CZ415, a Highly Selective mTOR Inhibitor Showing in Vivo Efficacy in a Collagen Induced Arthritis Model

Article abstract of DOI:10.1021/acsmedchemlett.6b00149

CZ415, a potent ATP-competitive mTOR inhibitor with unprecedented selectivity over any other kinase is described. In addition to a comprehensive characterization of its activities in vitro, in vitro ADME, and in vivo pharmacokinetic data are reported. The suitability of this inhibitor for studying in vivo mTOR biology is demonstrated in a mechanistic mouse model monitoring mTOR proximal downstream phosphorylation signaling. Furthermore, the compound reported here is the first ATP-competitive mTOR inhibitor described to show efficacy in a semitherapeutic collagen induced arthritis (CIA) mouse model.

Full text of DOI:10.1021/acsmedchemlett.6b00149

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Letter
CZ415, a highly selective mTOR inhibitor showing in
vivo efficacy in a collagen induced arthritis (CIA) model
Andrew D. Cansfield, Tammy Ladduwahetty, Mihiro Sunose, Katie Ellard, Rosemary
Lynch, Anthea L. Newton, Ann Lewis, Gavin Bennett, Nico Zinn, Douglas W Thomson,
Anne J. Rüger, John T. Feutrill, Oliver Rausch, Alan P. Watt, and Giovanna Bergamini
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00149 • Publication Date (Web): 10 Jun 2016
Downloaded from http://pubs.acs.org on June 12, 2016
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CZ415, a highly selective mTOR inhibitor showing in vivo efficacy in
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a collagen induced arthritis (CIA) model
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Andrew D. Cansfield, Tammy Ladduwahetty, Mihiro Sunose, Katie Ellard, Rosemary Lynch,
†
■
†∆
†
‡●
‡●
Anthea L. Newton, Ann Lewis, Gavin Bennett, ° Nico Zinn, Douglas W. Thomson, Anne J.
‡
●
†≈
†×
†∆
‡●
Rüger, John T. Feutrill, Oliver Rausch, Alan P. Watt, and Giovanna Bergamini*
†
‡
Cellzome Ltd, Chesterford Research Park, Saffron Waldon, CB10 1XL, United Kingdom; Cellzome AG, Meyerhofstraße
, 69117, Heidelberg, Germany; Present Addresses under Author Information.
1
KEYWORDS. mTOR, kinase inhibitor, inflammation, collagen induced arthritis model.
ABSTRACT: CZ415, a potent ATPꢀcompetitive mTOR inhibitor with unprecedented selectivity over any other kinase is deꢀ
scribed. In addition to a comprehensive characterization of its activities in vitro, in vitro ADME and in vivo pharmacokinetic data
are reported. The suitability of this inhibitor for studying in vivo mTOR biology is demonstrated in a mechanistic mouse model
monitoring mTOR proximal downstream phosphorylation signalling. Furthermore, the compound reported here is the first ATPꢀ
competitive mTOR inhibitor described to show efficacy in a semiꢀtherapeutic collagen induced arthritis (CIA) mouse model.
The mammalian target of rapamycin (mTOR/FRAP1) is a
serine/threonine protein kinase, which belongs to a family of
phosphoinositide 3ꢀkinaseꢀrelated kinases (PIKK), which also
includes ATR, ATM, DNAPK and SMGꢀ1. FRAP1 is the
catalytic subunit of two signaling complexes called mTORC1
and mTORC2, which play a central role in regulation of cell
Quality criteria such as high selectivity, well characterized
mode of action and a favorable pharmacokinetic profile are a
requirement to generate meaningful biological data and enable
1
2
translation of in vitro findings to in vivo models.
We report here a highly selective drugꢀlike small molecule
mTOR inhibitor from a novel chemical series, which shows
efficacy in in vivo models.
13
1
metabolism, proliferation, survival and migration. It has been
reported that mTOR is a key modulator in ageing, and dysregꢀ
ulated mTOR signaling can promote metabolic diseases, canꢀ
The morpholine substituted pyrimidine core is a common
structural motif that has been extensively explored for the
2,3
cer and is often associated with inflammation. The broad
1
4
development of ATP competitive mTOR inhibitors. In preꢀ
vious efforts on discovering mTOR inhibitors, we have also
worked on this compound class and identified CZ830 (1) and
CZ109 (2) as leads from two different chemical series (Figure
interest in mTOR as a drug target is reflected by the growing
number of mTOR inhibitors that have already been approved
4
–9
or are under clinical development.
