Structure-Based Design of GNE-495, a Potent and Selective MAP4K4 Inhibitor with Efficacy in Retinal Angiogenesis

Article abstract of DOI:10.1021/acsmedchemlett.5b00174

Diverse biological roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors in order to study its function in various disease contexts. In particular, compounds that can be used to carry out such studies in vivo would be critical for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compound 13 (GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were observed in the inducible Map4k4 knockout mice.

Full text of DOI:10.1021/acsmedchemlett.5b00174

Letter
pubs.acs.org/acsmedchemlett
Structure-Based Design of GNE-495, a Potent and Selective MAP4K4
Inhibitor with Efficacy in Retinal Angiogenesis
Chudi O. Ndubaku,*,† Terry D. Crawford,† Huifen Chen,† Jason W. Boggs,†,§ Joy Drobnick,†
Seth F. Harris,† Rajiv Jesudason,† Erin McNamara,† Jim Nonomiya,† Amy Sambrone,† Stephen Schmidt,†
Tanya Smyczek,† Philip Vitorino,†,∥ Lan Wang,† Ping Wu,† Stacey Yeung,† Jinhua Chen,‡ Kevin Chen,‡
Charles Z. Ding,‡ Tao Wang,‡ Zijin Xu,‡ Stephen E. Gould,† Lesley J. Murray,† and Weilan Ye†
†Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
‡Wuxi Apptec Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, People’s Republic of China
S
* Supporting Information
ABSTRACT: Diverse biological roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated
the identification of potent inhibitors in order to study its function in various disease contexts. In particular, compounds that can
be used to carry out such studies in vivo would be critical for elucidating the potential for therapeutic intervention. A structure-
based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into
the CNS led to an advanced compound 13 (GNE-495) that showed excellent potency and good PK and was used to
demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were observed in the inducible Map4k4
knockout mice.
KEYWORDS: Angiogenesis, MAP4K4, naphthyridine, GNE-495, P loop
itogen-activated protein kinase kinase kinase kinase 4
(MAP4K4, or HGK), and its homologues Misshapen
in vivo PK showed that the brain-to-plasma ratio was
substantial (unbound brain concentration = 14.6 μM, Figure
1). The exact mechanism of toxicity is not well understood at
present, but there is the possibility of off-target toxicity as 1 also
inhibits the closely related kinases MINK and TNIK to an
equal extent. These kinases have been previously shown to
impact neuronal integrity.12 Due to the fact that even an
extremely selective MAP4K4 inhibitor would still harbor
significant inhibitory activity against these two highly
homologous kinases, we proceeded with a target profile lacking
brain penetration.
We focused further medicinal chemistry efforts to decrease
CNS penetration.13−15 It is well accepted that parameters such
as MW, logP, TPSA, and number of hydrogen bond donors
have a strong impact on the ability of compounds to cross the
blood−brain barrier (BBB). Since the properties of 1 fell within
the favorable range for brain penetration (MW = 241; logP =
1.6; TPSA = 77; #HBD = 1),16 we decided to redesign the
series in order to limit CNS penetration. At the same time, we
M
and MIG-15 have been shown to regulate a multitude of
biological processes, including embryonic development,1,2
metabolism,3,4 inflammation,5 neural regeneration,6 angio-
genesis,7 and cancer.8,9 Given its diverse roles in these
processes, we became interested in the therapeutic potential
of MAP4K4 inhibition and began an endeavor to identify an
inhibitor that would function in vivo.
Our group recently reported two types of MAP4K4
inhibitors that were derived from separate fragment-based
lead discovery efforts.10,11 Between the two chemical series, we
were particularly attracted to the series bearing a pyridopyr-
imidine scaffold because of the potency and broad kinase
selectivity that it conferred.10 An optimized compound 1
showed low nanomolar biochemical activity, excellent kinase
selectivity, high exposure in vivo and also demonstrated a
pharmacodynamic (PD) response in an HT-1080 human
fibrosarcoma xenograft mouse model (Figure 1).
While this compound represented a valuable tool for our
early in vivo experiments, a significant shortcoming was that 1
was poorly tolerated when dosed repeatedly in a multiday
study. We hypothesized that this issue can be attributed to the
ability of 1 to permeate into the CNS as analysis of the mouse
Received: April 24, 2015
Accepted: June 29, 2015
© XXXX American Chemical Society
A
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Letter
pocket. Importantly, modeling suggested that expansion off of
this vector would not impinge on the induced Tyr36
orientation and therefore would maintain the desired folded
P loop conformation.
