Irreversible inhibition of BoNT/A protease: proximity-driven reactivity contingent upon a bifunctional approach.

Botulinum neurotoxin A (BoNT/A) is categorized as a Tier 1 bioterrorism agent and persists within muscle neurons for months, causing paralysis. A readily available treatment that abrogates BoNT/A's toxicity and longevity is a necessity in the event of a widespread BoNT/A attack and for clinical treatment of botulism, yet remains an unmet need. Herein, we describe a comprehensive warhead screening campaign of bifunctional hydroxamate-based inhibitors for the irreversible inhibition of the BoNT/A light chain (LC). Using the 2,4-dichlorocinnamic hydroxamic acid (DCHA) metal-binding pharmacophore modified with a pendent warhead, a total of 37 compounds, possessing 13 distinct warhead types, were synthesized and evaluated for time-dependent inhibition against the BoNT/A LC. Iodoacetamides, maleimides, and an epoxide were found to exhibit time-dependent inhibition and their k GSH measured as a description of reactivity. The epoxide exhibited superior time-dependent inhibition over the iodoacetamides, despite reacting with glutathione (GSH) 51-fold slower. The proximity-driven covalent bond achieved with the epoxide inhibitor was contingent upon the vital hydroxamate-Zn2+ anchor in placing the warhead in an optimal position for reaction with Cys165. Monofunctional control compounds exemplified the necessity of the bifunctional approach, and Cys165 modification was confirmed through high-resolution mass spectrometry (HRMS) and ablation of time-dependent inhibitory activity against a C165A variant. Compounds were also evaluated against BoNT/A-intoxicated motor neuron cells, and their cell toxicity, serum stability, and selectivity against matrix metalloproteinases (MMPs) were characterized. The bifunctional approach allows the use of less intrinsically reactive electrophiles to intercept Cys165, thus expanding the toolbox of potential warheads for selective irreversible BoNT/A LC inhibition. We envision that this dual-targeted strategy is amenable to other metalloproteases that also possess non-catalytic cysteines proximal to the active-site metal center.

COVALENT INHIBITION OF BOTULINUM NEUROTOXIN A - EXPLORATION OF WARHEAD REACTIVITY AND FUNCTION USING A BIFUNCTIONAL APPROACH.

INTRODUCTION AND OBJECTIVES: Botulinum neurotoxin A (BoNT/A) is extremely toxic possessing an estimated intravenous LD50 of 1-2 ng/kg and as such has been designated a category A bioterrorism agent.1, 2 BoNT/A also possesses an extremely long half-life and persists within muscle neurons for months to >1 year.3 Because of BoNT/A longevity, we have utilized covalent inhibition as a means to abrogate BoNT/A's toxicity. To this end, we describe an approach to designing inhibitors that possess both electrophilic warheads and metal-binding groups for the bifunctional inhibition of BoNT/A. METHODS: Small molecule inhibitors that possessed electrophilic moieties were designed, using X-ray crystallography as guidance, to target both the zinc metal-binding region and Cys165 within the active site of BoNT/A. Synthesized compounds were evaluated for covalent inhibition using a continuous SNAPtide FRET assay4 and exhaustive dialysis. Compounds were also evaluated against a C165A variant. Compound reactivity, stability, MMP selectivity and cellular efficacy/toxicity was also evaluated. RESULTS: Several electrophilic warhead types were confirmed to inhibit BoNT/A LC covalently with substantial differences in time-dependent inhibition between the WT and C165A variant. A trend in warhead reactivity was reflected in inhibitor stability and toxicity. Compounds exhibited moderate potency in a BoNT/A neuronal cellular assay but were not further explored due to undesirable therapeutic potential. CONCLUSIONS: A fundamental framework for the bifunctional covalent inhibition of BoNT/A LC has been established. This approach has potential to be translated to other small molecule metal-binding inhibitors of BoNT/A LC with the vision that different pharmacophores, possessing improved physicochemical properties, will address BoNT/As toxicity and longevity within cells.

Catch and Anchor Approach To Combat Both Toxicity and Longevity of Botulinum Toxin A.

