Picolinic acids as β-exosite inhibitors of botulinum neurotoxin A light chain

Bremer, Paul T.; Xue, Song; Janda, Kim D.

doi:10.1039/c6cc06749b

Cited by 6 publications

(4 citation statements)

References 34 publications

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“…Lomofungin, which incorporates a quinolinol substructure capable of chelating zinc, also exhibits no zinc sequestration capability. In an attempt to further explore the chemical space for β-exosite binding, a picolinic acid pharmacophore was used as a mimetic of the lomofungin scaffold . Cross-coupling indolyl fragments to bromopicolinates via the Suzuki–Miyaura reaction allowed for efficient synthesis of derivatives.…”

Section: Exosite Inhibitorsmentioning

confidence: 99%

“…In an attempt to further explore the chemical space for β-exosite binding, a picolinic acid pharmacophore was used as a mimetic of the lomofungin scaffold. 52 Cross-coupling indolyl fragments to bromopicolinates via the Suzuki−Miyaura reaction allowed for efficient synthesis of derivatives. Gratifyingly, we determined through competition studies that these compounds do bind mutually exclusively with lomofungin.…”

Section: ■ Exosite Inhibitorsmentioning

confidence: 99%

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Strategies to Counteract Botulinum Neurotoxin A: Nature’s Deadliest Biomolecule

et al. 2019

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Conspectus Botulinum neurotoxin serotype A (BoNT/A), marketed commercially as Botox, is the most toxic substance known to man with an estimated intravenous lethal dose (LD50) of 1–2 ng/kg in humans. Despite its widespread use in cosmetic and medicinal applications, no postexposure therapeutics are available for the reversal of intoxication in the event of medical malpractice or bioterrorism. Accordingly, the Centers for Disease Control and Prevention categorizes BoNT/A as a Category A pathogen, posing the highest risk to national security and public health as a result of the ease with which BoNT/A can be weaponized and disseminated. BoNT/A-mediated lethality results from neurons impeded from releasing acetylcholine, which ultimately causes muscle paralysis and possible death by asphyxiation with the loss of diaphragm function. Currently, the only available respite for BoNT/A poisoning is antibody-based therapy; however, this intervention is only effective within 12–24 h postexposure. Small molecule therapeutics remain the only opportunity to reverse BoNT/A intoxication after neuronal poisoning and are urgently needed. Nevertheless, no small molecule BoNT/A inhibitors have reached the clinic or even advanced to clinical trials. This Account highlights the accomplishments and existing challenges facing BoNT/A drug discovery today. Using the comprehensive body of work from our laboratory, we illustrate our nearly two-decade endeavor to discover a clinically relevant BoNT/A inhibitor. Specifically, a discussion on the identification and characterization of new chemical leads, the development of in vitro and in vivo assays, and pertinent discoveries in BoNT/A structural biology related to small molecule inhibition is presented. Lead discovery efforts in our laboratory have leveraged both in vitro high-throughput screening and rational design, and an array of mechanistic strategies for inhibiting BoNT/A has been discovered, including noncovalent inhibition, metal-binding active site inhibition, covalent inhibition, and α- and β-exosite inhibition. We contrast the strengths and weaknesses of each of these mechanistic strategies and propose the most favorable approach for success. Finally, we discuss multiple serendipitous discoveries of antibotulism small molecules with alternative mechanisms of action. Remaining challenges facing clinically relevant BoNT/A inhibition are presented and analyzed, including the current inability to reconcile toxin half-life (months to greater than one year) in neurons with in vivo pharmaceutical lifetimes and reoccurring inconsistencies between in vitro, cellular, and in vivo translation. Our Account of BoNT/A chemical research emphasizes the present accomplishments and critically analyzes the remaining obstacles for drug discovery. Importantly, we call for an increased focus on the discovery of safe and effective covalent inhibitors of BoNT/A that compete with the inherent half-life of the toxin.

