**3. Results and discussion**

Screening for inhibitors of the genetically validated drug target *Tb*CK is problematic due to the difficulty in following the reaction either continuously or directly. A direct choline kin‐ ase activity assay assessing the production of phosphocholine, utilising a modified method of Kim *et. al.* [39], using *Tb*CK and radiolabelled choline has been performed previously [23]. However this is not suitable for screening purposes, so choline kinase activity was measured by a spectrophotometric coupled assay (Figure 1). This coupled enzyme assay utilises regen‐ eration of ATP from the ADP by-product of the choline kinase by pyruvate kinase, and sub‐ sequent oxidation of NADH as the resulting pyruvate is converted to lactate, by lactate dehydrogenase. This assay using coupled enzymes is also problematic, as a compound could potentially inhibit the coupled enzymes giving rise to a false positive.

An alternative approach for screening is differential scanning fluorimetry (Figure 2), allow‐ ing identification of compounds that interact with the *Tb*CK protein, either to stabilise or de‐ stabilise it, therefore influencing the protein's Tm (melting point) [38-40].

Initially *Tb*CK was subjected to differential scanning fluorimetry to ascertain if this approach was possible. Known components required for enzyme activity were tested to see if thermal shifts were observed.In the presence of 6 mM MgCl2, a Tm of 41.2°C was obtained (Figure 1C, solid dark line). The addition of 0.5 mM ATP resulted in a > 3°C Tm shift for *Tb*CK(Figure 1C, dashed-line). These encouraging results showed *Tb*CK was amenable to differential scanning fluorimetry and allowed validation of this screening method.It is worth noting the surprising low Tm of *Tb*CK, considering that these parasites live within the bloodstream of a mammalian host, i.e. 37°C, or higher with a fever. However, the presence of physiological

Differential fluorimetric scans were performed and analysed as described in Experimental. *Tb*CK + DMSO (control) solid dark line, *Tb*CK + 0.5 µM ATP (positive control) dashed line, *Tb*CK + 1 mM compound 242, solid light line, *Tb*CK + 1 mM compound 269, dotted line.Tm of *Tb*CK in the presence of 0.5%DMSO is 41.21 ± 0.03°C (control); Tm of *Tb*CK and 0.5 mM ATP is 44.46 ± 0.05°C (positive control). Insert: schematic representation of the thermal shift assay.A protein will unfold exposing hydrophobic domains as it is denatured due to the in‐ creasing temperature. Dyes such as sypro orange (star) are able to bind to these exposed hy‐ drophobic areas giving rise to fluorescence. A plot of this increased fluorescence versus temperature allows determination of Tm (melting point) of the protein. If a compound (squares) is able to interact with the protein it may alter the protein's Tm and thus a library of compounds can be screened to see if they stabilise (increase in Tm) or destabilise (decrease in

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An alternative approach for screening is differential scanning fluorimetry (Figure 2), allow‐ ing identification of compounds that interact with the *Tb*CK protein, either to stabilise or de‐

Initially *Tb*CK was subjected to differential scanning fluorimetry to ascertain if this ap‐ proach was possible. Known components required for enzyme activity were tested to see if thermal shifts were observed. In the presence of 6 mM MgCl2, a Tm of 41.2°C was obtained (Figure 1C, solid dark line). The addition of 0.5 mM ATP resulted in a > 3°C Tm shift for *Tb*CK(Figure 1C, dashed-line). These encouraging results showed *Tb*CK was amenable to differential scanning fluorimetry and allowed validation of this screening method.It is worth noting the surprising low Tm of *Tb*CK, considering that these parasites live within the blood‐ stream of a mammalian host, i.e. 37°C, or higher with a fever. However, the presence of physiological relevant levels of ATP does stabilize *Tb*CK by > 3 °C, which may prolong the

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

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

The ~630 compounds from the MayBridge Rule of 3 Fragment Library were assessed for their ability to inhibit the *Tb*CK coupled enzyme activity assay at a single concentration of 0.5 mM (Figure 4A). At this relative high concentration only 9 of the compounds (1.4%) showed > 70% inhibition. These primary hits were retested in triplicate at 0.5 mM (Table 1), 2 of the 9(compounds 320 and 635) were confirmed as being false positives, while the re‐ maining 7 were confirmed to show good inhibition (80-100%) against the *Tb*CK coupled en‐

stabilise it, therefore influencing the protein's Tm (melting point) [38-40].

