Discovery of an in Vivo Tool to Establish Proof-of-Concept for MAP4K4-Based Antidiabetic Treatment

Article abstract of DOI:10.1021/acsmedchemlett.5b00215

Recent studies in adipose tissue, pancreas, muscle, and macrophages suggest that MAP4K4, a serine/threonine protein kinase may be a viable target for antidiabetic drugs. As part of the evaluation of MAP4K4 as a novel antidiabetic target, a tool compound,

Full text of DOI:10.1021/acsmedchemlett.5b00215

Letter
pubs.acs.org/acsmedchemlett
Discovery of an in Vivo Tool to Establish Proof-of-Concept for
MAP4K4-Based Antidiabetic Treatment
Mark Ammirati,† Scott W. Bagley,† Samit K. Bhattacharya,*,† Leonard Buckbinder,‡ Anthony A. Carlo,§
Rebecca Conrad,‡ Christian Cortes,‡ Robert L. Dow,† Matthew S. Dowling,† Ayman El-Kattan,⊥
Kristen Ford,∥ Cristiano R. W. Guimaraes,† David Hepworth,† Wenhua Jiao,† Jennifer LaPerle,⊥
̃
Shenping Liu,† Allyn Londregan,† Paula M. Loria,∥ Alan M. Mathiowetz,† Michael Munchhof,†
Suvi T. M. Orr,† Donna N. Petersen,∥ David A. Price,† Athanasia Skoura,‡ Aaron C. Smith,†
and Jian Wang†
‡
§
∥
†Worldwide Medicinal Chemistry, Cardiovascular and Metabolic Research Unit, External Research Solutions, Primary
⊥
Pharmacology Group, and Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research & Development, 610 Main
Street, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: Recent studies in adipose tissue, pancreas,
muscle, and macrophages suggest that MAP4K4, a serine/
threonine protein kinase may be a viable target for antidiabetic
drugs. As part of the evaluation of MAP4K4 as a novel
antidiabetic target, a tool compound, 16 (PF-6260933) and a
lead 17 possessing excellent kinome selectivity and suitable
properties were delivered to establish proof of concept in vivo.
The medicinal chemistry effort that led to the discovery of
these lead compounds is described herein together with in vivo pharmacokinetic properties and activity in a model of insulin
resistance.
KEYWORDS: MAP4K4, HGK, ZC1, kinase inhibitor, MAP4K4 inhibitor, P-loop, PF-6260933
A virtual screening effort of the internal Pfizer file using an
in-house crystal structure of the kinase domain of MAP4K4,
identified compound 1 as a potent MAP4K4 inhibitor (Table
1). It was selective in a standard kinase panel and upon
cocrystallization the complex between 1 and MAP4K4 revealed
that the P-loop adopts a rare folded conformation state (Figure
1).10 The P-loop, which normally exists in an extended
conformation, is a stretch of nine amino acids that in MAP4K4
is delimited by residues Val31 to Val39. The binding site adopts
a tunnel-like shape that provides a significant hydrophobic
enclosure for the inhibitor. There are potential π−π
interactions between the MAP4K4 P-loop Tyr36 residue and
the three aromatic rings of compound 1. The primary amide is
the only group that escapes the hydrophobic tunnel, being
partially exposed to solvent. The structure suggests that it forms
a hydrogen bond with Asp115.
