Extended Data Fig. 6: Target engagement of Z218484536.
From: Pharmacological inhibition of PSPH reduces serine levels and epileptic seizures
(a) PSPH protein sequence alignment between 11 species showing high evolutionary conservation of the four PSPH binding sites (arrowheads). (b) Assays of PSPH activity for wild-type (WT) and carriers of the Asp22Ala, Ala51Val, Ala51Gly, Gly110Val, Gly110Ala, Lys158Ala or Ala51Val/Gly110Ala mutations. (c) MST analysis of Z218484536 binding to the PSPH Ala51Val/Gly110Ala mutant. (d) Immunoblot analysis of cultured astrocytes after lentivirus-mediated PSPH-MT (Ala51Val/Gly110Ala) OE or PSPH KD or their combination with or without Z218484536 treatment. The sequence encoding PSPH-MT was optimized to avoid interference by shRNAs that target the endogenous PSPH gene sequence. (e, f) Levels of L-serine in cellular lysates (e) and supernatants (f). (g, h) The effects of Z218484536 in mice after bilateral hippocampal injection of AAV5-gfa104-miPSPH-eGFP to silence PSPH gene expression. One month after AVV injection, the mice were injected with Z218484536 once a day for three consecutive days (4 mg/kg), and PTZ (55 mg/kg) was used to induce acute seizures. (i, j) Statistical comparisons of time to first seizure (onset; i) and the total number of seizures per mouse (j) among the four groups. The mice were injected with vehicle or 4 mg/kg Z218484536 daily for three days. Awake mice were preinjected with 0.25 μL of vehicle or D-serine (10 μM) into the unilateral hippocampus though a preimplanted guide cannula, followed by injection of 7 ng of KA (0.25 μL). The data are shown as the means ± SDs or means with individual points. Statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparisons post hoc test (b, e, f), two-tailed Student’s t test (g, h) and two-way ANOVA followed by Tukey’s multiple comparisons post hoc test (i, j).
