From on-target to off-target activity: Identification and optimisation of Trypanosoma brucei GSK3 inhibitors and their characterisation as anti-Trypanosoma brucei drug discovery lead molecules

Article abstract of DOI:10.1002/cmdc.201300072

Human African trypanosomiasis (HAT) is a life-threatening disease with approximately 30000-40000 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 invitro inhibitors of T.brucei proliferation. We show that the TbGSK3 pharmacophore overlaps with that of one or more additional molecular targets.

Full text of DOI:10.1002/cmdc.201300072

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DOI: 10.1002/cmdc.201300072
From On-Target to Off-Target Activity: Identification and
Optimisation of Trypanosoma brucei GSK3 Inhibitors and
Their Characterisation as Anti-Trypanosoma brucei Drug
Discovery Lead Molecules
Andrew Woodland,[a] Raffaella Grimaldi,[a] Torsten Luksch,[a] Laura A. T. Cleghorn,[a]
Kayode K. Ojo,[b] Wesley C. Van Voorhis,[b] Ruth Brenk,[a] Julie A. Frearson,[a] Ian H. Gilbert,*[a]
and Paul G. Wyatt*[a]
Human African trypanosomiasis (HAT) is a life-threatening dis-
ease with approximately 30000–40000 new cases each year.
Trypanosoma brucei protein kinase GSK3 short (TbGSK3) is re-
quired 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-effi-
cient TbGSK3 inhibitors. Following the hit validation process,
we optimised a series of diaminothiazoles, identifying low-
nanomolar inhibitors of TbGSK3 that are potent in vitro inhibi-
tors of T. brucei proliferation. We show that the TbGSK3 phar-
macophore overlaps with that of one or more additional mo-
lecular targets.
Introduction
Human African trypanosomiasis (HAT), also known as African
sleeping sickness, is a parasitic disease caused by protozoan
parasites of the species Trypanosoma brucei and is fatal if un-
treated. HAT is endemic in certain regions of sub-Saharan
Africa, with around 50 million people at risk of infection across
25 countries. The number of reported cases of HAT has fallen
recently and is now at about 10000 reported new cases per
year; however, the actual number of cases is estimated to be
much higher (30000–40000 new cases per year).[1–3]
daytime slumber and nighttime insomnia, with progressive
mental deterioration leading to coma and death. Generally the
disease is diagnosed only when it has already progressed to
the phase 2 CNS stage.
HAT is a neglected disease, because despite millions of
people being under the threat of infection, there is no com-
mercial market to justify funding drug development. There are
only two stand-alone drugs available for the treatment of late-
stage sleeping sickness: melarsoprol and eflornithine. However,
both drugs have serious limitations such as toxicity, complex
parenteral administration, which is poorly suited to a rural Afri-
can setting, low and variable brain penetration, the develop-
ment of resistant parasites,[4] and patient compliance.[5] A com-
bination therapy of nifurtimox and eflornithine was recently
approved for the treatment of stage 2 HAT primarily due to
a cost benefit and improved convenience of the new treat-
ment over eflornithine alone. Unfortunately, resistance to nifur-
timox develops rapidly in the laboratory.[6–8]
Following infection by the bite of a tsetse fly, patients initial-
ly suffer from phase 1 disease, in which they experience epi-
sodes of fever, headache, sweating, and swelling of the lymph
nodes. Phase 2 disease results from the spread of infection
into the central nervous system (CNS). Patients begin to experi-
ence a disturbance in their circadian rhythm, resulting in bouts
of fatigue alternating with manic periods, which progress to
[a] Dr. A. Woodland, R. Grimaldi, Dr. T. Luksch, Dr. L. A. T. Cleghorn,
Dr. R. Brenk, Prof. J. A. Frearson, Prof. I. H. Gilbert, Prof. P. G. Wyatt
Drug Discovery Unit (DDU)
In recent years a number of drug development initiatives
funded by foundations and/or governments have begun to ad-
dress the need for improved drugs to treat stage 2 HAT.[9] Two
new oral clinical candidates were recently developed: fexinida-
zole,[10] a nitroimidazole derivative that is currently in clinical
development, and SCYX-7158,[11] a benzoxaborole derivative
that has been selected for entry into clinical development.
However, owing to the high rates of attrition in drug discovery
and the requirement for multiple drugs to combat the devel-
opment of resistant parasites, the pipeline must be further en-
hanced.
Division of Biological Chemistry and Drug Discovery
College of Life Sciences, University of Dundee
Sir James Black Centre, DD1 5EH (UK)
E-mail: i.h.gilbert@dundee.ac.uk
p.g.wyatt@dundee.ac.uk
[b] Dr. K. K. Ojo, Prof. W. C. Van Voorhis
Division of Allergy and Infectious Diseases, Department of Medicine
University of Washington, Seattle, WA 98195 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300072.
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
There is a lack of validated drug discovery targets and lead
compounds for HAT and other neglected diseases.[12] Protein
kinases have been explored as possible targets for HAT, as they
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play important roles in virtually every cellular event from cell
division to stress response.[13] Kinases are druggable targets,
and crystal structures have been published for many of
them.[14] Bioinformatics searches of the T. brucei genome identi-
fied 176 parasite protein kinases,[15,16] making this family an at-
tractive source of novel drug discovery targets for the treat-
ment of HAT and other parasitic diseases.[17–19]
Human GSK3b (HsGSK3b) is involved in the regulation of
a vast array of cellular processes in eukaryotes: insulin signal-
ling, growth factors, nutrient levels, cell fates during embryonic
development, cell division, apoptosis, and microtubule func-
tion.[20] HsGSK3b has been investigated as a drug target for
many diseases, from diabetes to neurodegenerative diseases.
