Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors

Abstract

Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B’s therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits ~11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo

“…31,32 BoNT serotype B, the only alternative to A used currently in the clinic, displays lower efficacy in humans due to a mutation in the synaptotagmin 2 receptor which results in its poor affinity at this receptor. 33 Here, B1 was highly potent in mice both in the PNHD and in vivo models, consistent with previously reported data for this species in in vitro and ex vivo assays. 34 In rats, B1 was the least potent serotype in vivo as well as in the SCN assay when evaluating VAMP1 cleavage, in agreement with reports of VAMP1 being relatively resistant to cleavage by serotype B 35 BoNT serotypes C, E, and F have been used in humans in a small number of case studies.…”

“…BoNT serotype B, the only alternative to A used currently in the clinic, displays lower efficacy in humans due to a mutation in the synaptotagmin 2 receptor which results in its poor affinity at this receptor . Here, B1 was highly potent in mice both in the PNHD and in vivo models, consistent with previously reported data for this species in in vitro and ex vivo assays .…”

Building Blocks for Quantum Network Based on Group‐IV Split‐Vacancy Centers in Diamond

“…This is usually achieved in two ways: 1) discretizing phonon spectrum by utilizing nanodiamond with a size smaller than half of phonon wavelength, similar to the idea of suppressing spontaneous emission from atoms by employing a nanocavity; [145] 2) creating a phononic bandgap by fabricating a periodic nanostructure on diamond, [80,140,141,146] which becomes possible thanks to the recent advance in diamond nanofabrications. [147,148] Up till now, these techniques have not been applied to split-vacancy centers yet, but the second approach of strain engineering has made some progress on both SiV − [72,143] and GeV − [144] systems.…”

Anthrax Toxin as a Molecular Platform to Target Nociceptive Neurons and Modulate Pain

“…For the LFN-LC/A C699S construct, the N terminal domain of LF (residues 1 -262 of native LF) and the catalytic domain of Botulinum neurotoxin type A1 (BoNT/A1) (residues 1 -448 of native BoNT/A1) separated by a (GGS)2 linker was codon-optimized and cloned into the pK8 expression vector with a cleavable C-terminal His-tag. This was expressed in E .coli strain NiCo21 (DE3) using conditions previously described (96) and purified by IMAC using NiHP (GE Healthcare Life Sciences) and AEC using QHP (GE Healthcare Life Sciences) columns. The Histag was removed by O/N incubation with TEV protease (Millipore Sigma) at 4 °C followed by negative INAC, and the final product was desalted into PBS pH 7.2 and stored at -80 °C.…”