Human African trypanosomiasis (HAT) is a life-threatening disease with approximately 30 000–40 000 new cases each year. Trypanosoma brucei protein kinase GSK3 short (TbGSK3) is required for parasite growth and survival. Herein we report a screen of a focused kinase library against T. brucei GSK3. From this we identified a series of several highly ligand-efficient TbGSK3 inhibitors. Following the hit validation process, we optimised a series of diaminothiazoles, identifying low-nanomolar inhibitors of TbGSK3 that are potent in vitro inhibitors of T. brucei proliferation. We show that the TbGSK3 pharmacophore overlaps with that of one or more additional molecular targets.
antiprotozoal agents; GSK3; medicinal chemistry; protein kinases; Trypanosoma brucei
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.
The bifunctional enzyme methylenetetrahydrofolate dehydrogenase – cyclohydrolase (FolD) is identified as a potential drug target in Gram-negative bacteria, in particular the troublesome Pseudomonas aeruginosa. In order to provide a comprehensive and realistic assessment of the potential of this target for drug discovery we generated a highly efficient recombinant protein production system and purification protocol, characterized the enzyme, carried out screening of two commercial compound libraries by differential scanning fluorimetry, developed a high-throughput enzyme assay and prosecuted a screening campaign against almost 80,000 compounds. The crystal structure of P. aeruginosa FolD was determined at 2.2 Å resolution and provided a template for an assessment of druggability and for modelling of ligand complexes as well as for comparisons with the human enzyme. New FolD inhibitors were identified and characterized but the weak levels of enzyme inhibition suggest that these compounds are not optimal starting points for future development. Furthermore, the close similarity of the bacterial and human enzyme structures suggest that selective inhibition might be difficult to attain. In conclusion, although the preliminary biological data indicates that FolD represents a valuable target for the development of new antibacterial drugs, indeed spurred us to investigate it, our screening results and structural data suggest that this would be a difficult enzyme to target with respect to developing the appropriate lead molecules required to underpin a serious drug discovery effort.
CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens.
Quinols have been developed as a class of potential anti-cancer compounds. They are thought to act as double Michael acceptors, forming two covalent bonds to their target protein(s). Quinols have also been shown to have activity against the parasite Trypanosoma brucei, the causative organism of human African trypanosomiasis, but they demonstrated little selectivity over mammalian MRC5 cells in a counter-screen. In this paper, we report screening of further examples of quinols against T. brucei. We were able to derive an SAR, but the compounds demonstrated little selectivity over MRC5 cells. In an approach to increase selectivity, we attached melamine and benzamidine motifs to the quinols, because these moieties are known to be selectively concentrated in the parasite by transporter proteins. In general these transporter motif-containing analogues showed increased selectivity; however they also showed reduced levels of potency against T. brucei.
Inhibitors; Medicinal chemistry; Trypanosoma brucei; P2 transporter; Quinols
N-Myristoyltransferase (NMT) represents
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.
As the pharmaceutical industry continues to re-strategise and focus on low-risk, relatively short term gains for the sake of survival, we need to re-invigorate the early stages of drug discovery and rebalance efforts towards novel modes of action therapeutics and neglected genetic and tropical diseases. Academic drug discovery is one model which offers the promise of new approaches and an alternative organisational culture for drug discovery as it attempts to apply academic innovation and thought processes to the challenge of discovering drugs to address real unmet need.
academia; drug discovery; neglected disease; novel target
Chitin is an essential structural component of the fungal cell wall. Chitinases are thought to be important for fungal cell wall remodelling, and inhibition of these enzymes has been proposed as a potential strategy for development of novel anti-fungals. The fungal pathogen Aspergillus fumigatus possesses two distinct multi-gene chitinase families. Here we explore acetazolamide as a chemical scaffold for the inhibition of an A. fumigatus ‘plant-type’ chitinase. A co-crystal structure of AfChiA1 with acetazolamide was used to guide synthesis and screening of acetazolamide analogues that yielded SAR in agreement with these structural data. Although acetazolamide and its analogues are weak inhibitors of the enzyme, they have a high ligand efficiency and as such are interesting leads for future inhibitor development.
Chitinase; Aspergillus fumigatus
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.
A resazurin-based cell viability assay was developed for phenotypic screening of the LOPAC 1280 ‘library of pharmacologically active compounds’ against bloodstream forms of Trypanosoma brucei in vitro identifying 33 compounds with EC50 values <1 μM. Counter-screening vs. normal diploid human fibroblasts (MRC5 cells) was used to rank these hits for selectivity, with the most potent (<70 nM) and selective (>700-fold) compounds being suramin and pentamidine. These are well-known antitrypanosomal drugs which demonstrate the robustness of the resazurin cell viability assay. The most selective novel inhibitor was (+)-trans-(1R,2R)-U50,488 having an EC50 value of 60 nM against T. brucei and 270-fold selectivity over human fibroblasts. Interestingly, (−)-U50,488, a known CNS-active κ-opioid receptor agonist and other structurally related compounds were >70-fold less active or inactive, as were several μ- and κ-opioid antagonists. Although (+)-U50,488 was well tolerated by the oral route and displayed good pharmaceutical properties, including high brain penetration, the compound was not curative in the mouse model of infection. Nonetheless, the divergence of antinociceptive and antitrypanosomal activity represents a promising start point for further exploratory chemistry. Bioinformatic studies did not reveal any obvious candidate opioid receptors and the target of this cytostatic compound is unknown. Among the other potent, but less selective screening hits were compound classes with activity against protein kinases, topoisomerases, tubulin, as well as DNA and energy metabolism.
