Targeting the Plasmodium falciparum IspE Enzyme

Abstract

The enzyme IspE in Plasmodium falciparum is considered an attractive drug target, as it is essential for parasite survival and is absent in the human proteome. Yet it still has not been addressed by a small-molecule inhibitor. In this study, we conducted a high-throughput screening campaign against the PfIspE enzyme. Our approach toward a PfIspE inhibitor comprises in vitro screening, structure-activity relationship studies, examining the docking position using an AlphaFold model, and finally target verification through probe binding and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The newly synthesized probe containing a diazirine and an alkyne moiety (23) allowed us to demonstrate its binding to IspE in the presence of a lysate of human cells (HEK293 cells) and to get evidence that both probe 23 and the best inhibitor of the series (19) compete for the same IspE binding site.

Scheme 1. Fourth Step in the MEP Pathway

The substrate 4-diphosphocytidyl-2C-methyl-d-erythritol (CDP-ME) is phosphorylated to 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate (CDP-MEP) by the kinase IspE.

Figure 1

Chemical structures of reported IspE inhibitors.

Figure 2

(a) Binding mode of 9 in the CDP-ME binding site of Plasmodium falciparum IspE. The phenyl ring is involved in a π–π interaction with the side chain of Tyr378. (b) Binding mode of 19 in the CDP-ME binding site of P. falciparum IspE. The benzimidazole nitrogen atom is involved in an H-bond interaction with the backbone NH of Asn154. The benzimidazole ring is involved in a π–π interaction with the side chain of Tyr378.

Figure 3

Chemical structure of photo-crosslinkers 23 and 24.

Figure 4

Verification of probe 23 binding to IspE proteins of E. coli and P. falciparum. (a) Analytical SDS-PAGE experiments with column 1 verifying the affinity of the probe for the target proteins, columns 2 and 3 comparing binding to the native and denatured protein with additional human HEK cell lysate as a background, column 4 testing IspE affinity to the minimal aromatic photo-crosslinker 24 (chemical structure shown in figure), columns 5–8: comparison of probe 23 binding in the presence of competing active compound 19. The molar ratios of 23:19 are given above the respective lanes. (b) Fluorescence scan of a competition SDS-PAGE with HEK cell lysate without (lane 1) and with being spiked in PfIspE protein (lanes 2–5). Dark bands correspond to areas with a prevalent affinity for probe 23. In lanes 3–5, competitor 19 is present in increasing excess compared to the probe (lane 3:1:1, lane 4:1:5, lane 5:1:15). Below, the corresponding loading control is shown. PfIspE (UniProt entry A0A1B1TK84) is expected to run at a molecular weight of 63 kDa, with EcIspE (UniProt entry P62615) expected to run at 31 kDa. All depicted gel scans are included in their entirety in the Supporting Information, Section S7.6.