Abstract 2448: Towards structure-based drug design of 3-phosphoglycerate dehydrogenase inhibitors
Authors: Unterlass Judith E. , Aljufri Nabila , Bex Sophie , Cano Celine , Noble Martin E M , Curtin Nicola J. 
Published: August 2015

Abstract
Background
The NAD+-requiring enzyme 3-phosphoglycerate dehydrogenase (PHGDH) diverts glycolytic flux into serine production and folate metabolism by catalyzing the oxidization of 3-phosphoglycerate to phosphohydroxypyruvate. The PHGDH gene is located on chromosome 1(1p12), a region frequently amplified in melanoma and certain breast cancer forms. PHGDH knockdown in cells with amplified PHGDH or overexpressing PHGDH at the protein level resulted in cell growth inhibition. In addition ectopic overexpression of PHGDH in a non-tumorigenic cell line induced morphological changes characteristic of transformation. Therefore, inhibitors of PHGDH may be therapeutically valuable.
No PHGDH inhibitors have been reported to date, our aim is to develop PHGDH inhibitors targeting the cofactor (and substrate) binding site.
Methods
Full length PHGDH and the catalytic subunit (sPHGDH) were expressed in, and isolated from E. coli. PHGDH was found to co-purify with its cofactor, making it necessary to abolish the cofactor-protein interaction to investigate inhibitor binding at the co-factor site. To accomplish this, site-directed sPHGDH mutants were constructed and purified. To characterize the mutants, their enzymatic activity and ability to bind to NADH (assessed by thermal stability and isothermal titration calorimetry (ITC)) were measured, and their structures were characterized by circular dichroism (CD) spectroscopy and on-going x-ray crystallographic studies.
Results sPHGDH retained 60% of the enzymatic activity of the full length protein whereas introduction of single or double point mutations around the cofactor or substrate binding site resulted in complete abolition of enzymatic activity. The binding of NADH (1mM) to PHGDH and sPHGDH resulted in increases in melting temperature (ΔTm) of 8.4 ± 0.2 °C and 9.3 ± 0.5 °C respectively. Mutation of the cofactor binding site resulted in reduced ability to bind NADH (ΔTm = 0.8 ± 0.2 °C, 2.3 ± 0.1 °C) whereas mutation at the substrate binding site, directly adjacent to the cofactor binding site, had no significant effect on the ability to bind NADH. Congruent findings were obtained via ITC determining a binding affinity (Kd) of 0.66 μM for wt sPHGDH and a reduced binding affinity of 3-30 fold for the mutated proteins. CD measurements showed that the proteins mutated at the co-factor binding site had undergone minor changes in the secondary structure compared to the wt sPHGDH whereas mutation at the substrate binding site had no effect on the secondary structure elements.
Conclusion Although the mutations carried out were single or double point mutations only, cofactor binding could be reduced substantially, giving rise to markedly lower catalytic activity. These investigations illustrate a promising way of making proteins with large, tight binding ligands accessible to structure-based drug-design.
Citation Format: Judith E. Unterlass, Nabila Aljufri, Sophie Bex, Celine Cano, Martin E M Noble, Nicola J. Curtin. Towards structure-based drug design of 3-phosphoglycerate dehydrogenase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2448. doi:10.1158/1538-7445.AM2015-2448