Fig 2: ASS1 is indeed the target of SPA and LM-2I.a Crystal violet assay for the viability of MDA-MB-231 cells treated with LM-2I or LM-2I pre-incubated with different concentrations of ASS1 recombinant protein (mean ± s.d., n = 6 biologically independent experiments). ***P < 0.001, NS: not significant (P > 0.05) from two-tailed unpaired Student’s t-tests. b Western blot for ASS1 expression in control or stable ASS1 knockout (ASS1-KO) MDA-MB-231 cells (top). Crystal violet assay for the viability of control and ASS1-KO MDA-MB-231 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). c Western blot for ASS1 expression in control or stable ASS1 knockdown (ASS1-KD) MCF-7 cells (top). Crystal violet assay for the viability of control and ASS1-KD MCF-7 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). d Western blot for ASS1 expression in control or stable ASS1 overexpression (ASS1-OE) MDA-MB-231 cells (top). Crystal violet assay for the viability of control and ASS1-OE MDA-MB-231 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). e The correlation between the relative ASS1 expression in cells and the IC50 of these cells treated with LM-2I (n = 3 independent experiments from n = 14 cell lines). P values of mean ± s.d. were determined by Pearson Coefficient. f LC-MS analysis of ASP, UMP, CMP, and TMP in MDA-MB-231 cells treated with SPA, LM-2I or vehicles for 24 h (mean ± s.d., n = 6 biologically independent experiments). **P < 0.01, ***P < 0.001 from two-tailed unpaired Student’s t-tests. g Crystal violet assay for the viability of MDA-MB-231 cells preincubated with pyrimidines or purines, followed by treatment with SPA or LM-2I (mean ± s.d., n = 6 biologically independent experiments). ** P < 0.01, ***P < 0.001, NS: not significant (P > 0.05) from two-tailed unpaired Student’s t-tests.Source data are provided as a Source Data file.

Fig 3: SPA and LM-2I target ASS1 at C97 site.a The recombinant ASS1 protein was incubated with Biotin-SPA in the absence or presence of SPA, SPAH and LM-2I for 1 h at room temperature, followed by western blot using streptavidin-HRP or ASS1 antibody (n = 3 biologically independent experiments). b Recombinant WT and mutant ASS1 proteins were incubated with Biotin-SPA or Biotin-SPAH for 30 min, followed by western blot using streptavidin-HRP or ASS1 antibody (n = 3 biologically independent experiments). MS/MS analysis for the C97-containing peptide of recombinant ASS1 pre-incubated with vehicle (c), SPA (d), or LM-2I (e). (SPA), combined with SPA; (LM-2I), combined with LM-2I; (c), carbamidomethylation; (d), deamidation. f Binding model of LM-2I-ASS1. The left panel is the global view of the entire ASS1 structure, the right panel is the focused view of LM-2I in the binding site (ASS1 in gray while LM-2I in green). Source data are provided as a Source Data file.

Fig 4: SPA and LM-2I enhance ASS1 catalytic activity and its kinetics.The recombinant ASS1 protein was incubated with different concentrations of SPA (a) or LM-2I (b) overnight, and the ASS1 catalytic activity was measured. The initial increase in absorbance at 660 nm (A660) between 1 and 8 min is expanded in the middle panel. In the right panel, the Hill 1 model of Origin 2018 software was used to analyze the increase in enzymatic reaction speed at different concentrations. The maximal activation ratio and AC50 were calculated. (mean ± s.d.). c The determination of kobs for the interaction of ASS1 (0.5 µM) with Biotin-SPA (20 µM) was calculated at different time points (n = 3 biologically independent experiments). d Plotting the kobs values for binding ASS1 (0.5 µM) with different concentrations of Biotin-SPA. BIAcore diagram (left panel) and KD (right panel) of the recombinant ASS1 protein binding to SPA (e) or LM-2I (f).Source data are provided as a Source Data file.

Fig 5: SPA and LM-2I directly target ASS1.a Chemical structures of SPA (1) and its reductive form SPAH (3). b Crystal violet assay for the viability of MCF-7 cells treated with different concentrations of SPA and SPAH (mean ± s.d., n = 3 biologically independent experiments). c Chemical structures of biotin-labeled SPA [Biotin-SPA (4)] and biotin-labeled SPAH [Biotin-SPAH (5)]. d Crystal violet assay for the viability of MCF-7 cells treated with different concentrations of biotin-labeled SPA [Biotin-SPA (4)] and biotin-labeled SPAH [Biotin-SPAH (5)] (mean ± s.d., n = 3 biologically independent experiments). MCF-7 cell lysates were incubated with biotin or Biotin-SPA in the presence or absence of LM-2I at 4 °C overnight, followed by pulling-down with streptavidin magnetic beads. The proteins bound to the magnetic beads were separated by SDS-PAGE, followed by silver staining (e) or by western blot using ASS1 antibody (f) (n = 3 biologically independent experiments). g Fluorescent staining of MCF-7 cells incubated with or without Biotin-SPA for 4 h (n = 3 biologically independent experiments). Scale bars are 20 µm. h The recombinant ASS1 protein was incubated with Biotin-SPA for different time periods as indicated, followed by western blot using streptavidin-HRP or ASS1 antibody (n = 3 biologically independent experiments). i The recombinant ASS1 protein was incubated with Biotin-SPA in the absence or presence of LM-2I for 1 h at room temperature, followed by western blot using streptavidin-HRP or ASS1 antibody (n = 3 biologically independent experiments). Source data are provided as a Source Data file.