Fig. 6: Structure-activity relationship of HSD17B13.

a Representative enzymatic activity titration curves of human (brown triangles), dog WT (blue squares), dog quadruple mutant (G177E/V178G/T205A/I293V, red diamonds), and dog double mutant (G177E/V178G, green triangles) HSD17B13, using 12 μM β-estradiol as substrate and 500 μM NAD⁺ as cofactor. The signals in relative luminescent units (RLU) were plotted against titrated enzyme concentrations, and non-linear regression fittings were also shown. The means of replicated measurements ± associated standard errors (SEM) were plotted (n = 2 independent experiments). Regression fitting and statistics in this figure were calculated using the program GraphPad⁶⁷. b To test cofactor selectivity, NAD⁺, and NADP were used as cofactors in the human HSD17B13 enzyme assay, using β-estradiol and LTB4 as substrates. The protein concentrations used were 20 nM. For inhibited reactions, 30 μM 1 was added to the reaction mixtures. The statistics were calculated between uninhibited (blue circles) and inhibited reactions (red circles) of the same cofactor and substrate (n = 2 independent experiments). Data were presented as mean values ± SEM. The unpaired, two-sided t test p values are 0.003 and 0.001, between uninhibited and inhibited reactions, for β-estradiol and LTB4 as substrates respectively, with NAD⁺ as cofactor. The p values are 0.29 and 0.05, respectively with NADP as cofactor. c Thermal shift assay demonstrating NAD⁺ -dependent binding of compound 1 to human HSD17B13. The Tagg values in this figure were temperatures at which protein started to aggregate, recorded using static light scattering at 266 nm wavelength. The ΔTagg and statistics were calculated between 500 μM NAD⁺ or compound 1 or compound 1 + 500 μM NAD⁺ added samples and samples that contained only buffer plus DMSO (n = 8, 4, 4, and 6 independent experiments). Data were presented as mean values ± SEM. The p values are 0.058, 0.045, and <0.00001, respectively. d Compound 1 and 2 binding to dog WT (blue squares), dog mutant (red diamonds), human HSD17B13 (brown circles), and human HSD17B11 (purple triangles) in the presence of 500 μM NAD⁺. Data were presented as mean values ± SEM (n = 10, 10, 10, 12, 12, 12, 12, 10, 10, 8, 8 and 8 independent measurements, for buffer, 1 and 2, respectively, with dog WT, dog mutant, human HSD17B13 and human HSD17B11, respectively). ΔTagg and statistics were calculated between protein samples in the compound buffer and the same protein in the control buffer containing only DMSO. The p values are 0.16 and 0.21; <0.0001 and 0.084; <0.001 and <0.001; and 0.7 and 0.23, for dog WT, dog mutant, human HSD17B13, and human HSD17B11, respectively. Unpaired two-sided t test was used in statistics analysis. Statistics symbols: ****p < 0.0001; ***p < 0.0005; **p < 0.005; *p < 0.05; ns, p > 0.05.