the chemical space coverage in commercial fragment screening collections
revealed the overlap between bioactive medicinal chemistry substructures
and rule-of-three compliant fragments is only ∼25%. We recommend
including these fragments in fragment screening libraries to maximize
confidence in discovering hit matter within known bioactive chemical
space, while incorporation of nonoverlapping substructures could offer
novel hits in screening libraries. Using principal component analysis,
polar and three-dimensional substructures display a higher-than-average
enrichment of bioactive compounds, indicating increasing representation
of these substructures may be beneficial in fragment screening.
Protein kinases constitute an attractive
family of enzyme targets
with high relevance to cell and disease biology. Small molecule inhibitors
are powerful tools to dissect and elucidate the function of kinases
in chemical biology research and to serve as potential starting points
for drug discovery. However, the discovery and development of novel
inhibitors remains challenging. Here, we describe a structure-based de novo design approach that generates novel, hinge-binding
fragments that are synthetically feasible and can be elaborated to
small molecule libraries. Starting from commercially available compounds,
core fragments were extracted, filtered for pharmacophoric properties
compatible with hinge-region binding, and docked into a panel of protein
kinases. Fragments with a high consensus score were subsequently short-listed
for synthesis. Application of this strategy led to a number of core
fragments with no previously reported activity against kinases. Small
libraries around the core fragments were synthesized, and representative
compounds were tested against a large panel of protein kinases and
subjected to co-crystallization experiments. Each of the tested compounds
was active against at least one kinase, but not all kinases in the
panel were inhibited. A number of compounds showed high ligand efficiencies
for therapeutically relevant kinases; among them were MAPKAP-K3, SRPK1,
SGK1, TAK1, and GCK for which only few inhibitors are reported in
The small molecule blebbistatin is now a front line tool in the study of myosin function. Chemical modification of the tricyclic core of blebbistatin could deliver the next generation of myosin inhibitors and to help address this we report here on the impact of structural changes in the methyl-substituted aromatic ring of blebbistatin on its biological activity. Chemical methods for the preparation of isomeric methyl-containing analogues are reported and a series of co-crystal structures are used to rationalise the observed variations in their biological activity. These studies further support the view that the previously identified binding mode of blebbistatin to Dictyostelium discoideum myosin II is of relevance to its mode of action. A discussion of the role that these observations have on planning the synthesis of focused libraries of blebbistatin analogues is also provided including an assessment of possibilities by computational methods. These studies are ultimately directed at the development of novel myosin inhibitors with improved affinity and different selectivity profiles from blebbistatin itself.
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
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.
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.
The ChEMBL database was mined to efficiently assemble an ion channel-focused screening library. The compiled library consists of 3241 compounds representing 123 templates across nine ion channel categories. Compounds in the screening library are annotated with their respective ion channel category to facilitate back-tracing of prospective molecular targets from phenotypic screening results. The established workflow is adaptable to the construction of focused screening libraries for other therapeutic target classes with diverse recognition motifs.
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.
