Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ

Article abstract of DOI:10.1021/acs.jmedchem.5b01311

Type III phosphatidylinositol 4-kinase (PI4KIIIβ) is an essential enzyme in mediating membrane trafficking and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles and may lead to novel antiviral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design, we have designed novel potent and selective (>1000-fold over class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed antiviral activity against hepatitis C virus. The co-crystal structure of PI4KIIIβ bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIβ inhibitors, which may play significant roles as antiviral therapeutics.

Full text of DOI:10.1021/acs.jmedchem.5b01311

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Article
Design and structural characterization of potent and
selective inhibitors of phosphatidylinositol 4 kinase III#
Florentine U. Rutaganira, Melissa L. Fowler, Jacob A McPhail, Michael A Gelman, Khanh Nguyen, Anming
Xiong, Gillian L Dornan, Brandon Tavshanjian, Jeffrey S Glenn, Kevan M. Shokat, and John E. Burke
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01311 • Publication Date (Web): 17 Feb 2016
Downloaded from http://pubs.acs.org on February 23, 2016
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Design and structural characterization of potent and
selective inhibitors of phosphatidylinositol 4 kinase IIIβ
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Florentine U. Rutaganira1,a, Melissa L. Fowler2,a, Jacob A. McPhail2, Michael A. Gelman3%,
Khanh Nguyen3, Anming Xiong3, Gillian L. Dornan2, Brandon Tavshanjian1, Jeffrey S. Glenn3,4,
Kevan M. Shokat1*, and John E. Burke2*
1Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology,
University of California, San Francisco (UCSF), San Francisco, CA 94143, USA.
2Department of Biochemistry and Microbiology, University of Victoria, Victoria BC, V8W 2Y2,
Canada
3Departments of Medicine and Microbiology & Immunology, Stanford University, Palo Alto,
CA 94305, USA.
4Veterans Administration Medical Center, Palo Alto, CA, 94304.
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ABSTRACT
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Type III Phosphatidylinositol 4ꢀkinase (PI4KIIIβ) is an essential enzyme in mediating membrane
trafficking, and is implicated in a variety of pathogenic processes. It is a key host factor
mediating replication of RNA viruses. The design of potent and specific inhibitors of this
enzyme will be essential to define its cellular roles, and may lead to novel antiꢀviral therapeutics.
We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions
of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks.
Using structureꢀbased drug design we have designed novel potent and selective (>1000 fold over
class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed antiꢀviral activity
against Hepatitis C Virus. The coꢀcrystal structure of PI4KIIIβ bound to one of the most potent
compounds reveals the molecular basis of specificity. This work will be vital in the design of
novel PI4KIIIβ inhibitors, which may play significant roles as antiꢀviral therapeutics.
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Introduction
Lipid phosphoinositides are essential regulators of myriad cellular processes, including
signaling, membrane trafficking, and cytokinesis1. Phosphoinositides are generated through the
phosphorylation of the inositol ring of phosphatidylinositol. Phosphatidylinositol can be
phosphorylated and dephosphorylated by a diverse set of enzymes, and this results in a total of
seven different mono and poly phosphorylated phosphoinositides. The lipid species
phosphatidylinositol 4ꢀphosphate (PI4P) is generated by the action of phosphatidylinositol 4
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kinases (PI4Ks) . PI4P is the main biosynthetic route for the multiply phosphorylated signaling
lipids phosphatidylinositol 4,5ꢀbisphosphate (PIP2), and phosphatidylinositol 3,4,5ꢀtrisphosphate
(PIP3)3. In mammals there are four different PI4K enzymes, two type II enzymes (PI4KIIα and
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PI4KIIβ) and two type III enzymes (PI4KIIIα and PI4KIIIβ). PI4KIIIβ is a peripheral membrane
protein that is primarily localized at the Golgi and the Trans Golgi Network (TGN). This enzyme
plays key roles in mediating lipid transport4, cytokinesis5, maintaining lysosomal identity6, and
in tandem with Rab GTPases plays key roles in regulating membrane trafficking7. Interest in the
development of potent small molecules of PI4KIIIβ has been driven recently by the discovery of
the key role of this enzyme in both mediating viral replication8, as well as in mediating
Plasmodium development9.
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PI4KIIIβ is critical for mediating viral replication of a number of RNA viruses through
the generation of PI4P enriched viral replication platforms. These membranous webs enriched in
PI4P play essential roles in spatially concentrating viral replication proteins, and are key in
intracellular viral replication. This process is essential for many human pathogenic viruses
including Poliovirus, coxsackieviruses, Enterovirus 71, rhinovirus, and Aichi virus8,10ꢀ14. There is
also evidence that PI4KIIIβ together with PI4KIIIα play a key role in mediating viral replication
of Hepatitis C virus14.
Small molecule inhibitors of PI4KIIIβ are potent antiꢀviral agents8,15,16. We previously
reported the potent PI4KIIIβ inhibitor PIK93 (compound 1)17, and this compound has been used
extensively to decipher the cellular roles of PI4KIIIβ4,18, and its role in mediating viral
replication of pathogenic RNA viruses8,11ꢀ14. Compound 1 potently inhibits PI4KIIIβ; however, it
shows cross reactivity towards a number of other lipid kinases. Compound 1 has very similar
IC50 values for PI4KIIIβ, class III PI3 kinase (vps34), and class IB PI3Kγ (Fig 1A). We have
previously crystallized 1 in complex with PI4KIIIβ19, vps3420, and with PI3Kγ17 (Fig. 1BꢀE).