Tm) the target protein.

half-life of the protein in the parasite.

results [41].

being reliable.

**Figure 1.** Schematic of the *Tb*CK reaction and coupled assay.

*T. brucei* choline kinase (*Tb*CK) catalyses the ATP dependent phosphorylation of choline, the ADP is converted back to ATP by pyruvate kinase (PK), which converts phosphoenolpyru‐ vate (PEP) to pyruvate in the process. The resulting pyruvate is reduced to lactate by the NADH dependent lactate dehydrogenase (LDH). The resulting conversion of NADH to NAD+ is monitored, by measuring the reduction in absorbance at 341 nM.

**Figure 2.** Thermal shift assay; typical differential fluorimetry scans of TbCK.

Differential fluorimetric scans were performed and analysed as described in Experimental. *Tb*CK + DMSO (control) solid dark line, *Tb*CK + 0.5 µM ATP (positive control) dashed line, *Tb*CK + 1 mM compound 242, solid light line, *Tb*CK + 1 mM compound 269, dotted line.Tm of *Tb*CK in the presence of 0.5%DMSO is 41.21 ± 0.03°C (control); Tm of *Tb*CK and 0.5 mM ATP is 44.46 ± 0.05°C (positive control). Insert: schematic representation of the thermal shift assay.A protein will unfold exposing hydrophobic domains as it is denatured due to the in‐ creasing temperature. Dyes such as sypro orange (star) are able to bind to these exposed hy‐ drophobic areas giving rise to fluorescence. A plot of this increased fluorescence versus temperature allows determination of Tm (melting point) of the protein. If a compound (squares) is able to interact with the protein it may alter the protein's Tm and thus a library of compounds can be screened to see if they stabilise (increase in Tm) or destabilise (decrease in Tm) the target protein.

dashed-line). These encouraging results showed *Tb*CK was amenable to differential scanning fluorimetry and allowed validation of this screening method.It is worth noting the surprising low Tm of *Tb*CK, considering that these parasites live within the bloodstream of a mammalian

*T. brucei* choline kinase (*Tb*CK) catalyses the ATP dependent phosphorylation of choline, the ADP is converted back to ATP by pyruvate kinase (PK), which converts phosphoenolpyru‐ vate (PEP) to pyruvate in the process. The resulting pyruvate is reduced to lactate by the NADH dependent lactate dehydrogenase (LDH). The resulting conversion of NADH to

is monitored, by measuring the reduction in absorbance at 341 nM.

host, i.e. 37°C, or higher with a fever. However, the presence of physiological

**Figure 1.** Schematic of the *Tb*CK reaction and coupled assay.

**Figure 2.** Thermal shift assay; typical differential fluorimetry scans of TbCK.

NAD+

418 Drug Discovery

An alternative approach for screening is differential scanning fluorimetry (Figure 2), allow‐ ing identification of compounds that interact with the *Tb*CK protein, either to stabilise or de‐ stabilise it, therefore influencing the protein's Tm (melting point) [38-40].

Initially *Tb*CK was subjected to differential scanning fluorimetry to ascertain if this ap‐ proach was possible. Known components required for enzyme activity were tested to see if thermal shifts were observed. In the presence of 6 mM MgCl2, a Tm of 41.2°C was obtained (Figure 1C, solid dark line). The addition of 0.5 mM ATP resulted in a > 3°C Tm shift for *Tb*CK(Figure 1C, dashed-line). These encouraging results showed *Tb*CK was amenable to differential scanning fluorimetry and allowed validation of this screening method.It is worth noting the surprising low Tm of *Tb*CK, considering that these parasites live within the blood‐ stream of a mammalian host, i.e. 37°C, or higher with a fever. However, the presence of physiological relevant levels of ATP does stabilize *Tb*CK by > 3 °C, which may prolong the half-life of the protein in the parasite.