To quickly test the tractability of this new design direction,
we selected targets that utilized a 1-aminoisoquinoline core due
to synthetic accessibility. This core allowed for rapid establish-
ment of the SAR before transferring to the more synthetically
challenging naphthyridine core. From previous studies, we
knew that going from the quinazoline to pyridopyrimidine (by
insertion of a “5-aza” nitrogen) typically resulted in a 5- to 10-
fold improvement in potency.10 If this new vector was
productive, we believed that the corresponding naphthyridine
scaffold would show a similar potency increase. In that case, a
campaign to identify a suitable route to the corresponding 1,7-
naphthyridine would then be warranted.
An outline of the synthetic route for the isoquinolines is
shown in Scheme 1. The starting 7-bromo-1-hydroxyisoquino-
Scheme 1. Synthesis of Isoquinoline MAP4K4 Inhibitors
a
(3−9)
Figure 1. (A) Profile of pyridopyrimidine MAP4K4 inhibitor 1;10 (B)
X-ray structure of 1 in MAP4K4 (PDB: 4OBP).
recognized that the 2-fluoropyridyl back pocket group was a
potential safety liability due to its electrophilic nature. We
desired to install a more stable group here in order to remove
this liability. We previously showed that the 3-fluorophenyl was
well tolerated in this region. In the optimization process, our
intent was to maintain or improve all of the other favorable
attributes including potency, kinase selectivity, and pharmaco-
kinetics.
a
Reagents and conditions: (a) POCl3, 100 °C, 3 h, 80%; (b) aq. NH3,
Our previously reported cocrystal structure of 1 (PDB:
4OBP) served as a guide for further optimization (Figure 1B).
Compound 1 binds to a folded P loop conformation of
MAP4K4 that has been observed for only a small subset of
kinases.17 This conformation was therefore deemed advanta-
geous for maintaining adequate kinase selectivity. In the
cocrystal structure of 1 in this constricted ATP pocket, Tyr36
(contained within the P loop) rotates inward to make an edge-
to-face interaction with the pyridopyrimidine core, thus
producing a form-fitting enclosure for the ligand. Two potential
vectors for further expansion from the scaffold were evident.
We previously described ligand augmentation into the open
channel near the catalytic Lys54 (A) with groups that were
designed to favorably interact with protein residues surround-
ing those cavities.10 However, this pursuit led to losses in ligand
efficiency without favorable improvements to other features
including stability and permeability.
We next evaluated the area near the hinge residues (B). A
cavity was observed between the hinge Cys108, Phe107, and P-
loop Tyr36, which contained three crystallographically
observed waters toward the opening to bulk solvent.18
WaterMap calculations19,20 predicted relatively high free energy
of hydration values (ΔGhyd = 1.77−3.8 kcal/mol) at these
locations suggesting the potential to displace them concomitant
with displacement of these waters. This strategy included the
opportunity to replace a water-mediated hydrogen bond to the
hinge residue Cys108 backbone carbonyl. In particular, we
recognized that the N1 position of the pyridopyrimidine
presented an appropriate vector to extend into the available
NMP, 148 °C, 89%; (c) 3-fluorophenylboronic acid, Pd(dppf)Cl2,
Na2CO3, 1,4-dioxane/H2O, 99%; (d) NBS, DMF, 0 °C, 49%; (e) CO
(50 psi), Pd(dppf)Cl2, DMAP, MeOH, 70 °C; NaOH, H2O, MeOH,
THF, 44%, 2 steps; (f) RNH2, HATU, DIPEA, DMF or THF; (g) (i)
HCl, EtOAc/DCM; (ii) for 8, CH3CO2H, HATU, DIPEA, THF; (iii)
for 9, CDI, DIPEA, CH3NH2.
line (15) was readily converted to the 1-amino-4-bromoiso-
quinoline (17) intermediate. Further derivatization provided a
carboxylic acid intermediate that was then parlayed into a
variety of isoquinoline-4-amido analogues (3−9).
As seen in Table 1, we were highly encouraged that even the
simple 1° amide extension resulted in isoquinoline 3 that had
improved potency (IC50 = 18 nM) relative to the comparative
quinazoline compound 2 (IC50 = 58 nM). This result is
consistent with earlier WaterMap predictions suggesting that
potency gains could be achieved by substitutions in that area.