Development of PI4KIIIβ as an effective drug target for antiꢀviral therapeutics requires
the generation of highly potent and specific inhibitors. We report the development of a set of
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derivatives from compound 1, and these represent some of the most potent PI4KIIIβ inhibitors
reported to date. The selectivity profile of these compounds has been determined against vps34,
PI3Kδ and PI3Kγ, with the most selective compounds being >1000 fold selective over the
related PI3K family of lipid kinases. We have successfully determined the structure of PI4KIIIβ
bound to one of the most potent and selective compounds, and this structure reveals the
molecular basis for the increased selectivity and potency of these compounds.
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Results
Design of optimized PI4KIIIβ inhibitors
Compound 1 is highly selective for PI4KIIIβ over PI4KIIIα, however, it is similarly
potent for a number of phosphoinositide 3ꢀkinases (PI3Ks), specifically the class I isoforms
PI3Kγ (also referred to as p110γ) and PI3Kδ (also referred to as p110δ), as well as the class III
PI3K vps34 (Fig. 1A). The structures of 1 bound to vps3420, PI3Kγ17, and PI4KIIIβ19 revealed
that within the binding pocket there were significant opportunities to modify 1 to increase both
potency and selectivity for PI4KIIIβ.
From examining the structures of 1 bound to each enzyme, there were three regions of the
molecule that were focused on to optimize both potency and selectivity of novel PI4KIIIβ
inhibitors. These consisted of modifying the substituent on the central phenyl ring (colored
green, Fig. 1A), the substituent off the sulfonamide (colored blue, Fig. 1A), and the acetamide
moiety (colored red, Fig. 1A). The chloro substituent on the central phenyl ring of 1 fits into a
pocket partially composed of the activation loop of both PI3Ks and PI4KIIIβ (Fig. 1C). The
conformation of the activation loop of PI3Kγ when bound to compound 1 is positioned closer to
this substituent than in either vps34 or PI4KIIIβ. This suggested that modifying this group to a
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larger substituent might increase selectivity over class I PI3Ks. Changing this group to a bromo
substituent (compound 2) caused a slight increase in potency for PI4KIIIβ, PI3Kδ and vps34, and
a slight decrease in potency for PI3Kγ. Modifying this group to a methoxy substituent
(compound 3) caused both an increase in potency for PI4KIIIβ and a large increase in specificity
over PI3Kγ, PI3Kδ (>40 fold selective for both) and vps34 (Fig. 2A).
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The structures of 1 bound to PI4KIIIβ, PI3Kγ, and vps34 also suggested that modification
of the acetamide group derived from the central thiazole might lead to further gains in
specificity. PI4KIIIβ has a much more open pocket around the acetamide group of 1 (Fig. 1C),
compared to both PI3Kγ (Fig. 1D), and vps34 (Fig. 1E) which have a number of bulky
hydrophobic residues. The methyl group of 1 was replaced with either a tꢀbutyl group
(compound 4) or a cyclopentyl group (compound 5). Both compounds showed similar or slightly
better potency against PI4KIIIβ, however, both showed a very large increase in selectivity over
PI3Kγ (>600 fold for compound 4 and >90 fold for compound 5), PI3Kδ, and vps34 (Fig. 2A).
Finally the ethanolamine attached to the sulfonamide in compound 1 was also
derivatized. The ethanolamine was replaced with either a paraꢀhydroxy phenol group (compound
6) or a paraꢀfluoro phenyl (compound 7). The presence of the paraꢀhydroxy phenol group caused
a large increase in potency for all lipid kinases tested, with minimal differences in both potency
and specificity for the paraꢀfluoro phenyl substituent (Fig. 2A).
Combining this set of information we generated three compound 1 derivatives
(compounds 8+9+10) that contained derivations at multiple positions of 1. All of these
compounds contained a methoxy substituent off the central phenyl, with compound 9+10
containing a paraꢀhydroxy phenol off the sulfonamide, and either a cylopentyl (compound 9) or a
tꢀbutyl (compound 8+10) group at the acetamide position off of the central thiazole. Both of the
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compounds containing the para hydroxyl phenol were extremely potent against PI4KIIIβ (IC50s
of 7 nM and 3.6 nM for compound 9+10 respectively), and were >140 and >1000 fold selective
over PI3Kγ, were >20 and >200 fold selective over PI3Kδ and showed no inhibition of vps34 at
concentrations up to 20 ꢁM (Fig. 2B). Compound 8 was not as potent (IC50 of 36 nM), but
showed an excellent selectivity profile, with no inhibition of both PI3Kγ and vps34 (<20% at 50
ꢁM), and very little inhibition of PI3Kδ (<50% inhibition at 50 ꢁM). Compound 10 is the most
potent PI4KIIIβ inhibitor currently reported; with very minor offꢀtarget inhibition of PI4KIIIβ
related lipid kinases. Both compound 9+10 were further characterized against a panel of six
additional lipid kinases (Fig. S1). Compound 9 showed weak inhibition of PI3KC2γ (IC50 ~1
ꢁM), PI3Kα (~2 ꢁM), and PI4KIIIα (~2.6 ꢁM) and <50% inhibition at concentrations up to 20
ꢁM for PI4K2α, PI4K2β, and PI3Kβ. Compound 10 showed weak inhibition of PI3KC2γ (IC50
~1 ꢁM), PI3Kα (~10 ꢁM), and PI4KIIIα (~3 ꢁM), and <20% inhibition at concentrations up to
20 ꢁM for PI4K2α, PI4K2β, and PI3Kβ.
