title: Retargeted clostridial endopeptidases: inhibition of nociceptive neurotransmitter release in vitro, and antinociceptive activity in in vivo models of pain.
abstract: 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.
authors.affiliation: Health Protection Agency, Porton Down, Salisbury, Wiltshire, United Kingdom. john.chaddock@hpa.org.uk
authors.forename: John A
authors.initials: JA
authors.lastname: Chaddock
authors.forename: John R
authors.initials: JR
authors.lastname: Purkiss
authors.forename: Frances C G
authors.initials: FC
authors.lastname: Alexander
authors.forename: Sarah
authors.initials: S
authors.lastname: Doward
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Fooks
authors.forename: Lorna M
authors.initials: LM
authors.lastname: Friis
authors.forename: Yper H J
authors.initials: YH
authors.lastname: Hall
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Kirby
authors.forename: Nicola
authors.initials: N
authors.lastname: Leeds
authors.forename: Hilary J
authors.initials: HJ
authors.lastname: Moulsdale
authors.forename: Anthony
authors.initials: A
authors.lastname: Dickenson
authors.forename: G Mark
authors.initials: GM
authors.lastname: Green
authors.forename: Wahida
authors.initials: W
authors.lastname: Rahman
authors.forename: Rie
authors.initials: R
authors.lastname: Suzuki
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Duggan
authors.forename: Conrad P
authors.initials: CP
authors.lastname: Quinn
authors.forename: Clifford C
authors.initials: CC
authors.lastname: Shone
authors.forename: Keith A
authors.initials: KA
authors.lastname: Foster
mesh_terms: 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
chemical_list: 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