Improved PIEZO1 agonism through 4‐benzoic acid modification of Yoda1

Background and Purpose The protein PIEZO1 forms mechanically activated, calcium‐permeable, non‐selective cation channels in numerous cell types from several species. Options for pharmacological modulation are limited and so we modified a small‐molecule agonist at PIEZO1 channels (Yoda1) to increase the ability to modulate these channels. Experimental Approach Medicinal chemistry generated Yoda1 analogues that were tested in intracellular calcium and patch‐clamp assays on cultured cells exogenously expressing human or mouse PIEZO1 or mouse PIEZO2. Physicochemical assays and wire myography assays on veins from mice with genetic disruption of PIEZO1. Key Results A Yoda1 analogue (KC159) containing 4‐benzoic acid instead of the pyrazine of Yoda1 and its potassium salt (KC289) have equivalent or improved reliability, efficacy and potency, compared with Yoda1 in functional assays. Tested against overexpressed mouse PIEZO1 in calcium assays, the order of potency (as EC50 values, nM) was KC289, 150 > KC159 280 > Yoda1, 600). These compounds were selective for PIEZO1 over other membrane proteins, and the physicochemical properties were more suited to physiological conditions than those of Yoda1. The vasorelaxant effects were consistent with PIEZO1 agonism. In contrast, substitution with 2‐benzoic acid failed to generate a modulator. Conclusion and Implications 4‐Benzoic acid modification of Yoda1 improves PIEZO1 agonist activity at PIEZO1 channels. We suggest naming this new modulator Yoda2. It should be a useful tool compound in physiological assays and facilitate efforts to identify a binding site. Such compounds may have therapeutic potential, for example, in diseases linked genetically to PIEZO1 such as lymphatic dysplasia.


Materials
All purchased chemicals and solvents were used without further purification unless otherwise stated.All compounds were at least 95% pure by 1 H NMR.

1
H Nuclear Magnetic Resonance spectra were recorded at 500 MHz using a Bruker DRX 500 instrument or at 400 MHz using a Bruker DPX 400. 1 H spectra are referenced based on the residual proton in the solvent (e.g. the CHCl3, 0.01 % in 99.99 % CDCl3).Coupling constants (J) are reported to the nearest 0.1 Hz. 13 C NMR spectra were recorded at 125 MHz on 500 MHz spectrometers or at 100 MHz on 400 MHz spectrometers.HRMS was performed on a Burker Daltonics micrOTOF using positive electrospray ionisation (ES+).Automated column chromatography (ACC) was carried out using a Biotage Isolera Four EXP with Spektra.

Figure S1 .
Figure S1.SI 2. In-house solubility comparisons for Yoda1, KC159 and KC289.Estimated solubility of Yoda1, KC159, and KC289 in universal aqueous buffer.Median values with data range from 5 independent experiments are shown (n=5).The lower limit of detection for the assay was 0.1 µM.Data for Yoda1 were below this limit, suggesting that Yoda1 was poorly soluble in these conditions.Results of a two-tailed Mann-Whitney rank comparison of the median values of the collated experiments are shown as: * p < 0.05.

Figure S4 .
Figure S4.SI 1. Supporting data for Figure 4 Background-subtracted (Δ) peak intracellular Ca 2+ measurements from individual experiments in which mPIEZO1-TREx cells were exposed to the indicated concentrations of; (i) Yoda1, (ii) KC159, (iii) KC289.Mean values from 2 or 3 technical replicates are shown for each of the individual experiments (n=12 for all compound tests).

Figure S8 .
Figure S8.SI 1. Effects of L-NAME on vessel response to PE and resting basal tension Paired comparisons of (i) tension responses to PE and; (ii) isometric basal tension measurements before and after a 30-minute pre-incubation of vessels with 100 µM Nω-nitro-L-arginine methyl ester (L-NAME).Each plot shows values from independent experiments (n=5).

Figure S10 .
Figure S10.SI 1. Validation of transfection efficiency in HEK 293 and HeLa cells(A) mRNA abundance of (i) mPIEZO1 and (ii) mPIEZO2 (n=5) transcripts relative to hGAPDH mRNA in HEK 293 cells overexpressing mPIEZO1, mPIEZO2 or neither ("non-transfected cells").(B) mRNA abundance of (i) hPIEZO1 or (ii) hPIEZO2 transcripts (n=5) relative to hGAPDH expression in HeLa cells after siRNA mediated knockdown of hPIEZO1 (siPIEZO1), hPIEZO2 (siPIEZO2), both (siPIEZO1 + siPIEZO2) or transfected with a non-targeting (Control, Ctrl) siRNA (siCtrl).Data expressed as Mean ± SD except in Aii, which is Median with Range because the data could not be confirmed as normally distributed.One way ANOVA followed by a Tukey's post-hoc test for multiple comparison was performed on the mean values of the collated experiments (Ai, Bi and Bii) and Friedmann test followed by a Dunn's post-hoc test for multiple comparison for (Aii): n.s (p > 0.05), * p < 0.05 for comparison against non-transfected and siCtrl only.

Figure S10. SI 2 .
Figure S10.SI 2. Detection of mechanically activated ionic current in HEK 293 cells overexpressing mouse PIEZO2 (A) Example (n=1) outside-out patch current recordings showing the response to (i) 100-ms 75 mmHg positive pressure pulse for (ii) non-transfected HEK 293 cells and (iii) HEK 293 cells overexpressing mPIEZO2.Traces in (ii) and (iii) are average currents from 3 consecutive pressure pulses.(B) Mean ± SD for peak inward currents activated by the pressure pulse as exemplified in (A): non-transfected cells (N=17 independent recordings) and HEK 293 cells overexpressing mPIEZO2 (N=11 independent recordings).