As shown in figure 4, NSC84087 showed dose dependent inhibition of endopeptidase activity of rBoNT/A-LC (10 nM), the IC50 is estimated to be 40 nM (63.7 nM). This indicates that 50% BoNT/A-LC inhibition is achieved when the inhibitor: rBoNT/ALC molar ratio is 4:1; however 100% inhibition was recorded at 6:1 molar ratio. The extent of inhibition and the IC50 values of these leads were comparable to or even better than those of previously reported small-molecule inhibitors [(10000:1) Park et al. [28]; (3000:1) Burnett et al. 2007 [35]; (800:1) Tang et al. 2007 [21]; (150:1) Eubanks et al. 2007 [36]; (320:1) Cai et al. 2010 [52]; (2000:1) Pang et al. 2010 [31]; (3000:1) Ruthel et al. 2011 [37]; (250:1) Burnet et al. 2010 [32]; (350:1) Thomson et al. 2011 [53], molar ratios given in parenthesis are of inhibitor: BoNT/A-LC]. The molar ratio between inhibitor and BoNT/A-LC molecules at 50% inhibition provides better parameter for comparison between assays, since it normalizes the neurotoxin used. The lower molar ratio in synaptosome based assay reflects the inhibitor’s efficiency is more relevant to in vivo conditions. In fact, the IC50 values of the compounds increased (worsened) 4- to over 50-fold when the concentration of one of the model enzymes, b-lactamase, was increased 10-fold, from 1 nM to 10 nM [54]. The authors also suggested for the unifying mechanism to be followed for the discovery of the inhibitors. Major advantages of a synaptosome based assay are: (i) it provides full length SNAP-25 substrate to b-exosite recognition and subsequent conformational changes in the enzyme to facilitate efficient substrate cleavage. It has been considered that, without exosite binding, BoNT/A is a significantly less efficient enzyme; and (ii) it simulates the microenvironment of the neurons. The other advantages are considerable reduction in the number of animals used (millions of reactions can be performed from synaptosomes isolated from single rat brain), substrate stability (synaptosomes isolated are stable for use as substrate for more than year), instrumentation requirement and cost incurred, especially when used for assessing large numbers of target molecules. We suspect the use of small peptide substrates (,17mer peptides/fluorescent substrate which is minimum substrate required for BoNT/A) in high-throughput screening and identification of small-molecule inhibitors is the reason for in vitro and in vivo disconnect which is reported by Eubanks et al. [36].
Figure 2. Initial screening of different compounds by western blotting using specific antibodies. RRGC and twelve other compounds (CB7969312, CB7967495, CB7925339, CB7887535, CB6378306, CB6376015, CB6377128, NSC1010, NSC84096, NSC84090, NSC84087 and NSC84093 were tested for their inhibition activity. To determine inhibition activity of these compounds, 2.05 mg synaptosomes prepared from rat brain was incubated with 200 nM of rBoNT/A-LC and 100 mM of each compounds solubilized in DMSO at 37uC for 15 min as described in materials and methods. Proteins were visualized using appropriate HRP-labeled second antibody by ECL. Nine molecules (CB796312, CB7925339, CB7887535, CB6378306, CB6376015, CB6377128, NSC1010, NSC84096, and NSC84087) showed complete inhibition while CB 7967495 & NSC84093 showed 87 and 85% of inhibition respectively. Significant inhibition was not observed with RRGC and NSC 84090 molecules. molecule inhibitors against botulinum neurotoxins, higher potency of small-molecule inhibitors observed during evaluation in the in vitro assay systems using small peptide substrate, may have resulted due to the suboptimal activity of BoNT/A-LC. As these small substrate peptides used for in vitro screens are not long enough to simultaneously occupy cleavage site and either of the exosite, hence it is clear inhibition in fluorescent peptides based assays relies on the interactions in the enzyme active site. However, our findings argue that in the context of BoNT therapeutics, caution should be used in extrapolating in vivo potency from these assays.
3. Evaluation of Small-molecule Inhibitors in Mouse Model
To be useful as therapeutics, the newly identified inhibitors must: (i) be able to enter neurons; (ii) inhibit the toxin within the neuronal cytosol; and (iii) be tolerated by animals (i.e. possess acceptable toxicity profiles). A true test or ultimate goal for inhibitors evaluated in both cell-free systems and even for cell based assays is whether their effectiveness holds true in vivo. The effectiveness of small-molecule inhibitors in the in vitro and ex vivo assays was only demonstrated when the compound was premixed with BoNT/A; thus far, pre-loading the inhibitor did not protect cells/tissues against BoNT intoxication [24]. The small-molecule inhibitor that was reported to be active in primary neurons [22] was demonstrated to show a dose-dependent inhibition of SNAP25 cleavage in a non-pre-loading system (cells were pretreated with inhibitor for 45 minutes followed by incubation with BoNT/A in the continuous presence of inhibitor). Additionally, the inhibitors reported by Eubanks et al. [36] and Boldt et al. [34] were characterized in cell culture assays that involved mixing BoNT/A toxin and varying concentrations of inhibitor. To our knowledge, no group has been able to provide experimental evidence showing that their inhibitors work in a pre-loading system. After completion of our in vitro screening, three compounds were deemed to have suitable activity and were advanced into animal models (CB 7887535, CB6378306 and NSC 84087). The fourth compound, included for comparison, was a molecule previously well characterized and reported to inhibit specifically BoNT/ALC (NSC 1010). To examine the lead compounds in vivo, a wellestablished mouse bioassay was used. This model is the Food and Drug Administration (FDA) standard for assessing BoNT levels and the universally accepted method for the study of BoNT antagonists (e.g., antibodies, small-molecules) [55]. For evaluation of inhibitory potential of small-molecules, they were injected into test animals as described in materials and methods. All animals were monitored continuously for a period of 4 days for signs of botulism, and the time of death was recorded. Of the compounds studied, one compound (NSC 84087) showed efficacy in preventing BoNT/A-induced death in all three modes of injections and an injection dosage of 100 ml of 1 mM per animal survived the BoNT challenge with no obvious signs of botulism upto 20 h (Fig. 5). All the mice of inhibitor followed by BoNT/A group, showed symptoms after 20 h and died within 30 h of injection. Mice of other two groups, BoNT/A followed by inhibitors and inhibitors toxin premixed, survived upto 48 h. In similar in vivo studies, conducted by Eubanks et al. [36] and Pang et al.