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Study of the therapeutic potential of new modulators of the lysosomal calcium channel hTRPML1

Grant number: 21/04853-4
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): September 01, 2021
Effective date (End): August 31, 2023
Field of knowledge:Health Sciences - Pharmacy
Principal researcher:Katlin Brauer Massirer
Grantee:Micael Rodrigues Cunha
Home Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Associated research grant:14/50897-0 - Open-acess Medicinal Chemistry Centre (OpenMedChem), AP.TEM


The human genome encodes three TRPML (Transient Receptor Potential cation channel, mucolipin subfamily) proteins. These are evolutionarily related six-transmembrane, Ca2+-permeable, and non- selective cation channels that belong to the large family of Transient Receptor Potential (TRP) ion channels. The TRPML1 is thought to be involved in endo/lysosomal homeostasis and its malfunction is linked to Lipid Storage Diseases, such as Mucolipidosis IV (MLIV). Until now, there is a lack of potent small molecules that can be used to investigate the cellular functions and the therapeutic potential of modulating the activity of the TRPML1 channel. The reason for that relies on the difficulty to obtain recombinant membrane proteins and experimental limitations which evaluate compound binding in the right cellular localization. In the human cell, the TRPMLs are embedded in the membrane of endosomes and lysosomes and thus, compounds need to traverse the plasmatic cellular membrane to reach these organelles. A major challenge in developing TRPML1 modulators is a lack of cell-based assays to investigate the in-cell binding of compounds to the TRPML1 channel in its physiological localization, appropriately embedded in endo/lysosomes. Here we propose to address the challenge of understanding the biology of TRPML1 in terms of its druggability. Initially, we will develop specific synthetic "tracer" molecules to create a novel cellular target-engagement assay for TRPML1, using Bioluminescence Resonance Energy Transfer (BRET). This technique relies on the proximity of a donor (TRPML1 fused to a luciferase) and an acceptor (a fluorescent version of a TRPML1 agonist). The BRET assay will then be used to identify cell-permeable TRPML1 modulators from the literature. This assay will also serve to optimize initial hits for TRPML1 binding while maintaining cell permeability. We will acquire and synthesize advanced candidate molecules to evaluate in BRET and follow up with biological experiments of activation/inactivation of TRPML1 using Ca2+ mobilization assays and address off-target effects suggested by polypharmacology prediction tools. The optimized TRPML1 modulators generated in this project should allow elucidating the pharmacological relevance of the TRPML1 channel in disease-related conditions such as lysosomal disorders. (AU)

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