Abstract

The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.

摘要

磷酸甘油酸脱氢酶的小分子抑制剂 NCT-503在体外减少葡萄糖衍生的碳与丝氨酸的结合. 在这里，我们描述了 NCT-503 在表达不同磷酸甘油酸脱氢酶 (PHGDH) 水平的神经母细胞瘤细胞系和具有 CRISPR-Cas9 定向 PHGDH 敲除或其各自野生型对照的单细胞克隆中的脱靶效应。与非活性药物对照相比，NCT-503 处理大大减少了所有研究的细胞模型中葡萄糖衍生的柠檬酸盐的合成，并且与 PHGDH 表达水平无关。NCT-503 处理增强了葡萄糖衍生碳通过丙酮酸羧化酶进入 TCA 循环的掺入。NCT-503 处理未改变柠檬酸合酶的活性。我们还在 NCT-503 处理的细胞的细胞热位移测定中检测到柠檬酸合酶的热稳定性没有变化。因此，观察到的脱靶效应的直接原因仍然是个谜。