However, compound 3 showed equally high metabolic
clearance in rat liver microsomes despite being less lipophilic.
We explored additional substituents that reach deeper into this
pocket and span a wide range of MW, cLogP, and TPSA with
the goal of limiting penetration into the CNS. We evaluated
brain penetration using the rat IV cassette brain-to-plasma
assay.21 Gratifyingly, we observed that further substitution in
this pocket with a cyanomethyl amide (4) led to decreased total
B/P ratio while retaining good potency. Introducing conforma-
tionally constrained substituents in the pocket including
cyclopropyl (5), 3-hydroxycylobutyl (6), and several azetidines
(7−9) led to improved potency and stability (except for the
B
DOI: 10.1021/acsmedchemlett.5b00174
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ACS Medicinal Chemistry Letters
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Table 1. Structure−Activity Relationships of Isoquinoline MAP4K4 Inhibitors (2−9)
a
IC50 data are an average of at least 3 independent experiments (Z’LYTE, see Supporting Information). Values are given as the mean standard
b
deviation. Compounds were incubated with rat liver microsomes for 1 h, and percent remaining was determined by LC−MS/MS analysis.
c
d
e
Compounds were formulated in 10%DMSO/60%PEG400/30%SQ-H2O. Not determined. IC50 was determined using Caliper LabChip 3000
(LC3K) technology (see Supporting Information). Only one data point was obtained.
unsubstituted azetidine 7, which reduced potency). We also
saw markedly reduced brain penetration for 6 and 7
presumably due to the presence of an additional H-bond
donor. From this survey, we discovered that we could indeed
expand the molecular size of our inhibitors, reduce the
lipophilicity, and increase TPSA without sacrificing the potency
or otherwise favorable properties of our compounds. We also
saw improved metabolic stability that showed that the 2-
fluoropyridine right-hand side substituent in compound 1 was
not required for stability. These promising findings led us to
explore further increasing potency by reinsertion of the “5-aza”
moiety in the form of the 1,7-naphthyridine scaffold.
preparation of compounds contained in Table 2. We readily
applied lessons from the SAR survey conducted with the
isoquinoline compounds described in Table 1, and the resulting
compounds (10−14) showed even greater increases in
biochemical potency than the expected 5−10-fold shifts that
had been observed in earlier series (Table 2). These optimized
compounds were within desirable molecular property space
with TPSA values that are expected to prevent entry into the
CNS but still had sufficient MDCK permeability (Papp) to allow
for distribution into peripheral tissues. Consistent with the
good permeability properties, these compounds exhibited
adequate potency preventing migration of HUVEC cells in
vitro, an assay that is used to evaluate antiangiogenic
behavior.23 Notably, compound 13 showed the best balance
As a result of the successful SAR exploration with the
isoquinoline scaffold, we were eventually able to invent a robust
chemical route to 1,7-naphthyridines.22 This route enabled the
C
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a
Table 2. Structure−Activity Relationships of Naphthyridine MAP4K4 Inhibitors (10−14)
a
b
See Table 1 for details. Compounds were incubated with MDCK cells at 10 μM test concentration over 60 min. ER is efflux ratio, which is
determined by rate of apical−basolateral/basolateral−apical flux.
of MAP4K4 inhibition, permeability, microsomal stability, and
cellular potency.
The X-ray cocrystal structure of compound 13 was solved
and maintained all of the expected interactions that were
present in the original pyridopyrimidine scaffold including
multiple hinge binding interactions and the folded P-loop
conformation (Figure 2). Additionally, the amide linker
extending from the naphthyridine scaffold effectively replaces
two of the water molecules and engages in a hydrogen-bonding
interaction to the hinge Cys108 carbonyl. Overall, it was
evident that the binding pocket was completely occupied by the
optimized ligand.
Given the favorable overall profile of 13 we proceeded to
evaluate its in vivo PK behavior in three preclinical species:
mouse, rat, and dog (Table 3). The compound was
administered by intravenous (IV) and oral (PO) routes at 1
and 5 mg/kg, respectively, in each of the species (total of three
animals per group). Compound 13 showed good in vivo profile
in all species tested, with low clearances, moderate terminal
half-lives, and reasonable oral exposure levels (F = 37−47%).
