Potent, Selective, and Orally Bioavailable Inhibitors of VPS34 Provide Chemical Tools to Modulate Autophagy in Vivo

Article abstract of DOI:10.1021/acsmedchemlett.5b00335

Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools, but selective inhibitors are now available to modulate the PI 3-kinase VPS34, which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models.

Full text of DOI:10.1021/acsmedchemlett.5b00335

Letter
pubs.acs.org/acsmedchemlett
Potent, Selective, and Orally Bioavailable Inhibitors of VPS34 Provide
Chemical Tools to Modulate Autophagy in Vivo
Ayako Honda,† Edmund Harrington,† Ivan Cornella-Taracido,† Pascal Furet,§ Mark S. Knapp,‡
Meir Glick,† Ellen Triantafellow,† William E. Dowdle,† Dmitri Wiedershain,† Wieslawa Maniara,†
Christine Moore,† Peter M. Finan,† Lawrence G. Hamann,† Brant Firestone,† Leon O. Murphy,†
and Erin P. Keaney*,†
†Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
‡Novartis Institutes for BioMedical Research, 4560 Horton Street, Emeryville, California 94608, United States
§Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland
S
* Supporting Information
ABSTRACT: Autophagy is a dynamic process that regulates
lysosomal-dependent degradation of cellular components.
Until recently the study of autophagy has been hampered by
the lack of reliable pharmacological tools, but selective
inhibitors are now available to modulate the PI 3-kinase
VPS34, which is required for autophagy. Here we describe the
discovery of potent and selective VPS34 inhibitors, their
pharmacokinetic (PK) properties, and ability to inhibit
autophagy in cellular and mouse models.
KEYWORDS: VPS34, autophagy, phosphoinositide 3-kinase
small molecule inhibitors have been developed.7−9 The small
he autophagy pathway is important for the transport of
Tcytoplasmic constituents such as damaged mitochondria molecule 3-methyladenine is widely used as an inhibitor of
VPS34 and autophagy, but this also inhibits PI3Kα.10 We
and protein aggregates to lysosomes where they are degraded
and recycled back into the cytoplasm.1 Progress over the last 10
recently described a highly selective inhibitor of VPS34, PIK-III,
and its use to discover NCOA4 as an autophagy substrate.8
years supports an essential role for autophagy in cellular
Herein, we describe our efforts leading to the identification of
PIK-III and novel derivatives that are orally bioavailable and
inhibit autophagy in mice.11
homeostasis and in some human diseases where mutations in
core pathway components have been identified.2,3 Given the
role that autophagy plays in normal physiology and disease,
there is interest in identifying new pharmacological agents to
modulate pathway activity. Several steps regulate the formation
of new autophagosomes and subsequent fusion with lysosomes.
The initiation step takes place following redistribution of the
ULK1-ATG13-FIP200 protein complex to the endoplasmic
reticulum (ER) surface, which triggers recruitment of the
vacuolar protein sorting 34 (VPS34)-VPS15-Beclin1-ATG14
complex. VPS34 is a phosphatidylinositol-3-kinase (PI3K) and
its presence at the ER generates local production of
phosphatidylinositol-3-phosphate (PtdIns(3)P), which provides
a high affinity binding site to recruit additional factors that
contain PtdIns(3)P-binding domains.4−6 Subsequently, the
ATG5-ATG12-ATG16L1 complex redistributes from the
cytoplasm to the autophagosome formation site, which
promotes expansion of the organelle structure. The LC3
protein is then directly conjugated to phosphatidylethanol-
amine on the forming membrane to help recruit cytoplasmic
cargos and eventual autophagosome maturation.
Novel bisaminopyrimidine compound 1 (Table 1) was
identified in a high-throughput screen as a potent inhibitor of
VPS34. In addition to enhancing potency against VPS34, our
efforts focused on developing selectivity against the other lipid
kinases (PI3Kα, β, δ, and γ) as well as identifying a compound
with suitable properties for in vivo studies. Initial metabolic
identification (met ID) and in vivo pharmacokinetic (PK)
studies also highlighted a number of metabolic liabilities within
1 that required further optimization (CL in vitro mouse = 82.6
mL min−1 kg−1).