itogen-activated protein kinase kinase kinase kinase 4
M(MAP4K4), also called HGK or ZC1, is a serine/
threonine protein kinase that belongs to the mammalian
STE20/MAP4K family.1 MAP4K4 is overexpressed in human
cancers and cancer cell lines and appears to play an important
role in cell transformation, invasiveness, adhesion, and
migration.2 More recently, studies in adipose tissue, pancreas,
muscle, and macrophages suggest that MAP4K4 could also be a
potential target for antidiabetic drugs.3,4 For example,
suppression of the expression of MAP4K4 using small
interfering RNA in human skeletal muscle cells from both
healthy subjects and type II diabetic patients prevented TNF-α
induced insulin resistance of glucose uptake.5 Furthermore, a
recent genetic analysis suggests that common genetic variants
in the MAP4K4 locus are associated with type II diabetes
underlying pathomechanisms, insulin resistance, and β-cell
failure.6 As part of the evaluation of MAP4K4 as a novel
antidiabetic target, it was desirable to deliver a tool compound
with greater kinome selectivity than the existing chemical
matter (unpublished results) and reasonable properties to
establish proof of concept in a mouse diabetes model. Herein
we describe the medicinal chemistry that led to the discovery of
such a compound. Since our work was conducted, alternative
series of MAP4K4 inhibitors for oncology and neurodegener-
ative indications have recently been described.7−9
Compound 1 had low molecular weight (MW = 299) and a
measured LogD of 3.6. It was a weak inhibitor of MAP4K4
(Table 1) but with good ligand efficiency (LE = 0.48). The
kinase activity is measured using a FRET-based assay that
utilizes catalytic domain of human recombinant MAP4K4. The
cell assay is designed to identify compounds inhibiting
Received: May 29, 2015
Accepted: September 28, 2015
© XXXX American Chemical Society
A
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Table 1. MM-GB/SA Score, Enzyme and Cell Potencies, and
Permeability Data of Analogues of Compound 1
Figure 1. Crystal structure of the complex between MAP4K4 and (a)
compound 1 (in green) (PDB code: 4ZP5) and (b) compound 13 (in
green) (PDB code: 5DI1).
Figure 2. Hydration sites obtained using WaterMap simulations. The
crystal structure of the complex between MAP4K4 and compound 1
was used. The compound is omitted in the simulations (compounds 1
and 2 were added to the figure above for comparison). The most
favorable and unfavorable water molecules with respect to a water
molecule with bulk-like properties are colored green and red,
respectively.
a
MM-GB/SA relative score in kcal/mol. The best scoring analogue
according to the MM-GB/SA method is used as reference (0.0 kcal/
b
mol). Arithmetic mean and standard error of ≥3 measurements
MAP4K4’s phosphorylation of serine/threonine residues on
traf2 (see Supporting Information for full details). In an
attempt to improve the enzyme and cell potencies of 1, several
heterocyclic alternatives to the oxazole hinge binding group
were evaluated by the MM-GB/SA scoring procedure.11 The
MM-GB/SA method involves simulations of a ligand in its
bound and unbound states and computes its free energy of
binding from the energy differences between these two states.
The energy terms include intramolecular and intermolecular
interactions, solvation energy, and conformational entropy. The
most promising analogues from this analysis were synthesized
and evaluated for MAP4K4 inhibitory activity (Table 1). The
best scoring compounds, 1−4, were the most potent in the
enzyme assay, whereas heterocycles 5−7, which had higher
scores (and hence predicted to be less potent), displayed
weaker potencies. The pyridine analogue 2 was found to be the
most potent inhibitor in the enzyme assay but displayed weak
inhibition in the cell-based assay. After the identification of
pyridine as a competent hinge binder, the WaterMap
methodology12 was employed to characterize unstable water
c
(unless noted otherwise). Less than three independent replicates.
d
Passive permeability (Papp in 10−6 cm/s) measured using low-efflux
MDCKII cells.13
molecules that could lead to incremental increase in potency
when liberated to the bulk upon ligand binding. The hydration
sites obtained from a simulation using the crystal structure of
the complex between MAP4K4 and 1 are illustrated in Figure
2; the compound is omitted in the simulation (compounds 1
and 2 are included in the figure for comparison). The overlap
between the map and compounds 1 and 2 suggests that they
replace the favorable waters (colored green) with the primary
amide and displace most of the unfavorable waters (colored
red), except for three moderately unstable waters that sit in a
deep pocket close to the methionine gatekeeper residue.
This map inspired the design of analogues 8 and 9 (Table 1).
