Designing, synthesis and evaluation of derived analogues of selected small molecule non-peptidic inhibitors against serotype BoNT/ F.

Botulinum neurotoxins are lethal Biowarfare categorized in group A of selected agents, by CDC USA. The unavailability of counter-measures against these neurotoxins has been a matter of extensive research. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. We have reported 8-HQ compounds NSC1014 and NSC1011 as potential small molecule inhibitors against BoNT/F. In the present study, analogues of NSC84087 and NSC1014 were designed, synthesized and studied for their inhibitory role against BoNT/F intoxication through in silico study, in vitro and in-vivo assays. ∼25 in-house synthesized small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay and then further assessed through endopeptidase assay. The binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in-vivo efficacy of these compounds was evaluated in mice model. Analogues C87.9, C87.10 and C87.12 of compound NSC84087 and C14.10, C14.11 and C14.13 of NSC1014 showed promising results through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed KD values in sub-micromolar range signifying high affinity interaction. The IC50 of C14.10 was found to be the lowest of 3.016 ± 0.798 µM as determined through endopeptidase assay. Finally, efficacy of selected molecules was evaluated in mice, C14.10 and C14.13 protected 40% animals against 4X LD50 and extended survival time up to 200% at 10X LD50. The present study thus proposes the emergence of NSC84087 and NSC1014 analogues as lead compound against BoNT/F.

Targeted 8-hydroxyquinoline fragment based small molecule drug discovery against neglected botulinum neurotoxin type F.

OBJECTIVES: Botulinum neurotoxins are highly potent biological warfare agents. The unavailability of countermeasures against these neurotoxins has been a matter of extensive research. However, no clinical therapeutics has come to existence till date. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. METHODS: In present work, three course studies were performed involving in silico, in vitro and in vivo cascade to screen 8-HQ small molecule inhibitors against BoNT/F intoxication. ~800 molecules obtained from open repositories were screened in silico and commercially obtained twenty-four 8-HQ derived small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay. Selected compounds were further evaluated through endopeptidase assay. Further binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in vivo efficacy of these compounds was evaluated in mice model. RESULTS: Three compounds NSC1011, NSC1014 and NSC84094 were found to be highly inhibitory after screening of 8-HQ compounds through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed highest affinity binding of NSC1014 (KD: 5.58E-06) with BoNT/F-LC. NSC1011, NSC1014, and NSC84094 displayed IC50 of 30.47 ±â€¯6.24, 14.91 ±â€¯2.49 and 17.39 ±â€¯2.74 µM, respectively, in endopeptidase assay. NSC1011 and NSC1014 displayed marked extension of survival time in mice model. CONCLUSION: NSC1011 and NSC1014 have emerged as promising drug candidate against BoNT/F intoxication displaying higher potential than previously reported compounds.