Selective and potent small-molecule inhibitors of PI3Ks.

Future

Review

Medicinal Chemistry

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Selective and potent small-molecule inhibitors of PI3Ks

Class I PI3Ks are composed of four catalytic subunit variants (p110α, p110β, p110δ and p110γ). The PI3K pathway is among the most frequently activated pathways in many diseases, and has emerged as an attractive target for drug development, in particular for the treatment of many human cancers including breast, prostate, ovarian, gastric, colon and hepatocellular cancers. One of the challenges in the discovery of drugs that target kinases is designing small-molecule inhibitors that are sufficiently selective to minimize off-target activity and reduce the risk of potential toxicity. This review explores the current landscape of PI3K-selective inhibitor development and highlights recent advances in achieving selectivity for PI3Ks over other protein kinases, with an emphasis on available structural information.

PI3Ks are a family of lipid kinases involved in phosphorylating the 3´-hydroxyl group of phosphatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-tris phosphate (PIP3) (Figure 1) [1–3] . Members of the PI3K family are categorized as class I, II and III based on their structure and regulatory mechanisms [1–3] . Class I PI3Ks are composed of four catalytic subunit variants (p110α, p110β, p110δ and p110γ) and an SH2-containing 85 kDa regulatory subunit [2] . PIP3 regulates a remarkable array of cellular functions, including cell proliferation, differentiation, motility, survival and metabolism [4] . In response to growth factors, PI3Kα, PI3Kβ and PI3Kδ can be activated by receptor protein TKs, whereas PI3Kβ and PI3Kγ are regulated by G protein βγ subunits (Gβγ) [1,5] . Activation of downstream signaling proteins, including Akt and mTOR, by elevated PIP3 levels may be a significant contributor to the development of cancer, inflammation, autoimmune conditions and cardiovascular diseases [6] . Hyperactivation or mutations in the PIK3A gene encoding the catalytic subunit p110α have been observed in many human cancers, including breast, prostate, ovarian, gastric, colon and hepatocellular cancers [6–8] . In addition, negative regulation of phosphatase and

10.4155/FMC.14.28 © 2014 Future Science Ltd

Yujeong Jeong1,2, Daeil Kwon1 & Sungwoo Hong*,1,2 Center for Catalytic Hydrocarbon Functionalizations, Institute of Basic Science, Daejeon 305-701, Korea 2 Department of Chemistry, Korea Advanced Institute of Science & Technology, Daejeon 305-701, Korea *Author for correspondence: Tel.: +82 42 350 2811 Fax: +82 42 350 2812 [email protected] 1

tensin homolog, encoding the tumor suppressor, is a frequent occurrence in human tumor types resulting from activation of the PI3K/ Akt pathway (Figure 2) [6,9] . Furthermore, recent studies have reported that the PI3K/ Akt pathway plays an important role in the expression of HIF-1α and VEGF, which are ultimately involved in promoting the angiogenesis process [10] . In this context, inhibition of the PI3K/Akt pathway could lead to clinically useful drugs, particularly for the treatment of many types of cancer. PI3Kβ has been investigated as a therapeutic target for the treatment of thrombosis, and β isoformselective inhibitors have been reported [11,12] . Both PI3Kδ and PI3Kγ have been identified as attractive targets for chronic inflammation, and respiratory and immune disorders [13–15] . Overall, accumulating cellular and clinical evidence indicates that inhibition of PI3Ks is an appealing approach for the treatment of many types of human diseases. A number of small-molecule PI3K inhibitors have been identified [1] . These compounds are used routinely in experimental biology to elucidate cellular signaling pathways, and some are currently under development as promising therapeutic agents [16–18] . Two early inhibitors in particular have been

Future Med. Chem. (2014) 6(7), 737–756

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ISSN 1756-8919

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Review Jeong, Kwon & Hong

O

O O

O O O O HO P O 1'

(CH2)16CH3 O O

PI3K

5' O–PO 23 HO 23' 4' O–PO3 H3C(H2C)4

(CH2)16CH3 O

O O O P HO O 1' O3P–O

2-

Phosphatidylinositol 4,5-bisphosphate

3'

O

5' O–PO 23 4' O–PO 23 H3C(H2C)4

Phosphatidylinositol 3,4,5-trisphosphate

Figure 1. PI3K phosphorylation of phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol-3,4,5-trisphosphate.

widely used to elucidate the functional role of PI3Ks (Figure 3) [1] . Wortmannin, a natural product isolated from Penicillium wortmannii, was the first PI3K inhibitor identified [19,20] . Its high potency (IC50 = 4.2 nM) is attributable to the irreversible interaction of the inhibitor with PI3Ks. LY294002 (IC50 = 1.4 μM), the first synthetic PI3K inhibitor developed, is also widely used experimentally [21] . Many additional PI3K inhibitors have been derived based on the structural features of these prototypical inhibitors. However, these inhibitors

are not selective over other protein kinases and have additional drawbacks, including poor physicochemical properties and high toxicity in animals, limiting their potential therapeutic use [22,23] . Since the discovery of the first-generation PI3K inhibitors, remarkable improvements have been made, yielding different types of PI3K inhibitors with higher selectivity, nano- to pico-molar potency, and new allosteric binding modes [1,17] . However, achieving inhibitory selectivity for PI3K by rational design remains a significant challenge

Growth factor

PIP2 P

PTEN

P

PIP3 P

P

P

P

PH P

PI3K RTK

P

p85

P MDM2

p53

Cell cycle arrest Apoptosis DNA repair

Akt/PKB P

p110

P NF-κB

P FKHR

P

P BAD

GSK3β

P mTOR

Growth translation

Apoptosis

Cell cycle Glucose metabolism

Figure 2. The PI3K pathway. PH: Pleckstrin homology; PIP2: Phosphatidylinositol 4,5-bisphosphate; PIP3 : Phosphatidylinositol-3,4,5-trisphosphate; PTEN: Phosphatase and tensin homolog.

