Highly selective phosphatidylinositol 4-kinase IIIβ inhibitors and structural insight into their mode of action

Article abstract of DOI:10.1021/acs.jmedchem.5b00499

Phosphatidylinositol 4-kinase IIIβ is a cellular lipid kinase pivotal to pathogenesis of various RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular membranes and use them for the construction of functional repli

Full text of DOI:10.1021/acs.jmedchem.5b00499

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Article
Highly selective PI4K III# inhibitors and
structural insight into their mode of action
Ivana Mejdrová, Dominika Chalupská, Martin Kögler, Michal Sala, Pavla Pla#ková, Adriana
Baumlová, Hubert Hrebabecky, Eliška Procházková, Milan Dejmek, Rémi Guillon, Dmytro Strunin,
Jan Weber, Gary Lee, Gabriel Birkus, Helena Mertlíková-Kaiserová, Evzen Boura, and Radim Nencka
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00499 • Publication Date (Web): 21 Apr 2015
Downloaded from http://pubs.acs.org on April 25, 2015
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Highly selective phosphatidylinositol 4ꢀkinase IIIβ
inhibitors and structural insight into their mode of
action
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a‡
a,‡
a,‡
a
a
Ivana Mejdrová , Dominika Chalupská , Martin Kögler , Michal Šála , Pavla Plačková ,
a
a
a
a
a
Adriana Baumlová , Hubert Hřebabecký , Eliška Procházková , Milan Dejmek , Rémi Guillon ,
a
a
b
b
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Dmytro Strunin , Jan Weber , Gary Lee , Gabriel Birkus , Helena MertlíkováꢀKaiserová , Evzen
a,*
a,*
Boura , Radim Nencka
a
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
v.v.i, Gilead Sciences & IOCB Research Centre, Flemingovo nám. 2, 166 10 Prague 6, Czech
Republic.
b
Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, United States.
ABSTRACT
Phosphatidylinositol 4ꢀkinase IIIβ is a cellular lipid kinase pivotal to pathogenesis of various
RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular
membranes and use them for the construction of functional replication machinery. Selective
inhibitors of this enzyme are potential broadꢀspectrum antiviral agents, as inhibition of this
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enzyme results in the arrest of replication of PI4K IIIβꢀdependent viruses. Herein, we report a
detailed study of novel selective inhibitors of PI4K IIIβ, which exert antiviral activity against a
panel of singleꢀstranded positiveꢀsense RNA viruses. Our crystallographic data show that the
inhibitors occupy the binding site for the adenine ring of the ATP molecule and therefore prevent
the phosphorylation reaction.
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INTRODUCTION
Phosphatidylinositol 4ꢀphosphate (PI4P) is the most abundant mono phosphoinositide in
eukaryotic cell. It decorates the Golgi, transꢀGolgi network (TGN), and also has a role at the
1
plasma membrane and endosomes. In addition, PI4P is the precursor for the synthesis of other
phosphoinositides, most notably phosphatidylinositol 4,5ꢀbisphosphate (PI(4,5)P ) – the
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hallmark of the plasma membrane – and phosphatidylinositol 3,4,5ꢀtrisphosphate (PI(3,4,5)P ), a
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key secondary messenger molecule. The synthesis of PI4P is carried out by phosphatidylinositol
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, 3
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ꢀkinases (PI4Ks). Mammalian genomes contain two types of PI4Ks, type II (PI4K IIα and
PI4K IIβ) and type III (PI4K IIIα and PI4K IIIβ) kinases that share homology with
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, 5
phosphatidylinositol 3ꢀkinases (PI3Ks). Recently, crystal structures were solved for PI4K IIα
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and for PI4K IIIβ.
Type II PI4Ks are palmitoylated enzymes stably associated with the membrane. They are
present at various endomembranes such as the TGN, early and late endosomes as well as at the
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9, 10
plasma membrane. These enzymes are critical to intracellular trafficking
, and interact with
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clathrin adaptor complexes (PI4K IIα with APꢀ3 and PI4K IIβ with AP1).