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 *Tb*CK coupled enzyme activity assay at a single concentration of 0.5 mM (Figure 4A). At this relative high concentration only 9 of the compounds (1.4%) showed > 70% inhibition. These primary hits were retested in triplicate at 0.5 mM (Table 1), 2 of the 9(compounds 320 and 635) were confirmed as being false positives, while the re‐ maining 7 were confirmed to show good inhibition (80-100%) against the *Tb*CK coupled en‐ zyme activity assay. These 7 compounds were then tested against just the coupled enzymes, some inhibition was observed for some of the compounds, but this was insufficient to ac‐ count for the strong inhibition against the *Tb*CK, thus these 7 compounds were believed to show true *Tb*CK inhibition.

those occasions where this interaction destabilizes a protein, i.e. lowering Tm, a thermo‐ dynamic 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 de‐ stabilized by very similar ligands, this was exquisitely demonstrated by the changes in thermal stability of Acyl-CoA thioesterase, upon incubation with either CoA (destabilise)

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**Figure 4.** A) Percentage inhibition of *Tb*CK enzyme activity assay for each of the compounds tested. B) Observed ther‐

mal shifts of *Tb*CK for each of the compounds tested

or Acyl-CoA (stabilise) [45].

**Figure 3.** Quality control (Z factors) for the *Tb*CK thermal shift analysis and coupled enzyme activity assay.

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 *Tb*CK with the ~630 compounds from the MayBridge Rule of 3 Fragment Library showed that the vast majority of compounds had little or no affect on the Tm of *Tb*CK (Figure 4B). Relatively few compounds showed an increase in Tm (stabilisation), and only a handful of these showing an increase in Tm> 1°C, i.e. compound 269 (Figure 2, dotted line), this was rather surprising given that ATP stabilises *Tb*CK by > 3°C. Significant‐ ly more compounds showed a destabilisation affect, with 3 compounds having > 10°C de‐ crease in the Tm, i.e. compound 242 (Figure 2, solid light line). Most of the compounds observed in this screen that show significant destabilisation of *Tb*CK, do not cause similar destabilisation affects with other enzymes that we have screened in a similar manner, the only exceptions are compounds 68 (2-aminothiophene-3-carbonitrile) and 565 (4-(2-ami‐ no-1,3-thiazol-4-yl)phenol) (Table 1).

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 thermo‐ dynamic 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 de‐ stabilized 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].

zyme activity assay. These 7 compounds were then tested against just the coupled enzymes, some inhibition was observed for some of the compounds, but this was insufficient to ac‐ count for the strong inhibition against the *Tb*CK, thus these 7 compounds were believed to

**Figure 3.** Quality control (Z factors) for the *Tb*CK thermal shift analysis and coupled enzyme activity assay.

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

Thermal shift analysis of *Tb*CK with the ~630 compounds from the MayBridge Rule of 3 Fragment Library showed that the vast majority of compounds had little or no affect on the Tm of *Tb*CK (Figure 4B). Relatively few compounds showed an increase in Tm (stabilisation), and only a handful of these showing an increase in Tm> 1°C, i.e. compound 269 (Figure 2, dotted line), this was rather surprising given that ATP stabilises *Tb*CK by > 3°C. Significant‐ ly more compounds showed a destabilisation affect, with 3 compounds having > 10°C de‐ crease in the Tm, i.e. compound 242 (Figure 2, solid light line). Most of the compounds observed in this screen that show significant destabilisation of *Tb*CK, do not cause similar destabilisation affects with other enzymes that we have screened in a similar manner, the only exceptions are compounds 68 (2-aminothiophene-3-carbonitrile) and 565 (4-(2-ami‐

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

show true *Tb*CK inhibition.

420 Drug Discovery

screening other ATP utilizing enzymes.

no-1,3-thiazol-4-yl)phenol) (Table 1).

**Figure 4.** A) Percentage inhibition of *Tb*CK enzyme activity assay for each of the compounds tested. B) Observed ther‐ mal shifts of *Tb*CK for each of the compounds tested


All of the compounds in the two data sets (the coupled enzyme activity assay and the ther‐ mal 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 *Tb*CK enzyme activity (Figure 5A). The vast majority of com‐ pounds showed little or no inhibition and little or no shift in Tm. Compounds showing < 40% inhibition of the enzyme activity were removed for clarification (Figure 5B), this highlighted that the majority of compounds that show *Tb*CK enzyme inhibition do not significantly alter the Tm of *Tb*CK. The exceptions are compound 565 with a decrease in Tm~9°C, and com‐ pound 68 and 95 with a decrease in Tm~2°C respectively, all show complete inhibition at 0.5 mM (Table 1). Twenty-one compounds showed > 40% enzyme inhibition, 9 of these (43%) displayed > 1°C change in *Tb*CK Tm. This is a substantial enrichment compared to the 7% of