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PI4KIIIβ has been shown to be a key host factor for the replication of the Hepatitis C
virus14,21. To test the potency of the compound 1 derivative inhibitors as antiꢀvirals we carried
out viral replication assays using a luciferase reporterꢀlinked infectious HCV clone. These results
(Fig. 2, S2) generally show a trend of the most potent molecules being the most effective antiꢀ
virals, with increased specificity leading to decreased toxicity. One noticeable trend in the assay
was that absence of the hydroxylꢀphenyl group led to a large decrease in the antiꢀviral potency of
the compounds. This may be due to differences in cell permeability caused by this group. The
compounds with the best combination of antiꢀviral efficacy, and lowest toxicity were compounds
9 and 10 (Fig. S2).
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Structural basis of specificity for PI4KIIIβ inhibitors
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To determine the molecular basis for the potency and specificity of these novel PI4KIIIβ
inhibitors we set out to crystallize them bound to PI4KIIIβ. We have previously crystallized a
truncated construct of PI4KIIIβ in complex with compound 1 and the GTPase Rab1119. This
structure revealed the molecular basis of its interaction with Rab11, as well as revealing the
binding of compound 1 to PI4KIIIβ, however, this construct only crystallized in the presence of
compound 1. To generate crystals of PI4KIIIβ bound to compound 9 we used a novel
crystallization construct generated through the use of a hydrogen deuterium exchange mass
spectrometry based approach22. This construct allowed us to generate crystals of PI4KIIIβ bound
to GDP loaded Rab11 in the absence of inhibitors. These crystals were used to soak compound 9,
and allowed us to solve the coꢀcrystal structure at 3.2 angstrom resolution. The binding mode of
compound 9 was unambiguous (Fig. S3). The residues mediating contacts with compound 9 are
shown in Fig. 3A, with the shape of the inhibitor in the active site pocket shown in Fig. 3B.
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Structure of PI4KIIIβ bound to compound 9
Compound 9 forms a crescent shape that conforms to the active site of PI4KIIIβ. This
molecule makes extensive contacts with PI4KIIIβ (Fig 3A). There are two putative hydrogen
bonds formed between the thiazole and the acetamide of compound 9 with both the amide and
carbonyl group of V598. The sulfonamide group also forms a hydrogen bond with Lys549. All
of these hydrogen bonds are similar to those reported in the structure of 1 bound to PI4KIIIβ19.
Intriguingly the paraꢀhydoxy group on the Nꢀphenol sulfonamide also makes a putative hydrogen
bond with the carbonyl of G660. The presence of this para hydroxyl group is important for
potency, as molecules lacking this group are less potent against PI4KIIIβ (compound 6 versus
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compound 7). The presence of this hydroxyl group also increases potency for both PI3Kγ and
vps34 suggesting that this putative hydrogen bond is most likely conserved across all three
enzymes.
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The structure of compound 9 also reveals the molecular mechanism for how the
acetamide and methoxy substituent mediate inhibitor selectivity over PI3Kγ and vps34. Aligning
the structure of PI4KIIIβ bound to compound 9 with structures of compound 1 bound to PI3Kγ
and vps34 (Figure 4AꢀC), reveals a number of steric clashes that explain the lack of potency of
compound 9 against the PI3K family kinases. The acetamide group clashes with residues W812
and M953 in PI3Kγ, and residues Y746 and F673 in vps34. The methoxy group off the central
phenyl group clashes with the aspartic acid from the DFG motif in the activation loop (D964 in
PI3Kγ and D823 in vps34).
Discussion
Phosphatidylinositol 4 kinase IIIβ plays both key physiological and pathological roles,
and the design of novel potent and selective inhibitors will be essential both in deciphering the
cellular functions of this enzyme, as well as in the development of potential future therapeutic
agents in diseases dependent on PI4KIIIβ activity. PI4KIIIβ mediates the replication of a variety
of pathogenic RNA viruses, including members of both the Picornaviridae and Flaviviridae
family of viruses10. These include viruses that pose significant threats to human health including
SARS, MERS, Hepatitis C, and Polio. Along with this key role in mediating pathological
conditions, PI4KIIIβ also plays key roles in a number of physiological roles including membrane
trafficking23, lipid transport24, and cytokinesis5 and the development of potent and selective
molecules will be essential in deciphering the isoform specific functions of this enzyme.
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Since PI4KIIIβ plays an essential role in mediating viral replication for a variety of
pathogenic viruses, the development of PI4KIIIβ inhibitors has been an attractive target for pan
antiꢀviral agents. A number of potent PI4KIIIβ inhibitors have recently been discovered9,12,15,21,25ꢀ
28, and experiments on a variety of RNA viruses show they are potent antiꢀviral agents. However,
a complication of this work has been the toxic effects of some of these inhibitors on host
function. There are conflicting results of the lethality of PI4KIIIβ inhibitors, with some showing
lethality in mice28, and others being well tolerated15. Deciphering if these effects are dependent
on the inhibition of PI4KIIIβ, or through other off target effects requires the generation of novel
more potent and specific PI4KIIIβ inhibitors. A specific goal for the development of antivirals is
to avoid off target effects on both PI3Kγ and PI3Kδ as both of these enzymes play key roles in
the immune system29.
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Here we describe a set of new compound 1 derivative inhibitors that are some of the most
potent and specific PI4KIIIβ inhibitors as yet reported. Through a detailed structure based drug
design approach we have designed derivatives that increased potency against PI4KIIIβ >5 fold
while increasing selectivity >1000 fold to the related lipid kinases PI3Kγ and vps34. The most
potent of these molecules (compound 10) was screened over a panel of nine related lipid kinases
and was >200 fold selective for PI4KIIIβ across all enzymes tested. Two of the most potent and
selective molecules (compound 9+10) were also the most effective antiviral compounds in a
cellular model of Hepatitis C virus replication, with the best balance of antiviral potency and low
cellular toxicity.