Prior to obtaining a crystal structure, a homology model of
VPS34 was generated based on an available crystal structure of
PI3Kγ (PDB code: 2CHZ) and a published protein sequence
alignment using the WHAT IF program.12,13 Utilizing this
model, the binding mode of 1 in the ATP binding site was
predicted (Figure 1) and was used as a guide for our medicinal
Received: August 14, 2015
The catalytic pocket of VPS34 is highly homologous to other
Accepted: November 13, 2015
PI3K enzymes such as PI3K alpha for which highly selective
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00335
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Table 1. VPS34 Inhibitory Activity and Lipid Kinase
Scheme 1. Synthesis of VPS34 Inhibitors
Selectivity Profile for HTS Hit 1
a
Conditions: (a) LHMDS, −10 °C to rt, then RCO2Bn or
RCONMeOMe, 3 h. (b) DMF-DMA, 80 °C, 2 h. (c) K2CO3,
amidine or guanidine, DMF, 120 °C, 2 h then rt, 18 h. (d) mCPBA,
DCM, rt 2 h. (e) DMSO, amine, microwave, 160 °C, 10 min or THF,
LHMDS, −78 °C, 30 min, then aniline, −78 °C to rt, 1 h.
enzyme
IC50 (nM)
VPS34
PI3Kα
PI3Kβ
PI3Kγ
PI3Kδ
PI4Kβ
110
6200
>9100
8500
7000
3800
Table 2. SAR of Cyclohexanol Replacement Analogues
Figure 1. Compound 1 docked into the VPS34 homology model.
chemistry efforts. Given the presence of two aminopyrimidine
moieties, one could envision either to act as the canonical hinge
binding group. However, the compound could only be docked
without steric clash with the cavity, when the aminopyrimidine
bearing the cyclohexanol group was used as the hinge binding
fragment. Thus, in this binding mode, the cyclohexanol moiety
is predicted to sit in the solvent exposed area of the pocket,
while the aminopyrimidine to which it is attached interacts with
residues I685 and F684 of the hinge region. A possible
interaction of the secondary amine with D761 of the DFG
motif was also noted.
Our chemistry optimization efforts initially centered on
replacement of the cyclohexanol portion of 1, which was
indicated to be partially responsible for high clearance in vivo
due to a glucuronidation mechanism. Compound 1 and
analogues 5 were synthesized as described in Scheme 1,
commencing with alkylation of 4-methyl-2-(methylthio)-
pyrimidine (2) with R-substituted Weinreb amides or
alternatively R-substituted benzyl esters to afford ketones 3.14
Further reaction of 3 with DMF-DMA and subsequent
cyclization led to bispyrimidine 4. Conversion to final
compounds 5 was accomplished by oxidation of the sulfide to
either the sulfoxide or sulfone, followed by amine displacement.
The biochemical inhibition of VPS34 (IC50) was measured
using a luminescence-based ATP detection assay (Table 2).
Initial results indicated that while measurable changes in IC50
values were observed, no modifications to the R2 moiety led to
complete loss of activity. This tolerability supports the binding
hypothesis (later confirmed by X-ray cocrystal, Figure 2) that
this portion of the molecule is solvent exposed. Within this
solvent exposed area, some structural features do confer more
potent activity. In general, unsaturated ring systems provide
additional potency over the corresponding saturated counter-
parts (10 and 8). A possible pi-stacking interaction between the
aromatic ring of 10 and Phe684 may lead to this enhancement
in activity. Also of note, the introduction of heteroatoms
provided an incremental enhancement in potency (7 cf. 8, and
10 cf. 13), while maintaining a similar lipid kinase selectivity
profile to compound 1 (Compound 13: PI3Kα = 6800 nM,
B
DOI: 10.1021/acsmedchemlett.5b00335
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 3. SAR of Methylamine Replacement Analogues
Figure 2. Cocrystal structure of compound 19 in the human VPS34
active site (PDB: 5ENN) with the active site surface of PI3Kα
overlaid.
PI3Kβ = >9100 nM, PI3Kδ = 2900 nM, and PI3Kγ = 1900
nM). It was hypothesized that a water mediated hydrogen bond
between the heteroatom and the backbone NH group of
Val690 could be the reason for this gain in potency, which was
later confirmed by X-ray crystallography.
Given the potent VPS34 inhibition of compounds in the
biochemical assay and the attractive lipid kinase selectivity
profile (Table 1 and compound 13 above), we further evaluated
the ability of compounds to inhibit VPS34 in cells. We
employed a GFP-FYVE reporter assay before and after
treatment of cells with compounds as reported previously.8
IC50 values generated in the GFP-FYVE cellular assay
correlated well with those generated in the VPS34 biochemical
assay (Table 3), indicating potent on target inhibition of VPS34
in cells, with no apparent diminishment of activity when
moving to a whole-cell context.
