The compounds from the Maybridge Rule of 3 library that show >70% inhibition of TbCK.
1. Introduction
Infectious diseases caused by parasitic protozoa affect approximately 15% of the global population, and more than 65% of the population in the Third and developing world, yet current drug therapies for protozoal infections are woefully inadequate. As protozoal infections take their toll predominantly in the developing world, market forces are insufficient to promote the development of novel anti-protozoal drugs. In 2000, only ca. 0.1% of global investment in health research was spent on drug discovery for tropical diseases [1].
One such neglected parasitic disease is Human African Trypanosomiasis (HAT) or African sleeping sickness, which is caused by the protozoan parasite
Treatment of HAT is dependent upon four drugs: suramin, melarsoprol, pentamidine and eflornithine.These therapies are often toxic, difficult to administer and increasingly have an acquired drug resistance [4, 5].Developed before the 1950s suramin and melarsoprol are used for chemotherapy of early stages of the disease, as is pentamidine. The arsenical melarsoprol is extremely toxic, with death for ~1 in 20 of cases and treatment failures as high as 30% in certain areas [4, 6]. Treatment of the second stage of the disease, where the parasites cross the blood-brain barrier and invade the central nervous system, is limited to melarsoprol and eflornithine [7].The WHO as a desperate measure recently introduced nifurtimox-eflornithine combination therapy for the treatment of HAT. This is despite nifurtimox, a drug often used to treat Chagas' disease (caused by the related protozoan the South American
Hence there is an urgent need for new, more effective, less toxic, cheap and easy to administer therapeutic agents to treat African sleeping sickness and other closely related parasitic diseases, e.g. Chagas’ disease and Leishmaniasis, whose current treatments suffer from similar limitations.
Phospholipids account for ~80% of total lipids in
Most eukaryotes have three alternative pathways by which PC can be synthesised [19 and reviewed in 20]. The first two pathways both involve three consecutive methylations of PE by S-adenosyl-L-methionemethyltransferases [20]. The PE can be derived from two alternative pathways, either from the concerted actions of the CDP-DAG dependantphosphatidylserine synthase and phosphatidylserine decarboxylase, or via the CDP-ethanolamine branch of the Kennedy pathway. This involves phosphorylation of ethanolamine by an ethanolamine kinase, its activation to CDP-ethanolamine by an ethanolamine-phosphate cytidyltransferase and its transfer to diacylglycerol by an ethanolamine phosphotransferase. The presence of this branch of the Kennedy pathway was demonstrated in
The trypanosomal genomes have revealed that
The third alternative pathway for
Collectively this evidence of an absence of redundancy of
In this study we interrogate ~630 compounds of the Maybridge Rule of 3 Fragment Library for compounds that interact with, and inhibit
2. Experimental
2.1. Materials
All materials unless stated were purchased either from Sigma/Aldrich or Invitrogen. An in house Maybridge Rule of 3 Fragment Library kept in master plates at 200 mM in DMSO (100%), was transferred into working plates with compounds occupying the central 80 wells of a 96-well plate, at 10 mM in 5% DMSO, allowing the two outside columns for positive and negative controls.
2.2. Recombinant expression and purification of Tb CK
Large-scale recombinant expression and purification of
Briefly, pelleted cells were suspended in buffer A (50 mMTris/HCl, pH 8.0, 300 mMNaCl and 10 mM imidazole) and lysed in the presence of DNase I by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTrap™ FF crude column column (GE Healthcare) pre-loaded with Ni2+. Unbound proteins were removed by washing the column with 15 column volumes of buffer A containing 32.5 mM imidazole and
Alternatively, pelleted cells were suspended in 50 mMTris/HCl, pH 8.0, 300 mMNaCl and 5 mM imidazole and lysed by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTALON Cartridge (Clontech).Unbound proteins were removed by washing the column with 10 column volumes of loading buffer,
Typical yields were > 10 mg per litre of bacterial culture,
2.3. T. brucei choline kinase activity assay
High throughput screening of the Maybridge Rule of 3 Fragment Library was carried out at a final test concentration of 0.5 mM in 96-well plates (final assay volume 200 μl) using a spectrophotometric assay that has been described previously [23]. The screened library working plates consisted of compounds arrayed in 96 well plates at 10 mM in 5% DMSO; columns 1 and 12 contained 5% DMSO only. For high throughput screening, 10 µl from each well of the working plates was added to 110 µl of buffer containing 50 mM MOPS (pH 7.8), 150 mMKCl and 6 mM MgCl2. 3 µg of purified
2.4. Differential scanning fluorimetry with Tb CK
Differential scanning fluorimetry was set up in 96 well PCR plates using a reaction volume of 100 µL. Samples contained 2.1 µM
Differential fluorimetric scans were performed in a realtime PCR machine (Stratagene Mx3005P with software MxPro v 4.01) using a temperature scan from 25°C to 95°C at 0.5°C min-1.Data were then exported to Excel for analysis using “DSF analysis” modified from the template provided by Niesen et al. [38].Tm values were calculated by non-linear regression, fitting the Boltzmann equation to the denaturation curves using GraFit.