In contrast, type
III kinases are soluble cytoplasmic proteins that are recruited to the membrane via
protein−protein interactions. Efr, YPP1/TTC7 and TMEM150 direct the PI4K IIIα recruitment to
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the plasma membrane where it is responsible for PI4P synthesis.
The Golgi resident proteins
where this enzyme is mostly
responsible for the biogenesis of the Golgi pool of PI4P although type II PI4Ks are also
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ACBD3 and GBF1/Arf recruit PI4K IIIβ to the Golgi
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contributing to this pool.
The type III PI4Ks are essential host factors for a plethora of
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singleꢀstranded positiveꢀsense (ss(+)RNA) viruses and are potential drug targets.
ss(+)RNA viruses hijack PI4Ks to produce membranous organelles (referred to as
“
membranous webs” or “replication factories”) highly enriched in PI4P, which facilitate the
assembly of functional viral replication machinery. For example, the Hepatitis C virus (HCV)
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hijacks both PI4K IIIα and PI4K IIIβ
, whereas various members of the Picornaviridae
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3ꢀ26
family, including coxsackieviruses and rhinoviruses
, and those of the Coronaviridae family
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,
were shown to depend on PI4K IIIβ. In spite of the broad dependence on PI4Ks, several
different molecular mechanisms of hijacking have recently been described. The NS5A protein of
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HCV directly binds to PI4K IIIα and stimulates its activity , whereas the 3A protein of the
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Aichi virus hijacks PI4K IIIβ through interaction with its binding partner, the ACBD3 protein.
The recruitment of PI4K IIIβ to coxsackievirus B3 replication factories occurs through an
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unknown mechanism that is independent of ACBD3. PI4Ks are therefore potential drug targets
as essential host factors for the replication of ss(+)RNA viruses. A possible impediment to these
30, 31
strategies, however, is that genetic inactivation of type III PI4Ks is detrimental for animals.
A window of possibility relies on the fact that RNA viruses require every bit of the cell’s PI4K
activity (and even up regulate PI4Ks) for replication, whereas partially compromised PI4K
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activity may be well tolerated in animals, especially if only for a short time period. To clarify
whether a therapeutic window exists for PI4K enzyme inhibition, potent and selective inhibitors
of the PI4K enzymes are needed.
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Although several potent inhibitors of PI4K type III enzymes have been discovered recently,
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most of them targeted PI4KIIIα.
The studies focused on PI4KIIIβ suffer from a lack of
detailed structure−activity relationship (SAR) and structural data, which would allow for a better
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understanding of the mode of action of these compounds.
We present an extensive study on a broad series of novel PI4K IIIβ inhibitors. We show that
these compounds are highly selective based on biochemical characterization. We also assess the
effects of these inhibitors against selected viruses in cellꢀbased assays. To gain detailed
knowledge of the molecular details of their inhibitory mechanisms, we solved the crystal
structures of PI4K IIIβ enzyme bound to an archetypical inhibitor and to ATP. These structures
revealed the mechanism of PI4K IIIβ enzyme inhibition by these compounds at the atomic level.
RESULTS
Chemistry
We prepared a series of 65 novel compounds based on the screening hit 2a (Tꢀ00127ꢀHEV1)
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discovered by Arita et al. We sequentially modified all three parts of the molecule – the amine
side chain 1 (Figure 1, in green), bicyclic central core (Figure 1, in red) and aromatic side chain 2
(Figure 1, in blue).
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Figure 1. Screening hit as a model compound for SAR study.
Firstly, we focused on the effect of diverse modifications of the side chain 1 using the original
pyrazolo[1,5ꢀa]pyrimidine central core. Most of the derivatives were prepared starting from the
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compound 1 by simple nucleophilic substitution (DIPEA, EtOH, 75 °C) of the chlorine atom at
position 7 of the bicyclic central core (Scheme 1), which proceeds smoothly with high to
excellent yields. An additional series of the inhibitors was prepared by derivatization of the
ethylenediamino chain with various substituents (Scheme 2, compounds 3ꢀ9). Analogous
derivatives 10 and 11 were obtained by acetylation from appropriate starting materials 2f and 2k,
respectively.
a
Scheme 1. Synthesis of C7ꢀmodified pyrazolopyrimidines.