While compound 13 proved to be a potent inhibitor of
VPS34 in vitro, the progression of this compound was limited
by its metabolic stability profile (in vitro CL mouse = 87 mL
min−1 kg−1). Met ID studies indicated N-demethylation and
cyclopropyl oxidation as major pathways of metabolism. We
first set out to investigate the amine substitution R1 in order to
identify more tractable analogues for in vivo studies.
Unmethylated analogue PIK-III (Table 3) does retain
activity, however, and showed only a modest improvement in
in vitro metabolic stability (in vitro CL mouse =73 mL min−1
kg−1), likely due to the presence of the cyclopropyl group. In
vivo PK studies confirmed high clearance and short elimination
half-life (data not shown).
a
IC50 values were measured against PI3K α, β, γ, and δ. Values for
PI3K α, β, and γ are higher than those shown for δ.
have solved the X-ray cocrystal structure of Vps34 with
compound 19, confirming the predicted binding mode and
providing support for the high selectivity observed (described
in detail below in Figure 2).
While removing this amine all together (compound 14) does
result in a drop off in activity, the consequent small but
measurable improvement in in vitro metabolic stability provides
a path forward for further modification to this purpose. Benzyl
and t-butyl analogues 20 and 17 both maintained potency
against VPS34, however, and showed no substantial improve-
ment in clearance.
Conversely amino-pyran derivative 16 showed an improve-
ment in clearance, but this modification led to a loss in potency.
A balanced solution was identified with branched analogue 18,
which exhibited much improved microsomal stability while
maintaining modest binding affinity for VPS34. Further
incorporation of a hydroxyl group (19) led to an additional
enhancement in both potency and metabolic stability providing
an excellent candidate for in vivo PK evaluation. In addition to
its high potency against VPS34, 19 is extraordinarily selective
over other lipid and protein kinases (>100-fold against >280
Initial SAR exploration of the R1 moiety (Table 3) identified
a number of modifications tolerated in this area of the molecule
and prior to obtaining an X-ray cocrystal structure also
provided us with insight into the potential binding mode.
The majority of the compounds prepared contained a
bisaminopyrimidine core and thus two possible bidentate
hinge binding sites. The modest loss in inhibitory potency with
replacement of one of the amino groups (R1), as in compounds
14 and 15, suggests that this amine makes important contacts
within the active site of VPS34 but is not essential for activity,
and therefore supports the binding mode hypothesized in the
docking model (Figure 1). As described in our recent
publication, the VPS34 X-ray cocrystal structure with PIK-III
confirms that the predicted aminopyrimidine is acting as the
hinge binding group in this class of compound.8 In addition, we
C
DOI: 10.1021/acsmedchemlett.5b00335
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
kinases evaluated, except TAK1 and PI3Kδ, 10-fold and 40-fold,
respectively).
Table 4. Pharmacokinetic Profile of Compound 19 in
C57BL/6 Mice Following a Single IV or Oral Dose
This selectivity can be rationalized by the structure of VPS34
in complex with compound 19 (Figure 2), which was solved
near the end of the medchem campaign. As is characteristic for
lipid and protein kinases, the active site of VPS34 is rather
compact and hydrophobic in nature, preferentially binding
coplanar aromatic molecules. As was predicted from the
homology model and SAR analysis, the aminopyrimidine
moiety attached to the pyridine acts as the hinge binder,
forming two hydrogen bonds with the backbone amide and
carbonyl oxygen of I685. Additionally, the aminopyridine
moiety forms an extended solvent-mediated hydrogen-bonding
motif with the side chains of Asp671 and Asp644. Interestingly,
the cyclopropyl group of 19 fits nicely into a hydrophobic
pocket formed by the side chains of Phe612, Pro618, and
Phe684 and appears to be the basis of selectivity. An overlay
with the PI3Kα active site surface shows this pocket in VPS34
to be closer in proximity to the hinge region compared to the
PI3Kα pocket. This shift is due to the presence of the Phe612
within VPS34, which allows the cyclopropyl group to fit into
the hydrophobic pocket while maintaining optimal interaction
of the donor/acceptor moiety of 19 with I685. Within PI3Kα,
the Phe612 of VPS34 corresponds to a methionine residue,
which does not allow for binding of the cyclopropyl group.
Additionally, the tertiary alcohol moiety of 19 would be
obstructed by the PI3Kα P-loop, which descends to the space
occupied by this moiety.