15 16
,
To define further potential therapeutic uses and guide the
development of safe and efficacious drugs it is crucial to fully
understand the biological role of mTOR and the effects of its
1
).
Both inhibitors showed moderate submicromolar affiniꢀ
ty for mTOR and exhibited high selectivity of more than 100
fold over other lipid kinases from the same family, namely
phosphatidylinositide 3ꢀkinases (PI3K) α/β/γ/δ and DNAꢀ
dependent protein kinase (DNAPK).
1
0
pharmacological inhibition. Ideally, target validation studies
using small molecules should be conducted with multiple
chemical probes to increase the confidence of target dependꢀ
ency of the observed phenotype and reduce the risk of misinꢀ
In order to discover more potent mTOR inhibitors, we comꢀ
bined both series, incorporating the sulfone moiety of comꢀ
pound 1 into the fused pyrimide core of 2.
11
terpretation
of
potential
offꢀtarget
effects.
13,15,16
Figure 1. mTOR inhibitors from different chemicals series. Starting from lead compounds 1 and 2, cyclic sulfone 3 was designed.
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In addition to what was previously reported on cyclic sulꢀ The potency and selectivity of 3 was assessed using Cellꢀ
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fones, we broadly explored methylꢀsubstitution on the 5ꢀ
membered ring. Compared to other 5ꢀmembered sulfones, the
introduction of a dimethylꢀsubstituent was advantageous in
terms of potency, selectivity or solubility and gave beneficial
PK profile as compared to the 6ꢀmembered sulfopyrimidines.
The morpholine substituent was found to be crucial for mainꢀ
taining high potency and selectivity, whereas multiple groups
were tolerated on the urea moiety. This is consistent with
reported mTOR crystal structure and models on close anaꢀ
zome’s chemoproteomic platform. Competition binding experꢀ
iments combined with a proteomic readout are a powerful tool
to determine the selectivity of small molecules against a large
1
9,20
portion of the proteome in a single experiment.
With this
approach, the compound’s binding affinities were measured
for approximately 285 protein kinases, including the family of
lipid and atypical kinases. The apparent dissociation constant
app
pKd for 3 against mTOR was found to be 8.2, while any
other kinase identified in the experiments, including other
PI3K family members – found to be frequent offꢀtargets of
other mTOR inhibitors – did not show any affinity within
1000 fold. The remarkable selectivity of 3 is depicted in Fig-
ure 2 (detailed list of proteins is provided in Supporting Inꢀ
formation).
logues showing morpholine making an important hinge bindꢀ
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ing interaction.
Exploring this chemical series, our efꢀ
forts culminated in the discovery of CZ415 (3), Figure 1. The
synthesis of 3 and related cyclic sulfones has been previously
13
reported and is described in the supporting information.
Figure 2. MSꢀbased kinaseꢀbinding profile of 3 across a set of protein kinases identified from mixed human cellꢀline lysates (285 kinases
app
identified). The bars indicate pKd values defined as the concentration of drug at which halfꢀmaximal competition of binding is observed,
21
corrected by the influence of the immobilized ligand on the binding equilibrium using the ChengꢀPrusoff equation. The dose dependent
binding curve for mTOR is also depicted.
app
To verify that binding of 3 to mTOR results in inhibition of
both mTORC1 and mTORC2 dependent signaling in the celluꢀ
lar context, 3 was tested in a cellular mechanistic assay. Inhiꢀ
bition of mTORC1 and mTORC2 protein complex activity
was assessed by monitoring the phosphorylation of downꢀ
stream targets, S6 Ribosomal Protein (S6RP, downstream of
mTORC1) and Protein Kinase B (=Akt, mTORC2 substrate).
Phosphorylation levels for both proteins, pS6RP (S240/244)
and pAkt (S473) were assessed in dose response format from
HEK293T cells after treatment with 3. As depicted in Figure
good cell permeability (Kd = 6.9 nM determined by chemoꢀ
proteomic binding assay from cellular lysate, see above).
