High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of Plasmodium falciparum

Abstract

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC₅₀ of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.

Keywords: DXPS; MEP pathway; Plasmodium falciparum; Polypharmacology Browser; malaria.

Scheme 1. Illustration of the MEP Pathway, Highlighting the Important Branch Point Enzyme 1-Deoxy-D-xylulose 5-Phosphate Synthase

d-GAP = glyceraldehyde 3-phosphate, DXPS = 1-deoxy-d-xylulose-5-phosphate synthase, B₁ = thiamine, B₆ = pyridoxine, DOXP = 1-deoxy-d-xylulose 5-phosphate, DXR = 1-deoxy-d-xylulose 5-phosphate reductoisomerase, MEP = 2-C-methylerythritol 4-phosphate, IDP = isopentenyl diphosphate, DMADP = dimethylallyl diphosphate.

Figure 1

Ligand-based virtual screening hits (1, 2, 3) were tested against M. tuberculosis DXPS. Minimum inhibitory concentrations (MICs) were determined against the Mycobacterium tuberculosis H37Rv strain.

Figure 2

Whole-cell antiplasmodial activity of DXPS compounds against Plasmodium falciparum 3D7 from three different laboratories and assays; following (A) method I, (B) method II, and (C) method III. IC₅₀ curves of 1 (blue triangles), 2 (green circles), and 3 (black squares). All data were averaged from two to four independent experiments conducted in duplicate or triplicate and is shown including SD (error bars). (D) For IC₅₀ determination, data were analyzed using nonlinear regression of the log-dose–response curves and interpolated from the sigmoidal curve. 95% CI is displayed as error measure. Please note that in Table 1, Table 2, and 3 the inhibition values from method III differ slightly as they have been calculated with a different program (see Methods section). As they are within the 95% CI, we have decided not to recalculate the values.

Scheme 2. General Reaction Scheme for the Synthesis of Oximes

O-A: sodium bis(trimethylsilyl)amide (4.0 equiv), dimethylformamide, −10 °C, 3–72 h. O-B: BBr₃ (10.0 equiv), dichloromethane, 25 °C, 5 h. O-C: KOAc (3.0 equiv), [NH₃OH]Cl (1.5 equiv), reflux, 2 h. R′ = OMe or H. R″ = Cl. R‴ = OMe or H or OH. X = C or N.

Scheme 3. General Reaction Scheme for the Synthesis of Indole Derivatives

I-A: POCl₃ (1.3 equiv), dimethylformamide, 80 °C, 15 min. I-B: NaH (1.8 equiv), dimethylformamide, 0 °C – 25 °C, 16 h. I-C: NaBH₄ (3.2 equiv), MeOH, 25 °C, 1 h. R′ = R³ in Table 2. R″ = H or Cl. X and Y = N or C.

Scheme 4. General Reaction Scheme for the Synthesis of Aminothiazoles

A-A: diisopropylethylamine (1.1 equiv), ethanol, 25 °C, 72 h.

Figure 3

(A) Binding mode and docking pose diagram of compound 1. (B) Binding mode and docking pose diagram of compound 47. Protein surface representation is clipped for clarity. Green and red spheres represent positive or negative contribution to the predicted affinity, respectively.

Figure 4

Aminothiazole hit compound 1 and similar compounds we found during the PolyPharmacology Browser search, with their reported target. KasA = β-ketoacyl ACP synthase; VCP = valosin-containing protein.