The structure of one of the most potent molecules bound to PI4KIIIβ was determined,
and reveals the molecular basis for specificity of the derivative molecules. The pocket that
accommodates the acetamide group of the inhibitors was found to be the most important region
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in mediating inhibitor selectivity, and the differences in the pocket accommodating this group in
PI4KIIIβ compared to PI3Kγ and vps34 explains this observation. The acetamide group
mediating specificity of PI4KIIIβ inhibitors strongly correlates with previous studies on
compounds very similar to compound 1. Addition of a bulky substituent in a similar position to
the acetamide group in compound 1 had a limited role in potency, but greatly enhanced
specificity27,30. Structural and computational models of a different class of inhibitors bound to
PI4KIIIβ suggested an important role of the pocket located near this region in mediating both
potency and selectivity25. The structural details of this compound bound in the active site pocket
will provide a future framework for the modification of these compounds to modify the
inhibitors in a way that will enhance their pharmacokinetic properties, but not lead to any
decreases in their potency and specificity.
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Experimental section
Protein Expression
Truncated human PI4KIIIβ (121ꢀ784 ꢂ249ꢀ287ꢂ408ꢀ507 S294A) (plasmid JM7) and fullꢀlength
human Rab11a(Q70L) (plasmid pJB88) were expressed in BL21 C41 (DE3) cells. For
Rab11a(Q70L) expression, cultures were grown to an OD600 of 0.7 and induced with 0.5 mM
IPTG for 3.5 hours at 37 oC. For truncated PI4KIIIβ expression, cultures were induced overnight
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at 16 C with 0.1 mM IPTG at an OD600 of 0.6. Cells were harvested by centrifugation, washed
with cold phosphateꢀbuffered saline (PBS), frozen in liquid nitrogen, and pellets were stored at ꢀ
80 oC.
Protein Purification
PI4KIIIβ (truncation) Purification:
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Cells were resuspended in 20 mM TrisꢀHCl pH 8.0, 100 mM NaCl, 10 mM imidazole, 5% (v/v)
glycerol, 2 mM βꢀmercaptoethanol, and a 1:1666 dilution of a protease inhibitor cocktail
(Milipore Protease Inhibitor Cocktail Set III, AnimalꢀFree), and were sonicated on ice for 5 min
with cycles consisting of 10 sec on, 10 sec off. Triton Xꢀ100 was then added to a final
concentration of 0.2%, and the lysate was centrifuged for 45 min at 20,000 x g. The supernatant
was then filtered through a 0.45 µm filter (Celltreat Scientific Products) and was loaded onto a 5
mL HisTrap FF column (GE Healthcare) equilibrated in buffer A (20 mM TrisꢀHCl pH 8.0, 100
mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, 2 mM βꢀmercaptoethanol). The column was
washed with 20 mL of buffer A, followed by 20 mL of 6% buffer B (20 mM TrisꢀHCl pH 8.0,
100 mM NaCl, 200 mM imidazole, 5% (v/v) glycerol, 2 mM βꢀmercaptoethanol), and was eluted
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with 100% buffer B. The His affinity tag protein was cleaved overnight at 4 C with TEV
protease. The cleaved protein was then diluted to 50 mM NaCl (using 20 mM TrisꢀHCl pH 8.0,
10 mM imidazole, 5% (v/v) glycerol, 2 mM βꢀmercaptoethanol) and was loaded onto a 5 mL
HiTrap Q HP column (GE Healthcare) equilibrated in buffer C (20 mM TrisꢀHCl pH 8.0, 50 mM
NaCl, 5% (v/v) glycerol, 2 mM βꢀmercaptoethanol). Protein was eluted with a gradient elution
using buffer D (20 mM TrisꢀHCl pH 8.0, 1.0 M NaCl, 5% (v/v) glycerol, 2 mM βꢀ
mercaptoethanol). Fractions containing the cleaved PI4KIIIβ were pooled and concentrated to
700 µL in an Amicon 50K centrifugal filter (Millipore). The protein was then loaded onto a
HiPrep 16/60 Sephacryl S200 column equilibrated in buffer E (20 mM HEPES pH 7.2, 150 mM
NaCl, 1 mM TCEP). The cleaved PI4KIIIβ was then concentrated to approximately 15 mg/mL in
an Amicon 50K centrifugal filter (Millipore), and aliquots were frozen in liquid nitrogen and
stored at ꢀ80 oC.
Rab11a(Q70L) Purification:
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Cells were resuspended in 20 mM TrisꢀHCl pH 8.0, 100 mM NaCl, 5% (v/v) glycerol, 2
mM βꢀmercaptoethanol, and a 1:1666 dilution of a protease inhibitor cocktail (Milipore Protease
Inhibitor Cocktail Set III, AnimalꢀFree). Cells were sonicated and centrifuged as described for
the truncated PI4KIIIβ. The supernatant was filtered through a 0.45 µm filter (Celltreat Scientific
Products) and incubated for 1 hour with 4 mL of Glutathione Sepharose 4B beads (GE
Healthcare) equilibrated in buffer F (20 mM TrisꢀHCl pH 8.0, 100 mM NaCl, 5% (v/v) glycerol,
2 mM βꢀmercaptoethanol) followed by a 3 x 15 mL wash in buffer F. The GST tag was cleaved
overnight on the beads with TEV protease. Anionꢀexchange chromatography was performed as
outlined above for the truncated PI4KIIIβ. Cleaved Rab11a(Q70L) were then concentrated to
between 5ꢀ15 mg/mL and nucleotide loaded by adding EDTA to 10 mM followed by 1 U of
phosphatase (Phosphatase, AlkalineꢀAgarose from calf intestine, Sigma P0762ꢀ100UN) per mg
of protein. Proteins were then incubated for 1.5 hours. The phosphatase was removed using a 0.2
µm spin filter (Millipore); the flowꢀthrough was collected, and a 10ꢀfold molar excess of GDP
was added followed by MgCl2 to a final concentration of 20 mM. Proteins were incubated for 30
min. Gel filtration was performed with cleaved GDPꢀloaded Rab11a(Q70L) as described above
for the truncated PI4KIIIβ.
