Selectivity analysis of diaminopyrimidine-based inhibitors of MTHFD1, MTHFD2 and MTHFD2L

The mitochondrial enzyme methylenetetrahydrofolate dehydrogenase (MTHFD2) is involved in purine and thymidine synthesis via 1C metabolism. MTHFD2 is exclusively overexpressed in cancer cells but absent in most healthy adult human tissues. However, the two close homologs of MTHFD2 known as MTHFD1 and MTHFD2L are expressed in healthy adult human tissues and share a great structural resemblance to MTHFD2 with 54% and 89% sequence similarity, respectively. It is therefore notably challenging to find selective inhibitors of MTHFD2 due to the structural similarity, in particular protein binding site similarity with MTHFD1 and MTHFD2L. Tricyclic coumarin-based compounds (substrate site binders) and xanthine derivatives (allosteric site binders) are the only selective inhibitors of MTHFD2 reported till date. Nanomolar potent diaminopyrimidine-based inhibitors of MTHFD2 have been reported recently, however, they also demonstrate significant inhibitory activities against MTHFD1 and MTHFD2L. In this study, we have employed extensive computational modeling involving molecular docking and molecular dynamics simulations in order to investigate the binding modes and key interactions of diaminopyrimidine-based inhibitors at the substrate binding sites of MTHFD1, MTHFD2 and MTHFD2L, and compare with the tricyclic coumarin-based selective MTHFD2 inhibitor. The outcomes of our study provide significant insights into desirable and undesirable structural elements for rational structure-based design of new and selective inhibitors of MTHFD2 against cancer.


Table of contents
Table S1.Average RMSD values for the MTHFD1compound 1 complex from the triplicate MD simulations.P6 Table S3.Average RMSD values for the MTHFD1compound 3 complex from the triplicate MD simulations.P10 Table S6.Average RMSD values for the MTHFD2compound 3 complex from the triplicate MD simulations.P14               Table S3.Average RMSD values for the MTHFD1compound 3 complex from the triplicate MD simulations.Table S4.Average RMSD values for the MTHFD2compound 1 complex from the triplicate MD simulations.Table S5.Average RMSD values for the MTHFD2compound 2 complex from the triplicate MD simulations.Table S6.Average RMSD values for the MTHFD2compound 3 complex from the triplicate MD simulations.Table S9.Average RMSD values for the MTHFD2Lcompound 3 complex from the triplicate MD simulations.

Figure
Figure S2.(A) Docking pose of compound 1 in the MTHFD1 binding site (ligand in orange, protein residues in pink).(B) Docking pose of compound 2 in the MTHFD1 binding site (ligand in green, protein residues in pink).(C) Docking pose of compound 3 in the MTHFD1 binding site (ligand in red, protein residues in pink).

Figure S6 .
Figure S6.RMSD analysis of the MTHFD1compound 1 complex from the triplicate MD simulations (A) Protein CA (B) Ligand.

Figure S7 .
Figure S7.Protein-ligand interaction histogram from the MD simulations of compound 1 in the MTHFD1 binding site.(H-bonds are shown in green, salt-bridge interactions are shown in pink, and lipophilic contacts are shown in grey).

Figure S8 .
Figure S8.RMSD analysis of the MTHFD1compound 1 complex during the 200ns simulation.Protein α-carbons are shown in pink while the inhibitor is hidden.

Figure S9 .
Figure S9.MD structure of the MTHFD1compound 1 complex at 15 th ns superposed with the X-ray structure of MTHFD1 without the substrate site inhibitor (PDB code: 6ECR).MTHFD1-compound 1 complex: protein ribbons in pink, loop 1 in dark blue and compound 1 in orange.MTHFD1 without the inhibitor: protein ribbons in cyan and loop 1 in green.

Figure S10 .
Figure S10.RMSD analysis of the MTHFD1compound 2 complex from the triplicate MD simulations (A) Protein CA (B) Ligand.