The methoxy and cyclopropoxy groups were expected to lead
to gains in potency due to partial or full displacement of one or
two of the hydration sites highlighted in Figure 2. These
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Table 2. Enzyme and Cell Potencies and Permeability Data of Aminopyridines
a
b
eLogD = measured logD at pH 7.4 [calculated logD (ACD Laboratories program v12) in italics]. Arithmetic mean and standard error of ≥3
c
d
measurements (unless noted otherwise). Less than three independent replicates. Passive permeability (Papp in 10−6 cm/s) measured using Low-
Efflux MDCKII cells as described.13
modifications unfortunately did not result in improved enzyme
potency when compared to compound 2. It is possible that the
gain resulting from displacing the unstable waters is offset by an
effect that opposes binding, such as a greater desolvation
penalty for the ether oxygen or that the WaterMap estimates
were not accurate.
Despite reasonable enzyme potency for some compounds
(1−4, 8−9), they all have weak or no activity in a cell assay. As
expected, enzyme potency is necessary but not sufficient to
ensure cell activity. Impaired membrane permeability, differ-
ences in protein constructs between enzyme and cell assays
(kinase domain vs full length), and competition with cellular
ATP concentrations may be some of the determining factors in
this enzyme-whole cell potency disconnect.. Passive perme-
ability data, obtained using the MDCKII-LE cell line, which
displays low expression of active transporters,13 indicated that
most of the compounds have moderate permeability (Table 1)
suggesting that it is not an impeding factor. Measured PgP
efflux ratios for many of these compounds showed no evidence
of efflux. To assess the impact of physiological ATP
concentrations on compound activity, 1, 3, and 9 were tested
in the enzyme assay using [ATP] = 1 mM instead of [ATP] =
Km, (∼10 μM for MAP4K4). The respective IC50 values under
these conditions were >10 μM for 1 and 3 and 9.8 μM for 9,
which are similar to their cell activities. It is plausible that this
chemotype relies heavily on the single hinge contact with the
protein for activity and, therefore, is less capable of competing
with ATP, which establishes two hinge contacts. To test this
hypothesis, an amino group was introduced to the C(2)
position of the pyridine ring in 2 to provide compound 10
(Table 2). This was accompanied by a modest 4-fold gain in
enzyme potency and led to a ∼30-fold gain in cell activity. The
H → NH2 change led to a drop of 0.5 units of logD but not
much of a change in permeability despite the increased
hydrogen bond donors. The efflux ratios were similar (MDR
BA/AB efflux ratio of 1.2 for 2 vs 1.4 for 10) and the kinetic
solubility was improved (<1.1 μM @ pH 6.5 for 2 vs 299 μM
for 10). Several analogues of 10 that preserved the amino-
C
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pyridine motif as the potential hinge-binder, but with
alterations of the substituents on the two phenyl rings, were
prepared (Table 2) in an effort to optimize overall properties
directed toward identification of an in vivo tool compound.
Compound 11, lacking the amide group, shows loss of kinase
potency accentuating the importance of the interactions
between the primary amide group of compound 10 and
Asp115 for achieving potency. Exchanging the Cl substituent in
10 for a SO2Me group (12) resulted in some loss of potency.
Replacement of the amide of 12 by a hydroxy group in 13
resulted in similar potency in the enzyme and cellular assays
despite the improved passive permeability. Crystallization of the
complex between 13 and MAP4K4 revealed the same folded
conformation state for the P-loop as the one adopted in the
complex with 1 (Figure 1).10 The crystal structure illustrates
the double hinge contact with the aminopyridine ring. It also
displays a direct hydrogen bond between the phenol OH and
Asp115, reinforcing the role of this interaction in MAP4K4
potency. The crystal structure also shows that the two flanking
aromatic rings are not in plane relative to the central aromatic
ring, thus mitigating ADME concerns of an otherwise planar
triaromatic scaffold. To alleviate metabolic issues of a phenolic
group, the pyridone analogue 14 was made, which led to a loss
in both the enzyme and cell potencies. The reversed pyridone
(15) regained some of the potency lost in 14 presumably due
to the interactions with Asp115.