Botulinum neurotoxins have remarkable persistence (∼weeks to months in cells), outlasting the small-molecule inhibitors designed to target them. To address this disconnect, inhibitors bearing two pharmacophores-a zinc binding group and a Cys-reactive warhead-were designed to leverage both affinity and reactivity. A series of first-generation bifunctional inhibitors was achieved through structure-based inhibitor design. Through X-ray crystallography, engagement of both the catalytic Zn2+ and Cys165 was confirmed. A second-generation series improved on affinity by incorporating known reversible inhibitor pharmacophores; the mechanism was confirmed by exhaustive dialysis, mass spectrometry, and in vitro evaluation against the C165S mutant. Finally, a third-generation inhibitor was shown to have good cellular activity and low toxicity. In addition to our findings, an alternative method of modeling time-dependent inhibition that simplifies assay setup and allows comparison of inhibition models is discussed.

Consequence of Hapten Stereochemistry: An Efficacious Methamphetamine Vaccine.

Recent trends in methamphetamine (METH) misuse and overdose suggest society is inadvertently overlooking a brewing METH crisis. In the past decade, psychostimulant-related lethal overdoses and hospitalizations have skyrocketed 127 and 245%, respectively. Unlike the opioid crisis, no pharmaceutical interventions are available for treating METH use disorder or reversing overdose. Herein, we report the first active vaccine that offers protection from lethal (+)-METH challenge in male Swiss Webster mice. This vaccine formulation of (S)MLMH-TT adjuvanted with CpG ODN 1826 + alum successfully raised anti-METH antibodies in high titers, reduced (+)-METH distribution to the brain, and lowered (+)-METH-associated stereotypies in a hyperlocomotion assay. A comparison of enantiomeric haptens and the racemate elucidated the importance of employing (S)-stereochemistry in METH hapten design for optimal protection.

Atropisomeric 4-phenyl-4H-1,2,4-triazoles as selective glycine transporter 1 inhibitors.

We report on the optimization of 4H-1,2,4-triazole derivatives to increase their activity and selectivity as glycine transporter 1 (GlyT1) inhibitors. Structure-activity relationship exploration resulted in the identification of a 3-[3-ethyl-5-(6-phenylpyridin-3-yl)-4H-1,2,4-triazol-4-yl]-2-methylbenzonitrile (14u) compound with markedly higher selectivity for GlyT1. Physiochemical studies revealed that 14u exists as a stable pair of atropisomers under physiological conditions. We successfully separated the atropisomers to obtain active enantiomer (R)-14u, which displayed favorable pharmacokinetic properties, as well as positive results in the mice Y-maze test.

Synthesis and biological evaluation of (4H-1,2,4-triazol-4-yl)isoquinoline derivatives as selective glycine transporter 1 inhibitors.

To identify novel glycine transporter 1(GlyT1) inhibitors with greater selectivity relative to GlyT2 and improved aqueous solubility, we synthesized a series of 4H-1,2,4-triazole derivatives with heteroaromatic rings at the 4-position and investigated their structure-activity relationships. Replacement of the 2-fluorophenyl group of lead compound 5 with various aromatic groups led to the identification of 5-(3-biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)isoquinoline (15) with 38-fold selectivity between GlyT1 and GlyT2. 15 also showed improved aqueous solubility and in vivo efficacy on (+)-HA966-induced hyperlocomotion in mice over the lead compound.

Synthesis and biological evaluation of 3-biphenyl-4-yl-4-phenyl-4H-1,2,4-triazoles as novel glycine transporter 1 inhibitors.

We describe the preparation and evaluation of a novel series of glycine transporter 1 (GlyT1) inhibitors derived from a high-throughput screening hit. The SAR studies resulted in the discovery of 3-biphenyl-4-yl-4-(2-fluorophenyl)-5-isopropyl-4H-1,2,4-triazole (6p). A pharmacokinetic study was also conducted and revealed that 6p had excellent oral bioavailability and ameliorated learning impairment in passive avoidance tasks in mice.