Ruth Brenk

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for ∼30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target—T. brucei N-myristoyltransferase—leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.

Lessons Learnt from Assembling Screening Libraries for Drug Discovery for Neglected Diseases

To enable the establishment of a drug discovery operation for neglected diseases, out of 2.3 million commercially available compounds 222 552 compounds were selected for an in silico library, 57 438 for a diverse general screening library, and 1 697 compounds for a focused kinase set. Compiling these libraries required a robust strategy for compound selection. Rules for unwanted groups were defined and selection criteria to enrich for lead‐like compounds which facilitate straightforward structure–activity relationship exploration were established. Further, a literature and patent review was undertaken to extract key recognition elements of kinase inhibitors (“core fragments”) to assemble a focused library for hit discovery for kinases. Computational and experimental characterisation of the general screening library revealed that the selected compounds 1) span a broad range of lead‐like space, 2) show a high degree of structural integrity and purity, and 3) demonstrate appropriate solubility for the purposes of biochemical screening. The implications of this study for compound selection, especially in an academic environment with limited resources, are considered.

Nucleophilic catalysis of acylhydrazone equilibration for protein-directed dynamic covalent chemistry

Dynamic covalent chemistry uses reversible chemical reactions to set up an equilibrating network of molecules at thermodynamic equilibrium, which can adjust its composition in response to any agent capable of altering the free energy of the system. When the target is a biological macromolecule, such as a protein, the process corresponds to the protein directing the synthesis of its own best ligand. Here, we demonstrate that reversible acylhydrazone formation is an effective chemistry for biological dynamic combinatorial library formation. In the presence of aniline as a nucleophilic catalyst, dynamic combinatorial libraries equilibrate rapidly at pH 6.2, are fully reversible, and may be switched on or off by means of a change in pH. We have interfaced these hydrazone dynamic combinatorial libraries with two isozymes from the glutathione S-transferase class of enzyme, and observed divergent amplification effects, where each protein selects the best-fitting hydrazone for the hydrophobic region of its active site.

Discovery of a novel class of orally active trypanocidal N-myristoyltransferase inhibitors.

N-Myristoyltransferase (NMT) represents a promising drug target for human African trypanosomiasis (HAT), which is caused by the parasitic protozoa Trypanosoma brucei. We report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which has potent activity against the enzyme (IC50 = 2 nM) and T. brucei (EC50 = 2 nM) in culture. The compound has good oral pharmacokinetics and cures rodent models of peripheral HAT infection. This compound provides an excellent tool for validation of T. brucei NMT as a drug target for HAT as well as a valuable lead for further optimization.

Nucleosomes can invade DNA territories occupied by their neighbors

Nucleosomes are the fundamental subunits of eukaryotic chromatin. They are not static entities, but can undergo a number of dynamic transitions, including spontaneous repositioning along DNA. As nucleosomes are spaced close together within genomes, it is likely that on occasion they approach each other and may even collide. Here we have used a dinucleosomal model system to show that the 147-base-pair (bp) DNA territories of two nucleosomes can overlap extensively. In the situation of an overlap by 44 bp or 54 bp, one histone dimer is lost and the resulting complex can condense to form a compact single particle. We propose a pathway in which adjacent nucleosomes promote DNA unraveling as they approach each other and that this permits their 147-bp territories to overlap, and we suggest that these events may represent early steps in a pathway for nucleosome removal via collision.

One Scaffold, Three Binding Modes: Novel and Selective Pteridine Reductase 1 Inhibitors Derived from Fragment Hits Discovered by Virtual Screening

The enzyme pteridine reductase 1 (PTR1) is a potential target for new compounds to treat human African trypanosomiasis. A virtual screening campaign for fragments inhibiting PTR1 was carried out. Two novel chemical series were identified containing aminobenzothiazole and aminobenzimidazole scaffolds, respectively. One of the hits (2-amino-6-chloro-benzimidazole) was subjected to crystal structure analysis and a high resolution crystal structure in complex with PTR1 was obtained, confirming the predicted binding mode. However, the crystal structures of two analogues (2-amino-benzimidazole and 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole) in complex with PTR1 revealed two alternative binding modes. In these complexes, previously unobserved protein movements and water-mediated protein−ligand contacts occurred, which prohibited a correct prediction of the binding modes. On the basis of the alternative binding mode of 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole, derivatives were designed and selective PTR1 inhibitors with low nanomolar potency and favorable physicochemical properties were obtained.

Novel Ligands for a Purine Riboswitch Discovered by RNA-Ligand Docking

Summary The increasing number of RNA crystal structures enables a structure-based approach to the discovery of new RNA-binding ligands. To develop the poorly explored area of RNA-ligand docking, we have conducted a virtual screening exercise for a purine riboswitch to probe the strengths and weaknesses of RNA-ligand docking. Using a standard protein-ligand docking program with only minor modifications, four new ligands with binding affinities in the micromolar range were identified, including two compounds based on molecular scaffolds not resembling known ligands. RNA-ligand docking performed comparably to protein-ligand docking indicating that this approach is a promising option to explore the wealth of RNA structures for structure-based ligand design.

DrugPred: A Structure-Based Approach To Predict Protein Druggability Developed Using an Extensive Nonredundant Data Set

Judging if a protein is able to bind orally available molecules with high affinity, i.e. if a protein is druggable, is an important step in target assessment. In order to derive a structure-based method to predict protein druggability, a comprehensive, nonredundant data set containing crystal structures of 71 druggable and 44 less druggable proteins was compiled by literature search and data mining. This data set was subsequently used to train a structure-based druggability predictor (DrugPred) using partial least-squares projection to latent structures discriminant analysis (PLS-DA). DrugPred performed well in discriminating druggable from less druggable binding sites for both internal and external predictions. The method is robust against conformational changes in the binding site and outperforms previously published methods. The superior performance of DrugPred is likely due to the size and composition of the training set which, in contrast to most previously developed methods, only contains cavities that have evolved to bind a natural ligand.

Identification of Inhibitors of the Leishmania cdc2-Related Protein Kinase CRK3

New drugs are urgently needed for the treatment of tropical parasitic diseases such as leishmaniasis and human African trypanosomiasis (HAT). This work involved a high‐throughput screen of a focussed kinase set of ∼3400 compounds to identify potent and parasite‐selective inhibitors of an enzymatic Leishmania CRK3–cyclin 6 complex. The aim of this study is to provide chemical validation that Leishmania CRK3–CYC6 is a drug target. Eight hit series were identified, of which four were followed up. The optimisation of these series using classical SAR studies afforded low‐nanomolar CRK3 inhibitors with significant selectivity over the closely related human cyclin dependent kinase CDK2.

Here Be Dragons: Docking and Screening in an Uncharted Region of Chemical Space

To compare virtual and high-throughput screening in an unbiased way, 50,000 compounds were docked into the 3-dimensional structure of dihydrofolate reductase prospectively, and the results were compared to a subsequent experimental screening of the same library. Undertaking these calculations demanded careful database curation and control calculations with annotated inhibitors. These ultimately led to a ranked list of more likely and less likely inhibitors and to the prediction that relatively few inhibitors would be found in the empirical screen. The latter prediction turned out to be correct, with arguably no validated inhibitors found experimentally. Subsequent retesting of high-scoring docked molecules may have found 2 true inhibitors, although this remains uncertain due to experimental ambiguities. The implications of this study for screening campaigns are considered.