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Section: Exosite Inhibitorsmentioning

confidence: 99%

Section: ■ Exosite Inhibitorsmentioning

confidence: 99%

Strategies to Counteract Botulinum Neurotoxin A: Nature’s Deadliest Biomolecule

et al. 2019

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“…In the search for inhibitors, the active site containing a zinc ion must be avoided because this metal is also present in human metalloproteases. For this reason, two specific exosites of the peptidase have shown great potential for inhibition [182,183], and several small molecules [184,185] and antibodies [186,187] have already been discovered that bind to either of the two. Similarly, the lethal metalloprotease of Bacillus anthracis has been inhibited by targeting its exosite [188].…”

Section: Regulation Via Exositesmentioning

confidence: 99%

Regulation of Peptidase Activity beyond the Active Site in Human Health and Disease

Obaha,

Novinec

2023

IJMS

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This comprehensive review addresses the intricate and multifaceted regulation of peptidase activity in human health and disease, providing a comprehensive investigation that extends well beyond the boundaries of the active site. Our review focuses on multiple mechanisms and highlights the important role of exosites, allosteric sites, and processes involved in zymogen activation. These mechanisms play a central role in shaping the complex world of peptidase function and are promising potential targets for the development of innovative drugs and therapeutic interventions. The review also briefly discusses the influence of glycosaminoglycans and non-inhibitory binding proteins on enzyme activities. Understanding their role may be a crucial factor in the development of therapeutic strategies. By elucidating the intricate web of regulatory mechanisms that control peptidase activity, this review deepens our understanding in this field and provides a roadmap for various strategies to influence and modulate peptidase activity.

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“…The most common inhibition strategy has been to employ zinc chelator groups such as hydroxamates , and quinolinols − or SNAP-25 peptidomimetics − with the goal of competing with SNAP-25 for the BoNT/A LC active site. Disruption of SNAP-25 binding at the LC exosites with small-molecule inhibitors has also been explored. − More recently, translocation inhibitors targeting endogenous TrxR such as ebselen and auranofin have been shown to be effective at preventing BoNT/A intoxication in cell-based and in vivo models. , The critical drawback to this therapeutic approach is that it provides minimal postsymptomatic relief because BoNT/A LC already translocated to the cytosol will continue to act unimpeded; therefore, direct LC inhibition remains an attractive, yet difficult strategy.…”

Section: Introductionmentioning

confidence: 99%

Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A

et al. 2017

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Botulinum neurotoxin serotype A (BoNT/A) causes a debilitating and potentially fatal illness known as botulism. The toxin is also a bioterrorism threat, yet no pharmacological antagonist to counteract its effects has reached clinical approval. Existing strategies to negate BoNT/A intoxication have looked to antibodies, peptides or organic small molecules as potential therapeutics. In this work, a departure from the traditional drug discovery mindset was pursued, in which the enzyme’s susceptibility to metal ions was exploited. A screen of a series of metal salts showed marked inhibitory activity of group 11 and 12 metals against the BoNT/A light chain (LC) protease. Enzyme kinetics revealed that copper (I) and (II) cations displayed noncompetitive inhibition of the LC (Ki ≈ 1 μM), while mercury (II) cations were 10-fold more potent. Crystallographic and mutagenesis studies elucidated a key binding interaction between Cys165 on BoNT/A LC and the inhibitory metals. As potential copper prodrugs, ligand-copper complexes were examined in a cell-based model and were found to prevent BoNT/A cleavage of the endogenous protein substrate, SNAP-25, even at low μM concentrations of complexes. Further investigation of the complexes suggested a bioreductive mechanism causing intracellular release of copper, which directly inhibited the BoNT/A protease. In vivo experiments demonstrated that copper (II) dithiocarbamate and bis(thiosemicarbazone) complexes could delay BoNT/A-mediated lethality in a rodent model, indicating their potential for treating the harmful effects of BoNT/A intoxication. Our studies illustrate that metals can be therapeutically viable enzyme inhibitors; moreover, enzymes that share homology with BoNT LCs may be similarly targeted with metals.

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Picolinic acids as β-exosite inhibitors of botulinum neurotoxin A light chain

Cited by 6 publications

References 34 publications

Strategies to Counteract Botulinum Neurotoxin A: Nature’s Deadliest Biomolecule

Strategies to Counteract Botulinum Neurotoxin A: Nature’s Deadliest Biomolecule

Regulation of Peptidase Activity beyond the Active Site in Human Health and Disease

Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A

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