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Updated: Jun 15, 2025
Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors
Published on: July 17, 2020
Phosphoenolpyruvate carboxykinase-2 (PCK2) is a therapeutic target in triple-negative breast cancer
Vignesh Gunasekharan1, Hao-Kuen Lin1, Michal Marczyk1,2
1Yale Cancer Center, Yale School of Medicine, 300 George Street, Suite 120, Rm 133, New Haven, CT, 06511, USA.
Purpose:
Metabolic rewiring in malignant transformation is often accompanied by altered expression of metabolic isozymes. Phosphoenolpyruvate carboxykinase-2 (PCK2) catalyzes the rate-limiting step of gluconeogenesis and is the dominant isoform in many cancers including triple-negative breast cancer (TNBC). Our goal was to identify small molecule inhibitors of PCK2 enzyme activity.
Methods:
We assessed the impact of PCK2 down regulation with shRNA on TNBC cell growth in vitro and used AtomNet® deep convolutional neural network software to identify potential small molecule inhibitors of PCK2-based structure. We iteratively tested candidate compounds in an in vitro PCK-2 enzyme assay. The impact of the top hit on metabolic flux and cell viability was also assessed.
Results:
PCK2 downregulation decreased growth of BT-549 and MDA-MB-231 cells and reduced metabolic flux through pyruvate carboxylase. The first AtomNet® in silico structural screen of 7 million compounds yielded 86 structures that were tested in PCK2 enzyme assay in vitro. The top hit (IC50 = 2.4 µM) was used to refine a second round of in silico screen that yielded 82 candidates to be tested in vitro, which resulted in 45 molecules with inhibition > 20%. In the second in vitro screen we also included 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, previously suggested to be PCK2 inhibitor based on structure, which emerged as the top hit. The specificity of this compound was tested in PCK1 and PCK2 enzymatic assays and showed IC50 of 500 nM and 3.5-27 nM for PCK1 and PCK2, respectively.
Conclusion:
3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a high affinity PCK2 enzyme inhibitor that also has significant growth inhibitory activity in breast cell lines in vitro and represents a potential therapeutic lead compound.
Insights
Researchers identified a potent inhibitor for Phosphoenolpyruvate carboxykinase-2 (PCK2), an enzyme crucial in triple-negative breast cancer (TNBC) metabolism. This compound shows promise as a therapeutic lead for TNBC treatment.
Area of Science:
- Biochemistry
- Oncology
- Drug Discovery
Background:
- Metabolic reprogramming is a hallmark of cancer, involving altered expression of metabolic enzymes.
- Phosphoenolpyruvate carboxykinase-2 (PCK2) is a key enzyme in gluconeogenesis and is upregulated in several cancers, including triple-negative breast cancer (TNBC).
Purpose of the Study:
- To identify small molecule inhibitors targeting PCK2 enzyme activity.
- To explore PCK2 as a therapeutic target in triple-negative breast cancer.
Main Methods:
- Downregulation of PCK2 using shRNA to assess its impact on TNBC cell growth.
- Utilized AtomNet® deep convolutional neural network for in silico identification of PCK2 inhibitors.
- Iterative in vitro enzyme assays and metabolic flux analysis to evaluate candidate compounds.
Main Results:
- PCK2 downregulation inhibited growth and reduced metabolic flux in TNBC cell lines.
- An initial in silico screen identified 86 compounds, with subsequent rounds yielding 45 molecules showing >20% inhibition.
- 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate emerged as a high-affinity PCK2 inhibitor (IC50 = 500 nM for PCK1, 3.5-27 nM for PCK2).
Conclusions:
- 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a potent and specific PCK2 inhibitor.
- This compound demonstrates significant in vitro growth inhibitory activity against breast cancer cell lines.
- It represents a promising therapeutic lead for further development in TNBC treatment.
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