Rational Design of Novel Highly Potent and Selective

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Rational design of novel highly potent and selective phosphatidylinositol 4-kinase III# (PI4KB) inhibitors as broadspectrum antiviral agents and tools for chemical biology Ivana Mejdrová, Dominika Chalupská, Pavla Pla#ková, Christin Müller, Michal Sala, Martin Klíma, Adriana Baumlová, Hubert Hrebabecky, Eliska Prochazkova, Milan Dejmek, Dmytro Strunin, Jan Weber, Gary Lee, Marika Matoušová, Helena Mertlíková-Kaiserová, John Ziebuhr, Gabriel Birkus, Evzen Boura, and Radim Nencka J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01465 • Publication Date (Web): 08 Dec 2016 Downloaded from http://pubs.acs.org on December 11, 2016

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Journal of Medicinal Chemistry

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Rational design of novel highly potent and selective phosphatidylinositol 4-kinase IIIβ (PI4KB) inhibitors as broad-spectrum antiviral agents and tools for chemical biology Ivana Mejdrováa,b‡, Dominika Chalupskáa,‡, Pavla Plačkováa, Christin Müllerc, Michal Šálaa, Martin Klímaa, Adriana Baumlováa, Hubert Hřebabeckýa, Eliška Procházkováa, Milan Dejmeka, Dmytro Strunina, Jan Webera, Gary Leeb, Marika Matoušováa, Helena Mertlíková-Kaiserováa, John Ziebuhrc, Gabriel Birkusd, Evzen Bouraa,*, Radim Nenckaa,* 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

Department of Chemistry of Natural Compounds, Institute of Chemical Technology Prague, Technická 5, Prague 166 28, Czech Republic

c

Institute of Medical Virology, Justus Liebig University Giessen, Schubertstrasse 81, D-35392 Giessen, Germany d

Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, United States.

ACS Paragon Plus Environment

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KEYWORDS Phosphatidylinositol 4-kinase IIIβ, picornaviruses, hepatitis C virus, selective inhibitor.

ABSTRACT

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is indispensable for the replication of various positive-sense single stranded RNA viruses, which hijack this cellular enzyme to remodel intracellular membranes of infected cells to set up the functional replication machinery. Therefore, the inhibition of this PI4K isoform leads to the arrest of viral replication. Here, we report on the synthesis of novel PI4KB inhibitors, which were rationally designed based on two distinct structural types of inhibitors that bind in the ATP binding side of PI4KB. These “hybrids” excel not only in outstanding inhibitory activity but they also show high selectivity to PI4KB compared to other kinases. Thus, these compounds exert selective nanomolar or even subnanomolar activity against PI4KB as well as profound antiviral effect against hepatitis C virus, human rhinovirus and coxsackievirus B3. Our crystallographic analysis unveiled the exact position of the side chains and explains their extensive contribution to the inhibitory activity.

INTRODUCTION Positive-sense single stranded RNA (+RNA) viruses replicate at specialized compartments termed replication factories or membranous webs 1. These membrane-derived compartments are specifically enriched in the phosphatidylinositol 4-phosphate (PI4P) lipid 2. Humans have four


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kinases that synthesize PI4P, two typical lipid kinases - PI4KA and PI4KB (also known as PI4K IIIα and PI4K IIIβ) and two type II (or atypical) kinases - PI4K2A and PI4K2B (also known as PI4K IIα and PI4K IIβ)3,4. In 2009 Berger and colleagues used siRNA screen to identify essential host factors for the replication of hepatitis C virus (HCV) and found PI4KA to be an essential host factor for HCV 5. Since then PI4KB has also been reported to be an essential host factor for a plethora of +RNA viruses including SARS coronavirus, kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus6-8. Surprisingly, so far PI4K2A or PI4K2B have never been reported as a viral co-factor. These enzymes are palmitoylated and can behave as integral transmembrane proteins, thus, it might be too difficult for a virus to manipulate them. Now it is generally accepted that most +RNA viruses hijack PI4K kinase to produce PI4Penriched membranes9. Viruses have evolved different strategies of PI4K hijacking. The aforementioned HCV uses its NS5A protein to directly hijack PI4KA10. Many picornaviruses use their non-structural 3A protein to hijack PI4KB. For instance, the 3A protein from the poliovirus, coxsackievirus, rhinovirus or Aichi virus binds to the Golgi residing ACBD3 (Acyl-CoA Binding Domain Containing 3) protein, a known binding partner of the PI4KB8, 11, 12. However, picornaviruses have also evolved additional means of PI4KB hijacking that are ACBD3 independent 13-15. PI4Ks are considered a potential pharmacological target because of their importance in the life cycle of various many +RNA viruses. The discovery of their importance for +RNA viruses aroused big interest in PI4Ks in the scientific community. During the last two years crystal structures of P4KB, PI4K2A and PI4K2B have become available 16-19 providing critical information for structure aided inhibitor design. The last missing piece is the structure of PI4KA


