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

Abstract

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.

Figure 1. The reaction catalyzed by FolD.

N ⁵,N ¹⁰-methylene-THF is converted to N ⁵,N ¹⁰-methenyl-THF and subsequently N ¹⁰-formyl-THF in a two-step reaction, initially in an NADP⁺ or NAD⁺ dependant oxidization to by N ⁵,N ¹⁰-methylenetetrahydrofolate dehydrogenase [DH, EC:1.5.1.5] and subsequent hydrolysis by N ⁵,N ¹⁰-methenyltetrahydrofolate cyclohydrolase [CH, EC:3.5.4.9].

Figure 2. Structure of PaFolD.

Cartoon representation of a homodimer of PaFolD with secondary structure labeled. The interface occurs between α5, α7 and β6 of partner subunits.

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. FolD assay development.

(A) N ⁵,N ¹⁰-methylene-THF K_m determination in the presence of 1 mM NADP⁺. (B) NADP⁺ K _M determination in the presence of 1 mM N ⁵,N ¹⁰-methylene-THF. All K _M 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. Replicate testing.

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. Ligand docking.

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.