Anna K. H. Hirsch

Structure‐Based Design of Inhibitors of the Aspartic Protease Endothiapepsin by Exploiting Dynamic Combinatorial Chemistry

Structure-based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein-bound library member(s) by saturation-transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization.

Biodynamers: Self-Organization-Driven Formation of Doubly Dynamic Proteoids

Polypeptide-type dynamic biopolymers (biodynamers) have been generated by polycondensation via acylhydrazone and imine formation of amino-acid-derived components that polymerize driven by self-organization. They have been characterized as globular particles, reminiscent of folded proteins, by cryo-TEM, LS, DOSY NMR, and SANS studies. The reversible polymers obtained show remarkably low dispersity and feature double covalent dynamics allowing for fine-tuning of both exchange and incorporation processes through pH control. In the course of build-up, they perform a selection of the most suitable building block, as indicated by the preferential incorporation of the more hydrophobic amino-acid component with increased rate and higher molecular weight of the polymer formed. The system described displays nucleation-elongation behavior driven by hydrophobic effects and represents a model for the operation of adaptation processes in the evolution of complex matter.

Nonphosphate Inhibitors of IspE Protein, a Kinase in the Non-Mevalonate Pathway for Isoprenoid Biosynthesis and a Potential Target for Antimalarial Therapy

The discovery of the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2) in the 1990s opened the way for new approaches in the fight against infectious diseases. This pathway starts with the condensation of pyruvate 3 and glyceraldehyde 3-phosphate 4 and is used exclusively by pathogenic bacteria such as Mycobacterium tuberculosis, and by the protozoan Plasmodium parasites (Scheme 1). Mammals, on the other hand, use the alternative mevalonate pathway. Hence, the development of small-molecule inhibitors for the enzymes of the nonmevalonate pathway constitutes a novel approach in the treatment of important infectious diseases. Malaria is without a doubt the most important and devastating tropical disease with 300–500 million clinical cases and between one and three million deaths a year. In light of the emergence of drug and insecticide resistance, the need for medicines with a novel mode of action is ever increasing. Inhibition of the enzymes of the non-mevalonate pathway by low-molecular-weight ligands constitutes a true challenge. The active sites for complexation and transformation of their phosphateand diphosphate-based substrates are highly polar and do not offer much concave hydrophobic surface. Correspondingly, most of the few inhibitors known today are phosphates or phosphonates, such as the best-known example, Fosmidomycin, which binds to IspC (1-deoxy-d-xylulose 5phosphate reductoisomerase, EC 1.1.1.267) and is currently in clinical trials. We selected the kinase IspE (4-diphosphocytidyl-2C-methyld-erythritol (CDP-ME) kinase, EC 2.7.1.148) in the center of the non-mevalonate pathway as a target for structure-based inhibitor design. IspE catalyzes the phosphorylation of the 2-OH group of 4-diphosphocytidyl-2C-methyl-d-erythritol (5) forming 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate (6) (Scheme 1). Two X-ray crystal structures have been published; the apoenzyme of Thermus thermophilus (1.7 A resolution, Protein Data Bank (PDB) code: 1UEK) and a ternary complex of

Development of inhibitors of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway enzymes as potential anti-infective agents

Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2), the universal precursors of isoprenoids, while humans exclusively utilize the alternative mevalonate pathway for the synthesis of 1 and 2. This distinct distribution, together with the fact that the MEP pathway is essential in numerous organisms, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents and herbicides. Herein, we review the inhibitors reported over the past 2 years, in the context of the most important older developments and with a particular focus on the results obtained against enzymes of pathogenic organisms. We will also discuss new discoveries in terms of structural and mechanistic features, which can help to guide a rational development of inhibitors.

Exploiting specific interactions toward next-generation polymeric drug transporters.

A generic method describes advanced tailoring of polymer drug carriers based on polymer-block-peptides. Combinatorial means are used to select suitable peptide segments to specifically complex small-molecule drugs. The resulting specific drug formulation agents render insoluble drugs water-soluble and enable precise adjustment of drug-release profiles beyond established block-copolymer carriers. While proof of principle is shown on chlorin as a partially approved drug for photodynamic cancer therapy, the concept is universal and applies to a broad spectrum of difficult drugs.