Furthermore, interferon gamma (IFNγ) release in whole blood
was used to test mTOR inhibition in primary cells. Treatment
of whole blood with monoclonal antibodies binding to cluster
of differentiation 3 and 28 (αCD3/αCD28) mimics antigen
receptor signalling and activates proliferation of Tꢀ
lymphocytes. Coꢀstimulation with Interleukin 2 (ILꢀ2) then
induces interferon gamma gene (IFNG) expression and IFNγ
2
2
is released. The immunosuppressive effect of 3 was measꢀ
ured by detecting secreted IFNγ after 18 hours in stimulated
human whole blood and the resulting IC50 was 226 nM
(Figure 3).
3
, inhibition of phosphorylation for both downstream targets
resulted in 14.5 nM IC50 for pS6RP and 14.8 nM for pAKT
(Western Blot analysis and Caspase 3/7 activity assay are
reported in the supporting information). Retaining high potenꢀ
cy against mTOR in the cellular system proves functional
inhibition
of
the
target
and
indicates
very
A:
B:
C:
Figure 3. Activity of 3 in cellular assays: A: Dose dependent inhibition of S6RP phosphorylation in HEK293T after 2 hours treatment of 3,
normalized to total S6RP levels. IC50 = 14.5 nM (95% CI 11.5 to 18.3 nM, n = 4). B: Dose dependent inhibition of Akt phosphorylation in
HEK293T after 2 hours treatment of 3, normalized to total Akt levels. IC50 = 14.8 nM (95% CI 10.4 to 21.0 nM, n = 4). C: Dose dependent
inhibition of IFNγ release in stimulated human whole blood after 18 hours treatment of 3. IC50 = 226 nM (95% CI 169 to 303 nM, n = 4).
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We have further investigated the properties of 3 in dedicated
in vitro assays for early drug safety prediction. The cytoꢀ
1
iv1mg/kg
po 3mg/kg
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chromes P450 (CYPs) are a family of enzymes that play a
major role in drug metabolism and interaction with them is
related to potential drugꢀdrug interactions and adverse drug
reactions. In human microsomes, no inhibition of the main
P450 isoforms CYP1A, CYP2C8, CYP2C9, CYP2C19,
CYP2D6 and CYP3A4 was observed within a 1000 fold winꢀ
dow of mTOR affinity. Further tests for cytochrome CYP3A4
also showed no induction at 10 µM and no time dependent
inhibition (TDI) at a concentration as high as 50 µM.
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.1
0.01
0
.001
Time (h)
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49 / 41 ml/min/kg (45% LBF)
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Cl / Clb
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Vdss
3.1L/kg
0.71 / 0.43 ꢀM
1.1h
0.76 / 1.0 ꢀM×h
44%
As a predictor for cardiotoxicity, the activity of 3 against the
human cardiac ion channel hERG was assessed in a wholeꢀcell
patchꢀclamp assay in HEK293 cells resulting in an IC50 of 48
µM. We were further pleased that 3 showed no genotoxic
potential. It was neither mutagenic in a bacterial mutation
assay (Ames test) nor did it show genotoxicity in the mouse
lymphoma assay (MLA), in either the presence or absence of
ratꢀliver S9 mix. Given the data reported above for 3, there
were no safety related liabilities predicted for this compound.
Next to potency/selectivity assessment and early safety predicꢀ
tion, physicochemical properties are important for the successꢀ
ful development of drugs as well as for high quality probes,
Cmax iv / po
MRT
AUC0ꢁINF iv / po
F (%)
Figure 4. Time dependent plasma concentration of 3 after intraꢀ
venous bolus (iv, circle) and oral solution (po, square) administraꢀ
tion to rats. Rats were dosed at 1 mg/kg (iv, n = 3) or 3 mg/kg (po,
n = 3). Vehicle: 5% DMSO / 95% (10% Kleptose).
To demonstrate the efficacy of 3 in vivo, modulation of
mTOR proximal downstream signaling has been monitored in
a mechanistic mouse model. In vivo treatment with antiꢀCD3
antibody specifically stimulates T cells upon binding to the
CD3 receptor and subsequent downstream activation of the
2
3,24
that can be used in vivo.