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Crystallography
Crystals of PI4KIIIβ (final concentration 7.4 mg/mL) with Rab11aꢀGDP (final
concentration 4.5 mg/mL) were obtained in 15% (w/v) PEGꢀ4000, 100 mM sodium citrate pH
5.6, 200 mM ammonium sulfate (protein:precipitant ratio of 3:1). Refinement plates were set by
gridding PEGꢀ4000, ammonium sulfate, and glycerol. Optimized crystals were obtained by
seeding using the Hampton Research Seed Bead Kit according to the manufacturer’s instructions
using a reservoir solution of 14.8% (w/v) PEGꢀ4000, 100 mM sodium citrate pH 5.6, 250 mM
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ammonium sulfate. The best crystals were obtained in 13ꢀ15% (w/v) PEGꢀ4000, 100 mM
sodium citrate pH 5.6, 250 mM ammonium sulfate, 2% glycerol with a 1/1000 or 1/10,000 seed
solution dilution, a Rab11aꢀGDP final concentration of 4.51 mg/mL and a PI4KIII final
concentration of 7.38 mg/mL. Crystals were frozen in liquid nitrogen using a 15% PEGꢀ4000
(w/v), 100 mM sodium citrate pH 5.6, 250 mM ammonium sulfate, 25% (v/v) glycerol cryo
solution.
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Inhibitor soaks were performed by incubating crystals with 0.5 µL of 10 µM inhibitor
stocks in cryo buffer (15% PEGꢀ4000 (w/v), 250 mM ammonium sulfate, 100 mM sodium
citrate pH 5.6, 25% glycerol (v/v)) for 30 min, followed by a 30 min incubation with 0.5 µL of
100 µM inhibitor stock in cryo buffer, and a final 30 min incubation in 1 mM inhibitor stock in
cryo buffer. Before the final addition, 1 µL was removed from the crystal drop and 1 µL of the 1
mM inhibitor in cryo buffer was added.
Diffraction data were collected at 100 K at the Canadian Macromolecular
Crystallography Facility (Canadian Light Source, CLS) beamline 08IDꢀ1. Data were integrated
using iMosflm 7.1.131 and scaled with AIMLESS32. Phases were initially obtained by molecular
replacement (MR) using Phaser33, with the structure of PI4KIIIβ bound to compound 1 and
Rab11 (pdb code: 4D0L) used as the search model. The final model of PI4KIIIβ bound to
compound 9 in complex with Rab11 was built using iterative model building in COOT34 and
refinement using Phenix35,36 to Rwork=23.87 and Rfree=26.79. The binding mode of compound 9
was unambiguous, and ligand geometry was generated using the elbow subset of Phenix37. Full
crystallographic statistics are shown in Table 1.
Biochemical Assays
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Lipid kinase assays were preformed using recombinant enzyme, phosphoinositides
purchased from Avanti Polar Lipids and γ32PꢀATP (Perkin Elmer Cat #BLU502A001MC) in a
membrane capture assay described previously38. Each inhibitor was diluted into 10% DMSO and
kinase assay buffer. Upon completion of the reaction, 4 µL was spotted onto 0.2ꢁm
nitrocellulose (BioꢀRad Catalog #162ꢀ0112). The membrane was dried for 5 minutes under a
heat lamp followed by 1x30second wash and 6x5min washes in 1M NaCl / 1% Phosphoric Acid.
The membrane was dried for 20 minutes under a heat lamp followed by overnight exposure to a
phosphor screen and phosphorimaging followed on a Typhoon 9500. Intensities were quantified
using SPOT38. Specifications for each enzyme follow.
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PI4KIIIβ
Recombinant enzyme was purchased from Life Technologies (Cat # PV5277, Lot
943589E). LꢀαꢀPhosphatidylinositol (PI, Cat # 840024P) and DOPS:DOPC lipids (Cat #
790595P) were sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction was setꢀup as
follows 1) kinase assay buffer, PI:DOPS:DOPC, BSA and PI4KIIIβ, were combined in a total
volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and
incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP were added
(2.5x solution) to initiate the reaction which ran for 30 minutes. Final conditions were as follows:
20mM BisꢀTris Propane pH 7.5, 10mM MgCl2, 0.075mM Triton Xꢀ100, 0.5mM EGTA, 1mM
DTT, 100ꢁM PI, 500ng/µL BSA, 2.5nM PI4KIIIβ, 2% DMSO, 10ꢁM ATP and 1uCi γ32PꢀATP.
PI3Kγ
Recombinant enzyme was purchased from Life Technologies (Cat # PV4786, Lot #
1638926A). LꢀαꢀPhosphatidylinositolꢀ4,5ꢀbisphosphate (PIP2, Cat # 840046P) and DOPS:DOPC
lipids (Cat # 790595P) were sonicated in water to generate 1mg/mL PIP2:DOPS:DOPC.
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Reaction was setꢀup as follows 1) kinase assay buffer, PIP2:DOPS:DOPC, BSA and PI3Kγ, were
combined in a total volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x
solution) and incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP
were added (2.5x solution) to initiate the reaction which ran for 15 minutes. Final conditions
were as follows: 50mM HEPES pH 7.5, 100mM NaCl, 0.03% CHAPS, 10mM MgCl2, 1mM
EGTA, 2mM DTT, 5nM PI3Kγ, 80ꢁM PIP2, 500ng/ µL BSA, 2% DMSO, 10ꢁM ATP and 1uCi
γ32PꢀATP.
