Development of a platform for crystallization of PLC³ SH2 domains bound to peptides to map target regions for selective inhibitors

• Grant number: 23/03290-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: July 24, 2023
• End date: May 23, 2024
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Deborah Schechtman
• Grantee: Allan Pradelli Roldão
• Supervisor: Marko Hyvonen
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of Cambridge, England
• Associated to the scholarship: 22/06958-0 - Structure-function Analysis of TrkA - PLC³ and the development of inhibitors based on such interactions that play an analgesic role, BP.DR

Abstract

The ongoing opioid crisis in developed countries has urged the importance of searching for new analgesics. Many pathways have been explored on the therapeutics of this area, but opioids are still the most potent class of drugs for this. Nevertheless, they have limited effects and may lead to addiction, tolerance, and even overdose. Our group, aiming to understand the genetics of a disease in which patients feel no pain (Congenital Insensitivity to Pain with Anhidrosis), unveiled the TrkA/ PLC³ pathway as a new target for pain management. In this disease, patients display several mutations along the TrkA coding gene, and the ones responsible for PLC³ anchoring to its partner proteins led us to rationally design a peptide that could modulate a protein-protein interaction (PPI) of this system. A 11- mer mimetic peptide of the C-terminus tail of the TrkA kinase domain, when injected into a mouse with previously inflamed tissue, inhibited TrkA binding to PLC³ and led to analgesia. This peptide, when tested in cells, modulated the activation of PLC³ but not of other TrkA scaffold proteins that normally engage with PLC³ once the receptor is activated by Nerve Growth Factor (NGF). The TrkA/ PLC³ pathway further leads to the activation of the membrane channel Transient Receptor Potential Vanilloid I (TRPV1), which triggers action potentials on peripheral neurons. PLC³ contains two tandem Src Homology 2 (SH2) domains, known to bind phospho-tyrosines, which mediate binding to TrkA. The aim of our study is to structurally characterize this interaction and rationally design inhibitors that are specific for this SH2 domain interactions and can be developed into non-opioid analgesic drug leads. In the present study we intend to crystallize N- and C-terminal SH2 domains of PLC³ to understand the specificity of the interactions of these domains with different kinases, and the mechanism of activation of PLC³. These studies will be important for future studies that aim at screening for fragments that bind the different SH2 domains of PLC³ using biophysical methods and by crystallography. (AU)