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Future Med. Chem. (2014) 6(7)

future science group

Selective & potent small-molecule inhibitors of PI3Ks

O O

O

O

O

O

O

N

H

O

O

O O Wortmannin

LY294002

Figure 3. Widely used first-generation PI3K inhibitors.

in the development of new inhibitors as drug candidates or as tools for chemical biology research. In this review, we consider the recent development of PI3K-selective small-molecule inhibitors, with an emphasis on available structural information, where available. These discussions address structural approaches for improving selectivity and binding modes, and highlight newly emerging features of PI3K-selective inhibitors. In addition, representative small molecules with hyperselectivity toward certain isoforms will be presented. Multiple kinase inhibi-

Review

tors, whether for biological or clinical applications, are beyond the scope of this review and will not be a subject of discussion. These inhibitors, which target multiple kinases and often owe their efficacy to the inhibition of one or more ‘off-target’ kinases, have been covered in some excellent recent reviews that present a basic overview of all types of PI3K inhibitors. This review begins with the structural lessons learned from previously developed inhibitors and our understanding of the molecular basis for improving selectivity for PI3K relative to other kinases. Structural approach for obtaining PI3K selectivity over other kinases Human cells contain more than 500 different kinases involved in transferring a phosphate group from ATP to the hydroxyl group of substrates [24] . These kinases are involved in regulating all aspects of cellular processes, including cell growth, metabolism and cell division. Thus, one of the challenges in the discovery of drugs that target kinases is designing small-molecule inhibitors that are efficacious in vivo, yet have selectiv-

Lys833

Glu880

Tyr867

Met804 Ser806

Val882

Asn951

Asp950

Figure 4. Co-crystal structures of PI3Kγ in complex with ATP, PDB ID: 1E8X, 2.2 Å.


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O

Hi

ng

er eg io

n

OH

NH

Glu880 H N

O

O

H N

Val882 O

H

N

N

H Adenine region N

Unique hydrophobicN pocket

N

O O

HO Tyr867

OH

OH

O

P O

O

O O +

P

O O P O O

NH3

O O Affinity pocket

Asp841

Lys833

Figure 5. Features of ATP binding site of PI3Ks.

Table 1. Summary of the properties of PI3K-selective inhibitors. Compound structure CH3 N O

O

Compound Activity

Selectivity profiles

E5E2

PI3Kγ IC50 = 39 nM

Assayed against five human protein kinase (MST1, PAK1, BRAFwt, and BRAF V600E, GSK3α and PIM1

HSW 178

PI3Kα Kd = 47 nM

Panel of 98 human protein kinase at 10 μM concentration

AMG 511

PI3Kα Ki = 9 nM PI3Kβ Ki = 5 nM PI3Kγ Ki = 4 nM PI3Kδ Ki = 2 nM

Panel of 372 protein kinase at 1 μM concentration

GNE-490

PI3Kα IC50 = 9 nM PI3Kβ IC50 = 5 nM PI3Kγ IC50 = 4 nM PI3Kδ IC50 = 2 nM

Screening in a 142 kinase panel at 1 μM concentration

HO F

N O

C

Ru

N

O NH

HO HO O S HN O N

O N

N

N N

F O

N

N N

N

N H N

N NH2

N

O N S OH

N N

N N

740

NH2

O S O




ity profiles that are sufficiently high to minimize offtarget activity and reduce the risk of potential toxicity. To achieve this goal, researchers in the drug-discovery field have been developing innovative strategies, including structural, computational, informatics and biophysical approaches. Many useful co-crystal structures of small molecular inhibitor-kinase complexes are available [25] . These structures enable novel compounds to be designed by H N

O

O

H N

O

N

N O

O

C

Ru

CH3 N O

O H

N

implanting some portion of a known inhibitor onto other chemical frameworks. For kinase activation, ATP first binds through its adenine moiety, making stable hydrogen bonds with a hinge region in a deep hydrophobic pocket of the kinase [26–28] . The ribose moiety and phosphate groups of ATP then interact hydrophobically with the ATP binding pocket in the active site of the kinase. A majority of PI3K inhibitors are ATPcompetitive and target the ATP-binding site of PI3Ks

O

HO

Review

O F

HO N

N

O

C

Ru

N

CH3 N O

HO N

O

O

NH

C

F Ru

N

O NH

O HO

NH Staurosporine

HO

HO

E5

DW12

E5E2

HO

Phe961

Tyr667

Val882 Asp964

Glu880

Ala805 Lys833

4.5 120

1.0 0.5 0.0

P13Kγ

GSK3β

40 20 0 MST1

1.5

60

PIM1

2.0

80

GSK3α

2.5

100

PAK1

IC50 (µM)

3.0

BRAFV60 0E

Remaining kinase activity

3.5

BRAF

DW12 E5

PI3Kγ

4.0

Figure 6. Deveopment of E5E2. (A) Structure of organoruthenium scaffold compounds. (B) Co-crystal structures of PI3Kγ in complex with E5E2, PDB ID: 3CST, 3.20 Å. (C) Comparison of inhibitory activity of DW12 and E5 over PI3Kγ and GSK3β [32] . (D) Selectivity profile of E5E2 over other kinases at 4 μM [32] .