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perhaps due to difficulties in the expression of the recombinant protein 20. So far only a study that used experiment guided homology modeling is available 21. In the last few years, both academia and pharma invested substantial amounts of time in developing small molecule inhibitors targeting PI4KA and PI4KB. One of the first compounds, 1 (PIK93), has attracted attention because of its selectivity towards PI4KB22, 23, however, 1 has also notable activity toward PI3 kinases24. Later it was used as a starting point for developing isoform specific inhibitors. A compound resembling 1 was reported to inhibit PI4KA with IC50 = 450 nM but the selectivity was rather poor (IC50 = 8000 nM for PI4KB)25. Recently, very potent inhibitors of PI4KA were reported – IC50 = ~1 nM for the best compound, however, the selectivity was not optimal (IC50 = 250 nM for PI4KB)26. Perhaps the best inhibitor of PI4KA was reported by GlaxoSmithKleine which had an IC50 of 5nM against PI4KA and negligible activity against PI4KB 27. We and others have also reported PI4KB inhibitors with varying degrees of potency and specificity28-32 with best compounds having an IC50 between 10−20 nM. Important questions regarding the drugablity of these enzymes remain as knockout of these enzymes is detrimental to the animals 25, 33. However, upregulated PI4K activity is needed for a successful viral infection. Total long time inhibition of either PI4KA or PI4KB might be toxic but a short time dose (few days or a week) that would inhibit the enzymes only partially might be well tolerated and still fatal for the virus. This is especially the case for PI4KB because PI4K2A can take over many of its roles at the Golgi 34, 35. Further studies are needed to decide whether PI4KB is a valid pharmacological target and these studies will need very potent and extremely specific inhibitors. In this study we present the design and synthesis of novel PI4KB inhibitors, which exert high inhibitory potency and outstanding selectivity in comparison to related lipid kinases.


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Furthermore, our prototypical compounds show absolutely no significant inhibition of any other kinases at 1 µM concentration in kinome-wide screen (Figure 3). RESULTS AND DISCUSION Design of inhibitors Generally, specific PI4KB inhibitors targeting ATP binding side can be divided into two structural groups. Firstly, several compounds derived from 1 exert both significant affinity and high selectivity towards this enzyme. Secondly, derivatives with bicyclic central core related to 2 (T-00127-HEV1)36 were also identified as potent and selective inhibitors of PI4KB.

Figure 1. Structures of PI4KB inhibitors. Structural studies reported by us and Burke et al.16, 28 proved that both these types of compounds bind in a similar manner in the ATP binding side and their structures can be easily overlaid and, in principle, merged. This observation, together with subsequent docking studies, led to the design of this second generation of PI4KB inhibitor. These newly designed inhibitors combine features of both these aforementioned compounds into a single molecule. Originally, these


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compounds were designed based on simple fusion of 1 and our compound 3 (MI14) (Figure 1 and Figure 2), which exerted a unique selectivity profile in our previous studies28. Subsequently, we tried to optimize the sidechains on both combinatorial as well as rational bases. Our previous crystal structures revealed a lateral pocket, which is occupied by the amino sidechain of the molecule (Figure 2A, in red). Evidently, this pocket can be accommodated by significantly more bulky substituent than the (2-acetamidoethyl)amino side-chain employed in our original study.