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**Figure 5.** Scatter graph representation showing the correlation between the observed thermal shifts and percentage inhibition of *Tb*CK enzyme activity. A) All of the compounds from the MayBridge Rule of 3 Fragment Library tested. B)

For those compounds with > 40% (dotted line) inhibition of *Tb*CK enzyme activity.

compounds with Tm shifts > 1°C observed for the entire library.

a Arbitrary library number

<sup>b</sup> CAS numbers are unique identifiers assigned by the "Chemical Abstracts Service" to describe every chemical descri‐ bed in open access scientific literature.

c Tm shift in °C, observed for TbCK in the presence of compound (1mM), value is mean ± SD from the Boltzman curve fitting, see Experimental for details.

<sup>d</sup> Cytotoxicity studies, see Major and Smith 2011 for details, values are percentage of controls in the absence of com‐ pound, either mean ± SD (n=3) or mean ± SE (n=2), the latter being in bold.

**Table 1.** The compounds from the Maybridge Rule of 3 library that show >70% inhibition of TbCK.

All of the compounds in the two data sets (the coupled enzyme activity assay and the ther‐ mal 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 *Tb*CK enzyme activity (Figure 5A). The vast majority of com‐ pounds showed little or no inhibition and little or no shift in Tm. Compounds showing < 40% inhibition of the enzyme activity were removed for clarification (Figure 5B), this highlighted that the majority of compounds that show *Tb*CK enzyme inhibition do not significantly alter the Tm of *Tb*CK. The exceptions are compound 565 with a decrease in Tm~9°C, and com‐ pound 68 and 95 with a decrease in Tm~2°C respectively, all show complete inhibition at 0.5 mM (Table 1). Twenty-one compounds showed > 40% enzyme inhibition, 9 of these (43%) displayed > 1°C change in *Tb*CK Tm. This is a substantial enrichment compared to the 7% of compounds with Tm shifts > 1°C observed for the entire library.

**Library numbera**

422 Drug Discovery

68 4651-82-5

95 933-67-5

278 199590-00-6

320 39270-39-8

346 57976-57-5

372 143426-51-1

565 57634-55-6

635 64354-50-3

Arbitrary library number

bed in open access scientific literature.

fitting, see Experimental for details.

pound, either mean ± SD (n=3) or mean ± SE (n=2), the latter being in bold.

a

c

**CAS numberb Molecular Structure**

**Compound Name**

2 aminothiophene-3-carbonitrile

> 7-methyl-1Hindole

(1-methyl-1Hindol-6 yl)methanol

2,3-dihydro-1,4 benzodioxin-6 ylmethanol

> 3-pyridin-3 ylaniline

[4-(1H-pyrrol-1-yl) phenyl] methanol

4-(2-amino-1,3 thiazol-4 yl)phenol

5-methyl-2 phenyl-3-furoic acid

**Table 1.** The compounds from the Maybridge Rule of 3 library that show >70% inhibition of TbCK.

<sup>b</sup> CAS numbers are unique identifiers assigned by the "Chemical Abstracts Service" to describe every chemical descri‐

Tm shift in °C, observed for TbCK in the presence of compound (1mM), value is mean ± SD from the Boltzman curve

<sup>d</sup> Cytotoxicity studies, see Major and Smith 2011 for details, values are percentage of controls in the absence of com‐

257 59147-02-3 4-(2-furyl)aniline 80 ± 3 ~234 63 0.46 ± 0.08 9.3 ± 8.5

101 ± 2

100 ± 0

80 ± 1

*Tb***CK activity % inhibition at 500μM Mean ± SD (***n***=3)**

*TbCK IC50* **(υM)**

> 25.45 ±1.16

12.35 ± 0.64

109.7 ± 10.6

**PK/LDH % inhibition at 500 υM mean (n=2)**

101 ± 3 ~758 40 -2.15 ± 0.08 10.3 ± 11

84 ± 4 ~380 49 -2.16 ± 0.14 4.1 ± 5.2

6 ± 4 ND ND -0.17 ± 0.07 63.1 ± 9.5

100 ± 0 ~120 85 -8.91 ± 0.49 28.7±19.3

23 ± 4 ND ND -2.94 ± 0.16 20.5 ± 7.4

*TbCK TmShiftc*

28 0.26 ± 0.09 8.8 ± 10.8

68 -1.18 ± 0.06 15.6 ± 9.3

26 1.24 ± 0.04 27 ± 14

*T. brucei % survivald*

**Figure 5.** Scatter graph representation showing the correlation between the observed thermal shifts and percentage inhibition of *Tb*CK enzyme activity. A) All of the compounds from the MayBridge Rule of 3 Fragment Library tested. B) For those compounds with > 40% (dotted line) inhibition of *Tb*CK enzyme activity.