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Review Jeong, Kwon & Hong by mimicking the structure of ATP [28,29] . Therefore, an efficient, general approach for attaining PI3K selectivity involves exploitation of structural differences in the periphery of the ATP-binding site between PI3Ks and other kinases. Improving selectivity through this approach depends on properly modifying the structure of inhibitors, which, in turn, requires an understanding of the key and unique interactions between PI3K kinase and inhibitors. An x-ray co-crystal structure of the PI3Kγ–ATP complex is available, and shows the binding mode of the adenine moiety adjacent to the loop linking the C- and N-terminal domains of PI3Kγ (Figure 4) [30] . The N1 of the adenine moiety in ATP acts as a hydrogen bond acceptor, forming a strong hydrogen bond with the backbone amide nitrogen of Val882. The NH2 group of the adenine moiety is a hydrogen bond donor, forming another hydrogen bond with the backbone amide carbonyl of Glu880. Note also that the side chain NH3 group of catalytic Lys833 interacts with the α-phosphate of ATP [31] . Many of the key interactions between ATP and the ATP-binding site of PI3Ks are essentially maintained in the binding mode of ATPcompetitive inhibitors. Despite the high degree of similarity among kinases, there are non-conserved residues in the active sites of kinases, and these differences may allow selectivity toward one kinase over another. The structural insights gained from the development of PI3K

inhibitors have highlighted the importance of exploiting divergent binding pockets, prompting a considerable effort to understand the binding modes between PI3Ks and small-molecule inhibitors. The emergence of several chemotypes of PI3K-selective inhibitors has made these studies more feasible and has guided the design of next-generation PI3K-selective inhibitors. Unique pocket in the hinge region of PI3K

To improve the selectivity of inhibitors for PI3K relative to other kinases, researchers compared the ATP binding site of PI3Ks with that of other protein kinases to identify key differences. Intriguingly, structural studies and sequence alignments of PI3K revealed that a non-conserved residue in the ‘gatekeeper’ region appeared to form a unique hydrophobic pocket in PI3Ks [32–35] . Thus, the gatekeeper residue (Tyr867 in PI3Kγ) has been considered one of the critical determinants for attaining high selectivity for PI3Ks. In the binding model of the PI3Kγ–ATP complex, the side of the adenine-binding region is oriented toward the small, unique pocket of PI3Kγ, consisting of Tyr867 and Glu880 (Tyr836 and Glu849 in PI3Kα). Because this unique pocket is not present in other kinases, inhibitors highly selective for PI3Ks can be generated by exploiting differences at the periphery of the ATPbinding site. In many cases, a small group (e.g., methyl,

Lys802

O S HN O

N

N

O N

N N X

HSW 104 X = H HSW 178 X = Me

Asp810 Tyr836

Val851

X=H

100 50 0

X = Me

100 50

PIK3C2B PIK3CA PIK3CG

CDK11 CDK2

0

Figure 7. Selectivity profiles of HSW 178. (A) Structure of PI3K inhibitor based on imiazo[1,2-a]pyridine core structure. (B) Calculated binding modes of HSW 178 in the ATP-binding sites of PI3Kα. (C) KINOMEscan® selectivity profile of HSW 178 tested at 10 μM in a high-throughput binding assay (Ambit Bioscience; CA, USA) [35] .

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Review

Glu880 Glu81

Val882

Leu83

Glu849

Glu81

Leu83 Val851

Figure 8. Calculated binding modes of HSW 178 in the ATP-binding sites. (A) PI3Kα and (B) CDK2.

chlorine or methoxy) is introduced in a position facing the unique pocket in the gatekeeper region (Figure 5) [33] . This method can provide a selectivity filter for inhibitors that bind PI3Ks, with the selectivity being rationalized by the interaction with the unique pocket of PI3Ks. Moreover, this approach for attaining selectivity can be generalized and applied to other scaffolds, provided the appropriate group(s) on inhibitors that allow interaction with the unique pocket space of PI3K, depicted in Figure 5, are introduced (Table 1) . PI3K-selective inhibitors E5E2: Organoruthenium scaffold

Staurosporine, a natural product of the bacterium Streptomyces staurosporeus, is a potent, but nonselective, ATP-competitive kinase inhibitor (PI3Kα: IC50 = 9 μM) [30] . Many organoruthenium derivatives have been designed based on staurosporine for the purpose of identifying GSK3 and PIM1 inhibitors [32,36,37] . During this design process, undertaken by the Wistar Institute, DW12, containing a hydroxyl group introduced into the indole moiety,


was discovered as a potent PI3K inhibitor (PI3Kα: IC50 = 0.75 μM). This modified derivative (DW12) eventually guided the design and identification of E5E2 (Figure 6) . Intriguingly, it was found that introduction of a methyl group at the N-atom of the maleimide moiety in E5E2 almost completely eliminated the inhibitory activity toward GSK3 and PIM1. The co-crystal structure of the PI3Kγ–E5E2 complex indicated that the space in the unique pocket of PI3K, composed of Tyr 867 and Glu880, could be filled in PI3Ks (Figure 6A). The methyl group on maleimide in E5E2 is directed toward the unique PI3K pocket near the gatekeeper region in the adenine pocket of PI3Kγ. On the other hand, a large amino acid, such as Phe or Ile, at this position in other protein kinases would cause unfavorable steric hindrance with the methyl group on the maleimide in E5E2 (Figure 6A) . The organoruthenium compound E5E2 exhibited a 100-fold improvement in selectivity relative to the six protein kinases tested: MST1, PAK1, BRAFwt, BRAFv600e, GSK3α and PIM1 (Figure 6C) .


