Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor Robert Gustafsson1, Ann-Soﬁe Jemth2, Nina M.S

Published OnlineFirst November 29, 2016; DOI: 10.1158/0008-5472.CAN-16-1476

Cancer Therapeutics, Targets, and Chemical Biology Research

Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor Robert Gustafsson1, Ann-Soﬁe Jemth2, Nina M.S. Gustafsson2, Katarina Farnega€ rdh3, Olga Loseva2, Elisee Wiita2, Nadilly Bonagas2, Leif Dahllund4, Sabin Llona-Minguez2, Maria Haggblad€ 5, Martin Henriksson2, Yasmin Andersson4, Evert Homan2, Thomas Helleday2, and Pa l Stenmark1

Abstract

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

Introduction scheme shown in Fig. 1A (3, 4). In mitochondria, the 1C unit usually derived from serine by serine hydroxymethyltransferase Rapidly dividing cells depend on a high and steady supply of (SHMT) or from glycine by the glycine cleavage system, is attached 10-formyltetrahydrofolate to sustain several vital anabolic reac- to tetrahydrofolate (THF; see Fig. 1C) yielding methylene-THF tions, for example the synthesis of purines. Targeting this pathway (CH2-THF), which is subsequently oxidized to formate. The is one way to specifically target cancer cells, and one successful formate is released to the cytoplasm, where it is again attached example is the antifolate drug methotrexate that has been used in to a THF molecule that is either used for de novo purine synthesis cancer therapies since the 1950s (1). or reduced further and used for thymidylate or methionine In eukaryotes, the folate-dependent one-carbon metabolism is synthesis (3). It has been shown that the majority of the 1C units highly compartmentalized between cytoplasm and mitochondria used in the cytoplasm are derived from the mitochondria (5). The (2, 3). These compartments are metabolically connected by the entire pathway is upregulated in cancer cells (6) as well as transport of the one-carbon (1C) donors serine, glycine, and embryonic cells (7). It is important for maintaining the ratios of formate across the mitochondrial membrane. Depending on the þ þ NAD to NADH and NADP to NADPH, thus affecting the redox different redox environments in the mitochondria and cytoplasm, balance of the cells and their ability to scavenge and reduce the metabolic flow occurs mostly in the clockwise direction in the reactive oxygen species (8). One enzyme of specific interest is MTHFD2, responsible for the oxidation of methylene-THF to 1Department of Biochemistry and Biophysics, Stockholm University, Stockholm, 10-formyl-THF in mitochondria, which is highly overexpressed in 2 Sweden. Science for Life Laboratory, Division of Translational Medicine and cancer cells and embryonic cells, but not in normal adult tissues. Chemical Biology, Department of Medical Biochemistry and Biophysics, Kar- 3 Thus, development of inhibitors targeting MTHFD2 is an attrac- olinska Institutet, Stockholm, Sweden. Drug Discovery and Development fi Platform, Science for Life Laboratory, Department of Organic Chemistry, Stock- tive opportunity to speci cally target cancer cells (9). holm University, Solna, Sweden. 4Drug Discovery and Development Platform, The enzyme family responsible for the conversion between Science for Life Laboratory, School of Biotechnology, Royal Institute of Tech- methylene-THF and formate is the methylenetetrahydrofolate nology, Solna, Sweden. 5Biochemical and Cellular Screening, Science for Life dehydrogenase (MTHFD) family that performs three main reac- Laboratory, Department of Biochemistry and Biophysics, Stockholm University, tions in the 1C metabolism: the 5,10-methylene-THF (CH2-THF) Stockholm, Sweden. þ dehydrogenase, 5,10-methenyl-THF (CH -THF) cyclohydrolase Note: Supplementary data for this article are available at Cancer Research and 10-formyl-THF (10-CHO-THF) synthetase activities Online (http://cancerres.aacrjournals.org/). (see Fig. 1B; refs. 10, 11). Corresponding Authors: Pa l Stenmark, Stockholm University, Svante In mitochondria, the 5,10-methylene-THF dehydrogenase Arrhenius vag€ 16C, Stockholm 106 91, Sweden. Phone: 46-816-3729; Fax: and 5,10-methenyl-THF cyclohydrolase activities are per- 46-815-5597; E-mail: [email protected]; and Thomas Helleday, formed by two enzymes, MTHFD2 and MTHFD2L (12). [email protected] MTHFD2 was first discovered in Ehrlich ascites tumor cells doi: 10.1158/0008-5472.CAN-16-1476 (13) already in 1960 and later described as a mitochondrial þ 2016 American Association for Cancer Research. NAD -dependent methylene-THF dehydrogenase and

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Figure 1. A, Mammalian 1C metabolism. Reactions 1–4 occur in both the cytoplasmic and the mitochondrial (m) compartments. Reactions 1, 2, and 3 are catalyzed by trifunctional MTHFD1 in the cytoplasm using 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase activity, respectively. In mammalian mitochondria, reaction 1m is catalyzed by monofunctional MTHFD1L, and reactions 2m and 3m are catalyzed by bifunctional MTHFD2 or MTHFD2L. Reactions 4 and 4m are catalyzed by serine hydroxymethyltransferase and reaction 5 by the glycine cleavage system. Hcy, homocysteine; Met, methionine; AdoMet, S-Adenosyl methionine; THF, tetrahydrofolate. Adapted from Shin and colleagues (36). B, The three activities, 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase, mediated by the MTHFD family. C and D, Chemical structures of tetrahydrofolate (THF; C) and LY345899 (D).