To aid development, the crystal structure of HsGSK3b has been
solved, and high-affinity small-molecule inhibitors of HsGSK3b
have been developed.[14,21–24] Whilst the precise role of the try-
panosome orthologue GSK3 short kinase (TbGSK3) in the
bloodstream form of T. brucei has yet to be determined in
terms of parasite biology, the importance of this enzyme has
been demonstrated by RNA interference experiments that
showed decreased growth rates for parasites in in vitro cul-
ture.[25,26]
Figure 1. Superposition of the HsGSK3b crystal structure (PDB code 1R0E)
with the homology model of TbGSK3. The human structure is shown in pink,
the TbGSK3 homology model in blue; both structures are shown in ribbon
representation. Binding pocket residues of TbGSK3 that differ from those of
HsGSK3b are represented as sticks. Regions of the kinase site are labelled ac-
cording to previously published conventions.[28]
Herein we report our studies on the identification and opti-
misation of TbGSK3 inhibitors with potent antiparasitic activity
and highlight their potential for the development of new
therapies for the treatment of HAT.
Table 1. Residue differences in the ATP binding sites of HsGSK3b and
TbGSK3.
Results and Discussion
Homology modelling
Kinase pocket region
Hinge region
HsGSK3b[a]
TbGSK3[b]
Crystal structures of Leishmania major[27] and human GSK3
have been published. The tertiary structure of LmGSK3 (PDB
code 3E3P) is very similar to that of HsGSK3b (PDB code 1R0E),
but several binding pocket residues are missing in the Leish-
mania crystal structure, as no clearly defined electron density
was present. In addition, no ligand is bound in the LmGSK3
crystal structure. Therefore, we selected the crystal structure of
HsGSK3b as the template to build a homology model for
TbGSK3. The TbGSK3 sequence is 52% identical and 71% simi-
lar to the sequence in the HsGSK3b structure (PDB code 1R0E).
Hence HsGSK3b provides a template for 91% of the TbGSK3
sequence (amino acids 20–348) which allowed a reliable model
to be built (Figure 1). Analysis of the ATP binding pockets re-
vealed amino acid differences that could be exploited to
design selective inhibitors (Table 1).
D133
Y134
L132
Y140
Q185
I62
Q72
N64
S66
E
F
Gatekeeper
E1
M
H
H
A
L
Adenine pocket
G-loop
Q
T
[a] PDB code 1R0E. [b] Homology model.
mined KM values for GSP2 and ATP are similar to those previ-
ously reported of 2.4 and 4.5 mm, respectively.[25]
For the primary screen, a 384-well KinaseGlo (Promega) lumi-
nescence-based assay was used as previously described.[25] In
this assay, luminescence is inversely related to kinase activity
and directly related to ATP depletion (Figure 3A). GW8510 was
used as a standard inhibitor[6] (figure S1, Supporting Informa-
tion). The DDU kinase set of 4110 compounds[29] was screened
in single point at 25 mm providing robust data (Z’=0.61ꢀ0.06;
Table 2). From this, 567 compounds with a percentage of in-
hibition >30% were retested in a duplicate-point screen, to
give 517 reconfirmed compounds with inhibition values
ꢁ30% (12.8% of the library; Figure 3B,C).
Hit discovery
Recombinant TbGSK3 was produced as previously described.[25]
The kinetic parameters were determined by measuring initial
reaction velocities in a matrix experiment of varied ATP and
peptide substrate concentrations. The KM value of TbGSK3 for
the substrate with sequence YRRAAVPPSPSLSAHSSPHQ[pS]E-
DEEE (GSP2) and ATP were 8.4ꢀ1.3 and 11.0ꢀ1.8 mm, respec-
tively, with no evidence of cooperativity (Figure 2). The deter-
For hit validation and all subsequent compound potency de-
terminations, a radiometric 96-well Flashplate assay (PerkinElm-
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Table 2. Assay conditions and screening statistics.
KinaseGlo
7.5 nm
Flashplate
TbGSK3
GSP2
2.5 nm
1 mm
3.2 mm
ATP
Z’
GW8510 IC50
1 mm (<KM)
0.61ꢀ0.06
10ꢀ0.2 nm
1 mm (<KM)
0.80ꢀ0.08
6ꢀ4 nm
tions. The Flashplate assay was performed at an ATP concen-
tration below the enzyme’s KM value for ATP, so that the Kiapp
approximated the measured IC50 value, aiding the assessment
of selectivity. Potency evaluation (10-point curves) was carried
out in duplicate for the 100 most potent compounds. As in
the primary assay, the potency assay format yielded highly
robust data (Z’=0.80ꢀ0.08; Table 2). The compounds exhibit-
ed a range of potencies for TbGSK3 which were highly repro-
ducible, with an r2 value of 0.99 for two replicates (Figure 4B),
with 15 compounds having IC50 values <1 mm. The identity
and purity of hits taken into potency determination were con-
firmed by LC–MS.
Figure 2. Determination of kinetic parameters for TbGSK3. KM values for the
ATP and GSP2 substrates were determined in a time-course matrix experi-
ment in which the initial velocities (v) were determined as product formed
(P, nm) per minute. The grid represents the best fit obtained by globally fit-
ting the data to the equation for the random-order rapid equilibrium model
with cooperation parameter (a) equal to 1.
Prioritisation of hits and series definition
To prioritise the hits, hit compounds were rank ordered by
er) was adopted (Figure 4A). Although the KinaseGlo format
has many advantages for screening of chemical libraries, its re-
liance upon ATP consumption means it requires a level of sub-
strate consumption >10% to achieve an acceptable signal
window; it is therefore unsuitable for accurate IC50 determina-
their ligand efficiencies against TbGSK3, in which ligand effi-
[30]
ciency=[ꢂRTln(IC50)]/Nnon-H atoms
.
Subsequently, the highest-
priority compounds were grouped into series based on struc-
tural similarity (Figure 5), resulting in a number of different
compound series. Several of the TbGSK3 inhibitors identified
have
ligand
efficiencies
>0.4 kcalmolꢂ1 per heavy atom,
beyond the commonly used
guideline of 0.3 kcalmolꢂ1 per
heavy atom, which relates to an
optimised lead with an IC50 value
of 10 nm and 38 non-hydrogen
atoms (Mr ~500 Da).[30] In particu-
lar, diaminothiazole 1 is a highly
ligand-efficient starting point,
with a TbGSK3 ligand efficiency
of 0.52 kcalmolꢂ1 per heavy
atom.
