Julie A. Frearson

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.

A novel multiple-stage antimalarial agent that inhibits protein synthesis

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

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.

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.

HTS and hit finding in academia – from chemical genomics to drug discovery

The liaison between academia and the pharmaceutical industry was originally served primarily through the scientific literature and limited, specific industry–academia partnerships. Some of these partnerships have resulted in drugs on the market, such as Vorinostat (Memorial Sloan-Kettering Cancer Centre and Merck) and Tenofovir (University of Leuven; Institute of Organic Chemistry and Biochemistry, Czech Republic; and GlaxoSmithKline), but the timescales from concept to clinic have, in most cases, taken many decades. We now find ourselves in a world in which the edges between these sectors are more blurred and the establishment and acceptance of high-throughput screening alongside the wider concept of ‘hit discovery’ in academia provides one of the key platforms required to enable this sector to contribute directly to addressing unmet medical need.

Dihydroquinazolines as a Novel Class of Trypanosoma brucei Trypanothione Reductase Inhibitors: Discovery, Synthesis, and Characterization of their Binding Mode by Protein Crystallography

Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR–ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.

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.

Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay.

ABSTRACT Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

Identification of a κ-opioid agonist as a potent and selective lead for drug development against human African trypanosomiasis

Graphical abstract Phenotypic screening of the LOPAC library identified several potent and selective inhibitors of African trypanosomes. The κ-opioid agonist (+)-U50,488 represents a novel lead for drug discovery against sleeping sickness.

Chemical Validation of Trypanothione Synthetase A POTENTIAL DRUG TARGET FOR HUMAN TRYPANOSOMIASIS

In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 × EC50 for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.