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F O

N N

N N

N H N

N N

Lys833

O S O

Asp964 Ala805

NH2

Met804

AMG 511 O

N

N N

N H N

N N

Tyr867

N

O N S O

HO

N H

N N

X

NH2

Glu880 N

N

2X=H 3 X = Me

1

N N H

Lys802 Phe961 Met953 Val882 Ala885

Figure 9. Chemical and co-crystal structures of AMG 511. (A) Structure of AMG 511 and related 1,3,5-triazine series. (B) Co-crystal structures of PI3Kγ in complex with AMG 511, PDB ID: 4FLH, 2.60 Å.

HSW 178: imidazo[1,2-a]pyridine scaffold

The approach for improving selectivity by occupying the unique pocket of PI3Ks was independently applied by engineering the imidazopyridine scaffold. HSW 178, an imidazopyridine derivative developed by Hong et al., has been shown to be a potent and selective inhibitor of PI3K [10] (Figure 7) . Although a previously identified compound, HSW 104, exhibited potent inhibitory activity toward PI3K, it displayed only moderate selectivity relative to other kinases. An evaluation of HSW 104 against a panel of 98 human kinases tested at a concentration of 10 μM (KINOMEscan®; Ambit Biosciences, CA, USA) showed that HSW 104 exhibited relatively high binding affinities (percent of control [POC] value < 10) to more than five protein kinases (CDK11, CDK7, CDK2, CSNK1G2 and mTOR) in addition to PI3Ks. Lower numbers of POC indicate stronger hits. HSW 104 was then modified using a knowledge-based design approach to obtain high selectivity for PI3K relative to other protein kinases [35] . The addition of a small group (e.g., F, 450 2

400

3

AMG 511

350 Ki (nM)

300 250 200 150 100 50 0 PI3Kα

PI3Kβ

PI3Kγ

PI3Kδ

BRAF

Figure 10. Comparison of selectivity profiles of 2, 3 and AMG 511.

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Me, Cl) at the C8 position of the imidazopyridine core resulted in remarkably enhanced selectivity over other protein kinases (all POCs > 60). A docking modeling study showed that the methyl group appears to be positioned toward the small, unique pocket of PI3K generated by Tyr836 and Glu849. A subsequent structural analysis of PI3K and CDK2 showed that the locations of the glutamate residues in the adenine-binding region (Glu849 in PI3Kα; Glu81 in CDK2) are different. In PI3Kα, the backbone carbonyl group of Glu849 is oriented above the imidazopyridine core, and a small pocket is available for substituent modifications at this position (Figure 8) . However, the backbone carbonyl group of Glu81 is oriented toward the imidazopyridine core in CDK2 (Figure 8B) . This difference in location of the glutamate may account for the high selectivity relative to other kinases, providing useful insight into the design of new PI3K-selective inhibitors. AMG 511: 4-amino-6-methyl-1,3,5-triazine sulfonamide scaffold

AMG 511 is a potent, orally available PI3K-selective inhibitor discovered by Amgen Inc., CA, USA [38] An x-ray co-crystal structure of PI3Kγ–AMG 511 complex revealed that AMG 511 has dual hydrogen bond interactions between the aminotriazine and Val882 in the hinge region. The nitrogen atom of the methoxypyridine appears to make favorable interactions with a water molecule located between Tyr867 and Asp841 in the region of the affinity pocket (Figure 9) . In addition, the oxygen atom of the methoxy group and the 3-fluoro atom of pyridine form hydrogen bonds with Lys833 in the affinity pocket. The oxygen atoms of methylsulfonamide make two hydrogen bonds: one to the amidic backbone N-H of Ala805 and one to



O

O

N S

N N

O

N S OH

OH

N S

N N

N N

4

Review

N N

OH

N

NH2

N

GNE-493

NH2

GNE-490

Figure 11. Development of GNE-490.


Applying a similar approach, scientists at Pfizer (NY, USA) engineered the pteridinone moiety to generate a series of PI3K-selective inhibitors [33] . The original lead compound 5 was a nonselective PI3K inhibitor. An induced-fit docking model predicted that the C-4 position of 5 is oriented towards the unique PI3K pocket. This methyl group in 6 occupies the critical space of the PI3K pocket, and this compound showed outstanding selectivity toward PI3K relative to other kinases in screens against a panel of 60 kinases. Interestingly, a slightly larger methoxy group is not as comfortably accommodated in the unique pocket of PI3Ks, 600 GNE-493

500

GNE-490

400 300 200 100

SYK

MLK1

AuroraA

mTOR

PI3Kγ

PI3Kδ

0 PI3Kβ

GNE-490 (Genentech; CA, USA) is a selective PI3K inhibitor designed based on a PI3Kγ-ligand co-crystal structure with GDC-0941 [41,42] . PI3K selectivity was improved by applying an approach similar to that described above, utilizing the unique pocket near the gatekeeping region. Introduction of a methyl group on the pyrimidine moiety at GNE-493 generated GNE-490, which was more selective than GNE-493 (Figure 11) . An analysis of the co-crystal structure of GNE-490 binding to PI3Kγ showed that the oxygen atom of the morpholine group appears to form a hydrogen bond with the amidic backbone Val882 at the hinge region. The NH2 group of the aminopyrimidine core makes another hydrogen bond with a water molecule bridging Tyr867 and Asp841 [21,43] . Again, the introduction of a methyl group at the aminopyrimidine moiety, which was directed toward the unique PI3K pocket (Tyr867, Asp841 and Val882), led to improved selectivity over other kinases. Introduction of this methyl substituent at the aminopyrimi-

Pteridinone scaffold

PI3Kα

GNE-490: (thienopyrimidin-2-yl) aminopyrimidine scaffold

dine also effectively decreased the inhibitory activity toward mTOR (GNE-490, IC50 = 740 nM; GNE493, IC50 = 32 nM). Both GNE-490 and GNE-493 were screened against a panel of 142 kinases (Invitrogen’s SelectScreen® service) at a concentration of 1 μM (Figure 12) . GNE-493 and GNE-490 exhibited meaningful off-target activity (>50% inhibition) against only a few kinases, including Aurora A, MLK1 and SYK (Figure 12) . Both GNE-490 and GNE-493 exhibited excellent efficacy in mouse xenograft models (MCF7.1 breast cancer, PC3 prostate cancer) and warrant further investigation as potential anticancer agents.