Compound 3 has a moderate moꢀ
lecular weight (MW 460) and polar surface area (PSA 114),
both in the desirable chemical space for bioavailable drugs.
25
PI3K/mTOR signalling pathway. To determine the effects of
on its pharmacological target, doseꢀdependent changes in
Despite a relatively high distribution coefficient (logDpH7.4
3
4
.31), 3 showed good kinetic solubility (CLND 149.5 µM) as
phosphorylation levels of S6 Ribosomal Protein and Akt –
both downstream targets of mTOR – were assessed. The inhibꢀ
itor was administered orally at 1, 3 and 10 mg/kg to mice 1 h
before antiꢀCD3 stimulus. 15 min after stimulation, spleens
were dissected and analyzed for pS6RP and pAKT levels. A
dose related significant inhibition of phosphorylation of both
S6RP and Akt was observed after compound administration
well as solubility in biorelevant media, which is important for
oral administration (FaSSIF 11.1 µg/mL, FeSSIF 60.1 µg/mL).
The permeability measured in a Cacoꢀ2 assay, predictive for in
vivo absorption of drugs across the gut wall, was moderate
(Papp (AꢀB) 0.47 nm/s, Papp (BꢀA) 1.02 nm/s). A table sumꢀ
marizing physicochemical properties of 3 is provided in the
supporting information.
(
Figure 5, A and C). In particular, 1 and 3 mg/kg 3 could fully
The pharmacokinetic (DMPK) properties of a compound
determine its ability to act over time on the molecular target. A
low rate of drug metabolism is desirable for long acting comꢀ
pounds to maintain sufficient compound levels in plasma. As
in vitro determination of the metabolic stability can help preꢀ
dicting in vivo PK, stability of 3 was assessed in microsomes
and hepatocytes from different species. Low to moderate
intrinsic clearance was measured, resulting in a calculated Clint
<0.6/<1.1/1.5 µL/min/g liver in microsomes and Clint
n.d./<0.8/≤0.5 µL/min/g liver in hepatocytes (rat/dog/human).
In addition to the metabolic stability, measurement of plasma
protein binding (PPB) enables estimation of the free fraction
of compound in the bloodstream. PPB of 3 in rat, mouse and
human plasma was determined to be 69/89/81% (r/m/h), indiꢀ
cating a sufficient free fraction of the compound being availaꢀ
ble for binding to the target.
inhibit S6RP phosphorylation induced by antiꢀCD3 stimulaꢀ
tion and 10 mg/kg additionally decreased the constitutive
phosphorylation levels as measured in the control group.
While phosphorylation of Akt was not increased upon antiꢀ
CD3 stimulation (Figure 5, C), 3 ꢀ already at the lowest dose
of 1mg/kg ꢀ significantly reduced pAkt constitutive levels, as
measured in the control group. The PK/PD relationship was
determined following measurement of compound concentraꢀ
tion in blood and the exposureꢀresponse correlation is depicted
in Figure 5, B and D. An EC50 of 0.22 µM was measured for
pS6RP and 0.055 µM for pAkt, which correlate well with the
nanomolar potency of 3 in the cell based assays.These results
showed 3 to be a potent mTOR inhibitor which retained its
activity in vivo
In vivo efficacy of 3 was then assessed in an inflammatory
context. In the collagen induced arthritis (CIA) model, mice
reliably develop polyarthritis when immunized against bovine
type II collagen and this model can be utilized to monitor antiꢀ
inflammatory effects of the tested compounds. It has been
For full characterization of 3 and to enable improved dose
predictions, the pharmacokinetic (PK) profile was assessed in
rat. PK and oral bioavailability were determined after of 1
mg/kg intravenous (iv) bolus and 3 mg/kg oral (po) adminꢀ
istration (Figure 4). The observed plasma clearance, correꢀ
sponding to 45% liver blood flow, suggested that sufficient
levels of free compound were circulating in the animal over
time. The oral uptake was rapid with a Tmax of 0.5 h and bioaꢀ
vailability F = 44% indicated very good absorption from the
gut. The plasma concentration peaked at 0.43 µM which was
above IC50 determined in cellular assays for 3.
shown, that treatment with Rapalogs can inhibit the clinical
severity of arthritis in mice,
26–28
but the effect of ATPꢀ
competitive mTOR inhibitors has not previously been reportꢀ
ed. A so called semiꢀestablished study design was utilized for
testing 3 at 10 mg/kg twice daily. Mice were challenged with
two injections of collagen, the first at day 0 and a boost injecꢀ
tion at day 21.
