Retargeted clostridial endopeptidases: inhibition of nociceptive neurotransmitter release in vitro, and antinociceptive activity in in vivo models of pain.
Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Previously reported data have demonstrated that the catalytically active LH(N) endopeptidase fragment of botulinum neurotoxin type A (termed LH(N)/A) can be retargeted to a range of cell types in vitro to lead to inhibition of secretion of a range of transmitters. Here, we report the synthesis of endopeptidase conjugates with in vitro selectivity for nociceptive afferents compared to spinal neurons. Chemical conjugates prepared between Erythrina cristagalli lectin and LH(N)/A are assessed in vitro and in in vivo models of pain. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH(N)/A, or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material is demonstrated. The duration of action of inhibition of neurotransmitter release by the conjugate in vitro is also assessed and is comparable to that observed with Clostridium botulinum neurotoxin. Selectivity of targeting and therapeutic potential have been confirmed by in vivo electrophysiology studies. Furthermore, the analgesic properties of the conjugate have been assessed in in vivo models of pain and extended duration effects observed. These data provide proof of principle for the concept of retargeted clostridial endopeptidases as novel analgesics.
Chaddock
John A
JA
Health Protection Agency, Porton Down, Salisbury, Wiltshire, United Kingdom. john.chaddock@hpa.org.uk
Purkiss
John R
JR
Alexander
Frances C G
FC
Doward
Sarah
S
Fooks
Sarah J
SJ
Friis
Lorna M
LM
Hall
Yper H J
YH
Kirby
Elizabeth R
ER
Leeds
Nicola
N
Moulsdale
Hilary J
HJ
Dickenson
Anthony
A
Green
G Mark
GM
Rahman
Wahida
W
Suzuki
Rie
R
Duggan
Michael J
MJ
Quinn
Conrad P
CP
Shone
Clifford C
CC
Foster
Keith A
KA
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000627:therapeutic use*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D010450:Endopeptidases / Q000737:chemistry / Q000502:physiology*; D005727:Ganglia, Spinal / Q000166:cytology; D005998:Glycine / Q000378:metabolism; D000922:Immunotoxins; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D036421:Nerve Fibers, Unmyelinated / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009465:Neuromuscular Agents / Q000737:chemistry / Q000627:therapeutic use*; D009474:Neurons / Q000187:drug effects*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010146:Pain / Q000188:drug therapy*; D010147:Pain Measurement / Q000187:drug effects; D011930:Reaction Time / Q000187:drug effects; D013116:Spinal Cord / Q000166:cytology; D013373:Substance P / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects; D050825:Synaptosomal-Associated Protein 25; D013997:Time Factors
D000922:Immunotoxins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009465:Neuromuscular Agents; D018377:Neurotransmitter Agents; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D013373:Substance P; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A; D005998:Glycine