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PI3Kδ
Recombinant enzyme was purchased from Life Technologies (Cat # PV6451, Lot #
1763224). LꢀαꢀPhosphatidylinositolꢀ4,5ꢀbisphosphate (PIP2, Cat # 840046P) and DOPS:DOPC
lipids (Cat # 790595P) were sonicated in water to generate 1mg/mL PIP2:DOPS:DOPC.
Reaction was setꢀup as follows 1) kinase assay buffer, PIP2:DOPS:DOPC, BSA and PI3Kδ, were
combined in a total volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x
solution) and incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP
were added (2.5x solution) to initiate the reaction which ran for 20 minutes. Final conditions
were as follows: 50mM HEPES pH 7.5, 100mM NaCl, 0.03% CHAPS, 10mM MgCl2, 1mM
EGTA, 2mM DTT, 2.5nM PI3Kδ, 80ꢁM PIP2, 500ng/ µL BSA, 2% DMSO, 10ꢁM ATP and
2.5uCi γ32PꢀATP.
vps34
Recombinant enzyme was purchased from Life Technologies (Cat # PV5126, Lot #
1555138A). LꢀαꢀPhosphatidylinositol (PI, Cat # 840024P) and DOPS:DOPC lipids (Cat #
790595P) were sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction was setꢀup as
follows 1) kinase assay buffer, PI:DOPS:DOPC, BSA and vps34, were combined in a total
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volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and
incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP were added
(2.5x solution) to initiate the reaction which ran for 1 hour. Final conditions were as follows:
50mM HEPES pH 7.5, 0.1% CHAPS, 2mM MnCl2, 1mM EGTA, 2mM DTT, 10nM vps34,
100ꢁM PI, 500ng/ µL BSA, 2% DMSO, 10ꢁM ATP and 2.5uCi γ32PꢀATP.
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PI4KIIIα
Recombinant enzyme was purchased from EMD Millipore (Cat # 14ꢀ908, Lot #
D9KN031NꢀB). LꢀαꢀPhosphatidylinositol (PI, Cat # 840024P) and DOPS:DOPC lipids (Cat #
790595P) were sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction was setꢀup as
follows 1) kinase assay buffer, PI:DOPS:DOPC, BSA and PI4KIIIα, were combined in a total
volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and
incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP were added
(2.5x solution) to initiate the reaction which ran for 25 minutes. Final conditions were as follows:
20mM BisꢀTris Propane pH 7.5, 10mM MgCl2, 0.075mM Triton Xꢀ100, 0.5mM EGTA, 1mM
DTT, 100ꢁM PI, 500ng/µL BSA, 1.2nM PI4KIIIα, 2% DMSO, 10ꢁM ATP and 2uCi γ32PꢀATP.
PI4KIIα
PI4KIIα was cloned from DNASU plasmid HsCD0000333239,40. Recombinant enzyme
was obtained through FLAG immunoprecipitation of FLAGꢀPI4KIIα transfected into HEK293T
cells (10ug of FLAGꢀPI4KIIα transfected with Life Technologies Lipofectamine and Plus
Reagent to a 15cm dish of HEK293Ts and 75uL elution with FLAG peptide). Lꢀαꢀ
Phosphatidylinositol (PI, Cat # 840024P) and DOPS:DOPC lipids (Cat # 790595P) were
sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction was setꢀup as follows 1)
kinase assay buffer, PI:DOPS:DOPC, BSA and PI4KIIα, were combined in a total volume of 10
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µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and incubated with
enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP were added (2.5x solution) to
initiate the reaction which ran for 25 minutes. Final conditions were as follows: 20mM BisꢀTris
Propane pH 7.5, 10mM MgCl2, 0.075mM Triton Xꢀ100, 0.5mM EGTA, 1mM DTT, 100ꢁM PI,
500ng/µL BSA, 2.9uL PI4KIIα, 2% DMSO, 10ꢁM ATP and 2uCi γ32PꢀATP.
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PI4KIIβ
PI4KIIβ was cloned from DNASU plasmid HsCD0000159239,40. Recombinant enzyme
was obtained through FLAG immunoprecipitation of FLAGꢀPI4KIIβ transfected into HEK293T
cells (10ug of FLAGꢀPI4KIIβ transfected with Life Technologies Lipofectamine and Plus
Reagent to a 15cm dish of HEK293Ts and 75uL elution with FLAG peptide). Lꢀαꢀ
Phosphatidylinositol (PI, Cat # 840024P) and DOPS:DOPC lipids (Cat # 790595P) were
sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction was setꢀup as follows 1)
kinase assay buffer, PI:DOPS:DOPC, BSA and PI4KIIβ, were combined in a total volume of 10
µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and incubated with
enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP were added (2.5x solution) to
initiate the reaction which ran for 25 minutes. Final conditions were as follows: 20mM BisꢀTris
Propane pH 7.5, 10mM MgCl2, 0.075mM Triton Xꢀ100, 0.5mM EGTA, 1mM DTT, 100ꢁM PI,
500ng/µL BSA, 2uL PI4KIIβ, 2% DMSO, 10ꢁM ATP and 2uCi γ32PꢀATP.
