A refined pharmacophore identifies potent 4-amino-7-chloroquinoline-based inhibitors of the botulinum neurotoxin serotype A metalloprotease.
TL;DR: Four new, potent inhibitors of a structurally diverse small molecule (non-peptidic) inhibitors of the botulinum neurotoxin serotype A (BoNT/A) light chain (LC) are identified, supporting the structure-based pharmacophore contribution to inhibition.
Abstract: We previously identified structurally diverse small molecule (non-peptidic) inhibitors (SMNPIs) of the botulinum neurotoxin serotype A (BoNT/A) light chain (LC). Of these, several (including antimalarial drugs) contained a 4-amino-7-chloroquinoline (ACQ) substructure and a separate positive ionizable amine component. The same antimalarials have also been found to interfere with BoNT/A translocation into neurons, via pH elevation of the toxin-mediated endosome. Thus, this structural class of small molecules may serve as dual-function BoNT/A inhibitors. In this study, we used a refined pharmacophore for BoNT/A LC inhibition to identify four new, potent inhibitors of this structural class (IC50's ranged from 3.2 to 17 μM). Molecular docking indicated that the binding modes for the new SMNPIs are consistent with those of other inhibitors that we have identified, further supporting our structure-based pharmacophore. Finally, structural motifs of the new SMNPIs, as well as two structure-based derivatives, were ...
Summary (2 min read)
- 1,2 As a result, these enzymes, which are responsible for the paralysis associated with botulism, are listed as category A (highest priority) biothreat agents by the Centers for Disease Control and Prevention (CDC).
- 2-6 Furthermore, as BoNTs are now used to treat a range of medical conditions, and in many cosmetic applications,3,7-14 they are being produced in increasing quantities, making their misuse, accidental overdosing, and/or instances of adverse side effects15 more likely.
- The authors build on their previous work by describing how the refined pharmacophore33 was used to discover new small molecule inhibitors possessing the ACQ substructure and a separate ionizable, aliphatic amine component.
Results and Discussion
- Based on the identification ofN,N-bis(7-chloroquinolin-4yl)diamines and five antimalarial drugs (amodiaquine, chloroquine, quinacrine, quinidine, and quinine) as SMNPIs of the BoNT/A LC,31 the authors have continued to pursue the identification of new inhibitors of this structural class possessing the weakly basic ACQ substructure and an ionizable amine; both of these components have been found to be key to activity.
- Å from the centroid of the ACQ ), and (3) at least one of the new components from their expanded pharmacophore, either F33 (a positive ionizable moiety located 11.7-16.7 Å from the quinoline centroid) or G33 (a hydrophobic moiety located 8.5-12.5 Å from the quinoline centroid) .
- Compounds1-3 were initially examined for percent inhibition of the BoNT/A LC at 50µM concentrations, while4 was tested at a 20µM concentration.
- Molecular Docking of SMNPIs 1-4 Demonstrates a Consistency with Previous Inhibitor Binding Modes, Reinforcing The authors Structure-Based Pharmacophore Approach.
- Substituents of1-3 and one of the ACQs of4 engage in favorable hydrophobic contacts with residues Phe 162, Phe 163, and Thr 219 of hydrophobic binding subsite 131-33 (also described as the S1′ binding site34).
Component E Dominates Potency for This Structural Class.
- The final analyses of this study involved determining the importance of the structural components of1-4 to BoNT/A LC inhibition.
- To determine if incorporating pharmacophore component E might transform8 into an inhibitor, the 3R-acetyloxy group of this molecule was replaced with an ionizable 3R-NH2 substituent , effectively creating a substructure of SMNPI3 (Scheme 1), the most potent molecule of the ACQ-cholate acetate congener series (Table 1).
- Finally, to determine if it would be possible to restore the inhibitory potency of10, the 3R-O-cholate-acetate and ACQ components were tethered with an extended linker incorporating a central, secondary amine.
- Specifically, the extended linker forces the cholate-acetate portion of11 to adopt a shallower binding mode, with its C23 methoxycarbonyl oriented outside of the substrate binding cleft .
- Amino ether11 (43% from 14) was obtained in a reductive amination reaction between 6 and aldehyde15 (Scheme 1).
- An expanded/refined pharmacophore for BoNT/A LC inhibition33 was used to identify four new, potent SMNPIs of the ACQ structural class.
- This provides additional support that molecular scaffolds, which are larger than many reported BoNT/A LC inhibitors,31,40-42 are needed to provide superior inhibition.
- Finally, examination of substructures composing1-4 (i.e., compounds5-9), as well as derivatives 10 and 11, revealed the critical importance of positive-ionizable pharmacophore component E to the potencies of inhibitors in the structural class.
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...For classic inhibition, data values using a candidate inhibitor of botulinum neurotoxin type A (17) are used as inputs: E, S, Km and IC50 (in micromolar units) 0....
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