Importantly, very minimal brain exposure was observed in mice
(measured unbound brain concentration at 1 h time point =
0.008 μM),24 consistent with our design hypothesis around
mitigating adverse effects. Compound 13 was also administered
intraperitoneally to neonatal mouse pups at high doses: 25 and
50 mg/kg (Supporting Information, Figure S2). In these
experiments, we also observed sustained exposures over the 24
h postdose. In order to determine if compound 13 (referred to
as GNE-495 herein) can inhibit MAP4K4 function in vivo, we
utilized the neonatal retinal vascular development model
because inducible knockout of Map4k4 inhibited retinal
vascular outgrowth and altered retinal vascular morphology.7
Figure 2. X-ray structure of 13 bound to MAP4K4 at 2.89 Å (PDB:
4ZK5). Select hydrogen bond (red), pi stacking (orange), and van der
Waals (yellow) interactions are highlighted. Proximal ordered waters
are shown in cyan spheres. The solvent-accessible protein surface in
the vicinity of the ligand is shown in white, demonstrating the
extensive contribution to the constricted enclosure created by the
approach of Tyr36 enabled by a buckled P loop conformation. The
ligand emerges toward solvent at the left, where the azetidine moiety
creates a bend around the Phe107 side chain to maintain close
contacts to the protein surface.
We found that IP injection of GNE-495 into newborn mice
dose-dependently delayed retinal vascular outgrowth (Figures
3A,B and S3) and induced abnormal retinal vascular
morphology (Figure 3C,D). These phenotypes recapitulated
the retinal vascular defects observed in the inducible Map4k4
knockout mice,7 indicating that GNE-495 is indeed active in
vivo. It is important to note that though GNE-495 also inhibits
D
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ACS Medicinal Chemistry Letters
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a
Table 3. Cross-Species Pharmacokinetics of 13 (GNE-495) Following Dosing via IV and PO Routes
b
c
IV bolus (1 mg/kg); solution
PO (5 mg/kg); suspension
species
%PPB
CL (mL/min kg−1
)
T1/2 (h)
Cmax (μM)
V (L/kg)
AUCinf (h·μM)
Cmax (μM)
Tmax (h)
F%
Cu,brain (μM)
mouse
rat
94.4
97.8
N.D.
19
1.5
3.4
1.8
2.7
4.0
3.9
1.6
1.2
1.1
5.3
1.4
1.5
1.2
1.0
4.0
1.7
47
40
37
0.008
N.D.
N.D.
7.5
8.9
12.0
21.0
dog
a
b
Mean values are shown. Further details provided in the Supporting Information. Vehicle: 10% EtOH, 30% PEG400, 60% 50 mM citrate, pH 3.0.
c
Vehicle: 0.5% (w/v) methylcellulose/0.2% (w/v) Tween 80 in sterile water (MCT).
Figure 3. (A) Representative images of Isolectin-B4 (vascular marker) stained flat-mounted retinas at postnatal day 6 (P6) from mice treated with
vehicle or 100 mg/kg GNE-495 daily from P1−P5. Areas in the retina without blood vessels (Avascular) are marked with dashed lines. Scale bar
represents 500 μm. (B) Quantification of avascular area normalized to total retina from similar images shown in A. Each dot represents one retina. P
value was calculated using Mann−Whitney unpaired test. (C) Representative images of isolectin-B4 stained P7 retinas at the vascular edges from
mice treated with vehicle or 100 mg/kg GNE-495 daily from P1−P6. Arrows indicate long membrane protrusions in vascular endothelial cells. Scale
bar represents 50 μm. (D) Numbers of long membrane protrusions (longer than 40 μm) along the vascular front per centimeter of vascular
perimeter. Each dot represents one retina. P value was calculated using Mann−Whitney unpaired test. Nonspecific fluorescence dusts in the images
shown in A and C were manually eliminated.
the related kinases MINK and TNIK, the observed in vivo
effects were attributable solely to MAP4K4 inhibition as has
been shown previously.7
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, kinase selectivity, details of in vitro
and in vivo assays, and characterization of compounds. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acsmedchem-
lett.5b00174.