IC50 (nM)

the side chain of Lys802. Again, the introduction of a methyl group on the aminotriazine core resulted in an improved PI3K-selectivity profile, and the methyl group appeared to be positioned toward the gatekeeper region – the unique pocket generated by Tyr867, Asp841 and Val882. The introduction of a methyl group to the aminotriazine core improved both PI3Kα enzymatic and cellular activities by 20-fold compared with parent compound 2 [39] . Tests of AMG 511 at a concentration of 1 μM against a panel of 372 protein kinases in a highthroughput binding assay (KINOMEscan) showed that AMG 511 exhibited high PI3K-selectivity profiles relative to other kinases (Figure 10) [40] . Moreover, AMG 511 exhibited good pharmacokinetic properties, with low clearance (0.4 l/h/kg), excellent oral bioavailability (F = 60%), and high oral delivery (area under the curve = 0.5 μM.h). AMG 511 was further evaluated for cellular and in vivo efficacy, and was shown to efficiently inhibit tumor growth in a mouse U87 MG glioblastoma xenograft model (ED50 = 0.6 mg/kg).

Figure 12. Comparison of selectivity profiles of GNE-490 and GNE-493 [41 ].


745


O N H2N

O

N N N

N

O

H2N

N

N N

N

O

6

5

PI3Kα Ki = 38 nM mTOR Ki = 5 nM Selective PI3K/mTOR inhibitor

Nonselective PI3K/mTOR inhibitors

O

O

N H2N

N

N

O

N

N H2N

N

N

O

O

8

7 PI3Kα Ki = 840 nM mTOR Ki > 4000 nM

PI3K Ki = 27 nM mTOR Ki = 974 nM

Figure 13. Development of pteridinone based PI3K-selective inhibitors.

and adding this group reduced the affinity for PI3Kα (7). Extending these results, an iterative process of optimization yielded the more potent and selective PI3K inhibitor 8 (Figure 13) . PI3K isoform-selective inhibitors One of the biggest challenges in PI3K research is attaining an optimal pattern of selectivity among the four structurally similar individual PI3K isoforms (Table 2) . Achieving high selectivity of one PI3K

isoform relative to another remains a significantly more difficult problem than creating inhibitors that are selective for PI3Ks over other kinase families, although resolution of the x-ray crystal structures of all isoforms (p110α, p110β, p110γ and p110δ) has contributed to our understanding of potentially useful structural differences (Figure 14) . Co-crystal structures of inhibitor-bound γ and δ isoform complexes have been reported, facilitating the development of isoform-selective inhibitors. However, x-ray structures

Table 2. Summary of the properties of isoform selective PI3K inhibitors. Compound structure

O O N N

HN N

N

N

Compound

Activity

Selectivity profiles

CNX-1351 PI3Kα selective inhibitor

PI3Kα IC50 = 6.8 nM PI3Kβ IC50 = 166.0 nM PI3Kγ IC50 = 240.3 nM PI3Kδ IC50 = 3020.0 nM

A panel of 70 kinases at 1 μM concentration

9 PI3Kβ selective inhibitor

PI3Kα IC50 = 2500 nM PI3Kβ IC50 = 0.6 nM PI3Kγ IC50 = 20 nM PI3Kδ IC50 = 790 nM

A panel of 290 kinases at 10 μM concentration

HSW 243 PI3Kβ selective inhibitor

PI3Kβ Kd = 0.23 μM PI3Kα POC = 92 PI3Kβ POC = 1.0 PI3Kγ POC = 25 PI3Kδ POC = 4.2

AS-252424 PI3Kγ selective inhibitor

PI3Kα IC50 = 940 ± 150 nM PI3Kβ IC50 = 20,000 nM PI3Kγ IC50 = 30 ± 10 nM PI3Kδ IC50 = 20,000 nM

80 protein kinases were assayed with 10 μM

S N O

O

O

N

S

N

N

N

CF3

O O S HN

N

N N

NH2

O S F

O

NH O

OH

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of PI3Kα- and PI3Kβ-inhibitor complexes have not yet been reported. Therefore, homology modeling of PI3Kα- and PI3Kβ-inhibitor complex has been frequently used for the design of α and β isoform-specific inhibitors [44,45] .

P13Kα: Arg852 P13Kβ: Ser855 P13Kδ: Leu829 P13Kγ: Lys883

CNX-1351: PI3Kα-selective inhibitor

CNX-1351 (Celgene Avilomics; MA, USA), which selectively inhibits the PI3Kα isoform, was identified by applying a rational drug-design approach [16] . Interestingly, biochemical and structural studies indicated that CNX-1351 is a covalent inhibitor of PI3Kα, owing to its electrophilic and linker functional groups. A docking model of GDC-0941 onto the structure of PI3Kα was constructed based on the binding mode of GDC-0941 to PI3Kγ, which had been determined by x-ray crystallography (Figure 15) [16,46] . A comparison of the PI3Kα model and the x-ray complex structure of GDC-0941 bound to PI3Kγ revealed that the Cys862 residue, which is unique to PI3Kα, plays a key role in attaining isoform selectivity. Cys862 in PI3Kα acts as a strong nucleophilic center, forming a covalent bond with CNX-1351. An examination of the biological activities of CNX-1351 showed that CNX-1351 exhibited high biochemical potency and selectivity for PI3Kα (20–400 greater than that for PI3K-β, -γ and -δ). CNX-1351 is a powerful tool for studying PI3K related biology because

