Symptomatic Relief of Botulinum Neurotoxin/A Intoxication with Aminopyridines: A New Twist on an Old Molecule

Abstract

Botulinum neurotoxins (BoNT) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNT/A is the most toxic protein known to man and has been classified by the Centers of Disease Control (CDC) as one of the six highest-risk threat agents for bioterrorism. Of particular concern is the apparent lack of clinical interventions that can reverse cellular intoxication. Efforts to uncover molecules that can act within an intoxicated cell so as to provide symptomatic relief to BoNT/A are paramount. Aminopyridines have shown clinical efficacy for multiple sclerosis treatment as well as BoNT/A intoxication; yet, aminopyridines for BoNT/A treatment has been abandoned because of blood brain barrier (BBB) penetration producing undesired neurotoxic side effects. Two aminopyridines, (5 and 11), exhibited inhibitory activity toward Shaker-IR voltagegated potassium (K V 1.x) channels with potencies similar to that of the previous "gold-standard", 3,4-diaminopyridine (3,4-DAP), including reversal of symptoms from BoNT-induced paralysis in phrenic nerve-hemidiaphragm preparations. Importantly, pharmacokinetic experiments revealed a lack of BBB penetration of 5, which is a significant advancement toward resolving the neurotoxicity issues associated with prolonged 3,4-DAP treatments. Finally, 5 was found to be as effective as 3,4-DAP in rescuing BoNT-poisoned mice in the mouse lethality assay, signifying an optimized balance between the undesired permeability across the BBB, and the required permeability across lipid cellular membranes. The results demonstrate that 5 is the most promising small molecule K + channel inhibitor discovered to date for the treatment of BoNT/A intoxication. KeywordsBotulinum Neurotoxin; aminopyridine; K + channel inhibitors * To whom correspondence should be addressed. Phone: (+1) 858-784-2516. Fax: (+1) 858-784-2595. kdjanda@scripps.edu. SUPPORTING INFORMATION AVAILABLE Synthetic and computational procedures, HPLC traces and 1 H/ 13 C NMR spectra of compounds 4-12; additional details for the biological evaluation of compounds 5 and 11. This material is available free of charge via the Internet at

Symptomatic treatment of botulism with a clinically approved small molecule

“…It has been proposed that neuromuscular paralysis can be overcome by drugs that increase cholinergic neurotransmission (22,23). Indeed, we and other groups have shown that aminopyridines such as 3,4-diaminopyridine (3,4-DAP) transiently reverse muscle paralysis in a concentration-dependent manner (24)(25)(26)(27). 3,4-DAP is a selective potassium channel blocker that prolongs neuronal action potential duration, thereby increasing Ca 2+ influx through presynaptic voltage-gated Ca 2+ channels (28)(29)(30).…”

Elucidating the Molecular Basis of Action of a Classic Drug: Guanidine Compounds As Inhibitors of Voltage-Gated Potassium Channels

“…The U.S. Food and Drug Administration has approved 4-aminopyridine as a drug for the treatment of multiple sclerosis under the trade name Ampyra (Acorda Therapeutics, Inc., Hawthorne, NY) (Hauser and Johnston, 2010). Another aminopyridine compound, 3,4 diaminopyridine, is used in the treatment of botulism (Mayorov et al, 2010;Chalk et al, 2011) and Lambert-Eaton myasthenic syndrome (Keogh et al, 2011). A major limitation of the therapeutic use of these compounds is the harmful side effects that accompany their administration.…”

“…A major limitation of the therapeutic use of these compounds is the harmful side effects that accompany their administration. Indeed, guanidine consumption can cause bone marrow suppression and renal failure (Blumhardt et al, 1977), and aminopyridines can penetrate the blood-brain barrier and cause neurotoxicity (Mayorov et al, 2010). To overcome these detrimental side effects, it would be desirable to design analogs of aminopyridines and guanidines that exhibit high potency for their biological targets, enabling administration at low dosage levels.…”

“…This information has been judiciously used for rational drug design on the aminopyridine template. For example, docking simulations on the binding site (Caballero et al, 2007) have facilitated structure-activity studies on aminopyridines, leading to the generation of molecules with better therapeutic indices (Mayorov et al, 2010). In contrast, the mechanism of inhibition of Kv channels by guanidine has never been investigated thoroughly, and there are no published structure-activity studies on the guanidine scaffold.…”

Formulating a new basis for the treatment against botulinum neurotoxin intoxication: 3,4-Diaminopyridine prodrug design and characterization

“…14 By synthesizing structural analogues, we hoped to discover more potent, yet, less toxic forms of the pharmacophore for treatment against BoNT intoxication. Previous reports indicate that 3,4-DAP and 4-AP, when administered at high doses, demonstrate toxicity issues such as seizures due to the molecules’ ability to penetrate the blood-brain barrier (BBB).…”