■
S
We previously reported the discovery of a potent and highly
selective MAP4K4 tool compound 1. However, due to the
possibility that high brain penetration precluded our ability to
achieve long-term administration of the compound, we
successfully optimized the molecular properties to limit the
level of brain exposure in subsequent molecules. We were able
to identify a new class of isoquinoline and naphthyridine-based
MAP4K4 inhibitors that reduced brain exposures but
maintained potent activity and good kinase selectivity. GNE-
495 demonstrates high exposure in peripheral tissues but
minimal brain penetration validating our design strategy. The
fact that IP injection of GNE-495 recapitulated the phenotypes
in Map4k4 inducible knockout mice confirms the notion that
GNE-495 is active in vivo. This compound provides an
opportunity to investigate the multitude of MAP4K4 functions
in animal models and ultimately in patient diseases.
AUTHOR INFORMATION
Corresponding Author
*Tel: 650-225-2923. E-mail: chudin@gene.com.
■
Present Addresses
§WIL Research, Ashland, Ohio 44805, United States.
∥Gilead Sciences, Foster City, California 94404, United States
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
E
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Is Broadly Expressed in Human Tumor Cells and Can Modulate
Cellular Transformation, Invasion, and Adhesion. Mol. Cell. Biol. 2003,
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Funding
Diffraction data were collected at beamline 08ID-1 at the
Canadian Light Source, which was supported by the NSERC,
the NRC, the Canadian Institutes of Health Research, the
Province of Saskatchewan, Western Economic Diversification
Canada, and the University of Saskatchewan at beamline 5.0.2
of the Advanced Light Source. The Berkeley Center for
Structural Biology was supported in part by the NIH, the
NIGMS, and the Howard Hughes Medical Institute. The
Advanced Light Source was supported by the Director, Office
of Basic Energy Sciences, of the U.S. Department of Energy
under Contract No. DE-AC02−05CH11231.
(10) Crawford, T. D.; Ndubaku, C. O.; Chen, H.; Boggs, J. W.;
Bravo, B. J.; Delatorre, K.; Giannetti, A. M.; Gould, S. E.; Harris, S. F.;
Magnuson, S. R.; McNamara, E.; Murray, L. J.; Nonomiya, J.;
Sambrone, A.; Schmidt, S.; Smyczek, T.; Stanley, M.; Vitorino, P.;
Wang, L.; West, K.; Wu, P.; Ye, W. Discovery of Selective 4-Amino-
Pyridopyrimidine Inhibitors of MAP4K4 Using Fragment-Based Lead
Identification and Optimization. J. Med. Chem. 2014, 57, 3484−3493.
(11) Wang, L.; Stanley, M.; Boggs, J. W.; Crawford, T. D.; Bravo, B.
J.; Giannetti, A. M.; Harris, S. F.; Magnuson, S. R.; Nonomiya, J.;
Schmidt, S.; Wu, P.; Ye, W.; Gould, S. E.; Murray, L. J.; Ndubaku, C.
O.; Chen, H. Fragment-Based Identification and Optimization of a
Class of Potent Pyrrolo[2,1-F][1,2,4]Triazine MAP4K4 Inhibitors.
Bioorg. Med. Chem. Lett. 2014, 24, 4546−4552.
Notes
The authors declare no competing financial interest.
(12) Hussain, N. K.; Hsin, H.; Huganir, R. L.; Sheng, M. MINK and
TNIK Differentially Act on Rap2-Mediated Signal Transduction to
Regulate Neuronal Structure and AMPA Receptor Function. J.
Neurosci. 2010, 30, 14786−14794.
(13) Wager, T. T.; Villalobos, A.; Verhoest, P. R.; Hou, X.; Shaffer, C.
L. Strategies to Optimize the Brain Availability of Central Nervous
System Drug Candidates. Expert Opin. Drug Discovery 2011, 6, 371−
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(14) Young, R. C.; Mitchell, R. C.; Brown, T. H.; Ganellin, C. R.;
Griffiths, R.; Jones, M.; Rana, K. K.; Saunders, D.; Smith, I. R.; Sore, N.
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ACKNOWLEDGMENTS
■
We thank Mengling Wong, Chris Hamman, Michael Hayes,
and Amber Guillen for compound purification. We also thank
Baiwei Lin, Deven Wang, and Yutao Jian for analytical support.
ABBREVIATIONS
■
MAP4K4, mitogen-activated protein kinase kinase kinase kinase
4; CNS, central nervous system; TPSA, topological polar
surface area; HUVEC, human umbilical vein endothelial cells
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