Hit validation
Five promising hit series contain-
ing ligands with ligand efficien-
cies >0.3 kcalmolꢂ1 per heavy
atom were progressed into hit
series validation. Series 2 was
based around a diaryl urea and
exemplified by compound 2.
Compounds of this series were
also identified as broad-spec-
trum kinase inhibitors with toxic-
Figure 3. Format and results of the primary screen. A) A KinaseGlo assay format was adopted for the primary
screen based on luminescence detection. B) Distribution of the percent inhibition of the focused kinase set. Hits
were selected by setting 3 standard deviation units from the average of high controls as threshold (ꢁ30% inhibi-
tion). C) 567 selected hits were retested in duplicate, and the two replicate values showed high correlation.
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pound series were commercially available at the time of our
study, and more than 100 compounds were selected for pur-
chasing and testing. However, none of these compounds dem-
onstrated activity in the T. brucei cell assay. This, combined
with the relatively poor TbGSK3 IC50 value of 0.1 mm observed
within the series after testing more than 140 examples, as well
as the flat SAR, it was decided that this series would not be
pursued any further.
Compound 1 (series 1) was the only 2,4-diaminothiazol-5-
carbaldehyde present in the DDU kinase screening set at the
time of screening (Figure 5). Although 1 was a singleton, it has
good predicted physical properties (Mr =288 Da, logP=2.1,
TPSA=68 ꢁ2), a ligand efficiency of 0.52 kcalmolꢂ1 per heavy
atom for TbGSK3, and demonstrated promising activity in
a T. brucei proliferation assay (EC50 2 mm). Of slight concern is
the presence of a ketone functionality, which has the potential
to interact with nucleophiles within the cell; this would have
to be monitored during compound development. Based on
these considerations, it was decided to progress this com-
pound to hit validation. As a side note, compound 1 is also
a very effective HsGSK3 inhibitor, with an IC50 value of 5 nm
(n=2) and an outstanding ligand efficiency of 0.67 kcalmolꢂ1
per heavy atom. Thus, 1 may also be an excellent starting
point for a human GSK3 drug discovery programme.
Structure-guided design
A potential binding mode for compound 1 in the homology
model of TbGSK3 was generated using Moloc (Gerber Molecu-
lar Design, Switzerland; Figure 6). In the proposed binding
mode the 2,4-diaminothiazole moiety forms three hydrogen
bonds with the protein backbone of the TbGSK3 hinge
(Glu102, Phe103, and Val104). Furthermore, the thiazole group
Figure 4. Format and results of the potency test. A) A flashplate radiometric
assay format was adopted for the potency determination based on selective
capture of the biotinylated phosphorylated product by the streptavidin-
coated plates. B) The 100 most potent hits were tested in 10-data point
curves in two independent determinations; the correlation plot between the
logIC50 values of the replicates is reported.
ity toward mammalian cell lines during our previous-
ly published investigations of the CRK3 kinase, so no
further work was conducted with this compound
series.[31]
Series 3 was based around 2-amino-1,3,5-triazines;
36 examples of this series were contained in the
screening library with compound 3 being the only
active example (Figure 5). Due to the limited com-
mercial availability for representatives of this series
and the apparent requirement for specific substitu-
ents, this series was not pursued further.
The 8-aminoimidazo[1,2-a]pyrazines (series 4) were
also identified in a previous DDU project and were
found to be broad-spectrum kinase inhibitors that
are toxic to human cell lines (MRC5 cells). In addition,
the lead compound 4 has an unfavourable calculated
logP value of 4.8 (Figure 5). Therefore, no further
work was carried out on this series.
Eleven oxazole-4-carboxamides (series 5) were
identified in the high-throughput screen (HTS), with
compound 5 inhibiting TbGSK3 at a sub-micromolar
Figure 5. Series classification. Common features representative of each series are high-
IC50 value (Figure 5). Over 900 examples of this com- lighted in red. Ligand efficiencies are given as kcalmolꢂ1 per heavy atom.
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A series of 112 commercially available 2,4-diaminothiazoles
with various substituents were docked into the homology
model of TbGSK3 using FlexX.[32] The docking solutions were
visually inspected, and 21 compounds were selected for pur-
chase (Table 3).
Biological results and structure–activity relationships
The biological data for early hit optimisation are summarised
in Table 3. The hit compound 1 has an ethyl group at R1 which
may form hydrophobic interactions with Leu159 and the hy-
drophobic part of Thr107 (Figure 6, Table 1). Although no
direct analogues were available, the closely related methyl ana-
logue 6 is around 50-fold less active than 1, suggesting that
lipophilic bulk in the R1 region is beneficial. Introducing an aro-
matic group into R1 and truncating R2 to methyl was tolerated,
with an IC50 value of 1 mm for 7. Increasing the size of R1 while
maintaining a lipophilic group at R2 gave a 10-fold improve-
ment in activity, with several examples demonstrating IC50
values of <100 nm for TbGSK3, such as compounds 8, 9, and
10.
In general the 2,4-diaminothiazol-5-carbaldehydes tested
were found to be more potent inhibitors of HsGSK3b than
they are of TbGSK3, confirming that selectivity between Hs and
TbGSK3 can be achieved, albeit initially in the undesired direc-
tion. In contrast, in cellular assays, the lead molecules are selec-
tive antiparasitic agents as exemplified by 9, which has an EC50
value of 0.13 mm against T. brucei, but no activity against MRC5
cells (EC50 >50 mm).
Figure 6. Proposed binding mode for 1 in the homology model of TbGSK3.
