Crotalphine: fluorescent analogues and study of mechanism of action in cultures and co-cultures of neurons and keratinocytes

• Grant number: 11/09333-7
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: July 01, 2011
• End date: December 31, 2015
• Field of knowledge: Biological Sciences - Pharmacology - General Pharmacology
• Principal Investigator: Yara Cury
• Grantee: Cesar Manuel Remuzgo Ruiz
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling, AP.CEPID
• Associated scholarship(s): 14/24245-5 - Effects of crotalphine on aggregating rat brain cell cultures, BE.EP.PD

Abstract

Crotalphine, a 14-mer peptide, isolated from the venom of C. d. terrificus snake, present long lasting antinociceptive activity (3-5 days) when administered by p.o., i.v. or s.c. routes. The antinociceptive effect is mediated by activation of peripheral kappa opioid receptor (acute pain models), or kappa and delta opioid receptors (chronic pain). Preliminary results obtained in binding assays using deuterated naloxone indicate that crotalphine is not able to directly activate opioid receptors. Additionally, recent data obtained in our laboratory have shown the involvement of cannabinoid receptors in this antinociceptive action. Despite the results, the molecular targets of this peptide are not yet known. Thus, the aim of this project is to extend the characterization of the molecular mechanisms involved in the action of crotalphine evaluating: (a) the possible interaction with opioid and cannabinoid receptors. Therefore, studies of co-localization between crotalphine and these receptors will be performed through immunofluorescence assays using carboxyfluorescein labeled crotalphine and specific antibodies to opioid and cannabinoid receptors present in neurons and keratinocyte; (b) using the same experimental approach, we will investigate its internalization and trafficking in neurons and keratinocytes cultures and the mechanisms involved in these processes. Hence, we will synthesize crotalphine, D-amino acid crotalphine analogue and fluorescent labeled analogue; (c) the possible release of endogenous opioid peptides induced by crotalphine. Therefore, the release of these peptides will be quantified in the supernatant of culture and co-cultures of neurons and keratynocytes treated with crotalphine using liquid chromatography and mass spectrometry. (AU)