Table 1 Binding affinity of GC373 and NMV to MPro^WT, MPro^1–199, MPro^1–196 and MPro^10–306 as determined by ITC.

Compound	Construct	N	K_d = K_i (µM)	ΔH (kcal/mol)	ΔS (cal/mol/K)	ΔG (kcal/mol)
GC373	MPro^WT	0.99 ± 0.01	0.15 ± 0.03	−6.7 ± 0.1	9.1	−9.4
	MPro^M	1.07 ± 0.02	6.13 ± 0.30	−6.0 ± 0.2	3.9	−7.2
	MPro^1–199	0.9 ± 0.03	32 ± 5	−2.4 ± 0.1	12.7	−6.2
	MPro^1–196	0.88 ± 0.09	45 ± 20	−1.48 ± 0.24	15	−6
	MPro^10–306	0.54 ± 0.03	44 ± 8	−1.38 ± 0.1	15.3	−6
NMV	MPro^WT	0.99 ± 0.003	0.007 ± 0.003	–10.75 ± 0.7	1.57	–11.2
	MPro^C145A	0.96 ± 0.04	2.7 ± 0.9	–3.89 ± 0.2	12.6	−7.7
	MPro^1–199	0.97 ± 0.04	19 ± 3	−3.9 ± 0.1	8.8	−6.5
	MPro^1–196	1.05 ± 0.03	14 ± 3	−1.8 ± 0.1	16.3	−6.7

ITC experiments were carried out in buffer C at 28 °C. Data were processed and plots were generated with the Origin software provided with the instrument. Titrations of MPro^1–199 with NMV shown in Figs. 3c and S6a were fit to a 2 sites model. The mean values obtained for the major isotherm are listed. Thermodynamic parameters derived for MPro^WT and MPro^M titrated with GC373, and MPro^WT titrated with NMV are cited from refs. ^{12, 13}, respectively, solely for comparison with MPro analogs. No thermal response was observed when NMV was titrated with MPro^1–199 (Fig. S6b) and MPro^10–306 (Fig. S5d), and GC373 with MPro^C145A as listed in Table S1.

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