Of the compounds identified that alter the Tm of *Tb*CK by > 1°C, ~20% of them inhibit *Tb*CK enzyme activity by > 40%. This suggests that for *Tb*CK thermal shift analysis has al‐ lowed significant enrichment, but not total capture of the potential inhibitors of *Tb*CK. However, if a direct assay for a potential drug target was very problematic, prior thermal shift analysis could significantly streamline the number of compounds to be screened, thereby increasing the potential to identify lead compounds. Thermal shift analysis has the disadvantage that good inhibitors could be missed if they do not significantly alter the Tm of the protein.

This raises an interesting question, is it a viable option to target compounds that specifi‐ cally 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 com‐ pounds 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 com‐ plex and several others, reviewed by Wells and McClendon [47] and more recently by Coyne and colleagues [48].

**Figure 6.** Correlation of the percentage inhibition of *Tb*CK enzyme activity assay and *T. brucei* survival.

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**Figure 7.** IC50 curves of the promising compounds (278 and 346) that show significant TbCK inhibition.

selectivity index.

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

The techniques utilized to study the formation / disruption of protein-protein complexes are driven by high throughput drug discovery, including fragment based approaches, these in‐ clude X-ray crystallography, NMR, dynamic light scattering, differential static light scatter‐ ing, 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 *Tb*CK, which we know exists as a dimer, one of several potential mechanisms of destabilisation could be dis‐ ruption of the dimer interface, whereby a ligand is able to bind to freshly exposed hydro‐ phobic surfaces on the protein, and this interaction allows further destabilisation of the monomer structure.

As it was clear that compounds that inhibit *Tb*CK enzyme activity do not necessarily show a significant increase or decrease in Tm, it was decided to compare inhibition of *Tb*CK enzyme activity with previously determined trypanocidal activities for the com‐ pounds [51]. From this comparison (Figure 6) a group of compounds above a threshold of > 70% inhibition of *Tb*CK showed significant trypanocidal activity (circled), suggesting a direct correlation.

Compounds from the May Bridge Rule of 3 Fragment Library with greater than 70% inhibi‐ tion (dotted line) of the TbCK enzyme activity are circled and numbered. Numbers corre‐ spond to arbitrary compound library numbers; see Table 1 for chemical structures and extra data. *T. brucei* survival data was previously determined [51].

Coupled Enzyme Activity and Thermal Shift Screening of the Maybridge Rule of 3 Fragment Library... http://dx.doi.org/10.5772/52668 425

**Figure 6.** Correlation of the percentage inhibition of *Tb*CK enzyme activity assay and *T. brucei* survival.

Of the compounds identified that alter the Tm of *Tb*CK by > 1°C, ~20% of them inhibit *Tb*CK enzyme activity by > 40%. This suggests that for *Tb*CK thermal shift analysis has al‐ lowed significant enrichment, but not total capture of the potential inhibitors of *Tb*CK. However, if a direct assay for a potential drug target was very problematic, prior thermal shift analysis could significantly streamline the number of compounds to be screened, thereby increasing the potential to identify lead compounds. Thermal shift analysis has the disadvantage that good inhibitors could be missed if they do not significantly alter the

This raises an interesting question, is it a viable option to target compounds that specifi‐ cally 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 com‐ pounds 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 com‐ plex and several others, reviewed by Wells and McClendon [47] and more recently by

The techniques utilized to study the formation / disruption of protein-protein complexes are driven by high throughput drug discovery, including fragment based approaches, these in‐ clude X-ray crystallography, NMR, dynamic light scattering, differential static light scatter‐

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 *Tb*CK, which we know exists as a dimer, one of several potential mechanisms of destabilisation could be dis‐ ruption of the dimer interface, whereby a ligand is able to bind to freshly exposed hydro‐ phobic surfaces on the protein, and this interaction allows further destabilisation of the