Phenotypic screening; African trypanosomiasis; Target identification; Target validation; U50,488
Drug discovery is a high-risk, expensive and lengthy process taking at least 12 years and costing upwards of US$500 million per drug to reach the clinic. For neglected diseases, the drug discovery process is driven by medical need and guided by pre-defined target product profiles. Assessment and prioritisation of the most promising targets for entry into screening programmes is crucial for maximising chances of success. Here we describe criteria used in our drug discovery unit for target assessment and introduce the ‘traffic light’ system as a prioritisation and management tool. We hope this brief review will stimulate basic scientists to acquire additional information necessary for drug discovery.
African sleeping sickness or human African trypanosomiasis (HAT), caused by Trypanosoma brucei spp., is responsible for ~30,000 deaths each year. Available treatments for this neglected 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 discovery of associated lead compounds with potential to address this unmet need. Inhibition of this target, T. brucei N-myristoyltransferase (TbNMT), 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 very promising pharmaceutical properties and represent an exciting opportunity to develop oral drugs to treat this devastating disease. Our studies validate TbNMT as a promising therapeutic target for HAT.
Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.
Phosphorylation/Kinases/Serine-Threonine; Signal Transduction/Protein Kinases/Serine/Threonine; Cell Division; Enzyme Kinetics; Parasitology; RNA Interference (RNAi); Trypanosoma brucei; Cytokinesis; Drug Target; NDR Kinase
Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase–thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.
Drug discovery; Screening; Pteridine reductase; Dihydrofolate reductase
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.
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 188.8.131.52; 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.
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.
compound selection; high-throughput screening; kinase inhibitors; neglected diseases; virtual screening
To enable the establishment of a drug discovery operation for neglected diseases, out of 2.3 million commercially available compounds 222552 compounds were selected for an in silico library, 57438 for a diverse general screening library, and 1697 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.
compound selection; high-throughput screening; kinase inhibitors; neglected diseases; virtual screening
Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process.
There is an urgent need for new drugs for the treatment of tropical parasitic diseases such as human African trypanosomiasis, which is caused by Trypanosoma brucei. The enzyme trypanothione reductase (TryR) is a potential drug target within these organisms. Herein we report the screening of a 62000 compound library against T. brucei TryR. Further work was undertaken to optimise potency and selectivity of two novel-compound series arising from the enzymatic and whole parasite screens and mammalian cell counterscreens. Both of these series, containing either a quinoline or pyrimidinopyrazine scaffold, yielded low micromolar inhibitors of the enzyme and growth of the parasite. The challenges of inhibiting TryR with druglike molecules is discussed.
human African trypanosomiasis; pyrimidopyridazines; quinolines; trypanosoma brucei; trypanothione reductase
Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP (1, 1-(1-benzo[b]thiophen-2-yl-cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (Ki=1 μm) and biologically active against bloodstream T. brucei (EC50=10 μm), but with poor selectivity against mammalian MRC5 cells (EC50=29 μm). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.
BTCP; inhibitors; medicinal chemistry; trypanosoma brucei; trypanothione reductase
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.
Genetic studies indicate that the enzyme pteridine reductase 1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimisation of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivatives. Data are reported on 33 compounds. This series was initially discovered by a virtual screening campaign (J. Med. Chem., 2009, 52, 4454). The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chemistry and structure-based approaches, we were able to derive compounds with potent activity against T. brucei PTR1 (=7 nm), which had high selectivity over both human and T. brucei dihydrofolate reductase. Unfortunately, these compounds displayed weak activity against the parasites. Kinetic studies and analysis indicate that the main reason for the lack of cell potency is due to the compounds having insufficient potency against the enzyme, which can be seen from the low Km to Ki ratio (Km=25 nm and Ki=2.3 nm, respectively).
antiprotozoal agents; drug discovery; pteridine reductase; structure-based drug design; Trypanosoma brucei
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.
Screening of the Sigma–Aldrich Library of Pharmacologically Active Compounds (LOPAC) against cultured Trypanosoma brucei, the causative agent of African sleeping sickness, resulted in the identification of a number of compounds with selective antiproliferative activity over mammalian cells. These included (+)-(1R,2R)-U50488, a weak opioid agonist with an EC50 value of 59 nm as determined in our T. brucei in vitro assay reported previously. This paper describes the modification of key structural elements of U50488 to investigate structure–activity relationships (SAR) and to optimise the antiproliferative activity and pharmacokinetic properties of this compound.
antiprotozoal agents; human African trypanosomiasis; medicinal chemistry; U50488
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.
CRK3; cyclin-dependent cdc2-related kinases; leishmaniasis; triazolopyridines; ureas