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Reagents and conditions: (a) amine, EtOH, heating.
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Scheme 2. Preparation of various Nꢀmodified ethylenediamino derivatives 3ꢀ11.
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a
Reagents and conditions: (a) isobutyric acid, DCC, CH Cl , 0 °C to rt, 20 h, 74% ; (b)
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cyclohexanecarbonyl chloride, Et N, CH Cl , 0 °C, 2 h, 72%; (c) 2ꢀphenylpropionic acid, DCC,
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isocyanate, CH Cl , 24 h, 89%; (f) ClCOOEt, Et N, CH Cl , 0 °C, 1.5 h, 61%; (g)
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methanesulfonyl chloride, Et N, CH Cl , 0 °C, 5 h and rt 4 h, 83%; (h) Ac O, Et N, CH Cl , 0
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2 3 3
We also prepared compounds with sulfur and oxygen atom instead of the NHꢀgroup at position
in order to investigate the influence of putative hydrogen bond with the protein. The thio
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derivatives were prepared either by reaction with thiourea and subsequent alkylation with
chloroethyl morpholine to obtain analogue 13 or by direct nucleophilic substitution using
appropriate thiophenol yielding derivative 14. The oxygen analogue 15 of the parent compound
2a was prepared also by a nucleophilic substitution under basic conditions (NaH/DMF) (Scheme
3).
a
Scheme 3. Synthesis of C7ꢀmodified pyrazolopyrimidines.
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Reagents and conditions: (a) thiourea, EtOH, reflux, 15 h, 85%; (b) 4ꢀ(2ꢀchloroethyl)ꢀ
morpholine hydrochloride, K CO , DMF, 77%; (c) 4ꢀ(morpholinꢀ4ꢀyl)benzenethiol, K CO ,
DMF, 43%; (d) 4ꢀ(2ꢀhydroxyethyl)morpholine, NaH, DMF, 58%.
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Secondly, we turned our attention to the modification of the central bicyclic core. We prepared
analogues of the parent compound with imidazo[1,2ꢀa]pyridine (16) (Scheme 4), imidazo[1,2ꢀ
b]pyridazine (17) (Scheme 5), and pyrazolo[1,5ꢀa][1,3,5]triazine (18) central core (Scheme 7).
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The synthesis of imidazo[1,2ꢀa]pyridine derivative started from compound 19 , which was
easily iodinated using NIS (Scheme 4). Suzuki crossꢀcoupling reaction followed by microwave
assisted BuchwaldꢀHartwig reaction with 4ꢀ(2ꢀaminoethyl)morpholine yielded the desired
analogue 16. However, this compound is surprisingly unstable and decomposes quite rapidly.
a
Scheme 4. Preparation of bicyclic analogue imidazo[1,2ꢀa]pyridine 16.
a
Reagents and conditions: (a) NIS, DMF, rt, 30 min., yield 89%; (b) 3,4ꢀdimethoxyꢀ
phenylboronic acid, Pd(dppf)Cl , Na CO , dioxane/H O (4:1), 16 h, 60%; (c) 4ꢀ(2ꢀ
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aminoethyl)morpholine, Pd (dba) , MeDalPhos, tꢀButONa, dioxane, MW, 30 min, 150 °C, 72%.
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The preparation of the compound 17 started from dichloro derivative 22. Since the reactivity of
the chlorine atoms differs significantly, treatment with 4ꢀ(2ꢀaminoethyl)morpholine afforded the
desired monosubstituted product 23 in high yield. This compound was utilized for subsequent
preparation of the methylated derivative 24 (Scheme 5). A number of diverse methylation
reactions failed to give this derivative. Finally, the transfer of a methyl group was accomplished
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by reacting compound 23 with DABALꢀMe (a reactive complex between one molecule of
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DABCO and two molecules of AlMe3) under Pdꢀcatalyzed crossꢀcoupling conditions. This
way, key intermediate 24 was prepared in moderate 42% yield. Iodination and Suzuki coupling
with 3,4ꢀdimethoxyphenyl boronic acid afforded title compound 17 (Scheme 5).
