Extended Data Fig. 1. Characterization of peptidomimetic inhibitors.

(A) Backbone structure of peptidomimetic compounds used in this study along with the groups used in the N-terminal R1 position, R2, R3 and the C-terminal warhead (ketobenzothiazole; kbt) and alcohol warhead (Kbt(OH)). The P1 position is Arginine in all compounds. (B) Cytotoxicity of compounds in Vero E6 cells. Cellular viability was evaluated after 24 h exposure to 10 μM of the indicated compounds (n = 4 independent experiments performed in duplicate). Results are background corrected and presented as the mean viability (%) ± standard deviation (SD) compared to one replicate of vehicle treated cells (DMSO 0.01%) in each experiment. Triton X-100 0.01% was used as a toxicity control. (C) N-0385 inhibition of mouse TMPRSS2 (mTMPRSS2). Vero E6 cells were transfected with either an empty vector or mTMPRSS2 for 24 h. N-0385 was added concomitantly with a fluorogenic substrate on cells for an additional 24 h before fluorescence reading. Relative activity was measured using the mock-subtracted fluorescence and reported as the percentage of residual activity. Dose–response curves were generated and IC₅₀ values were determined using nonlinear regression analysis. One representative IC₅₀ curve is shown. The IC₅₀ value shown represents the mean ± SD from n = 3 independent experiments. (D, E) Real-time PCR analysis for relative expression of (D) ACE2 and (E) TMPRSS2 expression in Calu-3 cells and colonoids. Relative expression levels normalized using 3 housekeeping genes (YWHAZ, PUM1, MRPL19) are shown for one sample (RNA extract) performed in technical triplicates

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