Targeting Botulinum A Cellular Toxicity: A Prodrug Approach

Abstract

The botulinum neurotoxin light chain (LC) protease has become an important therapeutic target for post-exposure treatment of botulism. Hydroxamic acid based small molecules have proven to be potent inhibitors of LC/A with nanomolar Ki values, yet, they lack cellular activity conceivably due to low membrane permeability. To overcome this potential liability, we investigated two prodrug strategies, 1,4,2-dioxazole and carbamate, based on our 1- adamantylacetohydroxamic acid scaffold. The 1,4,2-dioxazole prodrug did not demonstrate cellular activity, however, carbamates exhibited cellular potency with the most active compound displaying an EC50 value of 20 μM. Cellular trafficking studies were conducted using a “fluorescently silent” prodrug that remained in this state until cellular uptake was complete, which allowed for visualization of the drug’s release inside neuronal cells. In sum, this research sets the stage for future studies leveraging the specific targeting and delivery of these prodrugs, as well as other antibotulinum agents, into neuronal cells.

Probing BoNT/A Protease Exosites: Implications for Inhibitor Design and Light Chain Longevity

“…In seeking to develop therapeutics for the reversal of BoNT/A intoxication, the design of inhibitors against the protease has become a centerpiece of research efforts. 10 Indeed, several nonpeptide active site ligands, 11 , 12 as well as compounds binding to LC exosites, such as caftaric acid, chicoric acid, and lomofungin, 13 , 14 have been shown to possess excellent protease inhibitory activity. The active site of BoNT/A LC is governed by zinc and while metalloprotease inhibitors are plentiful, their liabilities are well-documented.…”

Discovery of a para-Acetoxy-benzyl Ester Prodrug of a Hydroxamate-Based Glutamate Carboxypeptidase II Inhibitor as Oral Therapy for Neuropathic Pain

“…The concept of hydroxamate prodrugs has been only recently reported in the literature, including carbamates, 17,18 O -acyl derivatives, 19 and 1,4,2-dioxazol-5-one. 20 However, little has been investigated on the oral pharmacokinetics of these prodrugs to assess their pharmacological advantages over the parent compounds.…”

Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging

“…As a result, probe technologies developed using live-cell and in vivo models translate more readily to therapeutic, diagnostic, and clinical applications. 23,24 Indeed, fluorogenic probes and masking groups are often inspired by prodrug and inhibitor design strategies. 15,25,26 …”

“…Probe 29 is a combination therapeutic and diagnostic agent, or “theranostic”, 72 with a dual-purpose masking group that is also a potent inhibitor of botulinum neurotoxins. 23 A hydroxamate linker enables 29 to undergo esterase-catalyzed activation of the prodrug and fluorogenic probe after freely diffusing across the plasma membrane, with enhanced intracellular delivery and effectiveness in live neurons. Probe 30 is a far-red sensor with variable lipid tails for profiling bacterial esterase and lipase activities.…”