Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor.

To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD+ and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. Cancer Res; 77(4); 937-48. ©2017 AACR.

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli.

Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrahdrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (EcoFolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (CpeFolD) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of CpeFolD is about five times more efficient than that of EcoFolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is ∼ 10 times more efficient than that of EcoFolD. Kinetic parameters for CpeFhs were also determined for utilization of all of its substrates. Both CpeFolD and CpeFchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of CpeFolD and CpeFchA is also necessary to rescue an E. coli folD deletion strain (harbouring CpeFhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.

Assessment of Pseudomonas aeruginosa N5,N10-methylenetetrahydrofolate dehydrogenase-cyclohydrolase as a potential antibacterial drug target.

The bifunctional enzyme methylenetetrahydrofolate dehydrogenase - cyclohydrolase (FolD) is identified as a potential drug target in Gram-negative bacteria, in particular the troublesome Pseudomonas aeruginosa. In order to provide a comprehensive and realistic assessment of the potential of this target for drug discovery we generated a highly efficient recombinant protein production system and purification protocol, characterized the enzyme, carried out screening of two commercial compound libraries by differential scanning fluorimetry, developed a high-throughput enzyme assay and prosecuted a screening campaign against almost 80,000 compounds. The crystal structure of P. aeruginosa FolD was determined at 2.2 Å resolution and provided a template for an assessment of druggability and for modelling of ligand complexes as well as for comparisons with the human enzyme. New FolD inhibitors were identified and characterized but the weak levels of enzyme inhibition suggest that these compounds are not optimal starting points for future development. Furthermore, the close similarity of the bacterial and human enzyme structures suggest that selective inhibition might be difficult to attain. In conclusion, although the preliminary biological data indicates that FolD represents a valuable target for the development of new antibacterial drugs, indeed spurred us to investigate it, our screening results and structural data suggest that this would be a difficult enzyme to target with respect to developing the appropriate lead molecules required to underpin a serious drug discovery effort.

The crystal structure of Leishmania major N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclohydrolase and assessment of a potential drug target.

Three enzyme activities in the protozoan Leishmania major, namely N(5),N(10)-methylenetetrahydrofolate dehydrogenase/N(5),N(10)-methenyltetrahydrofolate cyclohydrolase (DHCH) and N(10)-formyltetrahydrofolate ligase (FTL) produce the essential intermediate N(10)-formyltetrahydrofolate. Although trypanosomatids possess at least one functional DHCH, the same is not true for FTL, which is absent in Trypanosoma brucei. Here, we present the 2.7 Å resolution crystal structure of the bifunctional apo-DHCH from L. major, which is a potential drug target. Sequence alignments show that the cytosolic enzymes found in trypanosomatids share a high level of identity of approximately 60%. Additionally, residues that interact and participate in catalysis in the human homologue are conserved amongst trypanosomatid sequences and this may complicate attempts to derive potent, parasite specific DHCH inhibitors.

Crystal structure of bifunctional 5,10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum.

Folate co-enzymes play a pivotal role in one-carbon transfer cellular processes. Many eukaryotes encode the tri-functional tetrahydrofolate dehydrogenase/cyclohydrolase/synthetase (deh/cyc/syn) enzyme, which consists of a N-terminal bifunctional domain (deh/cyc) and a C-terminal monofunctional domain (syn). Here, we report the first analogous archeal enzyme structures, for the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum (TaMTHFDC) as the native protein and also as its NADP complex. The TaMTHFDC structure is a dimer with a polar interface, as well as a NADP binding site that shows minor conformational change. The orientations of the residues in the NADP binding site do not change on ligand binding, incorporating three water molecules which are hydrogen bonded with phosphate groups of NADP in the structure of the complex. Our structural information will contribute to an improved understanding of the basis of THF and one-carbon metabolism.

Disruption of the mthfd1 gene reveals a monofunctional 10-formyltetrahydrofolate synthetase in mammalian mitochondria.

The Mthfd1 gene encoding the cytoplasmic methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase enzyme (DCS) was inactivated in embryonic stem cells. The null embryonic stem cells were used to generate spontaneously immortalized fibroblast cell lines that exhibit the expected purine auxotrophy. Elimination of these cytoplasmic activities allowed for the accurate assessment of similar activities encoded by other genes in these cells. A low level of 10-formyltetrahydrofolate synthetase was detected and was shown to be localized to mitochondria. However, NADP-dependent methylenetetrahydrofolate dehydrogenase activity was not detected. Northern blot analysis suggests that a recently identified mitochondrial DCS (Prasannan, P., Pike, S., Peng, K., Shane, B., and Appling, D. R. (2003) J. Biol. Chem. 278, 43178-43187) is responsible for the synthetase activity. The lack of NADP-dependent dehydrogenase activity suggests that this RNA may encode a monofunctional synthetase. Moreover, examination of the primary structure of this novel protein revealed mutations in key residues required for dehydrogenase and cyclohydrolase activities. This monofunctional synthetase completes the pathway for the production of formate from formyltetrahydrofolate in the mitochondria in our model of mammalian one-carbon folate metabolism in embryonic and transformed cells.