Table 3. Physicochemical/ADME Properties for 16/17
properties
16
17
a
MW/eLogD/TPSA
296/3.3/78
297/2.8/91
13
b
HLM Clu (mL/min/kg)
89
solubility, pH 1.2, mg/mL
solubility, pH 6.5, mg/mL
13.0
5.8
2.7
3.96
0.002
1.1
c
MDR BA/AB ratio
d
e
drug−drug interactions (DDI)
>30 μM
>30 μM
CYP inhibition IC50
3 μM (1A2)
54% (30 μM)
61% (60 μM)
1.8 μM
f
TDI, % inhibition (dose)
8% (30 μM)
24% (60 μM)
>10 μM
g
hERG IC50
h
THLE IC50
>119 μM
>300 μM
i
AMES
Positive
Negative
a
b
eLogD = measured logD at pH 7.4. Free clearance calculated using
c
measured protein binding in human liver microsomes (HLM). MDR
BA/AB efflux ratio using MDCK cell line transfected with human
d
MDR1. <15% Inhibition at 3 μM for isoforms (3A4, 2D6, 2C8, and
e
2C9). <15% Inhibition at 3 μM for isoforms (3A4, 2D6, 1A2, 2C8,
f
and 2C9). Change in midazolam metabolism after incubation with
g
compound.16 Measurement of potassium current in human
embryonic kidney 293 (HEK293) cells stably transfected with the
h
human ether-a-go-go-related gene (hERG) channel. Transformed
i
human liver epithelial cell assay (THLE).17 Activity in a bacterial
mutagenesis assay.18
stomach and intestinal pH15 and a reasonably clean DDI
profile. It also showed cytotoxicity (THLE assay) only at levels
above 119 μM. Thus, 16 was progressed to pharmacokinetic
(PK) evaluation in the mouse. Following oral dosing (10 mg/
kg), it displayed a plasma exposure that provided free drug
concentrations above the cell IC50 value for about 4−6 h after
administration (Figure 4). Based on this profile, compound 16
was selected as a fit-for-purpose tool for in vivo pharmacological
characterization.
To optimize the hydrogen bond with Asp115, compound 16
was prepared, as modeling indicated that the NH2 group of the
newly introduced aminopyridine group interacts with Asp115
in a similar fashion as the phenol OH in 13. Compound 16
(PF-6260933) was very potent against the enzyme with a very
good ligand efficiency (LE = 0.57) and was the most potent
analogue in the cell assay.
The kinome selectivity of 16 was assessed at a dose of 1 μM
in a panel of kinases (Figure 3). Bolstered by the promising
Figure 3. Heat map obtained from an Invitrogen selectivity panel (%
inhibition of kinases tested @1 μM).
selectivity, this compound was advanced to an ActivX screen,
which allows for profiling protein and lipid kinase interactions
in cell lysates.14 Remarkably, the initial in vitro kinome
selectivity profile translated to cell lysates; with 16 demonstrat-
ing very good selectivity against more than 150 native kinases
that could be quantified in human peripheral blood
mononuclear cells; with only MAP4K4 (ZC1, HGK) and
closely related GCK family members (ZC2, TNIK and ZC3,
MINK) showing >70% inhibition at 1 μM (see Supporting
Information). The intrafamily lack of selectivity with an ATP-
site inhibitor was expected as the ATP-site similarity is >95%
between the three kinases (MAP4K4, TNIK, and MINK).
Indeed, compound 16 showed a kinase activity IC50 (tested at
Km) of 15 and 8 nM for TNIK and MINK, respectively.
Compound 16 demonstrated good physicochemical proper-
ties as well as moderate human liver microsomal (HLM)
clearance (Table 3). Compound 16 had good solubility at both
Figure 4. Total and free plasma concentration of 16 after PO dosing
(10 mg/kg) to mice. Mouse plasma protein fraction unbound (fu =
0.038).