As it was clear that compounds that inhibit *Tb*CK enzyme activity do not necessarily show a significant increase or decrease in Tm, it was decided to compare inhibition of *Tb*CK enzyme activity with previously determined trypanocidal activities for the com‐ pounds [51]. From this comparison (Figure 6) a group of compounds above a threshold of > 70% inhibition of *Tb*CK showed significant trypanocidal activity (circled), suggesting a

Compounds from the May Bridge Rule of 3 Fragment Library with greater than 70% inhibi‐ tion (dotted line) of the TbCK enzyme activity are circled and numbered. Numbers corre‐ spond to arbitrary compound library numbers; see Table 1 for chemical structures and extra

data. *T. brucei* survival data was previously determined [51].

Tm of the protein.

424 Drug Discovery

Coyne and colleagues [48].

monomer structure.

direct correlation.

ing, differential scanning fluorimetry [42-50].

**Figure 7.** IC50 curves of the promising compounds (278 and 346) that show significant TbCK inhibition.

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 com‐ pound 278 (1-methyl-(1H-indol-6-yl)methanol), which highlight that the N-methyl indole moiety seems necessary to have any *Tb*CK inhibition and the methanol portion of the mole‐ cule can not be replaced by a carboxylic acid. Investigation of the CHEMBL database for similar compounds identified 1-Methyl-1H-pyrrolo[2,3-c]pyridine (CHEMBL594467) which was screened as one of a library of tricyclic and bicyclic analogues of indoloquinoline alka‐ loids against a variety of protozoan parasites. The analogue mentioned here showed weak trypanocidal activity (624 µM) against *Trypanosoma brucei rhodesiense*, but significantly better (37 µM) against *Plasmodium falciparum* [52].

be investigated by undertaking various *in vivo* biochemical phenotyping experiments to as‐ certain if they are inhibiting *Tb*CK, thus causing a lack of *de novo* PC synthesis, known to be

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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

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

Biomedical Sciences Research Centre, The North Haugh, The University, St. Andrews, Fife,

[3] Priotto, G.; Kasparian, S.; Mutombo, W.; Ngouama, D.; Ghorashian, S.; Arnold, U.; Ghabri, S.; Baudin, E.; Buard, V.; Kazadi-Kyanza, S.; Ilunga, M.; Mutangala, W.; Poh‐ lig, G.; Schmid, C.; Karunakara, U.; Torreele, E.; Kande, V. Lancet 2009, 374, 56.

[4] Delespaux, V.; de Koning, H. P. Drug resistance updates : reviews and commentaries

[6] Bouteille, B., O. Oukem, S. Bisser, and M. Dumas. 2003. Treatment perspectives for

[7] Priotto, G., C. Fogg, M. Balasegaram, O. Erphas, A. Louga, F. Checchi, S. Ghabri, and P. Piola. 2006. Three drug combinations for late-stage *Trypanosoma brucei gambiense* sleeping sickness: a randomized clinical trial in Uganda. PLoSClin. Trials 1:e39

[1] WHO web site: http://www.who.int/topics/trypanosomiasis\_african/en/

in antimicrobial and anticancer chemotherapy 2007, 10, 30. [5] Baker, N.; Alsford, S.; Horn, D. MolBiochemParasit 2011, 176, 55.

human African trypanosomiasis. Fundam. Clin. Pharmacol. 17:171

essential for the parasite.

Third World diseases.

**Acknowledgements**

3 Fragment Library.

**Author details**

Scotland, U.K.

**References**

Louise L. Major, Helen Denton and Terry K. Smith

[2] Steverding, D. Parasites & vectors 2010, 3, 15.

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 *Leishmania* as a potential PTR1 inhibitor but was shown to be inactive at 500 µM [54]

For the relatively simple compound 346 (3-pyridin-3-ylaniline), there are several analo‐ gous structures in the library, including compound 262 (2-(1H-imidazo-1-yl)aniline) which shows ~55% *Tb*CK enzyme inhibition and is trypanocidal. Compound 347 (4-pyri‐ din-3-ylaniline) is a structural isomer of 346 but shows no *Tb*CK enzyme inhibition and is not trypanocidal. The only related structure in the CHEMBL database was 3-(pyri‐ din-3-yl)benzenaminium (CHEMBL1778131) which was shown to be a weakinhibitor of metallo-β−lactamase IMP-1 [55].
