Targeting the IspD Enzyme in the MEP Pathway: Identification of a Novel Fragment Class

Abstract The enzymes of the 2‐C‐methylerythritol‐d‐erythritol 4‐phosphate (MEP) pathway (MEP pathway or non‐mevalonate pathway) are responsible for the synthesis of universal precursors of the large and structurally diverse family of isoprenoids. This pathway is absent in humans, but present in many pathogenic organisms and plants, making it an attractive source of drug targets. Here, we present a high‐throughput screening approach that led to the discovery of a novel fragment hit active against the third enzyme of the MEP pathway, PfIspD. A systematic SAR investigation afforded a novel chemical structure with a balanced activity–stability profile (16). Using a homology model of PfIspD, we proposed a putative binding mode for our newly identified inhibitors that sets the stage for structure‐guided optimization.


Materials and Methods
Starting materials and solvents were purchased from commercial suppliers, and used without further purification. All chemical yields refer to purified compounds and were not optimized. Reaction progress was monitored using TLC silica gel 60 F254 aluminum sheets, and visualization was accomplished by UV at 254 nm. Column chromatography was performed using the automated flash chromatography system CombiFlash ® Rf (Teledyne Isco) equipped with RediSepRf silica columns.
Low resolution mass analytics and purity control of final compounds was carried out using an Ultimate 3000-MSQ LCMS system (Thermo Fisher Scientific) consisting of a pump, an autosampler, MWD detector and a ESI quadrupole mass spectrometer. Purity of all compounds used in biochemical assays was ≥ 95%. High resolution mass spectra were recorded on a ThermoFisher Scientific (TF, Dreieich, Germany) Q Exactive Focus system equipped with heated electrospray ionization (HESI)-II source. Final products were dried at high vacuum.

Preparative RP-HPLC purifications
Purifications via preparative RP-HPLC were carried out using the following condition:
The sample was dissolved in DMSO and manually injected to the HPLC system.

Synthetic Schemes
Scheme S1: General procedure for the synthesis of compounds 1ꟷ7.

S5
Internal Scheme S2: General procedure for the synthesis of compounds 12ꟷ18.

Experimental procedures
Compounds 8, 9, 10 and 11 were commercially available and were purchased directly. Na2CO3 (4 mmol). The reaction was heated in a microwave at 120 o C for 1 h. H2O (30 mL) was added followed by extraction with ethyl acetate (2 x 30 mL). The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure. Purification was done using preparative HPLC to yield compounds (12ꟷ18).

4'-Isopropoxy-4-nitro-[1,1'-biphenyl]-2-carboxylic acid (18)
Compound 18 was synthesized according to GPB by reacting 2-bromo-5-nitrobenzoic acid with (4isopropoxyphenyl)boronic acid. The crude product was purified by preparative HPLC (eluted at 62% The reactions were monitored photometrically (room temperature) at 340 nm in a plate reader (SpectraMax5, Molecular Dynamics). Initial rate values were evaluated with a nonlinear regression method using the program Dynafit. [1] MEP and CDP-ME were used as starting substrate in the IspD and IspE assay, respectively, and were synthesised and purified as described earlier. [2] All the compounds tested (1ꟷ18) showed no activity against the auxiliary enzyme.
This assay has been used in the initial HTS and as with most enzyme-based approaches, IspD hits may fall under PAIN-subcluster (pan-assay interference compounds), which indicate risk of non-specific interference with the assay, but sometimes also exclude valuable leads. The control reaction via the auxiliary IspE-enzyme done for the most active hits confirm that the described inhibitors are, in fact, selectively inhibiting the target enzyme and not the auxiliary enzymes of the IspD assay. Still, in follow up optimization the potential of non-specific assay interaction should be taken into account for further analogues S13 Internal

Molecular modeling
The PfIspD homology model was constructed using the sequence from 2-C-methyl-D-erythritol 4phosphate cytidylyltransferase from Plasmodium falciparum 3D7 (obtained from the NCBI (www.ncbi.nlm.nih.gov, sequence ID: XP_001351000.2, accessed 11/10/2021) and the structure from E. coli IspD (accessed 11/10/2021). The model was built using the PHYRE2 online homology modeling program. [3] Previous work showed the 1I52 E. coli IspD structure served as a good template. [4] The model created in PHYRE2 using 1I52 as the template structure showed a 99.77% degree of confidence.
Compounds of interest were docked into the homology model described above using the docking algorithm provided in SeeSAR 11.0 (BioSolveIT GPBH). Binding site definition was carried out manually from residues selected by SeeSAR's pocket finding function. The binding site selected is analogous to the CDP-ME binding site in the 1I52 E. coli IspD structure used as a template.