Cellular Entry

Abstract

EGA, 1, prevents the entry of multiple viruses and bacterial toxins into mammalian cells by inhibiting vesicular trafficking. The cellular target of 1 is unknown, and a structure− activity relationship study was conducted in order to develop a strategy for target identification. A compound with midnanomolar potency was identified (2), and three photoaffinity labels were synthesized (3−5). For this series, the expected photochemistry of the phenyl azide moiety is a more important factor than the IC 50 of the photoprobe in obtaining a successful photolabeling event. While 3 was the most effective reversible inhibitor of the series, it provided no protection to cells against anthrax lethal toxin (LT) following UV irradiation. Conversely, 5, which possessed weak bioactivity in the standard assay, conferred robust irreversible protection vs LT to cells upon UV photolysis. KEYWORDS: Photoaffinity labeling, semicarbazone, aryl azide, anthrax lethal toxin, endosomal trafficking A common mechanism by which viruses and bacterial toxins enter host cells involves trafficking from an early to a late endosome followed by release into the cytoplasm as triggered by endosomal acidification. 1−3 Inhibition of this process represents a host-targeted strategy for the development of broad-spectrum antiviral and antibacterial drugs. Recently, EGA, N 1 -(4-bromobenzylidene)-N 4 -(2,6-dimethylphenyl)-semicarbazone, 1, was identified from a high-throughput phenotypic screen to protect cells from anthrax lethal toxin (LT). 4 Further investigations showed that 1 protected cells from multiple viruses and bacterial toxins that rely on pHdependent endosomal trafficking to enter cells. Although the details of the biochemical mechanism have been studied, the cellular target(s) remain unknown. We have conducted a structure−activity relationship (SAR) study with the goal of improving the potency of 1 and developing a strategy for photoaffinity labeling and identification of the involved proteins.A series of derivatives of 1 were synthesized and tested, revealing precise requirements for activity in a tight and relatively flat structure−activity landscape. The 2,6-dimethyl substitution in the N 4 -ring as well as an unaltered semicarbazone core were shown to be optimal for activity while certain modifications to the N 1 -phenyl ring were tolerated. From the SAR, a compound more potent than the original hit was generated, namely the N 1 -2-fluoro-4-bromophenyl analogue (IC 50 = 0.4 μM), 2. In addition, three photoaffinity probes, 3−5, two of which possess low micromolar activity, were designed and synthesized. Our key finding is that the expected photochemistry of the phenyl azide moiety is a more important factor than the IC 50 of the photoprobe in achieving successful photolabeling. While 3 was the most potent inhibitor of intoxication by LT in the standard assay, it did not provide protection to the cells upon UV photolysis. Conversely 5, which possesses two fluorine atoms ortho to the azido function, was a poor inhibitor in the stand...

A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins

“…We would like to conclude by pointing out that the search for novel EGA-derived analogues is made simpler by the design of the novel method of synthesis of this compound described here, which provides a much higher yield with respect to the recently described method 29 . This procedure allowed us to rapidly and efficiently synthesize large quantities of EGA, an essential pre-requisite to produce the considerable amount necessary for a possible employment of this or related compounds in humans.…”

“…Together, our results suggest that EGA represents a new tool for studying BoNTs trafficking and a good candidate for the development of new inhibitors. Notably, we also report an optimized procedure for the synthesis of EGA, which involves milder reaction conditions and provides much higher overall yield than previously reported 29 .…”

A New Efficient Route to 2-Alkylsemicarbazides

“…Clearly, this alkylation can proceed only via formation of a conjugate base of the starting material. Various base/solvent combinations for deprotonation of semicarbazones (e.g., MeONa/DMF [25,26], Et4NOH/THF [27][28][29], NaOH/EtOH-H2O [30], t-BuOK/THF [5,[31][32][33], NaH/DMF [34], K2CO3/DMF [35][36][37], Cs2CO3/MeCN [38], tert-butylimino-tri(pyrrolidino)phosphorane/THF [39]), followed by treatment with alkylating reagents, were reported. We tested some base/solvent combinations for the alkylation of semicarbazones of aromatic aldehydes (E)-1a-c as model compounds (Scheme 2).…”

Multifluorinated Aryl Azides for the Development of Improved H2S Probes, and Fast Strain‐promoted Azide‐Alkyne Cycloaddition and Staudinger Reactions

“…Furthermore, the enhanced reaction kinetics caused by multifluorination also inspire us to investigate whether this strategy could also help to improve other azido‐related reactions . For example, Ting and co‐workers, Jung and co‐workers have reported perfluorinated aryl azides as better photoaffinity labeling reagents over phenyl azides, also highlighting multifluorinated aryl azides for the development of advanced bioorthogonal reactions.…”