PK parameter
mean IV 2 mg/kg
mean PO 10 mg/kg
6725
AUCinf (nM·h)
CL (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
2855
30
1.5
1.2
Tmax (h)
0.7
Cmax (nM)
F (%)
2994
47
the cellular activity described above, compound 19 constitutes a
suitable candidate for in vivo studies.
We next investigated the ability of compound 19 to inhibit
autophagy in vivo by using a PK/PD model in RKO colon
cancer tumor bearing nude mice. Upon oral administration of
19 at 50 mg/kg twice a day (BID) for 7 days, we observed
time-dependent accumulation of LC3-II consistent with
reduced autophagic capacity (Figure 4). Although no reduction
Figure 4. Seven-day BID treatment of compound 19 in tumor bearing
mice. Each lane represents tumor samples from individual mice.
Such selectivity is ideal for a tool compound to
unambiguously assess the pharmacological consequences of
VPS34 inhibition in vivo. Moreover, the ability of 19 to prevent
the degradation of autophagy substrates p62, NCOA4, NBR1,
NDP52, and FTH1 was similar to PIK-III (Figure 3). In
addition, treatment of cells with compound 19 leads to an
increase in the lipidated and nonlipidated forms of LC3 (Figure
3) similar to previous reports using PIK-III.8
in tumor volume was observed, a clear effect on autophagy was
evident. Additional studies with chronic dosing may be required
to see an effect on tumor size.
Through medicinal chemistry optimization of an HTS hit, we
have been able to develop highly potent and selective VPS34
inhibitors exhibiting nanomolar potency biochemically and in
cellular assays. These inhibitors of VPS34 display high
selectivity against both lipid kinases and protein kinases and
in cellular assays are capable of robustly inhibiting autophagy.
Further optimization of the pharmacokinetic properties of this
class of molecules led to the development of an orally
bioavailable compound that is the first disclosed selective
inhibitor of the autophagy process in vivo. While Ronan and co-
workers have recently reported a compound shown to inhibit
VPS34 in vivo, compound 19 constitutes the first example of a
VPS34 inhibitor shown to inhibit autophagy in vivo.15 Thus,
compound 19 provides a selective in vivo tool to probe this
process further and ultimately may provide therapeutically
relevant treatments for a number of indications.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00335.
■
S
Figure 3. DLD1 cells treated for 24 h with PIK-III and compound 19.
Biological assays, in vivo methods, and experimental
procedures (PDF)
The pharmacokinetic profile of analogue 19 was determined
in C57BL/6 mice (Table 4). After oral administration at 10
mg/kg, the compound was rapidly absorbed and showed
moderate mean systemic clearance (30 mL/min/kg, approx-
imately 33% of hepatic blood flow), with good oral
bioavailability (F% = 47). Based on these PK parameters and
AUTHOR INFORMATION
Corresponding Author
*E-mail: erin.keaney@novartis.com.
■
D
DOI: 10.1021/acsmedchemlett.5b00335
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Garcia-Echeverria, C.; Ronan, B. Discovery of (2S)-8-[(3R)-3-
methylmorpholin-4-yl]-1-(3-methyl-2-oxobutyl)-2-(trifluoromethyl)-
3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a novel potent and
selective inhibitor of Vps34 for the treatment of solid tumors. J. Med.
Chem. 2015, 58, 376−400.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
PI3K, phosphoinositide 3-kinase; PK, pharmacokinetic; ND,
not determined
REFERENCES
■
(1) Boya, P.; Reggiori, F.; Codogno, P. Emerging regulation and
functions of autophagy. Nat. Cell Biol. 2013, 15, 713−720.
(2) Green, D. R.; Levine, B. To Be or Not to Be? How Selective
Autophagy and Cell Death Govern Cell Fate. Cell 2014, 157, 65−75.
(3) Choi, A. M. K.; Ryter, S. W.; Levine, B. Autophagy in Human
Health and Disease. N. Engl. J. Med. 2013, 368, 651−662.
(4) Axe, E. L.; Walker, S. A.; Manifava, M.; Chandra, P.; Roderick, H.
L.; Habermann, A.; Griffiths, G.; Ktistakis, N. T. Autophagosome
formation from membrane compartments enriched in phosphatidyli-
nositol 3-phosphate and dynamically connected to the endoplasmic
reticulum. J. Cell Biol. 2008, 182, 685−701.