PI3Kα
Recombinant enzyme was purchased from EMD Millipore (Cat # 14ꢀ602, Lot #
2150294ꢀA). LꢀαꢀPhosphatidylinositolꢀ4,5ꢀbisphosphate (PIP2, Cat
#
840046P) and
DOPS:DOPC lipids (Cat # 790595P) were sonicated in water to generate 1mg/mL
PIP2:DOPS:DOPC. Reaction was setꢀup as follows 1) kinase assay buffer, PIP2:DOPS:DOPC,
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BSA and PI3Kα, were combined in a total volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor
solution was added (5x solution) and incubated with enzyme mixture for 15 minutes; 3) 10 µL
cold ATP and γ32PꢀATP were added (2.5x solution) to initiate the reaction which ran for 15
minutes. Final conditions were as follows: 50mM HEPES pH 7.5, 100mM NaCl, 0.03% CHAPS,
10mM MgCl2, 1mM EGTA, 2mM DTT, 3.3nM PI3Kα, 80ꢁM PIP2, 500ng/ µL BSA, 2%
DMSO, 10ꢁM ATP and 2uCi γ32PꢀATP.
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PI3Kβ
Recombinant enzyme was purchased from SignalChem (Cat # P28ꢀ10Hꢀ10, Lot # Fꢀ532ꢀ
3). LꢀαꢀPhosphatidylinositolꢀ4,5ꢀbisphosphate (PIP2, Cat # 840046P) and DOPS:DOPC lipids
(Cat # 790595P) were sonicated in water to generate 1mg/mL PIP2:DOPS:DOPC. Reaction was
setꢀup as follows 1) kinase assay buffer, PIP2:DOPS:DOPC, BSA and PI3Kβ, were combined in
a total volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and
incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP were added
(2.5x solution) to initiate the reaction which ran for 15 minutes. Final conditions were as follows:
50mM HEPES pH 7.5, 100mM NaCl, 0.03% CHAPS, 10mM MgCl2, 1mM EGTA, 2mM DTT,
0.6nM PI3Kβ, 80ꢁM PIP2, 500ng/ µL BSA, 2% DMSO, 10ꢁM ATP and 2uCi γ32PꢀATP.
PI3KC2γ
Recombinant enzyme was purchased from EMD Millipore (Cat # 14ꢀ910, Lot #
2023057ꢀA). LꢀαꢀPhosphatidylinositol (PI, Cat # 840024P) and DOPS:DOPC lipids (Cat #
790595P) were sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction was setꢀup as
follows 1) kinase assay buffer, PI:DOPS:DOPC, BSA and PI3KC2γ, were combined in a total
volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and
incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32PꢀATP were added
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(2.5x solution) to initiate the reaction which ran for 15 minutes. Final conditions were as follows:
50mM HEPES pH 7.5, 100mM NaCl, 0.03% CHAPS, 10mM MgCl2, 1mM EGTA, 2mM DTT,
1nM PI3KC2γ, 100ꢁM PI, 500ng/ µL BSA, 2% DMSO, 10ꢁM ATP and 2uCi γ32PꢀATP.
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Organic Synthesis
Materials obtained commercially were reagent grade and were used without further purification.
1H NMR spectra were recorded on a Varian 400 spectrometer at 400 MHz . Highꢀresolution
electron impact mass spectra were recorded on a Thermo Fisher Exactive EMR and Thermo
Fisher LTQ Orbitrap Velos at the University of CaliforniaꢀSan Francisco center for Mass
Spectrometry. Reactions were monitored by thin layer chromatography (TLC), using Merck
silica gel 60 F254 glass plates (0.25 mm thick). Flash chromatography was conducted with Grace
Reveleris flash cartridges with 40ꢁm silica on an Agilent 971ꢀFP. All RPꢀHPLC were performed
with a Waters 2545 binary gradient module equipped with an XBridge prep C18 column using
H2O + 0.1% formic acid and CH3CN + 0.1% formic acid (5ꢀ95% gradient) while monitoring at
254 nm. All final compounds were >95% pure as measured by Liquid Chromatography Mass
Spectrometry (LCMS). Full compound characterization details are presented in the supporting
information.
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Scheme 1. Synthesis of compound 1 and derivatives.
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SO3ClH
X
X
S
X
S
R-NH
THF
O
O
O
O
O
O
O
Cl
NH
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11
12
R
PH3PCH2COOH Br3
THF
O
X
S
O
Cl
Y
S
S
O
O
Toluene
H2N
NH2
H2N
N
Y
O
NH
H
Br
R
14
13
Ethanol
S
X
S
O
NH
O
NH
R
Y
N
O
Compound 1/ Compound 1 Derivative
Synthesis of compound 1 and derivatives was conducted similarly to methods described
previously17, and is shown in scheme 1. To commercially available acetaphenone chlorosulfuric
acid (11eq) was added dropwise in an ice bath and the reaction was heated to 40°C for 2 hours.
The reaction was stopped by dropwise transfer to iceꢀwater. The aqueous phase was extracted 3x
with ethyl acetate and the combined organic is dried with sodium sulfate, filtered and
concentrated in vacuo to give a brown oil, the crude corresponding to sulfonyl chloride, 11. The
sulfonyl chloride was dissolved in THF (1.5M), the appropriate amino alcohol (4.5eq) was added
and the reaction was allowed to stir overnight at room temperature. The reaction was
concentrated in vacuo, water is added and the aqueous phase was extracted 3x with ethyl acetate.
The combined organic phase was concentrated and intermediate 12 was purified using silica gel
flash chromatography (2% ꢀ 10% methanol in dichloromethane). If pure intermediate was not
obtained, the intermediate was subjected to a second purification by reverse phase HPLC using
acetonitrile/water/0.1% formic acid as a solvent system.
