Figure 1.
The reaction catalyzed by FolD.
N5,N10-methylene-THF is converted to N5,N10-methenyl-THF and subsequently N10-formyl-THF in a two-step reaction, initially in an NADP+ or NAD+ dependant oxidization to by N5,N10-methylenetetrahydrofolate dehydrogenase [DH, EC:1.5.1.5] and subsequent hydrolysis by N5,N10-methenyltetrahydrofolate cyclohydrolase [CH, EC:3.5.4.9].
Figure 2.
Cartoon representation of a homodimer of PaFolD with secondary structure labeled. The interface occurs between α5, α7 and β6 of partner subunits.
Table 1.
Crystallographic statistics.
Figure 3.
Different loop conformations at the active site.
Superposition of a subunit of E.coli FolD (PDB code: 1B0A black) against PaFolD. A loop in the PaFolD structure (red residues 231–243) adopts a different orientation compared to the EcFolD structure (blue) with equivalent residues (Gln235 Pa and Leu235 Ec) shifting by as much as 16.7 Å and an angle of nearly 60°. In the orientation seen for the PaFolD structure, the loop sits over the active site.
Figure 4.
Analysis of active site residues.
Superposition of the HsDHCH (green) - NADP+ (yellow) - LY354899 (black) complex (PDB code: 1DIB) onto the remodeled PaFolD structure (grey). Residues that interact with either NADP+ or LY354899 molecules in HsDHCH and their counterparts in PaFolD structure are shown as sticks.
Figure 5.
(A) N5,N10-methylene-THF Km determination in the presence of 1 mM NADP+. (B) NADP+ KM determination in the presence of 1 mM N5,N10-methylene-THF. All KM measurement data are presented as mean ± SD (n = 4) (C) Representative IC50 determination for LY374571. Data points are mean ± SD (n = 14). This representative example returns an IC50 for LY374571 of 27±3 nM.
Figure 6.
Correlation between replicate pIC50 values for each of the 24 compounds advanced to potency testing. Linear regression of these data returned a correlation coefficient of 0.92.
Figure 7.
Confirmed hit compounds from hit discovery campaign.
Summary of the compounds and series identified through the HTS and their respective potencies and Hill slope values.
Figure 8.
Stereoview of the docking of DDD32388 (cyan) into the active site of PaFolD. Potentially important residues are highlighted as sticks and with hydrogen bonds shown as dashes.