Both, ligand and protein are represented in sticks and colour-coded by atom
type. Ligand carbon atoms are shown in salmon, and protein carbon atoms
in light blue. Putative hydrogen bonds to the ligand are shown as yellow
dotted lines (other hydrogen bonds within the active are not shown for
clarity).
is sandwiched between Leu159 and Ala47 and undergoes hy-
drophobic interactions with both amino acids. In addition, the
ligand carbonyl group is proposed to be in plane with the
core thiazole and forms an intramolecular hydrogen bond with
the primary amino group. A water molecule, observed in many
HsGSK3b structures, was kept for the binding mode generation
and is predicted to mediate a hydrogen bond between the in-
hibitor carbonyl lone pair and the backbone amide NH group
of Asp171. The presence of an
A correlation plot of cell efficacy (bloodstream form (BSF)
T. brucei logEC50) against enzyme potency (TbGSK3 logIC50)
gave a strong correlation for the early members of this series
(correlation coefficient: 0.90) with a 5-fold drop off between
the TbGSK3 and T. brucei activities (Figure 7 and Supporting In-
formation table S1). Considering that the physiological level of
analogous water molecule in the
recently solved X-ray crystal
structure of LmGSK3 (PDB code
3E3P) gives further evidence that
the water molecule is conserved
and plays an important role in
GSK3–ligand binding. The 2-
chlorothiophene moiety is pre-
dicted to form lipophilic interac-
tions with Phe31 and Cys170,
while the ethyl group points
toward Leu159, and the hydro-
phobic part of Thr107 potentially
contributes van der Waals inter-
actions to the binding affinity.
Docking was carried out in
order to guide the hit expansion,
with the aim of confirming the
Table 3. Activity of key compounds from series 1.
IC50 [mm]
EC50 [mm]
Compd
R1
R2
TbGSK3
0.4
HsGSK3b
T. brucei
MRC5
>15
>50
25
1
6
7
8
0.005
ND[a]
ND[a]
0.007
2
7
25
1
13
0.03
0.2
13
9
0.05
0.1
0.003
0.007
0.1
0.7
>50
10
4
aminothiazoles as
a series of
TbGSK3 inhibitors, improving in
[a] Not determined.
vitro potency, and to build SAR.
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Hit-to-lead development
Based on the promising data obtained for the series we decid-
ed to progress the project into hit-to-lead development. A
stepwise solid-supported two-step synthetic route to diamino-
thiazoles has been published, starting from the non-commer-
cial reagent, benzyl carbamimidothioate hydrobromide 11.[40]
We modified the synthesis so that both steps are carried out in
a one-pot, solution-phase reaction (Scheme 1). Benzyl bromide
Figure 7. Correlation between inhibition of TbGSK3 and inhibition of
T. brucei cell growth for the initial set of compounds. Supporting Information
table S1 lists the compounds used to derive the correlation plots along with
the TbGSK3 logIC50 and T. brucei logEC50 values.
Scheme 1. Reagents and conditions: a) thiourea (1 equiv), EtOAc, 1208C,
5 min, b) isothiocyanate (1.1 equiv), DIPEA (1.2 equiv), DMF, RT, 24 h, then 2-
bromoketone (1.2 equiv), DIPEA (2.1 equiv), RT, 1 h.
ATP in T. brucei is in the millimolar range, whilst in our TbGSK3
potency assay, the concentration was 1 mm, we expected the
IC50 value for TbGSK3 in cells to drop off by at least 100-fold
according to the Cheng–Prusoff equation [Eq. (1)].[33] In addi-
tion, other factors such as protein binding or the requirement
for a high level of enzyme inhibition to achieve a phenotypic
effect, as observed previously for other T. brucei targets, could
even result in a >100-fold drop off.[34] The much lower ob-
served difference between IC50 and EC50 suggested that the
mode of action of series 1 may not be just through inhibition
of TbGSK3.
12 was treated with thiourea to give benzyl carbamimido-
thioate hydrobromide 11 in 89% yield. Benzyl carbamimido-
thioate 11 was allowed to react with an isothiocyanate in the
presence of Hꢂnig’s base, 2-bromoketones were then added
along with an additional equivalent of Hꢂnig’s base to give
the desired 2,4-diaminothiazoles 13 in a three-component
one-pot synthesis with yields ranging from 3 to 69%.
The most potent compounds identified in the early work
were 2,4-diamino-5-ketothiazoles 13 bearing aromatic groups
in both the R1 and R2 position, such as compound 8 (IC50 =
0.03 mm, Table 3). Although we had identified several TbGSK3
inhibitors with IC50 values less than 1 mm, we wished to im-
prove the selectivity over HsGSK3b. Our design explored R1
and R2 groups of varying size, shape, and polarity to probe the
limits of the ATP binding pockets of the TbGSK3 and HsGSK3b
enzymes to identify regions where selectivity could be ach-
ieved (Table 4).
Initial work focused on the R2 group. Introduction of ortho
substituents, which would be expected to twist the R2 group
out of plane with the ketothiazole core, was well tolerated for
small groups such the fluorine-substituted analogue 14 (IC50 =
0.02 mm). The larger and more electron-rich 2,6-dimethoxy-
phenyl 15 (IC50 =0.4 mm) was over 10-fold less active than 14
against TbGSK3. Lipophilic bulk in the meta position of the R2
substituent was tolerated, but provided no significant potency
gains, with 3-bromo analogue 16 (IC50 =0.1 mm) showing simi-
lar activity to the phenyl analogue 8 (IC50 =0.03 mm).
The R1 substituent was then investigated. The 2,6-dimethyl-
phenyl derivative 17 (IC50 =0.2 mm) is approximately 10-fold
less active against TbGSK3 than 8 (IC50 =0.03 mm). Increasing
the meta and para lipophilic bulk at R1 had no benefit, with
3,4-dimethylphenyl 18 similar to 8. Replacing the R1 phenyl
ring with cyclohexyl 19 (IC50 =0.19 mm) caused a small (sixfold)
decrease in potency against the enzyme. Insertion of one or
two methylene units between the amine and the cyclohexyl
and aromatic groups gave small decreases in activity against
TbGSK (20, IC50 =0.7 mm; 21, IC50 =0.5 mm). As part of our strat-
ꢀ
ꢁ
½ATPꢃ
IC50 ¼ Ki 1 þ
ð1Þ
ATP
KM
The small difference between potency against the enzyme
and the cell activity for this series led us to consider that there
may be more than one mechanism of action driving the cell
activity. Substituted 2,4-diaminothiazoles have been described,
and examples are known to be potent inhibitors of
HsGSK3b,[35] several CDKs,[36,37] p25,[37] and PDE4B.[38] Interest-
ingly, there are also reports of a 2,4-diaminothiazole (DAT1)
which binds to and disrupts microtubules.[39] Homologues of
these targets are present in T. brucei and could therefore be
modulated by compounds of this series. Additionally, prolific
kinase inhibitors often show toxicity toward cells in culture.