Figure S11 .
Figure S11.MD snapshots of the MTHFD1compound 2 complex at (A) 0 ns.(B) 41 ns: Hbond interaction with Tyr240 (C) 80 ns: H-bond/salt-bridge interaction with Arg250.MTHFD1 protein ribbons are shown in pink, loop 1 in dark blue and compound 2 in green.

Figure S12 .
Figure S12.Protein-ligand interaction histogram from the MD simulations of compound 2 in the MTHFD1 binding site.(H-bonds are shown in green, salt-bridge interactions are shown in pink, and lipophilic contacts are shown in grey).

Figure S13 .
Figure S13.RMSD analysis of the MTHFD1compound 3 complex from the triplicate MD simulations (A) Protein CA (B) Ligand.

Figure S14 .
Figure S14.Protein-ligand interaction histogram from the MD simulations of compound 3 in the MTHFD1 binding site.(H-bonds are shown in green, salt-bridge interactions are shown in pink, and lipophilic contacts are shown in grey).

Figure S15 .
Figure S15.RMSD analysis of the MTHFD2compound 1 complex from the triplicate MD simulations (A) Protein CA (B) Ligand.
Figure S16.(A) Co-crystallized pose of compound 1 in the MTHFD2 binding site (ligand in orange, protein residues in blue, PDB code: 6S4A).(B) Co-crystallized pose of compound 2 in the MTHFD2 binding site (ligand in green, protein residues in blue, PDB code: 6S4E).(C) Cocrystallized pose of compound 3 in the MTHFD2 binding site (ligand in red, protein residues in blue, PDB code: 6S4F).

Figure S20 .
Figure S20.Protein-ligand interaction histogram from the MD simulations of compound 1 in the MTHFD2 binding site.(H-bonds are shown in green, salt-bridge interactions are shown in pink, and lipophilic contacts are shown in grey).

Figure S21 .
Figure S21.RMSD analysis of the MTHFD2compound 2 complex from the triplicate MD simulations (A) Protein CA (B) Ligand.

Figure S22 .
Figure S22.Protein-ligand interaction histogram from the MD simulations of compound 2 in the MTHFD2 binding site.(H-bonds are shown in green, salt-bridge interactions are shown in pink, and lipophilic contacts are shown in grey).

Figure S23 .
Figure S23.RMSD analysis of the MTHFD2compound 3 complex from the triplicate MD simulations (A) Protein CA (B) Ligand.

Figure S24 .
Figure S24.Protein-ligand interaction histogram from the MD simulations of compound 3 in the MTHFD2 binding site.(H-bonds are shown in green, salt-bridge interactions are shown in pink, and lipophilic contacts are shown in grey).

Figure
Figure S25.(A) Docking pose of compound 1 in the MTHFD2L binding site (ligand in orange, protein residues in grey).(B) Crystallographic pose of compound 2 in the MTHFD2L binding site (PDB code: 7QEI, ligand in green, protein residues in grey).(C) Docking pose of compound 3 in the MTHFD2L binding site (ligand in red, protein residues in grey).

Figure S29 .Figure S30 .
Figure S29.RMSD analysis of the MTHFD2Lcompound 1 complex from the triplicate MD simulations (A) Protein CA (B) Ligand.

Figure S31 .Figure S32 .
Figure S31.RMSD analysis of the MTHFD2Lcompound 2 complex from the triplicate MD simulations (A) Protein CA (B) Ligand.

Figure S34 .
Figure S34.RMSD analysis of the MTHFD2Lcompound 3 complex from the MD simulations (A) Protein CA (B) Ligand.

Figure S35 .
Figure S35.Protein-ligand interaction histogram from the MD simulations of compound 3 in the MTHFD2L binding site.(H-bonds are shown in green, salt-bridge interactions are shown in pink, and lipophilic contacts are shown in grey).

Table S1 .
Average RMSD values for the MTHFD1compound 1 complex from the triplicate MD simulations.

Table S2 .
Average RMSD values for the MTHFD1compound 2 complex from the triplicate MD simulations.