Review

P13Kα: Arg770 P13Kβ: Ser777 P13Kδ: Leu750 P13Kγ: Lys802

P13Kα: His855 P13Kβ: Glu858 P13Kδ: Asp832 P13Kγ: Thr886

P13Kα: Ser773 P13Kβ: Asp780 P13Kδ: Asp753 P13Kγ: Ala805

P13Kα: Gln859 P13Kβ: Asp862 P13Kδ: Asn836 P13Kγ: Lys890

Figure 14. General features and nonconserved residues of PI3K isoforms.

of its α-isoform selectivity and prolonged inhibition resulting from covalent bond formation. These desirable features of CNX-1351 will allow continuing investigations of the role of PI3Kα in a variety of tumor models. However, CNX-1351 exhibits disappointing pharmacokinetic properties; thus, further optimization of the compound is required to improve its oral bioavailability while maintaining its potency and selectivity.

O O N N

HN N

N

N S

N CNX-1351 O

3500

1500

SPHK2

SPHK1

PI4Kβ

PI4Kα

PI3KC3

PI3KC2A

PI3Kδ

1000 500 0 PI3Kγ

Asp810

2000

PI3Kα

Cys862

3000 2500

PI3Kβ

Tyr836

IC50 (nM)

Val851

Figure 15. Structure and selectivity profiles of CNX-1351. (A) Chemical structure of CNX-1351. (B) Co-crystal structures of PI3Kγ in complex with CNX-1351, PDB ID: 3ZIM, 2.85 Å. (C) Selectivity profiles of CNX-1351 [16] .



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Review Jeong, Kwon & Hong Thiazolopyrimidinone series: PI3Kβ-selective inhibitor

was reported that a pyridyl sulfonamide core structure could increase potency by occupying the back pocket of PI3K (Figure 17) [49–51] . To enhance the selectivity of PI3Kβ over PI3Kα, a variety of heterocyclic groups were screened while leaving the a N-(pyridine-3-yl)benzenesulfonamide moiety fixed. Results of a high-throughput binding assay showed that the combination of a 2-aminopyrimidine moiety with pyridylbenzenesulfonamide was optimum for both PI3Kβ potency and selectivity. To understand the enhanced β-isoform selectivity, interactions between the inhibitor and the unique PI3Kβ pocket generated by salt bridge residues (Lys782– Asp923) were further investigated. The calculated binding mode of benzenesulfonamide revealed that the phenyl group of inhibitors occupies the critical space of the unique PI3Kβ pocket (Lys782–Asp923 and Asp862). Even bigger groups, such as a naphthyl group, are well tolerated in PI3Kβ, indicating a favorable hydrophobic interaction within the unique PI3Kβ pocket.

The PI3Kβ isoform has been implicated in arterial thrombosis and platelet aggregation [11] . A thiazolopyrimidinone-based series of selective PI3Kβ inhibitors was developed based on derivatives of the previously reported PI3Kβ inhibitors, imidazo[1,2-a]-pyrimidin5(1H)-one and 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)one (9) [12,47,48] . Three key functional groups that contribute to binding interactions include a carbonyl group that interacts with Tyr839 in the back pocket, a morpholine that functions as a hinge binder and a lipophilic group that binds a selectivity pocket (Met779 and Trp787) (Figure 16) . Thiazolopyrimidinone derivatives containing an N1-benzyl substituent satisfy the above three conditions. Furthermore, Met779, Lys805, Asp923 and Asp937, which are located near the 2-position of the thiazolopyrimidinone, provide additional lipophilic or hydrogen bonding interactions that could increase the potency for PI3Kβ. One example is a compound containing a methyl group at the 2-position, which shows higher potency and selectivity. This compound potently inhibits PI3Kβ (IC50 = 0.6 nM) and is highly selective relative to other PI3K isoforms, for which its IC50 values are 30–4000 times larger.

Furan-2-ylmethylene thiazolidinediones: PI3Kγ-selective inhibitor

PI3Kγ is known to play an important role in innate immune responses and mast cell degranulation [13] . Therefore, potent and selective inhibitors of PI3Kγ are potential drug candidates for the treatment of autoimmune and inflammatory diseases. AS-252424 (Merck Serono; Darmstadt, Germany), which contains a furan-2-ylmethylene thiazolidinedione moi-

Aminopyrimidine series: PI3Kβ-selective inhibitor

A pharmacophore-directed design was utilized to identify a new template for PI3Kβ-selective inhibitors [49] . It

O N

N

N

CF3

9

1000 100 10

DNA-PK

Val854 Val853

FRAP1

0.1

VPS34

1

PIK3C2B

Lys802

10000

PI3Kδ

Trp787

PI3Kγ

Lys782

IC50 (nM)

Met779

PI3Kβ

N

PI3Kα

O

S

Figure 16. Structure and selectivity profiles of compound 9. (A) Chemical structure of compound 9. (B) Binding mode and structure of 9 in the PI3Kβ homology model. (C) Selectivity profile of 9 [47] .

