Journal: iScience

Article Title: Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1 via a comprehensive screening funnel

doi: 10.1016/j.isci.2024.108907

Figure Lengend Snippet: Evaluation of cellular engagement by the putative SAMHD1 inhibitors using CETSA (A and B) Establishment of cellular SAMHD1 engagement CETSA assay, validated by thymidine (A) or dGTP (B) as positive controls on intact THP-1 cells (A) or cell lysate (B), respectively. Intact THP-1 cells (A) or cell lysates (B) were incubated with 10 mM thymidine (A) or 5 mM dGTP (B), before being heated at indicated temperatures and analyzed by western blot. [Left (A) or top (B) panels] Representative western blot images where SOD-1 protein served as the loading control. [Right (A) or bottom (B) panels] Densitometry analysis, where SAMHD1 or thymidylate synthase (TS) signals were normalized to SOD-1 signals and then relative to DMSO control samples heated at the lowest temperatures. Mean relative SAMHD1 signal ±SEM of n = 2 independent experiments are shown. (C and D) Engagement of cellular SAMHD1 in intact THP-1 cells (C) or cell lysates (D), interrogated by isothermal single-dose fingerprint CETSA. Intact THP-1 cells (C) or THP-1 cell lysates (D) were incubated with 100 μM putative SAMHD1 inhibitors or positive control compounds [10 mM thymidine in (C) and 5 mM dGTP in (D)]. Following heating at screening temperatures, soluble SAMHD1 was examined via western blot. (Top panels) Representative western blot images where SOD-1 protein served as the loading control; (bottom panels) densitometry analysis, where SAMHD1 signals were normalized to SOD-1 signals and then relative to DMSO control samples heated at the lowest temperatures. Mean relative SAMHD1 signal of n = 2 (C) or 3 (D) independent experiments are shown with values of individual experiments. (E and F) TH6342, but not TH7126, mildly engaged cellular SAMHD1, shown with CETSA melting curves. Clarified lysates were prepared from THP-1 cells expressing wild-type SAMHD1 (F) or a dimerization-defective (Y146S/Y154S) mutant under knockout background (E). The lysates were then incubated with SAMHD1 inhibitors, 5 mM dGTP, or equivolume of DMSO, before being heated at indicated temperatures. Remaining soluble and folded proteins in the cell lysates were then analyzed by western blot. (Top panels) Representative western blot images where SOD-1 protein served as the loading control; arrow indicates the band excluded from densitometry analysis. (Middle panels) Densitometry analysis, where SAMHD1 signals were normalized to SOD-1 signals and then relative to DMSO control samples heated at the lowest temperatures. Mean relative SAMHD1 signal of n = 2 (E) or 4 (F) independent experiments are shown with SEM. (Bottom panels) Summary of SAMHD1 T agg values, determined by curve-fitting SAMHD1 signals via a Boltzmann sigmoidal model (GraphPad Prism). Ordinary one-way ANOVA tests (E) or paired t test (F) were performed between SAMHD1 T agg in compound- versus DMSO-treated groups ( Figure 7 E—T agg (TH6342) vs. T agg (DMSO), p = 0.0381, t = 3.550, DF = 3; T agg (TH7126) Vs. T agg (DMSO), p = 0.7809, t = 0.3042, DF = 3; Figure 7 F—T agg (TH6342) Vs. T agg (DMSO), p = 0.0197, t = 4.567, df = 3.), where asterisk signifies statistical significance (∗ for p ≤ 0.05). (G) Proposed mechanism of action of TH6342 and analogs. We propose that the chemotypes identified herein, i.e., TH6342 and analogs, directly inhibited SAMHD1 hydrolase activities by deterring the enzyme dimerization, which is a prerequisite for formation of the catalytically competent SAMHD1 homotetramer. See also Figure S13 .

Article Snippet: Mean relative SAMHD1 signal of n = 2 (E) or 4 (F) independent experiments are shown with SEM. (Bottom panels) Summary of SAMHD1 T agg values, determined by curve-fitting SAMHD1 signals via a Boltzmann sigmoidal model (GraphPad Prism).

Techniques: Incubation, Western Blot, Control, Positive Control, Expressing, Mutagenesis, Knock-Out