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Figure 2. Design of new series of PI4KB inhibitors. A) Overlay of the crystal structure of PI4KB with 1 (yellow) and the docking model of the preferred conformation of 3 (magenta) unveils two sites of possible modifications. The red region can easily accommodate a large aromatic substituent. The blue region appears open for further optimization of the aromatic part of the molecule. B-D) Docking poses of derivatives 4, 5, and 8, respectively. The figure was prepared using PyMOL.


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Therefore, we investigated the effect of several related aromatic moieties that gave the best results in our docking experiments (Table 1 and Figure 2). Interestingly, our docking results suggest that compounds with various amino side-chains can exploit different sites of the binding canyon. In particular, the derivatives bearing the (2-acetamidoethyl)amino side-chain, e.g. 3, and compounds with pyridine attached at position 3 tend to form an interaction with Asn 600 whereas the analogs attached at position 4 bind to Tyr 385 on the opposite side of the cleft. Due to the significant effect on selectivity, the aromatic sidechain (Figure 2A, in blue) was more difficult to target by means of rational design and therefore this part of the molecule was elaborated by a combinatorial approach. Inhibition of phosphatidylinositol 4-kinases Initially we have focused our attention on the PI4KB potency and selectivity towards specific PI4K isoforms. The enzymatic activity was determined using a luminescent ADP-Glo kinase assay 37. Firstly, we determined the inhibitory activity of derivatives with the optimized “amine sidechain” of the molecule and it was encouraging to see similar trends in both the activity predicted by our docking experiments performed in AutoDock Vina software 38 and observed inhibition constants in enzymatic assay. The data obtained for derivatives with 3-(N,Ndimethylsulfamoyl)phenyl “aromatic side-chain” are summarized in the Table 1. This part of the study shows that the position of the nitrogen atom on the phenyl ring can significantly contribute to the efficiency of the inhibition, which significantly correlated with results obtained by the docking experiments. Thus the derivative connected to the central core at position 4 of the pyridine ring (compound 4) exerted significantly higher potency in comparison with its


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counterparts attached at position 2 or 3 – compounds 6 and 5, respectively. Subsequently, we also tried to optimize the substituent on the pyrimidine ring. Since there is significant space next to the pyrimidine core we gradually introduced methyl and ethyl group at the position 2 of the pyrimidine of the side chain, which seemed to fit the best into this cavity of the enzyme based on the docking experiments. Derivative 8 exerted significantly elevated inhibitory activity in comparison to 3 with retention of excellent selectivity towards PI4KB with respect to the most similar isoenzyme, PI4KA. Therefore, this motive was used in our subsequent studies focused on the “aromatic side-chain” of the molecule. Here, we focused primarily on the additional effect of introducing the substituent vicinal to the sulfonamide moiety and character of the sulfonamide moiety itself (both based on the 1/3 hybrid design) (Table 2, compounds 8 and 9). The installation of the sulfonamide moiety equivalent to 1 presented immediate success in enhancing the inhibitory activity and compound 10 exerted significant activity while still retaining the excellent selectivity. This series (Table 2, series A) gave a number of interesting compounds with single digit nanomolar inhibitory activity and showed that the substitution of the sulfonamide moiety has a significant impact on the selectivity towards PI4KB as well. The most active compounds of the series were derivatives 10, 17 and 22, which all exerted low nanomolar activity against PI4KB with outstanding selectivity. All these compounds shared the same sulfonamide substituent – a free hydroxy group. Series B of this study was mostly driven by our curiosity. Besides the derivatives with the optimized “amino side-chain”, we prepared derivatives with the original 2-acetamidoethylamino substituent as well in order to see how substantial is the impact of the more efficient but less


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flexible pyridine side-chain. To our surprise, some of the analogs within the series provided extremely high inhibitory activity against PI4KB with an outstanding PI4KB/PI4KA selectivity ratio with the most active compound being derivative 25, which exerts subnanomolar activity against PI4KB (IC50 = 0.98 nM) with PI4KA IC50 = 48 000 nM. Table 1. Correlation of the inhibitory potencies of the derivatives with modified “amino side chain” with the results obtained from the docking experiments using AutoDock Vina Software.