3. Results and discussion
Screening for inhibitors of the genetically validated drug target
An alternative approach for screening is differential scanning fluorimetry (Figure 2), allowing identification of compounds that interact with the
Initially
Differential fluorimetric scans were performed and analysed as described in Experimental.
An alternative approach for screening is differential scanning fluorimetry (Figure 2), allowing identification of compounds that interact with the
Initially
The respective controls in both assay types allowed Z-factors to be determined for all of the plates screened (Figure 3). Both the coupled enzyme activity assay and the thermal shift analysis showed Z-factors to be above 0.5 for all plates, except for plate 5 for the thermal shift assay (but still above 0.45), this is indicative of good reliable assays, with meaningful results [41].
The MayBridge Rule of 3 Fragment Library was distributed over 9 plates (80 compounds per plate) providing space for adequate positive and negative controls, allowing Z-factors to be determined. This was done for each plate for both the choline kinase assay (+) and thermal shift analysis (x). A Z-factor above 0.4 is acceptable and validates the data on that plate as being reliable.
The ~630 compounds from the MayBridge Rule of 3 Fragment Library were assessed for their ability to inhibit the
It is worth noting the lack of false positives arising from significant inhibition of either of the coupling enzymes, pyruvate kinase and lactate dehydrogenase this is encouraging when screening other ATP utilizing enzymes.
Thermal shift analysis of
Several drug discovery style studies have shown that an increase in the thermal stability of a protein is proportional to the concentration and affinity of the ligand to the protein in keeping with the equilibrium associated with ligand-protein binding [38, 41-44]. On those occasions where this interaction destabilizes a protein, i.e. lowering Tm, a thermodynamic model has been proposed which explains the how the same ligand can stabilise and destabilise different proteins [42]. While the same protein may be stabilized and destabilized by very similar ligands, this was exquisitely demonstrated by the changes in thermal stability of Acyl-CoA thioesterase, upon incubation with either CoA (destabilise) or Acyl-CoA (stabilise) [45].
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68 | 4651-82-5 |
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2-aminothiophene-3-carbonitrile | 101 ± 3 | ~758 | 40 | -2.15 ± 0.08 | 10.3 ± 11 |
95 | 933-67-5 |
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7-methyl-1H-indole | 84 ± 4 | ~380 | 49 | -2.16 ± 0.14 | 4.1 ± 5.2 |
257 | 59147-02-3 |
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4-(2-furyl)aniline | 80 ± 3 | ~234 | 63 | 0.46 ± 0.08 | 9.3 ± 8.5 |
278 | 199590-00-6 |
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(1-methyl-1H-indol-6-yl)methanol | 101 ± 2 | 25.45 ±1.16 | 28 | 0.26 ± 0.09 | 8.8 ± 10.8 |
320 | 39270-39-8 |
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2,3-dihydro-1,4-benzodioxin-6-ylmethanol | 6 ± 4 | ND | ND | -0.17 ± 0.07 | 63.1 ± 9.5 |
346 | 57976-57-5 |
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3-pyridin-3-ylaniline | 100 ± 0 | 12.35 ± 0.64 | 68 | -1.18 ± 0.06 | 15.6 ± 9.3 |
372 | 143426-51-1 |
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[4-(1H-pyrrol-1-yl) phenyl] methanol |
80 ± 1 | 109.7 ± 10.6 | 26 | 1.24 ± 0.04 | 27 ± 14 |
565 | 57634-55-6 |
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4-(2-amino-1,3-thiazol-4-yl)phenol | 100 ± 0 | ~120 | 85 | -8.91 ± 0.49 | 28.7±19.3 |
635 | 64354-50-3 |
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5-methyl-2-phenyl-3-furoic acid | 23 ± 4 | ND | ND | -2.94 ± 0.16 | 20.5 ± 7.4 |
All of the compounds in the two data sets (the coupled enzyme activity assay and the thermal shift analysis), were compared to assess any correlation between the two very different methods. In other words looking for compounds that showed a significant change in Tm and a significant inhibition in