748




ety, displays high potency and γ-isoform selectivity (Figure 18) . This key pharmacophore was identified by investigations of the x-ray crystal structure of the PI3Kγ–inhibitor complex and reiterative docking studies [13] . The acidic NH-group of the thiazolidinedione and a phenol hydroxyl group play important roles in improving the potency and PI3Kγ-selectivity of this inhibitor. The 4-fluoro substituent on the phenyl group of AS-252424, in particular, contributes to the increased selectivity ratio between the γ-isoform and other isoforms. Selectivity profiles revealed the high potency of AS-252424 for PI3Kγ (IC50 = 30 nM) and much larger IC50 value for α-, β- and δ-isoforms. Moreover, these enzymatic-level potencies and selectivities of the compound were reproduced in cellular assays [13] . In vivo, treatment of mice with AS-252424 produced results similar to those observed in a PI3Kγ-deficient murine peritonitis model [13] . Advanced PI3K-selective inhibitors in clinical trials BEZ235 is an imidazoquinoline derivative and a dual inhibitor of PI3K–mTOR (IC50 : PI3Kα = 4 nM; PI3Kβ = 75 nM; PI3Kδ = 7 nM; PI3Kγ = 5 nM; mTOR = 21 nM) [52] . Since the first clinical trial of BEZ235, different types of PI3K inhibitors have entered clinical trials (Table 3), some of which have

O O S HN

N

F

O OH AS-252424

N N

NH2

HSW 248

Asp862

Lys782 Met779

Asp923

Lys805 Val353 Val854 Tyr839

Figure 17. Structure and binding mode of HSW 248. (A) PI3Kβ-selective inhibitor, HSW 248. (B) Calculated binding mode of benzenesulfonamide with PI3Kβ homology model.

O S

Review

Lys833

NH

Asp964 O

Ile963 Asp841 Tyr867

Val882

Gly868

Abl AMPK Arg Aurora-A Axl Blk Bmx CaMlKll CaMKIV CDK1/cydinB CDK2/cydinA CDK2/cydinE CDK3/cydinE CDK5/p35 CDK6/cydinD3 CDK7/cydinH/MAT1 CHK1 CHK2 CK1 CK2 c-RAF CSK cSRC Fes FGFR3 Flt3 Fyn GSK3α GSK3β IGF-1R IKKα IKKβ IR JNK1α1 JNK2α2 JNK3 Lck Lyn MAPK1 MAPKAP-2 MEK1 MKK4 MKK6 MKK7β MSK1 P70S6K PAK2 PDGFRα PDGFRβ PDK1 P13Kγ PKA PKBα PKBβ PKBγ PKCα PKCβll PKCγ PKCδ PKCε PKCη PKC1 PKCµ PKCq PKD2 PRAK PRK2 ROCK-ll Rsk1 Rsk2 Rsk3 SAPK2a SAPK2b SAPK3 SAPK4 SGK Syk TrkB Yes ZAP-70

200 180 160 140 120 100 80 60 40 20 0

Figure 18. Structure and selectivity profiles of AS-252424. (A) PI3Kγ-selective inhibitor, AS-252424. (B) Calculated binding mode of AS-252424 in PI3Kγ. (C) Selectivity profiles of AS-252424 against 80 kinases tested at 10 μM [13] .



749

Review Jeong, Kwon & Hong displayed promising activity against a wide range of tumor types (advanced solid tumors, breast cancer, NSCLC, non-Hodgkin’s lymphoma [NHL], chronic lymphocytic leukemia [CLL]). Recently, a number of PI3K- or isoform-selective inhibitors have progressed to clinical trials. Because of their low toxicity, isoformselective inhibitors can potentially be used in combination therapy; this is of particular importance because kinase inhibitors are primarily cytostatic agents. GDC-0941: thieno[3,2-d]pyrimidine scaffold, pan-PI3K-selective inhibitor

The thieno[3,2-d]pyrimidine-based GDC-0941 (Genentech) is a pan-PI3K inhibitor (IC50 = 3 nM) with improved pharmacokinetic properties developed by iterative structural modifications [42] . The lead compound, containing a 3-hydroxyphenyl group was susceptible to glucuronidation; replacement of this group with a 4-indazolyl group dramatically diminished glucuronidation and resulted in improved pharmacokinetic properties, especially oral bioavailability. The crystal structure of the GDC-0941–p110γ complex indicated that the morpholine group of GDC-0941 is close to the hinge region of the active site (Figure 19). The oxygen atom of

the morpholine moiety forms a hydrogen bond with the amidic backbone of Val882, and the carbon atoms pack against the side chain of Ile881. The thienopyrimidine core structure interacts with M804, W812 and I831 residues from the N-terminal lobe, and Met953 and Ile963 residues from the C-terminal lobe of the enzyme. The indazole moiety is directed into a hydrophobic pocket consisting of residues Lys833, Glu836, Leu838, Try867 and Glu964. The two nitrogen atoms of the indazole moiety appear to form a hydrogen bond with the phenol oxygen of Try867 and the carboxyl group of Glu841. Finally, the 4-methanesulfonyl-piperazin-1-ylmethyl group extends toward the solvent region, with packing of the piperazine ring and the side chain of M804. The two oxygen atoms of the sulfonyl group appeared to form hydrogen bonds with the side chain of K802 and the amidic nitrogen of A805. GDC-0941 exhibited good selectivity for PI3K against a panel of 228 kinases (KinaseProfiler™ panel; Millipore, MA, USA) [53] . At a concentration of 1 μM, it inhibited only two kinases by more than 50% – Flt3 (59% inhibition) and TrkA (61% inhibition) – demonstrating a 300-fold selectivity for PI3K over other kinases (TrkA, IC50 = 2.85 μM).