Compound 8 was profiled at 10 mm against the mammalian
protein kinase panel at the University of Dundee, which at the
time of testing consisted of 76 mammalian kinases. In agree-
ment with our biochemical assays, compound 8 potently inhib-
ited HsGSK3b; it also inhibited CDK2, MKK1, ERK8, and HIPK2
by >90% at this concentration (Supporting Information
table S2). The human protein kinase profile was sufficiently
clean for an early-stage kinase inhibitor project, and we decid-
ed to monitor the kinase profile as the series was developed.
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During analysis of the SAR it
became apparent that the inhibi-
tion of cellular (T. brucei) growth
was no longer tracking with in-
hibition of the enzyme, TbGSK3.
This was particularly apparent
Table 4. Activity of key compounds from series 1.
IC50 [mm]
EC50 [mm]
Compd
R1
R2
TbGSK3
HsGSK3b
T. brucei
MRC5
13
when
a
correlation plot of
T. brucei logEC50 values against
TbGSK3 logIC50 values was pro-
duced for all of the compounds
generated by this stage in the
programme (Figure 9 and Sup-
porting Information table S1). At
the extremes of this plot, the
ratio of T. brucei EC50 values/
TbGSK3 IC50 values for 23 is 115,
whilst for 22 it is 0.017, a differ-
ence of ~6700-fold (Table 4). We
believe that the reason for this
variation is that an essential mo-
lecular target or targets is pres-
ent in the T. brucei parasite
which can be modulated by
compounds with a similar phar-
macophore to that required for
TbGSK3 inhibition.
8
0.03
0.007
0.005
0.2
0.2
14
15
0.02
0.4
1.6
20
2
0.4
16
17
0.1
0.2
0.005
0.2
0.2
9
>50
35
18
0.06
0.002
0.2
14
19
20
21
22
0.2
0.7
0.5
12
0.002
0.02
0.1
0.4
6
11
41
Earlier in the project we had
demonstrated that 8 was not
a prolific kinase inhibitor (Table 3
and Supporting Information
table S2). To determine whether
22 is a broad-spectrum inhibitor
of protein kinases we tested 22
along with 14 and 19 in the Uni-
versity of Dundee kinase panel
0.003
ND[a]
38
0.3
>50
23
0.04
0.007
4.1
0.3
[a] Not determined.
at
(Supporting
table S2). Most members of
a
concentration of 10 mm
Information
egy to test the limits of the TbGSK3 and HsGSK3b pockets, the
tetrahedral lipophilic tert-butyl ketone 22 was synthesised. This
compound is ~300-fold less active than 8 toward TbGSK3;
however, it is a potent inhibitor of T. brucei growth in vitro,
with an EC50 value of 0.3 mm, and is also highly selective (>
150-fold) over the human MRC5 cell line.
series 1 showed >90% inhibition against several of the kinases
tested. Interestingly, 22 was the least broad-spectrum kinase
inhibitor tested and it did not inhibit any kinase at the >90%
level; it only inhibited CDK2 and GSK3 at >70%. These en-
couraging data demonstrate that 22 selectively modulates the
activity of the unknown parasitic target(s) over a broad range
of mammalian kinases, and that these targets can be modulat-
ed by drug-like molecules. However, owing to the complex
pharmacology of these compounds, and as it is not clear what
the off-targets are, the only option will be to optimise these
compounds phenotypically, rather than through protein
screening.
Biological characterisation of 2,4-diaminothiazol-5-carbalde-
hydes
Studies of the mechanism of inhibition by compounds from
the diaminothiazole series (19 and 14) confirmed that they are
ATP-competitive inhibitors of TbGSK3, as evident from the
Lineweaver–Burk plots (Figure 8). The calculated Kiapp values
(0.25ꢀ0.03 and 0.05ꢀ0.01 mm for 19 and 14, respectively) cor-
related very well with the determined IC50 values in the bio-
chemical assay (Table 4) as expected, considering that the level
of ATP in the assay was below the KM value for ATP.
Conclusions
In summary, we have shown that a screen of a focused kinase
library led to the identification of ligand-efficient inhibitors of
TbGSK3 with sub-micromolar potency. The chemotypes we
identified have physicochemical properties consistent with the
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of
a clear understanding of
which molecular target(s) are re-
sponsible for anti-trypanosomal
activity, the only way to optimise
this compound is phenotypically.
We shall report results for this in
due course.
Experimental Section
Homology modelling: Sequence
alignments between TbGSK3b
(Tb927.10.13780 in GeneDB) and
HsGSK3b were generated with
ClustalW.[41] Subsequently, Model-
ler 9.2[42] was used to build homol-
ogy models of TbGSK3 short,
whereas the human GSK3b crystal
structure (PDB code 1R0E) served
as template.[43] Modeller was run
with default settings, and only the
highest-scoring structure was used
for further analysis and modelling.
The quality of the model was as-
sessed with QMEAN.[44,45] A total
QMEAN score of 0.726 and Z-score
of ꢂ0.50 were calculated, indicat-
ing that the model is of good qual-
ity.
Figure 8. Mechanism of inhibition was determined for A) 19 and B) 14. Rates (CPM) were determined at the re-
ported inhibitor concentrations (mm) with four varied concentrations of ATP at saturating concentration of the
other substrate. The resulting Lineweaver–Burk plots were examined for diagnostic patterns for competitive inhib-
ition and globally fitted to the equation for competitive inhibition.