To recapitulate the in vivo effects demonstrated by MAP4K4-
siRNA,4 compound 16 was dosed orally (15 mg/kg) in a
mouse model of LPS-induced TNFα levels in blood
(compounds were dosed 30 min before an intravenous
injection of LPS). Compound 16 showed >90% inhibition
(similar to the effect of rosiglitazone) of LPS-induced plasma
TNFα levels at 1.5 h with a corresponding plasma
D
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ACS Medicinal Chemistry Letters
Letter
concentration of 439 ng/mL (unbound levels of 56.2 nM,
which was lower than the cellular IC50).
compound 17 was negative in the AMES assay. Commensurate
with the reduced lipophilicity, 17 demonstrated a more
desirable physicochemical, metabolic (Table 3), and off-target
profile (Supporting Information). Compound 17 thus offered a
lead compound for further development of a MAP4K4
inhibitor with an improved overall profile.
In order to gain further understanding of the in vivo effect of
a MAP4K4 inhibitor, compound 16 and rosiglitazone, a
thiazolidinone-based antidiabetic drug, were tested in the ob/
ob rodent model of type II diabetes.19,20 Animals were treated
with vehicle (n = 12), rosiglitazone (n = 12) at 1 mg/kg, p.o.,
bid, or 16 (n = 12) at 10 mg/kg, p.o., bid for 4 weeks.
Compound 16 (10 mg/kg, bid) provided significant improve-
ment in fasting hyperglycemia (44% reduction in blood glucose,
Figure 5), which was comparable to the effect observed with
rosiglitazone and also consistent with the profile observed with
MAP4K4-siRNA knockdown.5
Compounds 1−9 were synthesized according to modified
literature procedures (Supporting Information). Scheme 1
a
Scheme 1. Synthesis of Pyridines 10−17
a
Conditions: (a) 4-ClPhB(OH)2 (21) or (4-SO2Me)PhB(OH)2 (22),
Pd(dppf)Cl2, Na2CO3, dioxane, H2O, 110 °C or KOH, PhCH3, EtOH,
H2O, 140 °C; (b) (4-CONH2) PhB(OH)2 (23) or PhB(OH)2 (24),
(4-OH)PhB(OH)2 (25), or (pyridin-2-NH2)-B2Pin2, (26), or
(pyrimidin-2-NH2)Bpin (27); catalyst: Ph(AmPhos)2Cl2, Pd(dppf)-
Cl2, or Pd(Ph3P)4; base: Na2CO3, K2CO3, Cs2CO3, or KOH; solvent:
EtOH, THF, PhCH3, or dioxane, H2O, heat; (c) B2Pin2 (26),
Pd(dppf)Cl2, KOAc, dioxane, 60 °C; (d) 5-bromopyridin-2-ol (28) or
4-chloropyridin-2-ol (29), Pd(dppf)Cl2, Na2CO3, dioxane, H2O, 110
°C.
Figure 5. (a) Effects of 16 on LPS-induced TNFα levels in C57-BL/6J
mice). (b) Effects of 16 on glycemic control in ob/ob mice.
illustrates the synthesis of the analogues having 3,5-
disubstituted-pyridine (or 2-aminopyridine) as the hinge
binding group. Commercially available halopyridine 18 under-
went sequential Suzuki couplings with a variety of substituted
phenylboronic acids or pinacol esters to provide compounds
10−13, 16, and 17. Compounds 14 and 15 were obtained from
bromide 19 via Pd-catalyzed borylation to provide a boronate
ester 28 that was then coupled with the appropriate
halogenated pyridones.
In summary, we describe two 2-aminopyridine leads 16 (PF-
6260933)22 and 17 that are potent and selective MAP4K4
inhibitors. Compound 16 had suitable PK properties in mouse
to be used as a tool in an in vivo model of diabetes. Compound
17 showed improved overall drug-like properties compared to
16 and thus offered a lead compound for advanced preclinical
development. Further studies with these leads will be the
subject of future publications.
