Characterization and Optimization of the Novel Transient Receptor Potential Melastatin 2 Antagonist tatM2NX

Abstract

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable channel activated by adenosine diphosphate ribose metabolites and oxidative stress. TRPM2 contributes to neuronal injury in the brain caused by stroke and cardiac arrest among other diseases including pain, inflammation, and cancer. However, the lack of specific inhibitors hinders the study of TRPM2 in brain pathophysiology. Here, we present the design of a novel TRPM2 antagonist, tatM2NX, which prevents ligand binding and TRPM2 activation. We used mutagenesis of tatM2NX to determine the structure-activity relationship and antagonistic mechanism on TRPM2 using whole-cell patch clamp and Calcium imaging in human embryonic kidney 293 cells with stable human TRPM2 expression. We show that tatM2NX inhibits over 90% of TRPM2 channel currents at concentrations as low as 2 μM. Moreover, tatM2NX is a potent antagonist with an IC₅₀ of 396 nM. Our results from tatM2NX mutagenesis indicate that specific residues within the tatM2NX C terminus are required to confer antagonism on TRPM2. Therefore, the peptide tatM2NX represents a new tool for the study of TRPM2 function in cell biology and enhances our understanding of TRPM2 in disease. SIGNIFICANCE STATEMENT: TatM2NX is a potent TRPM2 channel antagonist with the potential for clinical benefit in neurological diseases. This study characterizes interactions of tatM2NX with TRPM2 and the mechanism of action using structure-activity analysis.

Fig. 1.

Molecular modeling of tatM2NX with the human TRPM2 channel NUDT9-H domain. (A) Predicted secondary structure of tatM2NX after MD-based refinement. The peptide is oriented Nterm (left) to Cterm (right). (B) Top three predicted TPRM2–tatM2NX complexes; the arrowhead indicates the human ADPR binding site within the NUDT9-H domain. (C) Top scoring initial TPRM2–tatM2NX complex (left) and the same complex after 5 nanoseconds of MD simulation (right).

Fig. 2.

The peptide tatM2NX inhibits TRPM2 currents in a concentration-dependent manner. (A) Representative TRPM2 initial currents (ADPR_i, 3 to 4 nA) activated by 100 µM ADPR (red), with 2 µM tatM2NX (black) or 20 µM CTZ (gray). (B) TRPM2 current density at 0.15–10 µM tatM2NX compared with TRPM2 current at 0.05 µM tatM2NX (ineffective concentration control). (C) Dose-response curve showing normalized response (current density) vs. tatM2NX concentration. Potency was determined normalizing each concentration to TRPM2 current density with 0.05 µM tatM2NX. All data are represented as means ± S.D., and significance (*) was established at P < 0.05 for n ≥ 4–7 (at least three experimental days per condition) using one-way ANOVA.

Fig. 3.

TatM2NX inhibits TRPM2-mediated GSK3β signaling and competes with ADPR to antagonize TRPM2. (A) Coimmunoprecipitation of biotin-tagged tatM2NX with FLAG-tagged TRPM2 in Dox-inducible HEK293 cells (+Dox). No biotin–tatM2NX was observed in uninduced cells (−Dox). At least three independent experimental days (n = 3). (B) Western blot of HEK293 cells expressing TRPM2 treated with 250 µM H₂O₂ compared with untreated cells (Con) and cells preincubated with 2 µM tatM2NX for 30 minutes to 4 hours followed by H₂O₂ stimulation (10 minutes). (C) ADPR_f (control) currents with 500 µM ADPR in the presence of 0 or 2 µM tatM2NX. No significant differences (ns) in ADPR_i for control and 2 µM tatM2NX. All data are represented as means ± S.D., and significance was established at P < 0.05 for n ≥ 4 (at least three experimental days per condition) using Student’s t test. Con, untreated control cells; Dox, doxycycline.

Fig. 4.

The C terminus of tatM2NX is sufficient to antagonize TRPM2. (A) TRPM2 current density in the presence of 2 µM tat Cterm (blue), tatWV-AA (sky blue), or tat Nterm (green). All peptides were individually compared with control group (ADPR_f). (B) Representative images for Ca²⁺ imaging experiments at time (T) 0 and 20 minutes for 250 µM H₂O₂ (control) or H₂O₂ + 2 µM tatM2NX. Fluo5F, acetoxymethyl (5 µM, green) is the fluorescent Ca²⁺ indicator. (C) Quantification of changes in fluorescence/baseline fluorescence (F/F₀) for each peptide (2 µM). TatM2NX and tat Cterm significantly inhibit TRPM2 activity in HEK293 cells. F/F₀, corresponding to TRPM2 activity, is inhibited by 20 µM CTZ (positive control). (D) Area under the curve (AUC) analysis for each peptide: H₂O₂ control (black), tatM2NX (red), tat Cterm (blue), tatWV-AA (sky blue), tat Nterm (green). tatM2NX, tat Cterm, and CTZ significantly decreased AUC for TRPM2 activity, whereas tatWV-AA significantly increased AUC. All data are represented as means ± S.D., and significance (*) was established as P < 0.05 using one-way ANOVA with Dunnett’s post hoc for multiple group comparison, n ≥ 4–10 for electrophysiology, and n ≥ 25–53 cells for Ca²⁺ imaging (four to six independent experiments).