Generation of putative binding modes: An initial binding mode
for compound 1 was generated by manually docking the ligand
into the ATP binding pocket of the TbGSK3 homology model with
the requirement to establish hydrogen bonds to the hinge resi-
dues using Moloc. Its position was subsequently optimised with
the MAB force field as implemented in Moloc.[46] To allow for uncer-
tainties in the homology model, the amino acid side chains facing
the binding site were kept flexible during minimisation. For the hit
expansion, ligands were docked into the active site of the homolo-
gy model using FlexX.[32] A highly conserved water molecule (H20
82 in 1R0E) was kept in the structure used for docking. For the
active site determination, a radius of 13 ꢁ around the ligand
bound in 1R0E was selected, always using complete amino acids.
Ligand conformations were calculated using CORINA.[47] Com-
pounds were docked using default settings.
Figure 9. Correlation between inhibition of TbGSK3 and inhibition of
T. brucei cell growth for the initial set of compounds. The later compounds
show weaker correlation. Supporting Information table S1 lists the com-
pounds used to derive the correlation plots along with the TbGSK3 logIC50
and T. brucei logEC50 values. Figure 9 was generated using both the early
and later examples of series 1.
TbGSK3 biochemical characterisation: TbGSK3 short protein with
an N-terminal maltose binding protein fusion was cloned, ex-
pressed, and purified as previously reported.[25] For biochemical
characterisation of TbGSK3, the kinase assay buffer (25 mm Tris·HCl
pH 7.5, 10 mm MgCl2, 5 mm DTT, 0.02% CHAPS, 2 UmLꢂ1 heparin)
and mix of ATP/[g-33P]ATP and GSP2 substrate (YRRAAVPPSPSL-
SAHSSPHQ[pS]EDEEE; Pepceuticals) were used in a radiometric
format (filterplate assay). The KM values for the two substrates (ATP
and GSP2) were determined by varying the concentrations of both
substrates in a time-course matrix experiment.
development of orally bioavailable compounds. For the most
active series based on 2,4-diaminothiazoles, the compounds
were also active against the parasite T. brucei. Some of the ex-
amples had potencies in the order of 100 nm against the para-
site, which represents a very good starting point for a drug dis-
covery programme against HAT. However, it quickly became
apparent that this series has one or more molecular targets in
addition to TbGSK3, which contributes strongly to the trypano-
cidal activity of the compounds. Interestingly, although based
on a kinase scaffold, this series had a reasonably clean profile
against the 79 mammalian kinases investigated. In the absence
Compound library selection was described previously.[29]
Primary screen assays: For the primary screening of the focused
kinase inhibitor library, a 384-well KinaseGlo (Promega) lumines-
cence-based assay was used as previously described.[25] The reac-
tions contained 7.5 nm TbGSK3, 3.2 mm substrate peptide (Pepceut-
icals), 1 mm ATP, and 25 mm test inhibitor compound in optimised
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kinase assay buffer. DMSO and an eight-point titration of GW8510
(Sigma) from 1 mm to 50 pm were included as negative and posi-
tive controls. Reactions were incubated at room temperature for
1 h and stopped by the addition of KinaseGlo reagent. Plates were
then sealed, and the signal was left to stabilise for 1 h in the dark
before luminescence was determined using a TopCount NXT HTS
counter (PerkinElmer).
CH3CN (1:1)+0.1% formic acid as the mobile phase. High-resolu-
tion electrospray MS measurements were performed on a Bruker
MicroTof mass spectrometer. LC–MS analyses were performed with
an Agilent HPLC 1100 (Phenomenex Gemini Column 5 m C18 110A
50ꢃ3.0 mm, eluting with 20% MeOH/H2O, 0–3 min) and a diode
array detector in series with a Bruker MicroTof mass spectrometer.
Column chromatography was performed using RediSep 4 or 12 g
silica pre-packed columns.
Potency screen assays: For hit validation and all subsequent com-
pound potency determinations, a radiometric 96-well Flashplate
assay (PerkinElmer) was adopted. Compounds were solubilised in
DMSO at a top concentration of 3 mm and serially diluted to ach-
ieve 10-point titration of final assay concentrations from 30 mm to
0.3 nm with a final DMSO concentration of 1% (v/v). The reaction
mixtures contained 1 mm biotinylated GSP2 substrate, 1 mm ATP,
3.7 KBq [g-33P]ATP per well, and 2.5 nm TbGSK3 in the TbGSK3
kinase assay buffer. GSK3 inhibitors were screened for selectivity
assessment against HsGSK3b as well. For the HsGSK3b assay the re-
action mixes contained 1 mm biotinylated GSP2 substrate, 2 mm
ATP, 7.4 KBq [g-33P]ATP per well, and 15 nm HsGSK3b in the TbGSK3
kinase assay buffer (25 mm Tris·HCl pH 7.5, 10 mm MgCl2, 5 mm
DTT, 0.02% CHAPS, 2 UmLꢂ1 heparin).
The following compounds were purchased from commercial sup-
pliers and their purity and identity confirmed by LC–MS analysis
(all compounds were >85% pure based on a diode array detec-
tor). Compounds 1 and 2 were purchased from ChemBridge Cor-
poration; 3 was purchased from Maybridge; 4 was purchased from
BioFocus; 5 was purchased from ChemDiv Inc.; 6, 9, and 23 were
purchased from TimTec; 7 and 10 were purchased from Specs.
Benzyl carbamimidothioate hydrobromide (11): Benzyl bromide
(16.0 mL, 135 mmol), thiourea (10.0 g, 131 mmol), and EtOAc
(75 mL) were combined, and then heated at 1208C in a microwave
reactor for 5 min. The reaction was allowed to cool to room tem-
perature, and the resulting solid was collected by filtration to give
benzyl carbamimidothioate hydrobromide 11 as a white solid
1
Mammalian kinase profiling: Selected compounds were screened
against a panel of mammalian kinases routinely run by the Division
of Signal Transduction Therapy (DSTT) at the University of Dundee
in duplicate at 10 mm. Enzymes included in the panel and assay
conditions are reported.[48] All biochemical assays were carried out
(29.3 g, 118.6 mmol, 89% yield); H NMR (500 MHz, [D6]DMSO): d=
9.07 (brs, 4H), 7.44–7.32 (m, 5H), 4.49 ppm (s, 2H).