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a
Scheme 5.
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Pd/C, THF:MeOH, rt, 21 h; (d) NIS, AcOH/CH Cl or DMF, (49% for 25, 41% for 27, 41% for
9, 95% for 34) (e) 3,4ꢀdimethoxyphenylboronic acid, Pd(PPh ) , 1M K CO , dioxane, 95 °C,
overnight (60% for 17, 83% for 28, 77% for 30a).
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Since the compound 17 exerted interesting inhibitory activity in the PI4KIIIβ enzymatic assay,
the preparation of analogues with modified substituent at position 6 was the logical further step.
The derivative 23 served as a suitable precursor for the preparation of analogues bearing either
hydrogen or chlorine atom instead of the methyl group at this position (Scheme 5). The synthesis
of the former derivative (28) was accomplished by simple hydrogenolysis followed by iodination
and Suzuki reaction. The latter compound (30a) was obtained in the similar fashion without the
hydrogenolysis step. In this case, we prepared also several other related derivatives with this
structural pattern summarized in the Scheme 6 using intermediate 29 subdued to Suzuki crossꢀ
coupling reactions.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
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36
37
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39
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41
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Scheme 6. Suzuki crossꢀcoupling reaction of morpholino ethyl derivative 29.
a
Reagents and conditions: (a) ArꢀB(OH) , Pd(PPh ) ,1M K CO , dioxane, heating.
2
3 4
2
3
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6
Derivative 32 with the last bicyclic core, pyrazolo[1,5ꢀa][1,3,5]triazine, was obtained starting
4
1
from bromide 31 by cross coupling reaction with 3,4ꢀdimethoxyphenylboronic acid.
Subsequent chlorination followed by immediate substitution of the chlorine atom with the
appropriate amine afforded the desired analogue of 2a, compound 18, and derivative 33 (Scheme
7.)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
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5
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9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 7. Synthesis of pyrazolo[1,5ꢀa][1,3,5]triazine derivatives.
a
Reagents and conditions: (a) 3,4ꢀdimethoxyphenylboronic acid, Pd(dppf)Cl , Na CO ,
2
2
3
dioxane/H O (4:1), 16 h, 60%; (b) POCl , dimethylaniline, 120 °C, 20 h; (c) amine, DCM, 0 °Cꢀ
2
3
rt, 16 h (yields over two steps ꢀ 68% for 18, 65% for 33).
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9
Based on our preliminary results from PI4KIIIβ assay, with respect to synthetic feasibility and
potential metabolic decomposition, we selected 6ꢀchloroꢀ2ꢀmethylimidazo[1,2ꢀb]pyridazine as
the most suitable central core and [2ꢀ(acetylamino)ethyl]amino group as side chain 1 for further
optimization of the aromatic side chain 2. The key intermediate 35 for this study was prepared by
by iodination with NIS, giving the compound 34 (Scheme 5), followed by selective nucleophilic
substitution of the chlorine at position 8 with 2ꢀaminoethylacetamide.
10
11
12
13
14
15
16
17
18
19
20
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23
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29
30
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Majority of compounds form the last series was prepared by Suzuki crossꢀcoupling reaction
starting from the compound 35 (Scheme 8).
a
Scheme 8. Aryl derivatives prepared by Suzuki coupling reaction.
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0
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9
0
a
Reagents and conditions: (a) ArꢀB(OH) , Pd(PPh ) , K CO , dioxane/H O (4:1), 95 °C, 16 h.
2
3 4
2
3
2
b
Pinacol ester of boronic acid was used as reagent.
a
Scheme 9. Acylated derivatives preparation.
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49
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a
Reagents and conditions: (a) Ac O, DIPEA, CH Cl , 0 °C, 92%; (b) cyclohexanoyl chloride,
2
2
2
DIPEA, CH Cl , 0 °C, 63%; (c) pivaloyl chloride, DIPEA, CH Cl , 0 °C, 48%.