Despite its kinome selectivity and in vivo activity, compound
16 had liabilities that prevented its advancement beyond its
utility as a biological tool compound. Compound 16 displayed
inhibition in the hERG assay, nonkinase pharmacological
promiscuity (Cerep), and demonstrated time-dependent
inhibition (TDI)16 of CYP3A4, which led to drug accumulation
and toxicity when dosed beyond 10 mg/kg in mice. Toxicity
limited dosing to 10 mg/kg bid in chronic pharmacodynamic
models, though this was sufficient to provide ∼24 h coverage of
cellular IC50 levels. In addition, compound 16 had a mutagenic
risk demonstrated by the AMES assay.18
In order to understand the underlying causes of TDI,
compound 16 was subjected to incubation with human liver
microsomes. These studies showed that oxidation of the distal
aminopyridine ring leads to hydroxylation of the ring followed
by metabolism to a putative pyridoquinone-imine species that
was trapped with glutathione (Supporting Information). Thus,
a covalent adduct of the pyridoquinone-imine intermediate
with CYP3A4 could result in the observed TDI and may also be
contributing to the observed mutagenicity.
Various design strategies to attenuate the observed TDI were
explored.21 Addition of substituents ortho to the terminal amine
(F, CN CF3) or incorporation of additional nitrogen atoms to
the aminopyridine core provided no improvement. However,
conversion of the aminopyridyl tail to the corresponding 2-
aminopyrimidine (17) retained potency with a good ligand
efficiency (LE = 0.53) and kinome selectivity profile (Figure 3),
while eliminating the TDI risk (Table 3). Gratifyingly,
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00215.
■
S
Description of synthesis procedures and identification of
compounds, experimental protocols for the in vitro and in
vivo assays, ActivX results for compound 16 and
metabolite ID information, and Cerep promiscuity
profiles of compounds 16 and 17 (PDF)
E
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Letter
(11) Guimaraes, C. R. W.; Cardozo, M. MM-GB/SA rescoring of
docking poses in structure-based lead optimization. J. Chem. Inf. Model.
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(12) Abel, R.; Young, T.; Farid, R.; Berne, B. J.; Friesner, R. A. Role
of the Active-Site Solvent in the Thermodynamics of Factor Xa Ligand
Binding. J. Am. Chem. Soc. 2008, 130, 2817−2831.
̃
AUTHOR INFORMATION
Corresponding Author
*Tel: 617-551-3177. E-mail: samit.k.bhattacharya@pfizer.com.
Notes
■
The authors declare no competing financial interest.
(13) Di, L.; Whitney-Pickett, C.; Umland, J. P.; Zhang, H.; Zhang, X.;
Gebhard, D. F.; Lai, Y.; Federico, J. J., 3rd; Davidson, R. E.; Smith, R.;
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Development of a new permeability assay using low-efflux MDCKII
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ACKNOWLEDGMENTS
■
We thank Amit Kalgutkar and Tim Ryder for analysis of the
metabolite ID experiments, and John C. Kath, Michael Robbins
and Robert G. Linde for early work on this project.
(14) Patricelli, M. P.; Nomanbhoy, T. K.; Wu, J.; Brown, H.; Zhou,
D.; Zhang, J.; Jagannathan, S.; Aban, A.; Okerberg, E.; Herring, C.;
Nordin, B.; Helge, W.; Yang, Q.; Lee, J. − D.; Gray, N.; Kozarich, J. W.
In situ kinase profiling reveals functionally relevant properties of native
kinases. Chem. Biol. 2011, 18, 699−710.
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Whitney-Pickett, C.; Zhang, H.; Di, L.; Cram, M.; Fenner, K. S.; El-
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drug discovery. Mol. Pharmaceutics 2012, 9, 1199−1212.
(16) Orr, S. T.; Rip, S. L.; Ballard, T. E.; Henderson, J. L.; Scott, D.
O.; Obach, R. S.; Sun, H.; Kalgutkar, A. S. Mechanism-Based
Inactivation (MBI) of Cytochrome P450 Enzymes: Structure−Activity
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ABBREVIATIONS
■
MW, molecular weight; TPSA, topological polar surface area;
HLM, human liver microsomes; LPS, lipopolysaccharide; dppf,
1,1′-Bis(diphenylphosphino)ferrocene; B2Pin2, Bis(pinacolato)-
diboron; hERG, human ether-a-go-go-related gene; PK,
pharmacokinetic; TDI, time-dependent inhibition; MM-GB/
SA, molecular mechanics with generalized born/surface area
solvation; ND, not determined
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DOI: 10.1021/acsmedchemlett.5b00215
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