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Structure of Human MTHFD2 and Identiﬁcation of an Inhibitor

cyclohydrolase expressed in embryonic and transformed cells (40). MTHFD1L mRNA has also been found to be upregulated in (14–19). However, it has been demonstrated that MTHFD2 human colon adenocarcinoma (41). mRNA is expressed at low levels in all tissues but not confirmed In the cytosol, all three enzymatic functions are performed by to be translated (20). MTHFD2 has been shown to play an MTHFD1. MTHFD1 functions as a dimer where each monomer essential role in embryonic development for mammals because comprises two functional units, one DC-domain containing the gene inactivation in mice resulted in embryonic lethality (21). dehydrogenase (D) and cyclohydrolase (C) activities with a Theproteinhasbeenshowntobeimportantforrapidgrowing common active site (42) as demonstrated by both substrate cells, such as embryonic cells or cancer cells, mainly by sup- channeling (43, 44) and X-ray crystallographic structures with þ porting the high level of purine synthesis needed (21, 22). NADP (10) and with folate-analogues (45). The second domain MTHFD2-null mutant fibroblasts have previously been is responsible for the 10-formyl-THF-synthetase (S) activity. þ reported to be glycine auxotrophs (23), a condition that can MTHFD1 uses NADP as cofactor for the dehydrogenase activity þ þ be rescued by expression of an NAD -orNADP -dependent, (10) and the rate-limiting step of the D/C activities is the cyclo- mitochondrially targeted methylene-THF-dehydrogenase- hydrolase (46). MTHFD1 is expressed in all adult tissues exam- þ cyclohydrolase (24). For its dehydrogenase activity with NAD ined (47). MTHFD2 has an absolute requirement for inorganic phosphate Computer-generated homology models of both MTHFD2 (14) 2þ (Pi)andMg (14, 18, 19, 25). MTHFD2 displays activity also and MTHFD2L (9) have been published, based on the human þ with NADP , although lower, and in such case only requires MTHFD1 structure and in the case of the MTHFD2 homology 2þ thepresenceofMg and not Pi (25). It appears to have evolved model, also based on the Escherichia coli (48) and Saccharomyces from a tri-functional enzyme through the loss of the synthetase cerevisiae (49) homologs. So far no structure based on empirical þ þ domain and the change of specificity from NADP to NAD ,Pi data has been presented for any of these two proteins. þ and Mg2 (14, 26). Here we identify the first MTHFD2 inhibitor LY345899 MTHFD2 mRNA and protein are upregulated in many (Fig. 1D) and the target engagement of this substrate-based cancers and their overexpression is associated with tumor cell inhibitor, as well as present the first structure of the human þ proliferation (9). MTHFD2 depletion by RNA interference mitochondrial NAD -dependent methylene-THF dehydrogenase decreases cancer cell proliferation independent of tissue of and cyclohydrolase, MTHFD2. The MTHFD2 protein was cocrys- origin (27) and leukemia burden in xenografts (28). Upregu- tallized with the substrate-based inhibitor LY345899 and the þ lation of MTHFD2 is linked to poor prognosis in breast cancer cofactors NAD and inorganic phosphate. patients (27, 29) where it is associated with regulation of breast cancer cell migration and invasion (6, 30, 31). A SNP Materials and Methods study found that MTHFD2 variants are associated with a higher For all buffers, buffer composition can be found in Supple- risk for bladder cancer (32). MTHFD2 has been implicated in mentary Table S1. sensitivity of cancer cells toward artesunate, an anti-malarial drug (33). Recently, MTHFD2 was reported to have a nuclear localization in addition to its mitochondrial localization as Cloning, expression, and purification of human MTHFD2 and well as supporting cancer cell proliferation independently of its MTHFD1 dehydrogenase/cyclohydrolase domain dehydrogenase activity. Consistent with its role to support cDNAs encoding MTHFD2 and MTHFD1 (codon optimized cancer cell proliferation, MTHFD2 was found to be co- for E. coli expression) were purchased from Eurofins and expressed with proteins involved in cell-cycle progression, GeneArt. Bacterial expression constructs enabling His-tag puri- specifically in the S, G2, and M phases, and often overexpressed fication of human MTHFD2 lacking the N-terminal mitochon- in human tumors (34). In response to growth signals, the drial signal peptide (MTHFD2 AA36-350) and the dehydroge- mTORC1 activates the ATF4 transcription factor, which stimu- nase/cyclohydrolase (DC) domain of MTHFD1 (AA1-306) lates expression of MTHFD2 and other enzymes of the serine were generated. synthesis and mitochondrial THF cycle, thereby increasing the For MTHFD2 protein expression the construct was trans- production of formyl units required for de novo purine syn- formed into E. coli strain BL21(DE3). The transformed cells thesis (35). weregrowninLB-mediumþ 100 mg/mL ampicillin at 37C MTHFD2L, on the other hand, is described as a methylene-THF overnight. Fresh overnight culture inoculated into LB-medium þ dehydrogenase and cyclohydrolase (12) that can use either NAD was grown at 37 CtoOD600 1, protein expression was þ or NADP ; however, the catalytic efficiency, kcat/Km, is much induced by addition of 1 mmol/L IPTG. The bacteria were lower than for MTHFD2 (36). MTHFD2L is highly homologous harvested after 2 hours. Cells were dissolved in Lysis buffer to MTHFD2 (72% identity) and is thought to be a housekeeping A2. After incubation for 20 minutes at room temperature, the enzyme, because it is found in all adult tissues tested (12), as well suspension was centrifuged and the supernatant was loaded as in embryonic cells (36). onto Ni-Sepharose column HisTrap-HP (GE Healthcare) equil- In the mitochondria, the 10-formyl-synthetase activity is per- ibrated with Buffer B2. The column was washed with Buffer B2 formed by MTHFD1L, which is a homolog of the cytosolic þ 10 mmol/L imidazole, the bound proteins were eluted with trifunctional MTHFD1 (4, 37, 38). This enzyme consists of two 10–500 mmol/L imidazole gradient. The fractions containing functional units as for MTHFD1, but several key residues for the MTHFD2 were dialyzed against Buffer C2. domain responsible for dehydrogenase and cyclohydrolase activ- His-tag was cleaved off and removed by passing the protein ities differ, rendering it a monofunctional synthetase enzyme over a HisTrap column. MTHFD2 was dialyzed against Buffer D2 (37, 38). MTHFD1L is expressed in all embryonic and adult and loaded onto an anion-exchange monoQ-HP column (GE tissues examined (37, 39) and its deletion caused embryonic Healthcare) equilibrated with Buffer D2. The bound proteins were lethality as well as neural tube and craniofacial defects in mice eluted with 20 to 800 mmol/L NaCl gradient and analyzed by