2
2
2
2
Another subꢀseries of derivatives modified in the aromatic side chain was prepared by
derivatization of suitable precursors obtained by Suzuki couplings. Firstly, we modified the
amino group of the compound 36u. In particular, we obtained a few acylated derivatives (37ꢀ39)
either by reaction with acyl anhydrides or chlorides (Scheme 9). Subsequently, reaction with
isopropylisocyanate afforded the substituted urea derivative 40 and the compounds 41 and 42
were prepared by reductive amination and alkylation of the compound 36u, respectively
(
Scheme 10).
a
Scheme 10. Preparation of derivatives with substituted aromatic part of the molecule.
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0
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2
3
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6
7
8
9
0
a
Reagents and conditions: (a) acetone, NaBH CN, MeOH, 76%; (b) isopropylisocyanate, THF,
3
6
h, reflux, 86%; (c) Nꢀ(2ꢀchloroethyl)methanesulfonamide, K CO , toluene, 90 °C, 45%.
2 3
We also prepared 3ꢀ(3,4ꢀdihydroxyphenyl) derivative 43 by the demethylation of 30a, albeit in
very low yield (Scheme 11). Reduction of the formyl functions in compounds 36e and 36i with
NaBH gave the corresponding hydroxymethyl derivatives 44 and 45, respectively, in moderate
4
yields.
a
Scheme 11. Modification of the aromatic side chain.
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49
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a
Reagents and conditions: (a) BBr , CH Cl , ꢀ78 °C to rt, overnight, 13%; (b) NaBH ,
3
2
2
4
MeOH/CH Cl , rt, 1 h, 30% for 33 and 40% for 34; (c) i) H (3.5 bar), RaneyꢀNi, NH /EtOH, rt,
overnight; ii) HCl/Et O, CH Cl , 0 °C, 71%.
2
2
2
3
2
2
2
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2
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5
5
5
5
5
5
5
5
6
In order to introduce highly polar group, the cyano moiety in compound 36h was successfully
reduced to the primary amine 46 which was isolated as its hydrochloride salt.
0
1
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4
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7
8
9
0
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9
0
1
2
3
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5
6
7
8
9
0
The analogue of compounds 36a with hydrogen instead of the chlorine atom at the position 6
of the imidazo[1,2ꢀb]pyridazine core (47) was prepared by simple hydrogenolysis of 36a
(Scheme 12).
Finally, the derivative 49 was obtained by ammonolysis of compound 34 and subsequent
Suzuki coupling as described in the Scheme 12.
a
Scheme 12. Preparation of derivatives 47 and 49.
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Page 23 of 94
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43
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45
46
47
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49
50
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60
a
Reagents and conditions: (a) H (5 bars), Pd(OH) /C, Et N, THF:MeOH (1:1), 63%; (b)
2
2
3
NH /EtOH, MW, 140 °C, 1.5 h, 77%; (c) (3,4ꢀdimethoxyphenyl)boronic acid, Pd(PPh ) , 1M
K CO , dioxane, 90 °C, overnight, 80%..
2
3
3 4
3
Novel PI4KIIIβ inhibitors and their antiviral activity
To characterize the potency of this series of inhibitors in vitro we used the luminescent ADPꢀ
4
2
Glo kinase assay. For the presentation of the biological activities of the whole series, we have
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2
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5
5
5
5
5
5
5
6
selected 10 compounds, which exerted the highest inhibitory activities against PI4K IIIβ in vitro
(Figure 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
9
0
O
O
H
H
H
O
N
N
N S
O
HN
N
N
HN
N
HN
N
HN
N
HN
N
O
O
O
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O
2
c
3
9
10
28
H
H
H
N
H
N
NH2
N
HN
N
N
HN
HN
N
HN
N
O
O
N
O
N
N
O
N
N
N
N
N
Cl
N
Cl
N
N
Cl
Cl
N
O
S
O
N
O
O
O
O
O
O
O
F
3
6a
36p
36t
47
49
Figure 2. Structures of the selected subꢀseries of inhibitors. The amine side chains are in green,
bicyclic central core in red and aromatic side chain in blue.