Synthesis and Characterization of Cytidine Derivatives that Inhibit the Kinase IspE of the Non-Mevalonate Pathway for Isoprenoid Biosynthesis

The enzymes of the non‐mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. This pathway is used exclusively by the corresponding pathogens, but not by humans. A series of water‐soluble, cytidine‐based inhibitors that were originally designed for the fourth enzyme in the pathway, IspD, were shown to inhibit the subsequent enzyme, the kinase IspE (from Escherichia coli). The binding mode of the inhibitors was verified by co‐crystal structure analysis, using Aquifex aeolicus IspE. The crystal structures represent the first reported example of a co‐crystal structure of IspE with a synthetic ligand and confirmed that ligand binding affinity originates mainly from the interactions of the nucleobase moiety in the cytidine binding pocket of the enzyme. In contrast, the appended benzimidazole moieties of the ligands adopt various orientations in the active site and establish only poor intermolecular contacts with the protein. Defined binding sites for sulfate ions and glycerol molecules, components in the crystallization buffer, near the well‐conserved ATP‐binding Gly‐rich loop of IspE were observed. The crystal structures of A. aeolicus IspE nicely complement the one from E. coli IspE for use in structure‐based design, namely by providing invaluable structural information for the design of inhibitors targeting IspE from Mycobacterium tuberculosis and Plasmodium falciparum. Similar to the enzymes from these pathogens, A. aeolicus IspE directs the OH group of a tyrosine residue into a pocket in the active site. In the E. coli enzyme, on the other hand, this pocket is lined by phenylalanine and has a more pronounced hydrophobic character.

Fighting Malaria: Structure-Guided Discovery of Nonpeptidomimetic Plasmepsin Inhibitors

Plasmepsins (Plms) are aspartic proteases involved in the degradation of human hemoglobin by Plasmodium falciparum. Given that the parasite needs the resulting amino acid building blocks for its growth and development, plasmepsins are an important antimalarial drug target. Over the past decade, tremendous progress has been achieved in the development of inhibitors of plasmepsin using two strategies: structure-based drug design (SBDD) and structure-based virtual screening (SBVS). Herein, we review the inhibitors of Plms I-IV developed by SBDD or SBVS with a particular focus on obtaining selectivity versus the human Asp proteases cathepsins and renin and activity in cell-based assays. By use of SBDD, the flap pocket of Plm II has been discovered and constitutes a convenient handle to obtain selectivity. In SBVS, activity against Plms I-IV and selectivity versus cathepsins are not always taken into account. A combination of SBVS, SBDD, and molecular dynamics simulations opens up opportunities for future design cycles.

Druggability of the enzymes of the non-mevalonate-pathway

The non-mevalonate pathway constitutes a source of novel drug targets. This biosynthetic route is essential for pathogens but is absent in humans. Our systematic evaluation of the druggability of all enzymes provides a convenient way of selecting targets that should be most easily inhibited by small-molecule drugs. We found that not every target is equally druggable and we identified novel, druggable, potentially allosteric sites. These results should accelerate the development of anti-infective drugs with a novel mode of action, which are needed ever more urgently in light of the rapid emergence of drug-resistant strains.

The isoprenoid-precursor dependence of Plasmodium spp.

Due to the increase in resistance of Plasmodium spp. against available antimalarials, there is a need for new, effective and innovative drugs. The non-mevalonate pathway for the biosynthesis of the universal isoprenoid precursors, which is absent in humans, is suggested as an attractive source of targets for such drugs with a novel mode of action. The biological importance of this pathway to Plasmodium spp. is proven by the efficacy of the clinical candidate fosmidomycin, which inhibits the biosynthesis of isoprenoid precursors; it is, however, less clear which isoprenoid end products are essential for parasite survival. In this Highlight, we identify protein prenylation, isoprene-containing quinone production, N-linked glycosylation as well as carotenoid and vitamin-E biosynthesis as probably essential isoprenoid-dependent physiological processes in Plasmodium. Inhibition of any of these processes blocks parasite development. Furthermore, both protein prenylation of SNARE proteins and a protein tyrosine phosphatase as well as tRNA prenylation have been identified as isoprene-dependent processes for which the physiological role in Plasmodium remains unclear. Therefore, the biosynthetic route to the isoprenoid precursors presents attractive drug targets for the development of antimalarials with novel modes of action.

A Natural-Product Switch for a Dynamic Protein Interface**

Small ligands are a powerful way to control the function of protein complexes via dynamic binding interfaces. The classic example is found in gene transcription where small ligands regulate nuclear receptor binding to coactivator proteins via the dynamic activation function 2 (AF2) interface. Current ligands target the ligand-binding pocket side of the AF2. Few ligands are known, which selectively target the coactivator side of the AF2, or which can be selectively switched from one side of the interface to the other. We use NMR spectroscopy and modeling to identify a natural product, which targets the retinoid X receptor (RXR) at both sides of the AF2. We then use chemical synthesis, cellular screening and X-ray co-crystallography to split this dual activity, leading to a potent and molecularly efficient RXR agonist, and a first-of-kind inhibitor selective for the RXR/coactivator interaction. Our findings justify future exploration of natural products at dynamic protein interfaces.