Abstract

Simple SummaryThe mitochondrial enzyme serine hydroxymethyltransferase (SHMT2), which converts serine into glycine and generates 1C units for cell growth, is one of the most consistently overexpressed metabolic enzymes in cancer. Here, we reveal that the anti-diabetic biguanide metformin operates as a novel class of non-catalytic SHMT2 inhibitor that disrupts the pyridoxal-5′-phosphate (PLP)-dependent SHMT2 oligomerization process and ultimately SHMT2 activity. As SHMT2 inhibitors have not yet reached the clinic, these findings may aid the rational design of PLP-competitive SHMT2 inhibitors based on the biguanide skeleton of metformin.The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5′-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.

Highlights

  • A close relationship exists between the anti-cancer activity of the biguanide metformin and the folate-dependent functioning of the serine/glycine one-carbon (SGOC) network [1,2,3]

  • We first assessed the differential inhibitory effects of metformin on purified, human recombinant SHMT1 and SHMT2 isoforms using a spectrophotometric assay in which both isoenzymes are incubated with saturating concentrations of glycine, a fixed concentration of formyl-THF and a low concentration range of metformin (Figure 1A)

  • Enzyme activity was calculated by measuring the so-called quinonoid intermediate, which is generated when both glycine and formyl-THF bind the SHMT1/2 enzymes to form an serine hydroxymethyltransferases (SHMTs)–glycine– folate ternary complex [18,20,22] and displays an intense absorption band with a maximum at ~500 nm deriving from the deprotonation of glycine (Figure 1A, top)

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Summary

Introduction

A close relationship exists between the anti-cancer activity of the biguanide metformin and the folate-dependent functioning of the serine/glycine one-carbon (SGOC) network [1,2,3]. The elevation of homocysteine levels, an archetypal marker of one-carbon (1C) deficiency due to impaired flux through the serine catabolism pathway [4], is a pharmacodynamic marker that informs tumor response to metformin in non-diabetic patients with breast cancer [5]. Metformin treatment phenocopies the metabolic effects of hindering the incorporation of 1C units into folates by serine hydroxymethyltransferases (SHMTs). SHMT2-driven mitochondrial serine metabolism is an indispensable facet of the ability of metformin to promote S-adenosylmethionine (SAM) accumulation and global DNA methylation [10]. Metformin treatment leads to complex responses that can improve or impede the anti-tumor effects of serine and glycine starvation in vitro and in vivo [11,12]

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