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SDS-PAGE. Fractions with pure MTHFD2 (Supplementary Fig. The structure has been deposited in the protein data bank with S1) were combined and protein concentration was determined by accession code 5TC4. the Bradford assay. MTHFD1 DC was expressed in E. coli strain BL21(DE3). The Inhibition of MTHFD2 and MTHFD1 cells were grown in LB containing 50 mg/mL carbenicillin at 30 C – To determine IC50 values of LY345899, an eight-point dose until OD600 reached 0.55. The temperature was lowered to 16 C response curve with 3-fold difference in concentration between before protein expression was induced with 1 mmol/L IPTG. The points was generated. Each assay point was run in duplicate. cells were harvested after overnight expression. Bacteria were The starting concentration of LY345899 was 10 mmol/L for the resuspended in Lysis buffer A1 and homogenized using Emulsi- MTHFD2 assay and 1 mmol/L for the MTHFD1 assay. Serial Flex C3 (Avistin). After centrifugation, the supernatant was loaded dilution of compound LY345899 was transferred into a Perkin on a 1 mL HisTrapFF column (GE Healthcare) equilibrated with Elmer 384-Proxiplate, with DMSO as negative control. Buffer B1. After washing with Buffer B1, bound proteins were The assays were run in MTHFD2 assay buffer and MTHFD1 eluted using a gradient to 100% Buffer C1. The imidazole was assay buffer, respectively. A total of 2.5 mL enzyme was preincu- removed from the IMAC elute using a HiPrep 26/10 desalting bated with compound or DMSO for 10 minutes. The enzymatic column equilibrated and run with Buffer D1. His-tag was reaction was initiated by adding 2.5 mL folitixorin. For back- removed as described for MTHFD2. Finally, the nontagged ground control, 5 mL buffer was added to the well. Final con- MTHFD1DC pool was after desalting run on Superdex 16/60 centrations of the components in the MTHFD2 assay were þ equilibrated with Buffer E1. 3 nmol/L MTHFD2, 5 mmol/L folitixorin and 250 mmol/L NAD , and 25 nmol/L MTHFD1 DC, 30 mmol/L folitixorin, and 400 þ Crystallization mmol/L NADP in the MTHFD1 assay. After 15-minute reaction, 5 þ LY345899 (3 mmol/L), NAD (5 mmol/L), and MgCl2 mL NAD(P)H-Glo detection reagent (Promega) was dispensed in (6 mmol/L) were added to MTHFD2 and incubated for 50 all wells and the plate was incubated for 60 minutes. The dose– minutes. After 40 minutes, 10 mmol/L Na2HPO4 was added. response curve for MTHFD2 was run seven times, and twice for Proteases (1:50 ratio each of trypsin, a-chymotrypsin, pepsin, MTHFD1. For both assays, luminescence was measured on a papain, proteinase K, and subtilisin to MTHFD2) were added just Perkin–Elmer Envision reader. before crystallization. For crystallization at 20C, MTHFD2 at 5.9 mg/mL was mixed with 0.1 mol/L phosphate/citrate pH 4.1, 38% Differential scanning fluorimetry (v/v) PEG300 in 3:1 ratio. After 1 week, crystals were frozen in Differential scanning fluorimetry (DSF) was used to detect liquid nitrogen. Data collection were performed at beamline binding of LY345899 to purified MTHFD2 protein. MTHFD2 ID30A-3 at ESRF. Crystals diffracted to 1.9Å. Statistics can be (4 mmol/L) with LY345899 (100 mmol/L, 1% DMSO) or 1% fi found in Table 1. Details regarding data processing, re nement, DMSO was run with 5 SYPRO Orange dye (Ex492/Em610) in and model building can be found in Supplementary Methods. quadruplicate using DSF Buffer. The assay volumes were 20 mLin 96-well plates (HSP9655; Bio-Rad Laboratories). The temperature Table 1. Crystallographic data collection and refinement statistics was increased from 25Cto95C with 1C per minute. A real-time Data collection MTHFD2 PCR (Bio-Rad Laboratories) CFX96 Optical Reaction Module, Space group I4 C1000T chassis, Channel 6 FRET, was used. Cell dimensions a, b, c (Å) 74.3 74.3 98.6 a, b, g () 90, 90, 90 Cell culture Resolution (Å) 37.16–1.89 (1.93–1.89) U2OS human osteosarcoma cells, MRC-5 human lung fibro-