We found that the IC values for the top 10 compounds were in the range of 50 – 130 nM
5
0
against PI4K IIIβ. Further, we determined whether the compounds were isoform specific for
PI4K IIIβ, a necessity for evaluating therapeutic potential. We have thus used the ADPꢀGlo
kinase assay to measure the IC values for two structurally and functionally related enzymes,
5
0
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PI4K IIIα and PI4K IIα. We found that none of the top 10 compounds inhibited PI4K IIIα and
PI4K IIα with an IC less than 10 ꢀM (These data are summarized in Table 1 for the top 10
5
0
compounds, and in Supplementary Results, Supplementary Table 2 for the remaining 55).
Next we focused on determining the antiꢀviral activity of the compounds. For this, we used an
assay based on the virusꢀinduced cytopathic effect in HeLa cells (in detail in Experimental
Methods section). We tested the activity of the compounds against two important members of
Picornaviridae family, Coxsackievirus B3 (CVB3) and Human Rhinovirus (HRV), and two
distinct genotypes of HCV (Flaviviridae), genotype 1b and 2a. For each virus tested except for
HCV 2a several of the compounds exhibited IC values below 1 µM as summarized in the Table
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
0
2 and Supplementary Table 3.
Table 1. Inhibitory activity of selected compounds against members of PI4K family.
Compound
PI4KIIIβ
PI4KIIIα
PI4KIIα
IC (ꢀM)
IC (ꢀM)
IC (ꢀM)
5
0
50
50
2c
3
0.070±0.020
0.120±0.010
0.120±0.010
0.067±0.009
0.093±0.012
0.090±0.011
0.089±0.003
0.054±0.015
0.092±0.006
0.083±0.025
0.150±0.010
59.45±0.06
62.14±0.84
29.31±0.96
29.52±3.40
27.46±2.47
82.67±18.64
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
≥100
9
10
2
3
3
3
4
4
8
6a
6p
6t
7
>100
33.67±0.49
>100
9
a
Tꢀ00127ꢀHEV1
74.55±3.08
a
Ref. 24, 26.
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3
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3
4
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4
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4
4
4
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4
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5
5
5
5
5
5
5
5
5
6
Although the correlation between the results from the enzymatic assay and the cell based assay
was not very tight, there was a clear rise in antiviral potency as PI4K IIIβ inhibitory activity
increased. The sensitivity of various viruses to the inhibition of the PI4K IIIβ showed significant
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
differences. The HCV genotype 1b displayed some sensitivity, whereas the effects of PI4K IIIβ
inhibition on replication of HCV 2a were rather limited. The most potent compound in the series
was derivative 36t, which inhibited PI4K IIIβ with IC = 54 nM and exerted significant effect in
5
0
all cell based assays. Derivative 36t was the most active compound in HVC 2a assay (EC =
5
0
10.6 µM) and the second most active against HCV1b (EC50 = 0.087 µM). Furthermore, this
compound showed strong protection against CVB3ꢀinduced cytopathic effects in HeLa cells with
half maximal effective concentration (EC ) value of 145 nM. This compound was also evaluated
5
0
in a panel of phosphatidylinositol 3ꢀkinases (PI3Ka, PI3Kb, PI3Kd and PI3Kg). None of this
kinases was inhibited with IC up to 10 ꢀM.
5
0
Table 2. Antiviral activity against selected ss(+)RNA viruses.
Compound
CVB3
HRVM
HCV 1b
HCV 2a
Hela
EC (ꢀM)
EC (ꢀM) EC (ꢀM)
EC (ꢀM)
CC (ꢀM)
50
50
50
50
50
2c
3
1.23±0.03
11.0±0.1
1.43±0.01
0.86±0.08
0.50±0.07
0.75±0.03
>50
2.9±0.1
1.03±1.17
>44
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.62±0.04 1.21±0.22
3.0±0.1 0.91±0.44
>44
9
42.5±0.5
>44
10
28
1.38±0.05 0.96±0.67
0.88±0.10 0.37±0.06
1.09±0.14 0.29±0.07
17.9±0.1
31.8±0.2
13.6±0.1
36a
36p
36t
47
24.3±0.1
5.3±0.1
0.145±0.034 1.03±0.35 0.087±0.067 10.6±0.1
0.28±0.04 2.9±0.1 0.36±0.09 27.9±0.2
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0.023±0.004 0.89±0.10 0.21±0.09
26.5±0.21
>44
1.75±0.17
>50
a
Tꢀ00127ꢀHEV1
3.38±0.05
ND
2.5±0.1
5.1±1.5
1.03±0.12
0.21±0.07
b
MKꢀ0608
0.21±0.10
>50
a
24, 26
Ref.
