Development of Novel Inhibitors of Botulinum Neurotoxin A

 

Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria Clostridium botulinum are the most potent neurotoxins. BoNTs are classified by the Centers for Disease Control and prevention (CDC) as category A bioterrorism agents. They exist as seven distinct serotypes (A-G) out of which, Botulinum neurotoxin A (BoNT/A) is the most potent with an LD50 of 1.3 ng/kg. BoNT/A is a two-chain polypeptide made up of a heavy and a light chain, which are linked to each other by a disulfide linkage. The BoNT/A-light chain (LC) is a zinc metalloprotease that cleaves one of three SNARE proteins at the neuromuscular junction blocking the release of neurotransmitter acetylcholine, resulting in flaccid paralysis followed by respiratory failure (Figure 1). The limited availability of therapeutic treatment and their low LD50 makes BoNTs highly potential weapons for biological warfare. Thus there is a need for the development of agents that inhibit BoNTs.

 

BoNT_1 

Figure 1: Normal acetylcholine release versus the disease model of infection with Botulinum neurotoxin.

 

High throughput virtual screening

 

Molecular foot-print based rescoring methodology

 

Traditional screening programs such as DOCK utilize van der Waals (VDW) and electrostatic (ES) terms to rank order the compatibility of ligands with target. A new scoring function called the molecular foot print similarity (FPS) score was used to identify inhibitors for BoNT. The FPS scoring method identifies compounds that are energetically most similar to the known reference (Figure 2).

 BoNT_2

Figure 2: Use of FPS score in virtual screening (a) Reference ligand (red). (b) Comparison between the reference (red) and candidate molecule (green) from the virtual screen.

 

Peptidomimetics

 

Peptidomimetics was used for the initial attempts to identify inhibitors for BoNT/A. 8 of the available co-crystal structures of BoNT/A with inhibitors were overlaid to determine favorable vs unfavorable interaction energies. Polypeptide RRGC was found to have the best overall fit in 7 out of 8 co-crystal structures. Thus polypeptide RRGC, which has a Ki of 157 nM against BoNT-A, was used a reference ligand for virtual screening.

 

In order to identify new Botulinum neurotoxin light chain-A (BoNT/LC-A) inhibitors, virtual screening of over one million commercially available compounds was carried out of which 99 compounds were selected, purchased from Chem-Div. The 99 compounds were assayed in vitro using a high throughput SNAPtide assay. The results of the SNAPtide assay are summarized in Table 1.  Based on the IC50 values from the assay, compounds with a foot print score greater than 0.9 appeared to have better activity. This assay resulted in the identification of 2 hit compounds ChemDiv 5762-1843 and ChemDiv E843-1064. 3D docked structures of ChemDiv 5762-1843 and ChemDiv E843-1064 show that the compounds interact with Zinc ion and Aspartic acid 370. The coordination group for ChemDiv 5762-1843 is urea and that for ChemDiv E843-1064 is 1,2,4-oxadiazole. The structures and foot print overlay of these two hit compounds are shown in Figure 3.

 

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Table 1: Hit compounds screened with high-throughput SNAPtide fluorescence assay with their corresponding DOCK energy and FPS scores

aZINC database ID number.

bChemDiv ID number.

cDCE scores in kcal/mol.

dFPS score in units of normalized Euclidian distance where 0 represents the best overlap. FPSVDW and FPSES scores range from (0,2) and FPSVDW+ES range from (0,4).

eNumber of times the assay was run.

 

 BoNT_4

 

Figure 3: 2D chemical structure and 3D docked structures of hit compounds (A) ChemDiv ID 5762-1843 and (B) ChemDiv ID E843-1064. The Vander wals (VDW) and Electrostatic (ES) footprint overlay of RRGC (red) and hit compounds (blue) (C) ChemDiv ID 5762-1843 and  (D) ChemDiv ID E843-1064.

 

Several analogs of the 2 hit compounds ChemDiv ID 5762-1843 and ChemDiv ID E843-1064 were synthesized and their IC50 was determined using the SNAPtide assay as shown in Table 2.

 

BoNT_5

 

Table 2: IC50 values of resynthesized ChemDiv compounds and their analogs.

 

Cell based assay with hit compounds

 

The two hit compounds ChemDiv 5762-1843 and ChemDiv ID E843-1064 were screened using Neuro-2a cells inoculated with BoNT/LC. Both the compounds displayed graded inhibition at 10 µM in vivo. ChemDiv 5762-1843 and ChemDiv ID E843-1064 inhibited the quantity of SNAP25 cleaved in vivo at 10 µM after BoNT-LC was transfected into the cells as shown in Figures 4 and 5. The compounds were not cytotoxic at the concentrations used.

 

 BoNT_6

Figure 4: Western blot analysis of the inhibition of SNAP-25 cleavage by BoNT/A-LC in vivo in Neuro-2a cells by inhibitor ChemDiv 5762-1843. 1) 100 μM, 2) 50 μM, 320 μM, 4) 10 μM, 5) no inhibitor, 6) SNAP 25 marker.

 

 BoNT_7

Figure 5: Western blot analysis of the inhibition of SNAP-25 cleavage by BoNT/A-LC in vivo inNeuro-2a cells by inhibitor ChemDiv E843-1064. 1) no inhibitor, 2) 10 μM, 3) 20 μM, 4) 500 μM, 5)100 μM, 6) SNAP 25 marker.

 

Yu-Han Gary Teng, William Berger, Natasha NesBitt, Kunal Kumar, Robert C. Rizzo, Peter J. Tonge, Iwao Ojima and Subramanyam Swaminathan, Manuscript in preparation.