(5) Polson, H. E. J.; de Lartigue, J.; Rigden, D. J.; Reedijk, M.; Urbe,
S.; Clague, M. J.; Tooze, S. A. Mammalian Atg18 (WIPI2) localizes to
omegasome-anchored phagophores and positively regulates LC3
lipidation. Autophagy 2010, 6, 506−522.
(6) Matsunaga, K.; Morita, E.; Saitoh, T.; Akira, S.; Ktistakis, N. T.;
Izumi, T.; Noda, T.; Yoshimori, T. Autophagy requires endoplasmic
reticulum targeting of the PI3-kinase complex via Atg14L. J. Cell Biol.
2010, 190, 511−521.
(7) Miller, S.; Tavshanjian, B.; Oleksy, A.; Perisic, O.; Houseman, B.
T.; Shokat, K. M.; Williams, R. L. Shaping development of autophagy
inhibitors with the structure of the lipid kinase Vps34. Science 2010,
327, 1638−1642.
(8) Dowdle, W. E.; Nyfeler, B.; Nagel, J.; Elling, R. A.; Liu, S. M.;
Triantafellow, E.; Menon, S.; Wang, Z. C.; Honda, A.; Pardee, G.;
Cantwell, J.; Luu, C.; Cornella-Taracido, I.; Harrington, E.; Fekkes, P.;
Lei, H.; Fang, Q.; Digan, M. E.; Burdick, D.; Powers, A. F.; Helliwell,
S. B.; D’Aquin, S.; Bastien, J.; Wang, H.; Wiederschain, D.; Kuerth, J.;
Bergman, P.; Schwalb, D.; Thomas, J.; Ugwonali, S.; Harbinski, F.;
Tallarico, J.; Wilson, C. J.; Myer, V. E.; Porter, J. A.; Bussiere, D. E.;
Finan, P. M.; Labow, M. A.; Mao, X. H.; Hamann, L. G.; Manning, B.
D.; Valdez, R. A.; Nicholson, T.; Schirle, M.; Knapp, M. S.; Keaney, E.
P.; Murphy, L. O. Selective VPS34 inhibitor blocks autophagy and
uncovers a role for NCOA4 in ferritin degradation and iron
homeostasis in vivo. Nat. Cell Biol. 2014, 16, 1069−1079.
(9) Fruman, D. A.; Rommel, C. PI3K and cancer: lessons, challenges
and opportunities. Nat. Rev. Drug Discovery 2014, 13, 140−156.
(10) Wu, Y. T.; Tan, H. L.; Shui, G. H.; Bauvy, C.; Huang, Q.; Wenk,
M. R.; Ong, C. N.; Codogno, P.; Shen, H. M. Dual Role of 3-
Methyladenine in Modulation of Autophagy via Different Temporal
Patterns of Inhibition on Class I and III Phosphoinositide 3-Kinase. J.
Biol. Chem. 2010, 285, 10850−10861.
(11) Cornella-Taracido, I.; Harrington, E.; Honda, A.; Keaney, E. P.
Bi-heteroaryl compounds as vps34 inhibitors. WO2012085815, June
28, 2012.
(12) Maira, S. M.; Voliva, C.; Garcia-Echeverria, C. Class IA
phosphatidylinositol 3-kinase: from their biologic implication in
human cancers to drug discovery. Expert Opin. Ther. Targets 2008,
12, 223−238.
(13) Vriend, G. WHAT IF: a molecular modeling and drug design
program. J. Mol. Graphics 1990, 8, 52−6.
(14) Nahm, S.; Weinreb, S. M. N-methoxy-n-methylamides as
effective acylating agents. Tetrahedron Lett. 1981, 22, 3815−3818.
(15) Pasquier, B.; El-Ahmad, Y.; Filoche-Romme, B.; Dureuil, C.;
Fassy, F.; Abecassis, P. Y.; Mathieu, M.; Bertrand, T.; Benard, T.;
Barriere, C.; El, B. S.; Letallec, J. P.; Sonnefraud, V.; Brollo, M.;
Delbarre, L.; Loyau, V.; Pilorge, F.; Bertin, L.; Richepin, P.; Arigon, J.;
Labrosse, J. R.; Clement, J.; Durand, F.; Combet, R.; Perraut, P.;
Leroy, V.; Gay, F.; Lefrancois, D.; Bretin, F.; Marquette, J. P.; Michot,
N.; Caron, A.; Castell, C.; Schio, L.; McCort, G.; Goulaouic, H.;
E
DOI: 10.1021/acsmedchemlett.5b00335
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX