Table 1.

Estimated T_m, K_d and thermodynamic parameters of binding of various inhibitors to MPro^WT and its catalytic dyad mutants

Constructs	Tm (°C)	ΔTm (°C)	Kd (μM)	ΔH (kcal/mol)	ΔS (cal/mol/K)	ΔG (kcal/mol)
MProWT	53.9 ± 0.1
MProC145A	60.7 ± 0.1	6.8
MPro1-304/C145A	59 ± 0.1	5.1
MProH41A	53.9 ± 0.1	0
MProWT + NMV	75 ± 0.1	21.1	0.007 ± 0.003a	−10.8 ± 0.7	1.57	−11.2
MProWT + NMV (8.2)			0.012 ± 0.006	−11.8 ± 0.1	−2.86	−10.9
MProC145A + NMV	68.5 ± 0.1	7.8	2.7 ± 0.9b	−3.9 ± 0.2	12.6	−7.7
MPro1-304/C145A + NMV	66.5 ± 0.1	7.5	9.2 ± 1.7	−4.3 ± 0.2	8.9	−6.9
MProH41A + NMV	73.6 ± 0.1	19.7	0.14 ± 0.02	−8.4 ± 0.05	3.5	−9.5
MProH41A + NMV (8.2)			0.03 ± 0.02	−8.5 ± 0.17	6.07	−10.4
MProWT + NBH2	70.6 ± 0.1	16.7	0.026 ± 0.016a	−8.76 ± 0.17	5.63	−10.5
MPro1-304/C145A + NBH2	60.5 ± 0.1	1.5	nd
MProH41A + NBH2	67.1 ± 0.1	13.2	2.3 ± 0.9	−1.47 ± 0.01	20.9	7.76
MProWT + GC373c,d	73.7 ± 0.1	19.8	0.15 ± 0.03	−6.7 ± 0.1	9.1	−9.4
MProC145A + GC373	60.9 ± 0.1	0.2	nd
MPro1-304/C145A + GC373	58.9 ± 0.1	−0.1	nd
MProH41A + GC373d	70.8 ± 0.1	16.9	1.4 ± 0.5	−5.6 ± 0.2	8.3	−8.1
MProWT + ESV	72.9 ± 0.2	18.9	0.006 ± 0.003	−15.4 ± 0.01	−13.9	−11.2
MProC145A + ESV	71.8 ± 0.2	11.1	0.226 ± 0.061	−11.5 ± 0.2	−7.8	−9.2
MProH41A + ESV	71.9 ± 0.1	18	0.056 ± 0.031	−9.53 ± 0.23	1.53	−10

T_m values were extracted from DSF profiles shown in Figure 1, C and D. ΔT_m denotes the difference in T_m in the presence and absence of inhibitor. Inhibitor binding (K_d) to MPro^1-304/C145A along with corresponding MPro controls (^a (38)). Not determined (nd) because of no heat response when titrating 50 μM protein in the cell and 0.5 mM inhibitor, indicative of very weak binding, relative to the binding of NMV to MPro^C145A and MPro^1-304/C145A. DSF scans and ITC were not carried out with BBH1 because of poor solubility. ^{b, c, d} cited from references (15, 28, 29). All titrations were performed in buffer C (pH 7.2) except for two at pH 8.2, indicated in parantheses in the first column.