4-Amino-2-(phenylamino)thiazol-5-yl)(phenyl)methanone
(8):
General procedure A (used for the synthesis of compounds 8, 14–
22): Benzyl carbamimidothioate hydrobromide (100 mg, 0.4 mmol),
phenyl isothiocyanate (52 mL, 0.43 mmol), and N,N-diisopropylethyl-
amine (DIPEA; 76 mL, 0.43 mmol) were added to DMF (5 mL), and
the resulting mixture was stirred at room temperature for 24 h. 2-
Bromoacetophenone (96 mg, 0.48 mmol), DIPEA (139 mL,
0.80 mmol), and DMF (2 mL) were then added, and the mixture
was stirred at room temperature for 1 h, after which the reaction
was quenched by the addition of aqueous HCl (1m, 4 mL). The
product was extracted with EtOAc (3ꢃ3 mL), and the combined
extracts were back washed with LiCl (2ꢃ3 mL of a 5% w/w solu-
tion in H2O), brine (3 mL), and then dried with MgSO4, and the sol-
vent was removed under reduced pressure. Purification by column
chromatography on silica eluting with petroleum ether (PE) 40–
608C and EtOAc gave 8 as a yellow solid (47 mg, 0.15 mmol, 39%
app
below the KM value for ATP for each enzyme, allowing compari-
son of inhibition across the panel.
Trypanosome and MRC5 proliferation assay: Measurement of in-
hibition of the proliferation of MRC5 (human lung fibroblast) cells
and T. brucei bloodstream-stage cells was performed using a modifi-
cation of a cell viability assay previously described.[49] Compounds
(50 mm to 0.5 nm) were incubated with 2ꢃ103 cells per well in
0.2 mL of the appropriate culture medium (MEM with 10% foetal
bovine serum for MRC-5 cells) in clear 96-well plates. Plates were
incubated at 378C in the presence of 5% CO2 for 69 h. Resazurin
was then added to a final concentration of 50 mm, and plates were
incubated as above for a further 4 h before being read on a BioTek
flx800 fluorescent plate reader.
1
yield); H NMR (500 MHz, [D6]DMSO): d=10.80 (brs, 1H), 8.22 (brs,
Data analysis: Determination of the TbGSK3 kinetic parameters
was carried out as described previously.[50] IC50 values were deter-
mined using a four-parameter equation in XLFit 4.2. To establish
mode of inhibition, rates were determined at 10 inhibitor concen-
trations with four varied concentrations of ATP in saturating con-
centration of GSP2. The resulting Lineweaver–Burk plots were ex-
amined for diagnostic patterns for competitive, mixed, or uncom-
petitive inhibition. Graphs and analyses were carried out using
Grafit 6.0. The correlation between in vitro IC50 against TbGSK3 in
T. brucei cells and inhibitor potency (Ki) for ATP-competitive inhibi-
tors was determined according to the Cheng–Prusoff equation
[Eq. (1)].
2H), 7.69–7.67 (m, 2H), 7.62 (d, J=7.7 Hz, 2H), 7.51–7.46 (m, 3H),
7.39–7.36 (m, 2H), 7.09 ppm (tt, J=7.4 and 1 Hz, 1H); 13C NMR
(125 MHz, DMSO): d=182.7, 167.2, 165.6, 141.9, 139.5, 130.4, 129.1,
128.4, 126.7, 123.4, 119.0, and 92.2 ppm; LC–MS m/z=296 [M+H]+
, tR =4.28 min, purity 88% (Agilent, 20–90% CH3CN, ES+ on an
acidic method).
(4-Amino-2-(phenylamino)thiazol-5-yl)(2,6-difluorophenyl)me-
thanone (14): General procedure A gave 14 as a yellow solid
1
(32 mg, 0.10 mmol, 10% yield); H NMR (500 MHz, [D6]DMSO): d=
10.89 (brs, 1H), 8.31 (brs, 2H), 8.08 (brs, 1H), 7.57–7.50 (m, 3H),
7.39–7.35 (m, 2H), 7.23–7.19 (m, 2H), 7.13–7.10 ppm (m, 1H); LC–
MS m/z=332 [M+H]+, tR =4.25 min, purity 90% (Agilent, 20–90%
CH3CN, ES+ on an acidic method).
Chemistry
(4-Amino-2-(phenylamino)thiazol-5-yl)(2,6-dimethoxyphenyl)me-
thanone (15): General procedure A gave 16 as a yellow solid
(9 mg, 0.03 mmol, 3% yield); 1H NMR (500 MHz, [D6]DMSO): d=
10.58 (brs, 1H), 7.79 (s, 2H), 7.54 (d, J=8.0 Hz, 2H), 7.54–7.52 (m,
3H), 7.06 (t, J=7.3 Hz, 1H), 6.69 (d, J=8.4, 2H), 3.71 ppm (6H, s);
LC–MS m/z=356 [M+H]+, tR =4.10 min, purity 100% (Agilent, 20–
90% CH3CN, ES+ on an acidic method).
1H NMR spectra were recorded on a Bruker Avance DPX 500 instru-
ment unless otherwise stated. Chemical shifts (d) are expressed in
ppm. Signal splitting patterns are described as singlet (s), broad
singlet (brs), doublet (d), triplet (t), quartet (q), multiplet (m), or
combination thereof. Low-resolution electrospray (ES) mass spectra
were recorded on a Bruker MicroTof mass spectrometer, run in pos-
itive ion mode, using either MeOH, MeOH/H2O (95:5), or H2O/
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(4-Amino-2-(phenylamino)thiazol-5-yl)(3-bromophenyl)metha-
none (16): General procedure A gave 16 as a yellow solid (211 mg,
0.56 mmol, 69% yield); 1H NMR (500 MHz, [D6]DMSO): d=10.85
(brs, 1H), 8.29 (brs, 2H), 7.81 (t, J=1.7 Hz, 1H), 7.71 (ddd, J=1.0,
2.0, 8.0 Hz, 1H), 7.69–7.67 (m, 1H), 7.63 (d, J=7.8 Hz, 2H), 7.46 (t,
J=7.8 Hz, 1H), 7.39–7.36 (m, 2H), 7.12–7.08 ppm (m, 1H); LC–MS
m/z=376 [M+H]+, tR =4.58 min, purity 100% (Agilent, 20–90%
CH3CN, ES+ on an acidic method).