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A:
C:
B:
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Figure 5. Compound 3 in antiꢀCD3 mouse model. A: pS6RP levels (normalized to total S6RP) measured in spleens of compound treated
as compared to disease vehicle group (p<0.01 for 1 mg/kg of 3, p<0.001 for 3 and 10 mg/kg of 3ꢀ; one outlier removed in normal control
and disease vehicle group). B: Exposure response fit: pS6RP levels at terminal exposure. EC50 0.22 µM (95% CI 0.15 to 0.32 µM). C:
pAkt levels (normalized to total Akt) measured in spleens of compound treated as compared to disease vehicle group (p<0.001 for 1, 3 and
0 mg/kg of 3). D: Exposure response fit: pAkt levels at terminal exposure. EC50 0.055 µM (95% CI 0.048 to 0.065 µM). Legend: White –
normal controls (n = 4). Black – antiꢀCD3 / vehicle 5% DMSO / 95% (10% Kleptose) (n = 6). Grey – antiꢀCD3 / 3 (1, 3 or 10 mg/kg, of 3
1
PO) (n = 6).
Administration of 3 was started at day 22, immediately beꢀ
fore the onset of the disease and continued until day 34 at
termination of the study. The development of arthritis, which
reached 100% disease incidence in the vehicle group, was
determined by evaluation of clinical arthritis scores (from 0 to
No significant difference in body weight loss was observed
compared to vehicleꢀtreated, diseaseꢀinduced mice at end of
the study (Figure 6, B). Measurement of compound concenꢀ
trations in blood, determined on day 31, indicated that up to 8
hours after dosing the levels of circulating 3 were above EC50
as determined both in vitro in the whole blood assay and in
vivo in the PK/PD mouse model (Figure 6, C). Unlike many
antiꢀinflammatory molecules that require prophylactic dosing
in the CIA, this data demonstrates that 3 is active in a situation
more akin to human disease.
5
) that were measured by inspection of the paws for erythema
and swelling. The daily clinical arthritis scores of the treated
group differed from disease control group over time with
significant reductions (p<0.05) at day 33ꢀ34 (Figure 6, A).
Clinical arthritis scores expressed as area under the curve
(AUC) were reduced in the group treated with 3 by 59% as
compared to the disease vehicle group (data not shown).
5.0
10.000
A:
Normal Controls
Disease Vehicle
Compound 3
B:
C:
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.100
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0.0
Day 22 23 24 25 26 27 28 29 30 31 32 33 34
8
0.010
Time (h)
Day
22 24 26 28 30 32 34
0
4
8
12 16 20 24
Figure 6. Compound 3 in a mouse CIA model. A: Clinical arthritis score – all paws (Scored 0ꢀ5). Normal controls (white squares): signifiꢀ
cance (p≤0.05) from disease vehicle group (black squares) observed at day 27 through day 34; Compound 3 at 10 mg/kg, bid (grey dots):
significant reduction as compared to disease vehicle group at day 33ꢀ34 (p≤0.05). B: Body weight over time. Significant difference of
normal controls at day 26 through day 34 as compared to disease vehicle group, and of 10 mg/kg 3 at day 26 (p≤0.05 to disease vehicle).
C: Drug levels of 3 in blood on study day 31. Exposure: AUC 14 µM×h, Cmax 1.7 µM. Legend: Normal controls (n = 4), Disease vehicle
(5%DMSO/95% (10% kleptose), n = 10), Compound 3 (10 mg/kg, PO, n = 10).