PI4KB IC50 (nM)a

AutoDock Vina Score

4

26.4

9.9

5

74.6

9.8

6

145.4

9.7

33.7

10.2

Compd

7

R

N


10


1 2 3 4 5 6 7 8 9 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

8

12.5

10.4

3

54.0

9.3

a

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Determined by three independent experiments.

Table 2.

Compd

Ar

Series

PI4KΒ

PI4KΑ

PI4K2A

IC50 (nM)a

% of control

% of control

(IC50 (nM))

(IC50 (nM))

8

A

12.5

ND

ND

9

A

444.1

102

101


11

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A

6.1

42 (13 400)

106

11

A

15.7

9 (553)

101

12

A

17.6

70

97

13

A

59.2

80

99

14

A

9.0

14 (1 128)

100

A

17.2

50

104

A

19.1

77

112

10 S

O

O O N H

S

OH

O

O O N H

N

15 S

O

O O N H

16

O


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17

A

6.2

58

109

18

A

8.6

58

105

19

A

27.3

74

106

20

A

41.5

61

115

21

A

56.0

84

114

22

A

5.3

59

104

23

B

22.0

63

112

S

O

O O N H

OH


13

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24

B

26.9

13 (660)

102

25

B

0.98

93 (48 000)

88

26

B

189.4

62

60

27

B

93.5

32 (4 860)

105

B

134.8

80

107

29

B

163.3

98

83

30

B

43.8

72

88

S O O

O N O

S

O

O O N H

N

28 S

O

O O N H

O


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31

B

19.9

77

93

32

B

2.4

72

104

33

B

14.9

43 (8 190)

85

34

B

105.8

80

108

35

B

7.23

34 (12 280)

77

36

B

146.9

44 (17 070)

73

B

54

(>100 000)

(>100 000)

S

O

O O N H

3

a

H N

Determined by three independent experiments.

Antiviral screening.


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The obtained compounds were also screened against selected +RNA viruses - human rhinovirus 1 (HRV1), Coxsackie virus B3 (CVB3), hepatitis C virus (HCV - 1b and 2a genotype) and Middle East respiratory syndrome coronavirus (MERS-CoV). For clarity we present only the results for the compounds that had an IC50 lower than 20 nM in the enzymatic assays (Table 2, additional data can be found in Supplementary Information Table 2 and 3). Antiviral screening unveiled a significant difference in activity of the A and B series. Generally, the members of the A series exerted extremely high activity against HRV1, which in most cases reached the detection limit of the assay (EC50 lower than 100 nM for all presented compounds). Furthermore, most of the members of this series exerted significant inhibitory effect towards CVB3 and HCV 1b. It is noteworthy, that derivatives 10, 17, and 22 which were the most active members of this series in the enzymatic assay also exerted steady nanomolar activities in these three antiviral assays. Notably, the effect on HCV 2a is rather negligible, which is consistent with genotype dependent insensitivity of HCV to knockdown of PI4KB 39. In this respect, the inactivity can be regarded as proof of selectivity towards PI4KB, as HCV 2a is significantly more susceptible to inhibition of PI4KA. In contrast, the compounds from series B exerted significantly lower nominal activity in cellbased assays. However, their toxicities were shifted in the similar fashion suggesting that the compounds suffered from limited permeation through the cell membranes and therefore cannot reach the side of the action. However, compound 25, the derivative exerting the highest activity in the enzymatic assay, was potent enough to inhibit the sensitive viruses at the submicromolar or low micromolar level.


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None of the compounds exerted significant antiviral effect against MERS-CoV, which seems to be in contrast with the reported implication of PI4KB in the replication of pathogenic coronaviruses such as Severe acute respiratory syndrome coronavirus (SARS-CoV)6. Table 3. Compd

Series

HRV1

CVB3

HCV 1b

HCV 2a

HeLa

EC50 (nM)

EC50 (nM)

EC50 (nM)

EC50 (nM)

CC50 (nM)

8

A

62

1100

393

8689

15354

10

A

75

58

29

14402

18333

11

A

69

56

9196

26540

5757

12

A

Rational Design of Novel Highly Potent and Selective

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