Transformation of a Metal Chelate into a "Catch and Anchor" Inhibitor of Botulinum A Protease.

Targeting the botulinum neurotoxin light chain (LC) metalloprotease using small-molecule metal chelate inhibitors is a promising approach to counter the effects of the lethal toxin. However, to overcome the pitfalls associated with simple reversible metal chelate inhibitors, it is crucial to investigate alternative scaffolds/strategies. In conjunction with Atomwise Inc., in silico and in vitro screenings were conducted, yielding a number of leads, including a novel 9-hydroxy-4H-pyrido [1,2-a]pyrimidin-4-one (PPO) scaffold. From this structure, an additional series of 43 derivatives were synthesized and tested, resulting in a lead candidate with a Ki of 150 nM in a BoNT/A LC enzyme assay and 17 µM in a motor neuron cell-based assay. These data combined with structure-activity relationship (SAR) analysis and docking led to a bifunctional design strategy, which we termed "catch and anchor" for the covalent inhibition of BoNT/A LC. Kinetic evaluation was conducted on structures prepared from this catch and anchor campaign, providing kinact/Ki values, and rationale for inhibition seen. Covalent modification was validated through additional assays, including an FRET endpoint assay, mass spectrometry, and exhaustive enzyme dialysis. The data presented support the PPO scaffold as a novel candidate for targeted covalent inhibition of BoNT/A LC.

Investigation of Salicylanilides as Botulinum Toxin Antagonists.

Botulinum neurotoxin serotype A (BoNT/A) is recognized by the Centers for Disease Control and Prevention (CDC) as the most potent toxin and as a Tier 1 biowarfare agent. The severity and longevity of botulism stemming from BoNT/A is of significant therapeutic concern, and early administration of antitoxin-antibody therapy is the only approved pharmaceutical treatment for botulism. Small molecule therapeutic strategies have targeted both the heavy chain (HC) and the light chain (LC) catalytic active site and α-/ß-exosites. The LC translocation mechanism has also been studied, but an effective, nontoxic inhibitor remains underexplored. In this work, we screened a library of salicylanilides as potential translocation inhibitors. Potential leads following a primary screen were further scrutinized to identify sal30, which has a cellular minimal concentration of a drug that is required for 50% inhibition (IC50) value of 141 nM. The inquiry of salicylanilide sal30's mechanism of action was explored through a self-quenched fluorogenic substrate conjugated to bovine serum albumin (DQ-BSA) fluorescence, confocal microscopy, and vacuolar H+-ATPase (V-ATPase) inhibition assays. The summation of these findings imply that endolysosomal proton translocation through the protonophore mechanism of sal30 causes endosome pH to increase, which in turn prevents LC translocation into cytosol, a process that requires an acidic pH. Thus, the inhibition of BoNT/A activity by salicylanilides likely occurs through disruption of pH-dependent endosomal LC translocation. We further probed BoNT inhibition by sal30 using additivity analysis studies with bafilomycin A1, a known BoNT/A LC translocation inhibitor, which indicated the absence of synergy between the two ionophores.

Identification of Slow-Binding Inhibitors of the BoNT/A Protease.

Botulinum neurotoxin A (BoNT/A) is a lethal toxin, which causes botulism, and is categorized as a bioterrorism threat, which causes flaccid paralysis and death. Botulinum A neurotoxicity is governed through its light chain (LC), a zinc metalloprotease. Pharmacological investigations aimed at negating BoNT/A's LC have typically looked to inhibitors that have been shown to inhibit the light chain's activity by reversible zinc chelation within its active site. This report outlines the first examples of nonpeptidic inhibitors of the BoNT/A LC that possess slow-binding kinetics, a needed logic to counteract the longevity of BoNT/A. Cyclopropane, alkyl, and alkenyl derivatives of 2,4-dichlorocinamic hydroxamic acid (DCHA) were shown to possess both one-step and two-step slow-binding kinetics. Structure-kinetic relationships (SKRs) were observed and were rationalized with the aid of docking models that predicted improved interactions with residues within a hydrophobic cleft adjacent to the active site.

Synthesis, structure and photophysical properties of a 2D network with gold dicyanide donors coordinated to aza[5]helicene viologen acceptors.

A recently synthesized photoluminescent organic acceptor, 5,10-dimethyl-5,10-diaza[5]helicene is shown to react with dicyanoaurate anions to form a 2D network N,N-dimethylaza[5]helicene dicyanoaurate. The structure of the synthesized complex was investigated via X-ray crystallography showing the presence of [Au(CN)2]- dimers and monomers within the helicene framework. Photophysical measurements between 298 K and 10 K indicate quenching of the [Au(CN)2]- anion by 5,10-dimethyl-5,10-diaza[5]helicene via an electron transfer. A Stern-Volmer and Rehm-Weller analysis shows that this is a result of quenching from transfer of an electron from [Au(CN)2]- anions to 5,10-dimethyl-5,10-diaza[5]helicene as opposed to resonance energy transfer. DFT calculations were performed to support the assignment of an electron transfer.

Crystal structure of tris-(3-methyl-1H-pyrazol-1-yl)methane.

The title mol-ecule, C13H16N6, crystallizes from hexane as a mol-ecular crystal with no strong inter-molecular inter-actions (the shortest C-H⋯N contact is longer than 3.38 Å). A relatively short intra-molecular contact (3.09 Å) has a C-H⋯N angle of 118° which is quite small to be still considered a hydrogen bond. The three pyrazole rings form a propeller-like motif, with one methylpyrazole unit almost perpendicular to the mean plane of the three rings [82.20 (6)°]. The other two methylpyrazole units, with nitrogen donor atoms oriented in opposite directions, are oriented at 67.26 (6) and 72.53 (6)° to the mean plane.