Acknowledgements
We thank the Wellcome Trust for financial support for these stud-
ies (grant ref. 077705 and strategic award WT083481). We also
thank Iain Collie, Irene Hallyburton, and Bhavya Rao (DDU, Uni-
versity of Dundee) for carrying out the T. brucei and MRC5 prolif-
eration studies, and Daniel James for data management. W.C.V.V.
and K.K.O. were supported by US NIH grants R01AI089441 and
R01AI080625.
(4-Amino-2-((2,6-dimethylphenyl)amino)thiazol-5-yl)-
(phenyl)methanone (17): General procedure A gave 17 as a yellow
1
solid (35 mg, 0.11 mmol, 11% yield); H NMR (500 MHz, [D6]DMSO):
d=10.11 (brs, 1H), 8.40 (brs, 1H), 7.97 (brs, 1H), 7.54–7.16 (m,
8H), 2.20 ppm (s, 6H); LC–MS m/z=324 [M+H]+, tR =4.39 min,
purity 95.6% (Agilent, 20–90% CH3CN, ES+ on an acidic method).
Keywords: antiprotozoal agents · GSK3 · medicinal chemistry ·
protein kinases · Trypanosoma brucei
(4-Amino-2-((3,4-dimethylphenyl)amino)thiazol-5-yl)-
(phenyl)methanone (18): General procedure A gave 18 as a yellow
solid (100 mg, 0.30 mmol, 30% yield); 1H NMR (500 MHz,
[D6]DMSO): d=10.60 (brs, 1H), 8.31 (brs, 1H), 8.11 (brs, 1H), 7.67–
7.65 (m, 2H), 7.51–7.45 (m, 3H), 7.34 (d, J=8.3 Hz, 1H), 7.31 (s,
1H), 7.12 (d, J=8.2 Hz, 1H), 2.21 (s, 3H), 2.19 ppm (s, 3H); LC–MS
m/z=324 [M+H]+, tR =4.55 min, purity 88% (Agilent, 20–90%
CH3CN, ES+ on an acidic method); HRMS m/z [M+H]+ calcd for
C18H18N3OS: 324.1165, found: 324.1158.
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(4-Amino-2-(cyclohexylamino)thiazol-5-yl)(phenyl)methanone
(19): General procedure A gave 19 as a yellow solid (59 mg,
1
0.19 mmol, 19% yield); H NMR (500 MHz, [D6]DMSO): d=8.57 (brs,
1H), 8.49 (brs, 1H), 7.89 (brs, 1H), 7.63–7.61 (m, 2H), 7.47–7.42 (m,
3H), 3.70 (brs, 1H), 1.91 (d, J=10.7 Hz, 2H), 1.76–1.70 (m, 2H), 1.57
(d, J=12.9 Hz, 1H), 1.32–1.13 ppm (m, 5H); LC–MS m/z=302 [M+
H]+, tR =4.42 min, purity 88% (Agilent, 20–90% CH3CN, ES+ on an
acidic method); HRMS m/z [M+H]+ calcd for C16H20N3OS:
302.1322, found: 302.1321.
(4-Amino-2-((cyclohexylmethyl)amino)thiazol-5-yl)-
(phenyl)methanone (20): General procedure A gave 20 as a yellow
solid (136 mg, 0.43 mmol, 43% yield); 1H NMR (500 MHz,
[D6]DMSO): d=8.63 (brs, 1H), 8.43 (brs, 1H), 7.84 (brs, 1H), 7.63–
7.61 (m, 2H), 7.48–7.43 (m, 3H), 3.17–3.03 (m, 2H), 1.17–1.52 (m,
6H), 1.23–1.11 (m, 3H), 0.94–0.87 ppm (m, 2H); LC–MS m/z=316
[M+H]+, tR =4.63 min, purity 97% (Agilent, 20–90% CH3CN, ES+
on an acidic method); HRMS m/z [M+H]+ calcd for C17H22N3OS:
316.1478, found: 316.1472.
(4-Amino-2-(phenethylamino)thiazol-5-yl)(phenyl)methanone
(21): General procedure A gave 21 as a yellow solid (77 mg,
1
0.24 mmol, 24% yield); H NMR (500 MHz, [D6]DMSO): d=8.73 (brs,
1H), 8.47 (brs, 1H), 7.87 (brs, 1H), 7.63–7.61 (m, 2H), 7.48–7.43 (m,
3H), 7.32–7.29 (m, 2H), 7.26–7.21 (m, 3H), 2.90 (s, 1H), 2.87 (t, J=
7.2 Hz, 2H), 2.74 ppm (d, J=0.5 Hz, 1H); HRMS m/z [M+H]+ calcd
for C14H18N3OS: 324.1165, found: 324.1153.
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1
(d, J=7.1 Hz, 1H), 3.30–3.28 (m, 1H), 2.00 (dd, J=13.6, 3.3 Hz, 1H),
1.70 (dt, J=13.6, 4.0 Hz, 2H), 1.57 (dt, J=13.2, 4.0 Hz, 1H), 1.38–
1.33 (m, 2H), 1.20 (s, 9H, tBu-H), 1.19–1.17 (m, 1H), 0.81 (t, J=
7.1 Hz, 1H), 0.79–0.76 ppm (m, 1H); 13C NMR (DMSO, 125 MHz) d=
128.6, 128.4, 119.5, 53.4, 53.3, 32.0, 27.0, 24.9, 24.3, 17.9, 16.6,
12.2 ppm; LC–MS m/z=282 [M+H]+, tR =4.60 min, purity 96%
(Agilent, 20–90% CH3CN, ES+ on an acidic method); HRMS m/z
[M+H]+ calcd for C14H24N3OS: 282.1635, found: 282.1641.
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Received: February 17, 2013
Revised: May 15, 2013
Published online on June 14, 2013
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ChemMedChem 2013, 8, 1127 – 1137 1137