We have presented here a highly potent mTOR inhibitor
CZ415 (3) with exquisite selectivity over other lipid and proꢀ
tein kinases. It has a defined mechanism of action, which was
demonstrated in mechanistic and phenotypic cell based assays,
including whole blood assays. Beside favorable physicochemꢀ
ical properties, early in vitro safety assessment did not exhibit
any liabilities and pharmacokinetic properties of moderate
clearance and good oral bioavailability showed suitability of 3
for progression to in vivo studies. In an antiꢀCD3 mouse model
3 efficiently inhibited mTOR downstream signaling and in a
CIA mouse model showed significant antiꢀinflammatory efꢀ
fects. With its extraordinary selectivity, drugꢀlike properties
and proven efficacy in vivo, 3 represents an ideal molecule for
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the pharmacological investigation of mTOR pathophysiologiꢀ
cal role in vivo.
Author Contributions
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G. Bergamini and A.J.R. wrote the manuscript, T.L. and M.S.
designed and participated to the execution of the work, A.D.C.,
R.L, K.E. and J.F. participated to the chemical design and syntheꢀ
sis, A.L.N. designed and performed the cellular assays, A.L.
supervised and analyzed the DMPK studies, G. Bennett superꢀ
vised and analyzed the animal model studies, D.W.T. and N.Z.
supervised and analyzed the selectivity profiling work, A.P.W.
and O.R. contributed to the design of the studies, G. Bergamini
designed and supervised the work reported. All authors have
given approval to the final version of the manuscript.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
NMR and MS spectra, detailed selectivity profiling data, protoꢀ
cols for cellular assays, DMPK and supplemental information on
animal studies (PDF)
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AUTHOR INFORMATION
Corresponding Author
Notes
The human biological samples were sourced ethically and their
research use was in accord with the terms of the informed conꢀ
sents. All animal studies were ethically reviewed and carried out
in accordance with European legislation and guidelines for animal
welfare.
*
Eꢀmail: giovanna.2.bergamini@gsk.com.
Present Addresses
┴
Heptares Therapeutics, BioPark, Broadwater Road, Welwyn
Garden City, Hertfordshire, AL7 3AX, UK.
ACKNOWLEDGMENT
§
Charles River Laboratories, Early Discovery, Harlow, Essex CM
We thank Cellzome chemistry, biology, pharmacology, biochemꢀ
istry, informatics and mass spectrometry departments, the leaderꢀ
ship team and the whole mTOR program team for support and
advice.
1
9 5TR, UK.
║
Sygnature Discovery Ltd., BioCity, Pennyfoot Street, Nottingꢀ
ham, NG1 1GF, UK.
Joyce Frankland Academy, Newport, Saffron Walden, Essex,
#
CB11 3TR, United Kingdom.
ABBREVIATIONS
%
Aqdot Ltd., Iconix Park, London Road, Cambridge, CB22 3EG,
Akt, Protein Kinase B; ATM, Ataxia telangiectasia mutated kiꢀ
nase; ATR, ataxia telangiectasia and Rad3ꢀrelated protein;
αCD3/αCD28, monoclonal antibodies binding to cluster of differꢀ
entiation 3/28; CIA, collagen induced arthritis; DNAPK, DNAꢀ
dependent protein kinase; IFNγ, interferon gamma; IFNG, interꢀ
UK.
■
LGC Ltd., Newmarket Road, Fordham, Cambridgeshire, CB7
5
WW, UK.
∆
Xenovium, Chesterford Research Park, Saffron Waldon, CB10
XL, UK.
Bicycle Therapeutics Ltd., Babraham Research Campus, Camꢀ
1
°
app
feron gamma gene; ILꢀ2, Interleukin 2; Kd , apparent dissociaꢀ
tion constant; mTOR, mammalian target of rapamycin; PI3K,
phosphatidylinositide 3ꢀkinases; PIKK, family of phosphoꢀ
inositide 3ꢀkinaseꢀrelated kinases; PK/PD, pharmacokinetꢀ
ic/pharmacodynamic; SMGꢀ1, nonsense mediated mRNA decay
associated PI3K related kinase; S6RP, S6 Ribosomal Protein.
bridge, CB22 3AT, UK.
≈
SYNthesis med chem, 399 Royal Parade, Parkville VIC 3052,
Australia.
×
National Institute for Health Research, Translational Research
Partnerships, 80 London Rd, London SE1 6LH, UK.
●
Cellzome GmbH, a GSK company, Meyerhofstraße 1, 69117,
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mTOR
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pKdapp 8.2
Normal Controls
Disease Vehicle
Compound 3
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Day 22232425262728293031323334
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