► Using RNA-ligand docking, four new ligands were discovered for a purine riboswitch ► Two of the ligands were based on scaffolds not known to bind to this riboswitch ► Crystal structures were determined that confirm the binding modes of new ligands ► Molecular docking is a promising method for RNA-structure-based ligand design
A model binding site was used to investigate charge–charge interactions in molecular docking. This simple site, a small (180 Å3) engineered cavity in cyctochrome c peroxidase (CCP), is negatively charged and completely buried from solvent, allowing us to explore the balance between electrostatic energy and ligand desolvation energy in a system where many of the common approximations in docking do not apply. A database with about 5300 molecules was docked into this cavity. Retrospective testing with known ligands and decoys showed that overall the balance between electrostatic interaction and desolvation energy was captured. More interesting were prospective docking scre”ens that looked for novel ligands, especially those that might reveal problems with the docking and energy methods. Based on screens of the 5300 compound database, both high-scoring and low-scoring molecules were acquired and tested for binding. Out of 16 new, high-scoring compounds tested, 15 were observed to bind. All of these were small heterocyclic cations. Binding constants were measured for a few of these, they ranged between 20 μM and 60 μM. Crystal structures were determined for ten of these ligands in complex with the protein. The observed ligand geometry corresponded closely to that predicted by docking. Several low-scoring alkyl amino cations were also tested and found to bind. The low docking score of these molecules owed to the relatively high charge density of the charged amino group and the corresponding high desolvation penalty. When the complex structures of those ligands were determined, a bound water molecule was observed interacting with the amino group and a backbone carbonyl group of the cavity. This water molecule mitigates the desolvation penalty and improves the interaction energy relative to that of the “naked” site used in the docking screen. Finally, six low-scoring neutral molecules were also tested, with a view to looking for false negative predictions. Whereas most of these did not bind, two did (phenol and 3-fluorocatechol). Crystal structures for these two ligands in complex with the cavity site suggest reasons for their binding. That these neutral molecules do, in fact bind, contradicts previous results in this site and, along with the alkyl amines, provides instructive false negatives that help identify weaknesses in our scoring functions. Several improvements of these are considered.
molecular docking; electrostatic; solvation; cyctochrome c peroxidase; X-ray crystallography
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.
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.
We report the identification of novel inhibitors of Trypanosoma brucei 6PGDH enzyme by virtual fragment screening.
The enzyme 6-phosphogluconate dehydrogenase is a potential drug target for the parasitic protozoan Trypanosoma brucei, the causative organism of human African trypanosomiasis. This enzyme has a polar active site to accommodate the phosphate, hydroxyl and carboxylate groups of the substrate, 6-phosphogluconate. A virtual fragment screen was undertaken of the enzyme to discover starting points for the development of inhibitors which are likely to have appropriate physicochemical properties for an orally bioavailable compound. A virtual screening library was developed, consisting of compounds with functional groups that could mimic the phosphate group of the substrate, but which have a higher pKa. Following docking, hits were clustered and appropriate compounds purchased and assayed against the enzyme. Three fragments were identified that had IC50 values in the low micromolar range and good ligand efficiencies. Based on these initial hits, analogues were procured and further active compounds were identified. Some of the fragments identified represent potential starting points for a medicinal chemistry programme to develop potent drug-like inhibitors of the enzyme.
Virtual fragment screening; Trypanosoma brucei; 6-Phosphogluconate dehydrogenase
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. Since nucleosomes are spaced close together within genomes it is likely that on occasion they approach each other and or collide. Here we have used a dinucleosomal model system to show that the 147bp 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 147bp territories to overlap. These may represent early steps in a pathway for nucleosome removal via collision.
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
The nonmevalonate route to isoprenoid biosynthesis is essential in Gram-negative bacteria and apicomplexan parasites. The enzymes of this pathway are absent from mammals, contributing to their appeal as chemotherapeutic targets. One enzyme, 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), has been validated as a target by genetic approaches in bacteria. Virtual screening against Escherichia coli IspF (EcIspF) was performed by combining a hierarchical filtering methodology with molecular docking. Docked compounds were inspected and 10 selected for experimental validation. A surface plasmon resonance assay was developed and two weak ligands identified. Crystal structures of EcIspF complexes were determined to support rational ligand development. Cytosine analogues and Zn2+-binding moieties were characterized. One of the putative Zn2+-binding compounds gave the lowest measured KD to date (1.92 ± 0.18 μM). These data provide a framework for the development of IspF inhibitors to generate lead compounds of therapeutic potential against microbial pathogens.