Of the compounds identified that alter the Tm of
This raises an interesting question, is it a viable option to target compounds that specifically destabilise an enzyme, causing a decrease in enzyme activity? One could argue this is exactly what pharmaceutical companies are focusing their research efforts upon, but with a slightly different approach. Some of their therapeutic targets rely on finding compounds that disrupt various interactions; hetero- or homo-oligomeric protein-protein, DNA-binding protein and RNA polymerases, many of these are associated with signaling events. Success stories include the identification of HDM2 antagonists associated with P53 activation [46], the identification of anti-cancer agent for the BCL-XL protein-protein complex and several others, reviewed by Wells and McClendon [47] and more recently by Coyne and colleagues [48].
The techniques utilized to study the formation / disruption of protein-protein complexes are driven by high throughput drug discovery, including fragment based approaches, these include X-ray crystallography, NMR, dynamic light scattering, differential static light scattering, differential scanning fluorimetry [42-50].
In summary, destabilisation by a ligand could affect the oligomeric state of a protein, or in the case of a monomer disrupt intra-molecular interactions, i.e. between stacking α-helixes or β-sheets, causing partial unfolding and thus destabilisation.In the case of
As it was clear that compounds that inhibit
Compounds from the May Bridge Rule of 3 Fragment Library with greater than 70% inhibition (dotted line) of the TbCK enzyme activity are circled and numbered. Numbers correspond to arbitrary compound library numbers; see Table 1 for chemical structures and extra data.
IC50 values were determined for these compounds (Table 1, Figure 7) ranging from 100s of μM to low μM. For example, compound 278 (1-methyl-(1H-indol-6-yl)methanol) has an IC50 value of 25.45 ± 1.16 μM however the selectivity index is not very good, while compound 346 (3-pyridin-3-ylaniline) has an IC50 value of 12.35 ± 0.64 μM with a high selectivity index.
One of the strengths of the Maybridge Rule of 3 Fragment Library is the chemical diversity, additionally a range of analogous structures can normally be found within it allowing initial structure activity relationships to be formulated. There are several close analogues of compound 278 (1-methyl-(1H-indol-6-yl)methanol), which highlight that the N-methyl indole moiety seems necessary to have any
Another analogue, 1-Methyl-1H-indole (CHEMBL19912) has been shown to interact with human intracellular adhesion molecules and highlights the importance of selectivity [53]. 1H-indol-5-yl-methanol (CHEMBL1650258) has previously been screened against
For the relatively simple compound 346 (3-pyridin-3-ylaniline), there are several analogous structures in the library, including compound 262 (2-(1H-imidazo-1-yl)aniline) which shows ~55%
4. Conclusions
In this study, screening of a comparatively small fragment library by two different screening methods has allowed identification of several compounds that interact with and inhibit
This study highlights that if faced with a drug target that is problematic to screen, prior thermal shift analysis could significantly triage the number of compounds to be screened, thereby significantly increasing the potential to identify lead compounds. This approach obviously has the limitation that potential inhibitors could be missed if they do not significantly alter the Tm of the protein.
Future follow up work with
The ultimate goal is to identify new easy to make, affordable, easy to administer, drugs in the fight against African sleeping sickness and other closely related protozoan transmitted Third World diseases.
Acknowledgements
This work was supported in part by a Wellcome Trust Senior Research Fellowship (067441) and Wellcome Trust project grants 086658 and 093228. We thank the late and sorely missed Dr Rupert Russell (St Andrews), supported by SUSLA, for access to the May Bridge Rule of 3 Fragment Library.
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