A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate

Abstract

Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Figure 1. Identification and characterization of small molecule PHGDH inhibitors

a, Coupled PHGDH assay with diaphorase/resazurin readout used for the primary screen. b, Screening pipeline for PHGDH inhibitors. Following HTS, manual triage selected synthetically tractable compounds and eliminated promiscuous inhibitors. Remaining compounds were confirmed and counterscreened to eliminate false positives and pan-dehydrogenase inhibitors. The number of compounds remaining is listed beneath each step. c, Piperazine-1-carbothioamide PHGDH inhibitors. PHGDH-hit (1) was the initial hit in the screen; NCT-502 (2) was a derivative with improved potency, and NCT-503 (3) has improved solubility and in vivo characteristics. The structurally related inactive compound (PHGDH-inactive; 4) had no activity against PHGDH and served as a negative control. d, NCT-503 exhibits noncompetitive inhibition with respect to both 3-PG and NAD⁺. Data are average of three experiments and error bars represent standard deviations. e, Dilution data demonstrating in vitro reversibility of NCT-502 and NCT-503. Data are average of 96 experiments and error bars represent standard deviations. f, Melting temperature curves demonstrating NCT-502 and NCT-503-induced destabilization of PHGDH. Curves are representative of 3 experiments.

Figure 2. Target engagement and efficacy of PHGDH inhibitors

All data points are the average of 3 biological replicates. Error bars represent standard deviations. a, NCT-502 reduces intracellular serine concentrations in MDA-MB-231 cells expressing PHGDH in medium lacking serine and glycine. Inactive compound (PHGDH-inactive) has no effect on intracellular serine concentrations. b, NCT-502 treatment in media lacking serine and glycine decreases the concentrations of serine and glycine only in MDA-MB-231 cells expressing PHGDH, while sparing all other amino acids except for aspartate. c, NCT-503 does not affect the intracellular concentration of aspartate in MDA-MB-468 cells in complete RPMI. d, PHGDH inhibitors reduce M+3 serine produced from U-¹³C glucose while sparing the labeling of the glycolytic intermediates M+3 dihydroxyacetone phosphate (DHAP) and M+3 3-phosphoglycerate. e, C234S PHGDH is less sensitive to NCT-503 inhibition than wild type PHGDH in vitro. f, Expression of C234S PHGDH in MDA-MB-468 cells increases glucose-mediated serine flux in the presence of NCT-503. g, Intracellular synthesis of M+3-serine from U-¹³C glucose following washout of NCT-502 demonstrates PHGDH inhibitor reversibility. *, p<0.05, Student’s t-test.

Figure 3. In vitro and in vivo efficacy of PHGDH inhibitors

a, Selective toxicity of NCT-503 towards five cell lines that overexpress PHGDH relative to three cell lines with low PHGDH expression. Data points are the average of three independent biological experiments and error bars represent standard deviations. b, Compound cytotoxicity towards PHGDH-expressing MDA-MB-468 cells correlates with inhibition of M+3 serine production. Each data point represents an EC₅₀ that is the average of three independent experiments and a single IC₅₀ flux experiment comprised of 6 data points. c, NCT-503 reduces the volume of MDA-MB-468 orthotopic xenografts while sparing the growth of MDA-MB-231 xenografts. Data points are the mean of ten animals, and error bars represent standard error of the mean. *, p<0.05, Student’s t-test. d, NCT-503 reduces the weight of MDA-MB-468 xenografts but not the weight of MDA-MB-231 xenografts. Each data point is a single animal, n=10 in each arm. Horizontal bar indicates the mean of ten animals and error bars represent standard error of the mean. *, p<0.05, Student’s t-test. e, NCT-503 increases the fraction of necrosis in MDA-MB-468 orthotopic xenografts but not in MDA-MB-231 orthotopic xenografts. Scale bars, 2 mm. Images are representative of ten animals. Each data point is a single animal, n=10 in each arm. Horizontal bar indicates mean of ten animals and error bars represent standard error of the mean. *, p<0.05, Student’s t-test. f, Following infusion of U-¹³C glucose, NCT-503 reduces the fraction of M+3 serine (normalized to M+3 3-PG) in MDA-MB-468 orthotopic xenografts. Data are the mean of three independent experiments (3 animals in each arm) and error bars represent standard deviations. *, p<0.05, Student’s t-test.

Figure 4. PHGDH inhibition in a PHGDH-dependent cell line unexpectedly reduces the incorporation of exogenous serine into dTMP and AMP

All data are the mean of three biological replicates. Error bars represent standard deviations. *, p<0.05, Student’s t-test. a, Incorporation of ¹³C from glucose, glucose-derived serine, and exogenous serine into nucleotides. b, 10 µM NCT-503 treatment for four hours reduces the synthesis of glucose-derived serine and decreases the incorporation of ¹³C from glucose via serine into AMP. c, 10 µM NCT-503 treatment for four hours in the presence of exogenous U-¹³C-serine does not increases the proportion of labeled serine but increases the fraction of labeled glycine, consistent with decreased synthesis of unlabeled serine. Unexpectedly, NCT-503 reduces the incorporation of one-carbon units from exogenous U-¹³C-serine into AMP and dTMP.

Figure 5. SHMT1 mediates the loss of nucleotide labeling induced by PHGDH inhibition

All data are the mean of three biological replicates. Error bars represent standard deviations. *, p<0.05, Student’s t-test. a, NCT-503 induces increased synthesis of M+2 serine from M+2 glycine and unlabeled 5,10-CH₂-THF in a PHGDH-dependent cell line. b, Probable SHMT1-catalyzed synthesis of M+1-serine from unlabeled glycine and ¹³C-serine-derived 5,10-methylene THF (5,10-CH₂-THF) increases with PHGDH inhibition (10 µM NCT-503) and is suppressed by exogenous unlabeled formate. c, Serine synthesis pathway activity, or a serine synthesis pathway intermediate, represses SHMT1 activity. SHMT1 catalyzes serine synthesis from glycine and 5,10-CH₂-THF. d, SHMT1 deletion restores incorporation of carbon from U-¹³C serine into dTMP in the presence of PHGDH inhibitor. Mouse SHMT1 expression restores decreased dTMP labeling induced by PHGDH inhibition. SHMT2 knockout does not block PHGDH inhibitor-mediated loss of dTMP labeling. e, SHMT1 deletion restores incorporation of carbon from U-¹³C serine into AMP in the presence of a PHGDH inhibitor. Mouse SHMT1 restores PHGDH inhibitor-mediated loss of AMP labeling by U-¹³C serine. f, NCT-503 treatment induces G₁/S cell cycle arrest in MDA-MB-468 cells, consistent with a defect in nucleotide synthesis. g, Nucleoside supplementation partially rescues PHGDH inhibitor toxicity. h, Model of one-carbon unit wasting induced by PHGDH inhibition. Suppression of PHGDH activity increases the activity of SHMT1, which consumes one-carbon units to resynthesize serine but reduces the availability of one-carbon units needed for purine and dTMP synthesis.