Rmerge (%) 31.6 (434.8) blasts, and Hs-587T breast cancer cells were obtained from the I/s (I) 6.8 (0.9) ATCC and authenticated by the supplier using STR analysis. Cells Completeness (%) 99.8 (96.8) were cultured in DMEM GlutaMAX (Life Technologies) supple- CC(1/2) (%) 98.0 (29.7) Redundancy 6.9 (6.7) mented with 10% FBS, penicillin (50 U/mL), and streptomycin (50 mg/mL) and maintained in a humidified incubator at 37C fi Re nement with 5% CO . Resolution (Å) 37.16–1.89 2 No. unique reflections 21,322 (1,331) Rwork/Rfree 16.26/21.22 Cellular thermal shift assay No. atoms U2OS cells were harvested using trypsin, upon detachment Protein 2249 trypsination was inhibited by addition of cell media and cells Ligand 83 Water 214 spun down at 1,500 rpm, 10 minutes, 4 C. Pellet was resuspended 6 B-factors in TBS, cells were counted and adjusted to 2 10 cells per Protein 27.687 milliliter. Cells were lysed by a freeze–thaw cycle three times at Ligand 32.382 80C for 3 minutes followed by 37C for 3 minutes. Cell lysates Water 36.874 were cleared by centrifugation at 13,000 rpm at 4C for 20 min- R.M.S. deviations utes, supernatant was transferred to new tubes and 50 mmol/L Bond lengths (Å) 0.0231 Bond angles () 2.2764 LY345899 or DMSO was added and sample was incubated 30 Ramachandran plot, residues in (%) minutes at room temperature. Cell lysates were aliquoted and Most favorable region 97.25 heated to indicated temperatures for 3 minutes, insoluble pro- Additional allowed region 2.75 teins were removed by centrifugation at 13,000 rpm at 4C for 20 NOTE: Highest resolution shell is shown in parenthesis. minutes, supernatant transferred to new tubes and protein

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Structure of Human MTHFD2 and Identiﬁcation of an Inhibitor

concentration determined using BCA assay (Pierce). Western blot NAD(P)H-Glo assay (Promega). MTHFD2 showed clear activity analysis was performed according to standard procedures. The and the biochemical assay gave consistent results, with Z0 ¼ 0.8 antibodies were mouse anti-MTHFD2 (Abcam, ab56772), mouse (Supplementary Fig. S2; Supplementary Table S2). In vitro inhi- anti-Actin (Abcam, ab6276) followed by IRDye 800 CW donkey bition of MTHFD2 and MTHFD1 DC-domain using the substrate- anti-mouse (LI-COR, 926-32212). Images were taken with Odys- based inhibitor LY345899 was shown using the same assay. IC50 sey Fc imager (LI-COR; ref. 50). values of LY345899 were determined to 663 nmol/L for MTHFD2 (n ¼ 7) and 96 nmol/L for MTHFD1 (n ¼ 2; Fig. 2). Drug affinity responsive target stability assay U2OS cells grown to approximately 70% to 80% confluence Target engagement were lysed in mammalian protein extraction lysis buffer M-PER To further validate the interaction with MTHFD2, target (Thermo Scientific) supplemented with 1 complete protease engagement of LY345899 was next investigated by DSF. The inhibitor cocktail (Roche) and after centrifugation the super- temperature at which a protein unfolds is measured by an natant was diluted to a final concentration of 1 TN buffer. increase in the fluorescence of SYPRO Orange dye. The dye is Protein concentration of the cell lysate was determined by quenched in aqueous solution but emits strong fluorescence Bradford method. Aliquots of the cell lysate were incubated upon binding to exposed hydrophobic parts of the protein as with 1% DMSO or serial dilution of compound LY345899 in the protein unfolds with increasing temperature. LY345899 1% DMSO at room temperature for 1 hour and then with (100 mmol/L, 1% DMSO) was able to stabilize MTHFD2 pronase (Roche) solution for 30 minutes. For the nondigested (4 mmol/L) upon binding, increasing the protein melting point (ND) sample, TN buffer was added instead of protease. Pro- (Tm)from44C(1%DMSO)to55C(n ¼ 4; Fig. 3A). To teins were separated using SDS-PAGE and blotted onto nitro- investigate target engagement by LY345899 in a cellular con- cellulose membranes, and then probed with mouse anti- text, cellular thermal shift assay (CETSA) was performed. MTHFD2 antibody (Abcam, ab56772), followed by incubation CETSA is based on the principle that ligand binding leads with goat anti-IgG mouse antibody (Jackson ImmunoResearch, to a thermal stabilization of the target protein upon engage- 711-035-150), and protein was visualized using SuperSignal ment, which is detected using Western blotting with antibodies West Femto chemiluminescence substrate (Thermo Scientific). specifically recognizing the protein of interest. Following treat- The blot was probed with rabbit anti-GAPDH Ab (Santa Cruz ment of U2OS cell lysates with LY345899, the MTHFD2 pro- Biotechnology, sc25778) followed by incubation with donkey tein was stabilized as compared to DMSO (Fig. 3B and C). anti-rabbit IRDye 800CW Ab (LI-COR, 926-32213) and images In support of this, target engagement by LY345899 in cell lysate taken using LI-COR (51). was analyzed by drug affinity responsive target stability (DARTS). The DARTS assay is based on the principle that Structural sequence alignment interaction between target protein and a small molecular Structure-based sequence alignment of MTHFD1-DC (1A4I. ligand reduces susceptibility of the protein to protease diges- pdb) and MTHFD2 (5TC4.pdb) was generated using PDBe- tion. The inhibitor LY345899 was found to protect the FOLD (52). Graphic representation was generated using MTHFD2 protein from pronase digestion as compared with ESpript 3.0 (53). DMSO(Fig.3D).However,wecouldnotdetectanytarget engagement by LY345899 in CETSA using intact cells, thus Results LY345899 does not seem to be able to enter cells under the Activity and inhibition of MTHFD2 and MTHFD1 conditions used (Supplementary Fig. S3A and S3B). Consis- Human MTHFD2 and the DC domain of MTHFD1 were tently, no inhibition of cell viability could be observed upon purified from bacteria and activity was determined using the LY345899 treatment (Supplementary Fig. S3C).

