Structure‐Based Design and Synthesis of the First Weak Non‐Phosphate Inhibitors for IspF, an Enzyme in the Non‐Mevalonate Pathway of Isoprenoid Biosynthesis

Abstract

In this paper, we describe the structure-based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C 5 precursors to isoprenoids, isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2; Scheme 1). The nonmevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non-mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as potential drug-like inhibitors of IspF protein, and synthesized (Schemes 2 -5). Determination of the enzyme activity by 13 C-NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine-2,5-diamine derivatives in the upper micromolar range (IC 50 values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56', which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub-pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to Pocket III ) and rings that occupy the flexible hydrophobic region of Pocket II . The proposed binding mode remains to be further validated by X-ray crystallography.

2C-Methyl-d-erythritol 4-Phosphate Enhances and Sustains Cyclodiphosphate Synthase IspF Activity

“…25 Various inhibitors of IspF have been reported 25–27 including CDP. 26 For further validation of this HPLC-based assay, we tested inhibition of IspF by CDP.…”

Thiazolopyrimidine Inhibitors of 2‐Methylerythritol 2,4‐Cyclodiphosphate Synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum

“…[16] More recently, a series of non-phosphate, cytosine-type inhibitors with IC 50 values in the micromolar range has been reported. [17] Earlier, we showed that fluorescent ligands for IspF protein can serve as a basis for a fluorescence-monitored ligand binding competition assay for inhibitor screening. [16] More recently, we developed a dynamically monitored kinetic assay that is suitable for the screening of large compound libraries (Scheme 1).…”

Synthesis and evaluation of stable substrate analogs as potential modulators of cyclodiphosphate synthase IspF

“…Rationally designed IspF inhibitors incorporate the CDP moiety 14 or a cytidyl-like component 15 to bind in the rigid, well-conserved “pocket III” of the IspF active site, and a non-polar component to bind in the more flexible hydrophobic “pocket II”. 14 We reasoned that the unique methylerythritol scaffold could also drive recognition of selective IspF inhibitors.…”

“…15,16 These efforts by Diederich and colleagues to identify inhibitors underscore the challenges in targeting the highly polar active site of IspF.…”