Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor.

Characterizing the mode of action of non-covalent inhibitors in multisubunit enzymes often presents a great challenge. Most of the conventionally used methods are based on ensemble measurements of protein-ligand binding in bulk solution. They often fail to accurately describe multiple binding processes occurring in such systems. Native electrospray ionization mass spectrometry (ESI-MS) of intact protein complexes is a direct, label-free approach that can render the entire distribution of ligand-bound states in multimeric protein complexes. Here we apply native ESI-MS to comprehensively characterize the isoprenoid biosynthesis enzyme IspF from Arabidopsis thaliana, an example of a homomeric protein complex with multiple binding sites for several types of ligands, including a metal cofactor and a synthetic inhibitor. While standard biophysical techniques failed to reveal the mode of action of recently discovered aryl-sulfonamide-based inhibitors of AtIspF, direct native ESI-MS titrations of the protein with the ligands and ligand competition assays allowed us to accurately capture the solution-phase protein-ligand binding equilibria in full complexity and detail. Based on these combined with computational modeling, we propose a mechanism of AtIspF inhibition by aryl bis-sulfonamides that involves both the competition with the substrate for the ligand-binding pocket and the extraction of Zn2+ from the enzyme active site. This inhibition mode is therefore mixed competitive and non-competitive, the latter exerting a key inhibitory effect on the enzyme activity. The results of our study deliver a profound insight into the mechanisms of AtIspF action and inhibition, open new perspectives for designing inhibitors of this important drug target, and demonstrate the applicability and value of the native ESI-MS approach for deep analysis of complex biomolecular binding equilibria.

Aryl Bis-Sulfonamide Inhibitors of IspF from Arabidopsis thaliana and Plasmodium falciparum.

2-Methylerythritol 2,4-cyclodiphosphate synthase (IspF) is an essential enzyme for the biosynthesis of isoprenoid precursors in plants and many human pathogens. The protein is an attractive target for the development of anti-infectives and herbicides. Using a photometric assay, a screen of 40 000 compounds on IspF from Arabidopsis thaliana afforded symmetrical aryl bis-sulfonamides that inhibit IspF from A. thaliana (AtIspF) and Plasmodium falciparum (PfIspF) with IC50 values in the micromolar range. The ortho-bis-sulfonamide structural motif is essential for inhibitory activity. The best derivatives obtained by parallel synthesis showed IC50 values of 1.4 µm against PfIspF and 240 nm against AtIspF. Substantial herbicidal activity was observed at a dose of 2 kg ha(-1) . Molecular modeling studies served as the basis for an in silico search targeted at the discovery of novel, non-symmetrical sulfonamide IspF inhibitors. The designed compounds were found to exhibit inhibitory activities in the double-digit micromolar IC50 range.

Enantioselective synthesis of the C1-C11 fragment of tedanolide C.

A convergent synthesis of the protected C(1)-C(11) fragment 6 of the targeted enantiomer of tedanolide C is described. The key step of the synthesis is the Felkin-Ahn addition of vinyl iodide 7 to aldehyde 8 that proceeds in 80% yield with 4:1 diastereoselectivity.

Identification of 1,3-diiminoisoindoline carbohydrazides as potential antimalarial candidates.

A series of inhibitors of plant enzymes of the non-mevalonate pathway from herbicide research efforts at BASF were screened for antimalarial activity in a cell-based assay. A 1,3-diiminoisoindoline carbohydrazide was found to inhibit the growth of Plasmodium falciparum with an IC(50) value <100 nM. Synthesis of a variety of derivatives allowed an improvement of the initial antimalarial activity down to IC(50) =18 nM for the most potent compound, the establishment of a structure-activity relationship, and the evaluation of the cytotoxic profile of the diiminoisoindolines. Furthermore, interesting configurational and conformational aspects for this class of compounds were studied by computational and X-ray crystal structure analysis. Some of the compounds can act as tridentate ligands, forming 2:1 ligand-iron(III) complexes, which also display antimalarial activity in the nanomolar IC(50) range, paired with low cytotoxicity.

Thiazolopyrimidine inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum.

A library of 40,000 compounds was screened for inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high-throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC(50) values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC(50) values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.