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Intermediate 12 was dissolved in THF (1.5M). (2ꢀcarboxyethyl) triphenylphosphonium
bromide was dissolved in THF (1M) and intermediate 12 was added to the bromide solution
dropwise at room temperature. The reaction was allowed to proceed 1 hour at room temperature
and the solvent is removed in vacuo and the product was purified by silica gel flash
chromatography (0% ꢀ 10% methanol in dichloromethane) to yield intermediate 13.
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To ethanolꢀrecrystallized thiourea in dry toluene (0.5M) was added appropriate acyl
chloride and the reaction was heated to reflux for 18 hours. Toluene was removed in vacuo and
the reaction was diluted in ethyl acetate and filtered. The organic layer was washed 2x with
water and dried with brine and sodium sulfate and reduced in vacuo. The resulting acetylthiourea
14 was purified by silica gel flash chromatography (25% ꢀ 100% ethyl acetate in hexanes).
Intermediate 13 was dissolved in ethanol (0.17M) and appropriate acetylthiourea 14 was
added at room temperature. The reaction was heated to reflux for 30 minutes and then cooled to
room temperature and compound 1 or compound 1 derivative (compounds 2ꢀ10) was purified by
silica gel chromatography (0% ꢀ 10% methanol in dichloromethane) and if necessary, followed
by reverse phase HPLC using acetonitrile/water/0.1% formic acid as a solvent system.
Gaussia luciferaseꢀbased HCV reporter virus and stable cell line of Huh7.5 containing infectious
HCV reporter virus.
A fullyꢀinfectious HCVcc genotype 2a reporter virus encoding Gaussia luciferase
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between p7 and NS2 similar to Maurkian et al. was generated to monitor HCV replication
quantitatively. Briefly, Gaussia luciferase gene with an in frame foot and mouth disease virus
autoproteolytic 2A peptide sequence was inserted between HCV p7 and NS2 of a J6/JFH1
infectious HCV clone 42, resulting in the fullyꢀinfectious HCVcc reporter virus J6/JFH1ꢀGLuc
(p7ꢀNS2ꢀGLucFM2A). After Huh7.5 cells were infected with the Gaussia luciferaseꢀbased HCV
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reporter virus, the cells were maintained for three days and then passed every four days for
twenty passages. A stable cell line with consistent Gaussia luciferase secretion was established.
HCV2a antiviral assay
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Huh 7.5 cells stably infected with J6/JFH1ꢀGLuc were maintained and passaged in
DMEM (Mediatech, Manassas, VA) supplemented with 10% FBS (Omega Scientific, Tarzana,
CA), 2 mM glutamine (Mediatech, Manassas, VA), nonꢀessential amino acids (Mediatech,
Manassas, VA), 100 IU/mL penicillin (Mediatech, Manassas, VA), and 100 g/mL streptomycin
(Mediatech, Manassas, VA) in 37°C incubator with 5% CO2 and 95% relative humidity.
Ten thousand cells were plated in 96 well plates (E&K Scientific Products, Santa Clara,
CA) and supplemented with serially diluted compounds one hour after plating. After three days
incubation, viability was tested using the Presto Blue cell viability reagent (Life Technologies
Corporation, Grand Island, NY) per the manufacturer’s protocol and replication was measured
by determining the Gaussia luciferase activity in the supernatant using the luciferase reagent
(Promega Corporation, Madison, WI) according to the manufacturer’s protocol.
Viability and luminescence were read using a plate reader (Infinite 1000, Tecan Systems,
San Jose, CA). Values were imported into Prism (GraphPad Software, La Jolla, CA) for graphing
and calculations of EC50 and CC50 values.
ANCILLARY INFORRMATION
Supporting Information.
Supporting information including compound characterization, and three supplemental figures are
found in the supplemental information, and this material is available free of charge via the
Internet at http://pubs.acs.org.
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PDB ID Codes: The structure factors and pdb coordinates of PI4K bound to compound 9 have
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been deposited at the protein databank (PDB) with the coordinates 5EUQ.
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Corresponding Authors: Burke, John E. (jeburke@uvic.ca, 1ꢀ250ꢀ721ꢀ8732) and Shokat, Kevan
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M. (Kevan.Shokat@ucsf.edu, 1ꢀ415ꢀ514ꢀ0472).
%
Present/Current Author Addresses: Department of Medicine, Veterans Administration Medical
Center, Bronx, NY 10468
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. aThese authors contributed equally.
Acknowledgements
J.E.B. wishes to thank CIHR support (CIHR new investigator grant and CIHR open operating
grant FRN 142393). K.M.S. and J.S.G. wish to thank NIH support (R01 AI099245 and U19
AI109622). M.A.G. wishes to thank NIH support (K08 AI097322).
We thank the staff at the Canadian Light Source (CLS) CMCFꢀID beam line where diffraction
data were collected. The CLS is supported by the Natural Sciences and Engineering Research
Council of Canada, the National Research Council Canada, the Canadian Institutes of Health
Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the
University of Saskatchewan. High Resolution Mass Spectrometry was provided by the Bioꢀ
Organic Biomedical Mass Spectrometry Resource at UCSF (A.L. Burlingame, Director)
supported by the Biomedical Technology Research Centers program of the NIH National
Institute of General Medical Sciences, NIH NIGMS 8P41GM103481 and NIH 1S10OD016229
and the Howard Hughes Medical Institute.
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Abbreviations
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PI4K, Phosphatidylinositol
4
kinase, PI3K, phosphoinositide
3
kinase, PI4P,
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9
phosphatidylinositol 4ꢀphosphate, PIP2, phosphatidylinositol 4,5ꢀbisphosphate, PIP3,
phosphatidylinositol 3,4,5ꢀtrisphosphate.
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