7
.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
b
36
ꢀDeazaꢀ2'ꢀCꢀmethyladenosine (MKꢀ0608), Ref. .
Structural analysis of the inhibition mode of PI4K IIIβ
To determine how the inhibitors function at the atomic level, we solved the crystal structure of
PI4K IIIβ either with an inhibitor or bound to ATP. We selected several inhibitors of
archetypical chemical structure and reasonable solubility for crystallographic trials. The best
crystals were obtained with inhibitor 49 but still diffracted only to 3.5Å resolution. The structure
was solved by molecular replacement using chain A of the crystal structure of PI4K IIIβ with
Rab11 (pdb code: ID0L) without the PIK93 inhibitor to reduce bias. Because of the rather low
resolution the unbiased Fo – Fc map contoured at 3σ did not provide enough information to
unambiguously place the 49 inhibitor (Supplementary Figure 3). However, the density for the 49
inhibitor was clearly visible in the Fo – Fc map contoured at 2σ and allowed for unambiguous
placement of the 49 inhibitor (Figure 3a). We have refined the structure to R = 20.89% and Rfree
43
=
25.16%. To confirm that the result of manual docking and refinement in Coot is correct we
calculated the R factors for a structure where the 49 inhibitor is rotated by 180° and refined into
the Fo – Fc density. The obtained values (R = 21.19% and Rfree = 26.07%) are significantly
worse and argue for correct placement of the ligand.
Crystals with ATP bound diffracted to 3.3Å resolution, the structure was solved in the same
way as the structure with 49 inhibitor and refined to R = 18.25% and Rfree = 24.41%. However,
we didn’t observe density for the whole ATP molecule, only for the adenine ring (Figure 3b),
despite the better resolution. We, therefore, concluded that the ribose ring and the three
phosphate groups are disordered in our structure. The density for adenine at 3.3Å, in principle,
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5
5
5
5
5
5
5
6
allows positioning the adenine ring in multiple orientations. To confirm that the best fit obtained
4
3
in Coot was the right solution, we compared the structure of PI4K IIIβ with the structure of
4
PI4K IIα where the ATP molecule is clearly resolved. We have used the Cꢀlobes of PI4K IIα
0
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and PI4K IIIβ for superposition taking advantage of the fact that they are structurally very
similar. The superposition revealed that the adenine rings in both structures are in a similar
orientation (Supplementary Figure 2), supporting the placement of the adenine ring in the
presented structure.
Both 49 and the adenine ring lie in a binding canyon of PI4K IIIβ held in place by a
combination of hydrophobic interactions and hydrogen bonds, water bridges surely also
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contribute (for instance the hydroxyl group of Tyr and the N6 of the adenine ring are in perfect
distance and orientation for a water bridge) but water molecules were not modeled due to limited
resolution of our crystal structures (Figure 4a, Figure 4b). Superposition of the two structures
revealed that compound 49 and the adenine ring occupy the same pocket (Figure 4c) suggesting
that this family of inhibitors function by sterically blocking the binding of the ATP’s adenine
ring rather than the phosphate moiety and therefore interfering with the course of the reaction.
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Journal of Medicinal Chemistry
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Figure 3.Crystal structures of 49 and ATP bound to PI4K IIIβ.
a) Compound 49 in the unbiased Fo – Fc map contoured at 2σ. Top – view on the whole enzyme.
Bottom – detailed view of the binding pocket.
b) The adenine ring of the ATP in the unbiased Fo – Fc map contoured at 2σ. Top – view on the
whole enzyme. Bottom – detailed view of the binding pocket.
The Nꢀlobe is colored in orange, the Cꢀlobe in cyan and the helical domain and the FoꢀFc map in
green. In the detailed view carbon atoms are colored silver, nitrogen in blue, chlorine in green
and oxygen in red.
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