Figure 2.

LY345899 dose–response curves for MTHFD2 (A) and MTHFD1 DC-domain (B). IC50 for MTHFD2 ¼ 663 nmol/L (n ¼ 7) and MTHFD1 DC-domain ¼ 96 nmol/L (n ¼ 2).

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Figure 3. Target engagement by LY345899. A, DSF detection of MTHFD2 stabilization by LY345899. LY345899 (100 mmol/L, 1% DMSO) was able to stabilize MTHFD2 (4 mmol/L) upon binding, increasing the protein melting point (Tm) from 44 C (1% DMSO) to 54.75 C. Values are shown as an average, with standard deviation, of four measurements. B and C, CETSA, U2OS cell lysates were treated with 10 mmol/L LY345899 or DMSO. B, Thermal stabilization of MTHFD2 was detected using Western blotting. b-Actin was used as loading control. C, Quantiﬁcation of B using the program Image J, MTHFD2 levels were normalized to b-actin and fraction non-denatured protein was calculated. Data are shown as mean SEM of duplicate independent experiments and is representative of three independent experiments. D, DARTS, Western blot assay of U2OS cell lysate samples incubated with DMSO or LY345899 compound followed pronase digestion. ND, non-digested. GADPH was used as loading and digestion control.

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Structure of Human MTHFD2 and Identiﬁcation of an Inhibitor

The overall structure of MTHFD2 within 3.4Å of its symmetry-related mate and could form a To evaluate the detailed binding of LY345899 to MTHFD2, we disulfide bond under oxidizing conditions. solved the X-ray cocrystal structure of MTHFD2 with Ly345899, Overlay of MTHFD2 with the structure of MTHFD1 DC- þ NAD and Pi. The structure of MTHFD2 contains one monomer domain in complex with LY345899 published previously (45) of MTHFD2 in the asymmetric unit. The resolution is 1.9Å and shows considerable similarity (Fig. 5) between the structures with shows clear electron density for residues Glu36-Leu332, except the same general folds being present (Fig. 6). The structure of one break between His280 to Lys286. See Table 1 for relevant MTHFD1 does however only have LY345899 bound in one of the statistics of the structure. monomers. In the monomer with LY345899 bound, there are MTHFD2 is built up of two domains connected by two long a only small differences in backbone between MTHFD1 and helices (A and I in Fig. 4A) and a small helix (helix D2) ordered so MTHFD2 with Ca-RMSD of 0.96Å, except for the insertion of a that a large cleft is formed between the domains. MTHFD2 is loop between helix E and strand f. For the other monomer, the similar to DC-domain of MTHFD1 but has several distinct fea- changes are much larger due to the absence of bound LY345899 in tures. The overall structure and secondary sequence annotation is MTHFD1 with an overall Ca-RMSD of 1.70Å. shown in Fig. 4A. The structure-based sequence alignment to MTHFD1 DC-domain is shown in Fig. 5. The cofactor binding sites þ MTHFD2, like MTHFD1, is dimeric (Supplementary Fig. S4). The binding site for NAD (Fig. 4B) does not resemble a þ The monomer forms a homodimer with extensive contacts to a traditional NAD site, as many of the features described in þ crystallographic symmetry related molecule (Fig. 4A), showing a literature are missing or more similar to a NADP site. The buried surface area of 1609Å2. diphosphate binding loop sequence GXSXXXG is different from þ the conserved GXGXXG and has been described for the NADP þ binding MTHFD1 (10). The main difference from a traditional Binding of cofactors NAD and Pi þ The structure contains both the nicotine amide dinucleotide NAD site is the lack of an Asp or Glu sidechain hydrogen þ cofactor NAD as well as the inorganic phosphate needed for bonding to the ADP diol. Backbone nitrogens of Arg201 and Arg233, as well as the inorganic phosphate, instead bind the enzymatic activity. They are bound within the large cleft formed þ between the N- and C-terminal domains of MTHFD2 toward the adjacent hydroxyl groups in NAD bound to MTHFD2. The C-terminal domain, as can be seen in Fig. 4A and B. Detailed presence of Arg233 in front of the A side of adenine is a usual þ þ 0 depiction of the NAD binding site is shown in Supplementary sign of a NADP site as this residue often binds the 2 -phosphate. Fig. S5A. The adenine group is bound in a crevice formed by the Interestingly, in MTHFD1 this residue is a serine, but is still binding the 20-phosphate together with Arg173 (Arg201 in loops between strand g and 310-helix G and between strand f and þ MTHFD2; ref. 44). MTHFD2 has thus adapted a NADP site to helix F of the Rossmann domain. The classical dinucleotide þ bind NAD by the use of phosphate, which binds to the ADP diol binding motif GXGXXG cannot be found in the sequence of þ 200 of NAD . The phosphate mediates several hydrogen bonds to MTHFD2 but instead the residues G RSKNVG making up the þ þ NAD to bind it to the protein. In the NADP -dependent loop connecting strand e with helix E constitute the dinucleotide 0 200 202 205 MTHFD1, these hydrogen bonds instead go directly to the 2 - binding loop in MTHFD2. The G RS KNVG residues align þ þ structurally with the classical dinucleotide sequence GXGXXG phosphate and thus bind NADP to the site. The use of NAD as thus making the minimal sequence of this protein GXSXXXG, as cofactor for MTHFD2 favors the production of 10-formyl-THF from methylene-THF during rapid cell proliferation, as the previously shown for this protein family (10). þ The phosphate is bound close to the 20-OH group of the high ratio of NAD /NADH in the mitochondria drives the reac- þ sugar in the adenosine monophosphate moiety of NAD , tion in this direction. MTHFD1L uses the 10-formyl-THF to þ produce formate, which is transported into the cytosol, where where the phosphate would be covalently bound in NADP þ as can be seen in Fig. 4B. Detailed interactions are shown in the high ratio of NADPH/NADP drives the reaction of MTHFD1 Supplementary Fig. S5B. The phosphate interacts with both in the opposite direction forming 10-formyl-THF and methylene- monomers of the dimer. Especially the interaction to Asp216 is THF from formate to be used for purine and thymidylate syn- fl very short, only 2.4Å. thesis, respectively, and forcing the metabolic ow clockwise in Fig. 1A (3, 4). The inorganic phosphate is bound by 11 hydrogen bonds (Fig. Inhibitor binding site 4B and Supplementary Fig. S5B). No interaction with backbone The inhibitor LY345899 is clearly defined by electron density nitrogen is observed, otherwise a common mode of phosphate (Fig. 4C) and is positioned in the large cleft between the N- and binding (54). Instead all hydrogen bonds to the protein are to C-terminal domain with the protein bridging over the inhib- sidechains. The use of arginine sidechain to bind phosphates is itor, forming a tunnel as can be seen in Fig. 4C. The interactions common (54) and Arg201 has been shown to be responsible for with LY345899 can be seen in Supplementary Fig. S5C. The phosphate binding because conservative mutations to lysine terminal glutamate moiety of LY345899 is extending through result in loss of all enzymatic activity (14). Asp225 from the the tunnel of MTHFD2 and reaching the solvent. second monomer holds Arg201 in place, suggested to be important for positioning the Arg201 sidechain correctly (14). In Discussion contrast, Arg233 can be mutated to other residues whilst retaining MTHFD2 monomer and dimer structure dehydrogenase activity. The hypothesis is that this residue helps to The MTHFD2 protein is clearly a dimer as seen both by the bind and position the phosphate but is not strictly necessary as extensive dimeric interface and the size exclusion chromatogra- Arg201 (14). Asp216 and His219 are in the loop between helix E phy described herein, which is in agreement with previous studies and strand f and not found in MTHFD1 (Figs. 5 and 6) and (Supplementary Fig. S4; ref. 18). The sulfur atom of Cys166 is binding to the phosphate from the second monomer of the dimer.

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Figure 4. A, Overall structure of MTHFD2. Monomer from the asymmetric unit shown in rainbow cartoon with secondary structure annotation. Crystallographic symmetry related molecule of dimer shown in gray. Ligands (NADþ, LY345899, and inorganic phosphate) for both monomers are shown as sticks (cyan; phosphor, purple). B and C, Electron density: 2Fo-Fc map at 1.5s. Monomers shown in green and cyan. Important residues for hydrophobic interactions and hydrogen bonding are shown as sticks. Waters are omitted for clarity. Phosphor, purple. B,BindingofNADþ (gray) and inorganic phosphate. C,Bindingof LY345899 (yellow).

Phosphate binding to histidine is common, although not as MTHFD2 structure. This is believed to account for the preference common as to arginine, binding to negatively charged residues of dibasic phosphate present at physiological pH over tribasic is rare (54). When found, the bond is often short, as seen in the phosphate or sulfate for some proteins, where it would act as a

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Structure of Human MTHFD2 and Identiﬁcation of an Inhibitor

Figure 5. Structural sequence alignment of the MTHFD1 DC-domain and MTHFD2 from 1A4I.pdb and 5TC4.pdb. The gap between AA280-AA286 of MTHFD2 and AA240-AA251 of MTHFD1 is due to unresolved residues in the structures (Supplementary Fig. S7). Filled boxes mark conserved residues and white boxes weakly conserved residues. hydrogen bond acceptor (55). MTHFD2 has previously been binding (14). In the MTHFD2 structure, no magnesium is shown to have 8% activity with sulfate instead of phosphate found even though 6 mmol/L is present in the crystallization (18). The structure presented herein gives insights into the nature conditions. We have collected anomalous data on crystals þ of the cofactor-binding site of MTHFD2. grown in the presence of Mn2 . The anomalous difference þ þ þ In addition to phosphate, MTHFD2 requires Mg2 or Mn2 maps did not reveal any bound Mn2 . It is possible for the þ þ for activity (14, 18, 19, 25). Mg2 or Mn2 binding always magnesium to bind both Asp168 and His219 as well as the occurs through at least one negatively charged amino acid side inorganic phosphate (Fig. 4B). The structure presented here þ þ chain such as Asp or Glu. Mutational studies have shown that does not reveal why Mg2 or Mn2 is needed for activity, the phosphate and magnesium are likely interacting with each however, it does open for new approaches to investigate the other (14). There are only few aspartates or glutamates in the reasons for the apparent metal dependence of MTHFD2. vicinity of the inorganic phosphate, and only Asp168 (Fig. 4B) has been suggested to be conserved in related enzymes requir- Inhibitor binding site þ ing Mg2 (14). Furthermore, mutational studies of this posi- The good resolution and well-deﬁned electron density around tion indicate that this aspartate is involved in magnesium LY345899 clearly shows the ligand with R conﬁguration at the

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Gustafsson et al.

Figure 6. Overlay of MTHFD1 DC-domain (1A4I.pdb, gray) and MTHFD2 (5TC4.pdb; monomers, green and cyan). A, Dimer overlay. B, Overlay of MTHFD1 DC-domain NADPþ þ and MTHFD2 NAD þ Pi binding sites. Important residues for phosphate binding are shown as sticks and the MTHFD2 insertion loop Asp216 to Gly224 in dark þ þ blue. Phosphates of NAD þ Pi of MTHFD2 are purple and phosphates of NADP in MTHFD1 DC-domain orange.

chiral carbon in the fused tricyclic system (Fig. 4C). Attempts binding site of MTHFD1 and are conserved in MTHFD2 (Fig. 5 to fit the corresponding S-diastereomer to the electron density and Supplementary Fig. S7; ref. 10). The hydrophobic envi- were unsuccessful. The synthetic procedure employed to produce ronment of Lys56 (Lys88 in MTHFD2) is conserved and pro- LY345899, starting from (S)-folic acid (Supplementary Fig. S6), posedtodecreasethepKa of this residue to keep it deproto- in all likelihood produces a 1:1 mixture of the RS- and SS- nated, which is needed for activity (56). Asp155 in MTHFD2 is diastereomers. Thus, MTHFD2 has singled out the RS-diastereo- contributing strongly to binding of the pteridine of LY345899 mer as a ligand from the mixture, indicating that the SS-diaste- by two direct hydrogen bonds (Fig. 4C). The corresponding reomer probably is less active as a MTHFD2 inhibitor. Attempts residue in MTHFD1, Asp125, has been shown to be critical for to separate the diastereomers of LY345899 are currently in the binding and positioning of the substrate (56). progress. There are several conserved residues found in the sequence Protein activity and inhibition alignment of MTHFD1, MTHFD2, and MTHFD2L involved in LY345899 has been characterized as an MTHFD1 inhibitor. the binding of LY345899 (Supplementary Fig. S7). An To evaluate this inhibitor for MTHFD2, we synthesized Y52XXXK56 motif has been proposed for the binding and LY345899 as described in Supplementary Methods. We here cyclohydrolase catalytic activity of MTHFD1 (10, 45, 56), and show that LY345899 is a potent MTHFD2 inhibitor. This is the conserved for both MTHFD2 and MTHFD2L. Tyr84 of MTHFD2 first inhibitor to be identified for MTHFD2; LY345899 has an contributes with binding of the substrate analogue by pi-stack- IC50 value of 663 nmol/L. As a comparison, inhibition of the ing with the p-aminobenzoate moiety. Mutations of Tyr52 in DC-domain of MTHFD1 by LY345899 was tested, showing an MTHFD1 still have some cyclohydrolase activity; however, IC50 value of 96 nmol/L, clearly showing that LY345899 has a mutation of Lys56 abolishes cyclohydrolase activity, showing higher affinity for MTHFD1 compared to MTHFD2. The overall that this position is vital in the catalytic mechanism proposed binding mode of LY345899 is conserved between MTHFD1 (45, 56). In the proposed catalytic mechanism for MTHFD1, andMTHFD2(Fig.6A).Thereasonforthedifferenceinaffinity Lys56 is supporting the formation of methenyl-THF by hydro- of LY345899 to MTHFD1 and MTHFD2 likely depends on gen bonding to the pteridine carbonyl of the substrate. In the Ser83, Asn87, Phe157, Ala175, Asn204, and Leu289 that all cyclohydrolase reaction, Lys56 supports the attacking water by interact with the inhibitor. These residues are not conserved accepting and donating protons in several stages of the mech- between MTHFD1 and MTHFD2 (Figs. 4C and 5 and Supple- anism, thus having different roles in the two activities. Glu100 mentary Fig. S5C). supports Lys56 by hydrogen bonding and is important for Using CETSA we show that LY345899 engages MTHFD2 in cell activity (56). The water between Lys88 and methenyl-THF is lysates (Fig. 3B and C). DSF shows that LY345899 increases the likely not present in the MTHFD2 structure because the car- melting temperature (Tm) of MTHFD2 by 11 degrees (Fig. 3A). bonyl group of LY345899 displaces it. LY345899 is bound with LY345899 is reducing the susceptibility of MTHFD2 to protease the carbonyl directed toward Lys88, thus imitating the way a digestion (Fig. 3D). The stabilizing effect is however not seen in substrate methenyl-THF would belinedupforthecyclohydro- CETSA using intact cells, which could possibly be due to low lase activity. Structures of MTHFD1 with bound folate analo- cell permeability (57). This also explains the lack of inhibition gues, including LY345899, suggests that the pteridine moiety of cell viability by LY345899 as compared to depletion of needs to flip 180 between the dehydrogenase and cyclohy- MTFHD2 by siRNA (27). LY345899 is however a good starting drolase activities (45) or reorient (56). Ser49, Gln100, and point for optimization toward an inhibitor with favorable ADME Pro102 have been proposed to be important in the THF properties.

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Structure of Human MTHFD2 and Identiﬁcation of an Inhibitor

Biological implications Analysis and interpretation of data (e.g., statistical analysis, biostatistics, There is clear evidence of MTHFD2 upregulation and over- computational analysis): R. Gustafsson, A.-S. Jemth, N.M.S. Gustafsson, € expression in cancer cells and recent data show reduced leukemia K. Farnegardh, O. Loseva, E. Wiita, N. Bonagas, M. Henriksson, Y. Andersson, – E. Homan, P. Stenmark burden in xenograts upon MTHFD2 depletion by shRNA (9, 27 Writing, review, and/or revision of the manuscript: R. Gustafsson, A.-S. Jemth, 32, 34). The protein is expressed in embryonic cells and trans- N.M.S. Gustafsson, K. F€arnegardh, O. Loseva, N. Bonagas, S. Llona-Minguez, formed cells independent of tissue of origin, but importantly not M. Henriksson, Y. Andersson, T. Helleday, P. Stenmark in adult, healthy cells (14–19). Inhibition of MTHFD2 is thus an Administrative, technical, or material support (i.e., reporting or organizing attractive way to selectively target cancer cells to disrupt purine data, constructing databases): M. H€aggblad, M. Henriksson, Y. Andersson synthesis and 1C metabolism while sparing healthy normal cells. Study supervision: T. Helleday, P. Stenmark Other (designed, performed, and analyzed synthetic chemical experiments): A limiting factor of many antimetabolite drugs is that the target K. F€arnegardh enzymes are not only expressed in the cancer cells but also in healthy proliferative cells, causing adverse side-effects in patients Acknowledgments and limiting their use. Thus, the cancer-specific expression profile We thank the beamline scientists at ESRF, France; Max-Lab, Sweden; BESSY, of MTHFD2 holds the promise of fewer adverse side-effects in Germany; Diamond, United Kingdom; PETRA, Germany; and the Swiss Light proliferative tissues compared to many current anti-metabolite Source, Switzerland, for their support and Biostruct-X. We thank Nina Braun for drugs used in cancer therapy. Collectively, these aspects make the her contributions to the initial crystallizations. MTHFD2 protein highly relevant as an anticancer target. The crystal structure of MTHFD2 and the identification of the Grant Support first MTHFD2 inhibitor now provide a first starting point for This work was supported by the Swedish Research Council (T. Helleday, P. Stenmark), the Knut and Alice Wallenberg Foundation (T. Helleday, developing potent and selective MTHFD2 inhibitors. P. Stenmark), the Wenner-Gren Foundation, Åke Wiberg Foundation (P. Stenmark), the Goran€ Gustafsson Foundation, the Swedish Pain Relief Disclosure of Potential Conflicts of Interest Foundation, and the Torsten and Ragnar Soderberg€ Foundation (T. Helleday), the Swedish Children's Cancer Foundation (T. Helleday, N.M.S. Gustafsson), No potential conflicts of interest were disclosed. the Swedish Society for Medical Research, Karolinska Institute Foundations (N.M.S. Gustafsson), the Helleday Foundation (N. Bonagas), and the Swedish Authors' Contributions Cancer Society (T. Helleday, P. Stenmark). Conception and design: R. Gustafsson, E. Homan, T. Helleday, P. Stenmark The costs of publication of this article were defrayed in part by the payment of Development of methodology: A.-S. Jemth, L. Dahllund, S. Llona-Minguez, page charges. This article must therefore be hereby marked advertisement in M. Henriksson, Y. Andersson accordance with 18 U.S.C. 1734 solely to indicate this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Gustafsson, A.-S. Jemth, N.M.S. Gustafsson, Received June 2, 2016; revised September 23, 2016; accepted November 4, O. Loseva, N. Bonagas, M. Henriksson, Y. Andersson 2016; published OnlineFirst November 29, 2016.

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

Robert Gustafsson, Ann-Sofie Jemth, Nina M.S. Gustafsson, et al.

Cancer Res 2017;77:937-948. Published OnlineFirst November 29, 2016.

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