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
Molecular docking is widely used to predict novel lead compounds for drug discovery. Success depends on the quality of the docking scoring function, among other factors. An imperfect scoring function can mislead by predicting incorrect ligand geometries or by selecting nonbinding molecules over true ligands. These false-positive hits may be considered “decoys”. Although these decoys are frustrating, they potentially provide important tests for a docking algorithm; the more subtle the decoy, the more rigorous the test. Indeed, decoy databases have been used to improve protein structure prediction algorithms and protein–protein docking algorithms. Here, we describe 20 geometric decoys in five enzymes and 166 “hit list” decoys–i.e., molecules predicted to bind by our docking program that were tested and found not to do so–for β-lactamase and two cavity sites in lysozyme. Especially in the cavity sites, which are very simple, these decoys highlight particular weaknesses in our scoring function. We also consider the performance of five other widely used docking scoring functions against our geometric and hit list decoys. Intriguingly, whereas many of these other scoring functions performed better on the geometric decoys, they typically performed worse on the hit list decoys, often highly ranking molecules that seemed to poorly complement the model sites. Several of these “hits” from the other scoring functions were tested experimentally and found, in fact, to be decoys. Collectively, these decoys provide a tool for the development and improvement of molecular docking scoring functions. Such improvements may, in turn, be rapidly tested experimentally against these and related experimental systems, which are well-behaved in assays and for structure determination.
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. (Journal of Biomolecular Screening 2005:667-674)
high-throughput screening; HTS; virtual screening; molecular docking; database preparation
synthase kinase 3 (GSK3) is a genetically validated drug
target for human African trypanosomiasis (HAT), also called African
sleeping sickness. We report the synthesis and biological evaluation
of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high
selectivity over the off-target human CDK2 and good selectivity over
human GSK3β enzyme, have been prepared. These potent kinase
inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture.
(TbNMT) is an attractive therapeutic
target for the treatment of human African trypanosomiasis (HAT). From
previous studies, we identified pyrazole sulfonamide, DDD85646 (1), a potent inhibitor of TbNMT. Although
this compound represents an excellent lead, poor central nervous system
(CNS) exposure restricts its use to the hemolymphatic form (stage
1) of the disease. With a clear clinical need for new drug treatments
for HAT that address both the hemolymphatic and CNS stages of the
disease, a chemistry campaign was initiated to address the shortfalls
of this series. This paper describes modifications to the pyrazole
sulfonamides which markedly improved blood–brain barrier permeability,
achieved by reducing polar surface area and capping the sulfonamide.
Moreover, replacing the core aromatic with a flexible linker significantly
improved selectivity. This led to the discovery of DDD100097 (40) which demonstrated partial efficacy in a stage 2 (CNS)
mouse model of HAT.
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
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
The efficiency of automated compound
screening is heavily influenced
by the design and the quality of the screening libraries used. We
recently reported on the assembly of one diverse and one target-focused
lead-like screening library. Using data from 15 enzyme-based screenings
conducted using these libraries, their performance was investigated.
Both libraries delivered screening hits across a range of targets,
with the hits distributed across the entire chemical space represented
by both libraries. On closer inspection, however, hit distribution
was uneven across the chemical space, with enrichments observed in
octants characterized by compounds at the higher end of the molecular
weight and lipophilicity spectrum for lead-like compounds, while polar
and sp3-carbon atom rich compounds were underrepresented
among the screening hits. Based on these observations, we propose
that screening libraries should not be evenly distributed in lead-like
chemical space but be enriched in polar, aliphatic compounds. In conjunction
with variable concentration screening, this could lead to more balanced
hit rates across the chemical space and screening hits of higher ligand
efficiency will be captured. Apart from chemical diversity, both screening
libraries were shown to be clean from any pan-assay interference (PAINS)
behavior. Even though some compounds were flagged to contain PAINS
structural motifs, some of these motifs were demonstrated to be less
problematic than previously suggested. To maximize the diversity of
the chemical space sampled in a screening campaign, we therefore consider
it justifiable to retain compounds containing PAINS structural motifs
that were apparently clean in this analysis when assembling screening