Table 3. Summary of the properties of selective PI3K inhibitors in clinical trials. Compound structure O S N O N

HN N

N

N

Compound

Activity

Selectivity profile

Clinical phase

GDC- 0941 Pan-PI3K


A panel of 228 kinase 1 μM concentration

I–II

NVP-BKM120 Pan-PI3K

PI3Kα IC50 = 52 ± 32 nM PI3Kβ IC50 = 166 ± 29 nM PI3Kγ IC50 = 264 ± 94 nM PI3Kδ IC50 = 116 nM

Test more than 200 protein kinase

II–III

442 in different kinase panels at 10 μM concentration

I–II

A panel of 402 diverse kinases at 10 μM concentration

I–II

S N O

O

F

F

N F

N N

N H 2N

O

N

F

F F

O

N S

N

NH

N O

750

NVP-BYL719 PI3Kα selective


N

F

O

H2N

N N

N H

N

N

NH

CAL-101 PI3Kδ selective


PI3Kα IC50 = 820 nM PI3Kβ IC50 = 565 nM PI3Kγ IC50 = 89 nM PI3Kδ IC50 = 2.5 nM



Phe961 O S N O HN N

N N

N S

Review

Thr886

Val882 Tyr867 Glu880 Phe965

N O

Asp841

GDC-0941

Met804

Lys802 Ala805

Lys833 12

IC50 (µM)

10 8 6 4 2 0 p110α p110α p110α p110β p110δ p110γ C2β (class lA) (class lA) (class lA) (class lA) (class lA) (class lB) (class ll) mutant mutant E545-K H1047-R

Vps34 DNA-PK mTOR (class lll) (class lV) (class lV)

Figure 19. Structure and selectivity profiles of GDC-0941. (A) GDC-0941, pan-PI3K selective inhibitor in clinical trials. (B) Co-crystal structures of PI3Kγ in complex with GDC-0941, PDB ID: 3DBS, 2.80 Å. (C) Selectivity profile of GDC-0941 [42] .

GDC-0941 exerted potent growth inhibitory activity against various human tumor cell lines, and exhibited strong in vivo efficacy in the U87MG glioblastoma mouse xenograft model [42] . Clinical trials showed evidence of antitumor responses in 3 of 19 patients with advanced cancer [17] . Moreover, GDC-0941 demonstrated synergy in combination therapy in vivo, GDC-0941 is undergoing Phase II clinical trials in combination with fulvestrant in patients with estrogen receptor-positive breast cancer and is being tested for the treatment of advanced solid tumors, NHL and metastatic breast cancer [54,55] . NVP-BKM120: 2-morpholinopyrimidine scaffold, pan-PI3K-selective inhibitor NVP-BKM120 (Novartis; Basel, Switzerland) is an orally available pan-PI3K inhibitor that competes for the ATP-binding site of PI3K [56] . Modifying the substituent at the 4-position and the electronic characteristic of the heterocyclic ring at the 6-position of the original lead compound 10 created a profile more suitable for use as a drug, particularly improving pharmacokinetic properties. The co-crystal structure of NVP-BKM120 in the ATP binding site of PI3Kγ indicated that the


key binding interactions involve the aminopyrimidine moiety and the morpholine group (Figure 20). The aminopyridine moiety interacts with Asp836, Asp841 and Tyr867 through the formation of hydrogen bonds. The C4´ position of the aminopyridine moiety also plays an important role in the active site, where only small groups are tolerated. The oxygen atom of the morpholine group forms a hydrogen bond with the amidic backbone of Val882 in the hinge region [30,57] . The substituent at the C4 position of the pyrimidine core appears to be oriented towards the solvent-exposed surface. NVP-BKM120 was tested against a panel of more than 200 protein kinases and showed no significant activity against any kinases other than PI3Ks. NVP-BKM120 potently inhibited class I PI3Ks and the most common p110α mutants (IC50 value ranges from 50 to 300 nM). Oral administration of NVP-BKM120 inhibited the growth of human tumor xenografts (U87MG glioma and A2780 ovarian) in vivo through deregulation of the PI3K pathway. Based on its safety profiles in Phase I trials, NVP-BKM120 is currently undergoing evaluation in Phase II/III clinical trials as a potential anticancer treatment [54,58] . In addition, combinations of NVP-BKM120


751


O

O HN 4 N H2N

N

6 N

F

4'

2 N O

N

H2N

10

N 4

F

N 2

6 N

N O

N NVP-BKM120

30

Lys833 Asp841

F

25 Met804

IC50 (µM)

Asp984

20 15 10

PI4Kβ

DNAPK

mTOR

VPS34

P110γ

P110δ

P110β

P110α-E545K

Glu880

P110α-H1047R

0

P110α

5 Tyr867

Figure 20. Structure and selectivity profiles of NVP-BKM120. (A) Formation of NVP-BKM120. (B) Co-crystal structures of PI3Kγ in complex with NVP-BKM120, PDB ID: 3SD5, 3.20 Å. (C) Selectivity profile of NVP-BKM120 [55] .

F F

with cytotoxic agents that target the mitogen-activated protein kinase cascade component MEK or the receptor tyrosine kinase HER2 (docetaxel or temozolomide) have shown promising synergistic effects.

F O

H 2N N

O S

N

NH

NVP-BYL719: PI3Kα-selective inhibitor

N NVP-BYL719

Pro778 Lys802 Gln859

Asp933

Ser854 Tyr696

Val851

Figure 21. Structure and calculated binding mode of NVP-BYL719. (A) NVP-BYL719 and (B) calculated binding mode of NVP-BYL719 in PI3Kα.

752


The 2-aminothiazole-based PI3Kα-selective inhibitor, NVP-BYL719 (Novartis), entered clinical trials in 2010 [18] . NVP-BYL719 contains a 2-aminothiazole scaffold and an (S)-pyrrolidinecarboxamide urea moiety. The 2-aminothiazole moiety is a valuable privileged scaffold for delivering PI3K inhibitors, and the (S)-pyrrolidinecarboxamide urea moiety appears to be involved in enhancing selectivity for the α-isoform [18,59] . Interestingly, the amide group seems to form three stable hydrogen bonds with PI3Kα. Hydrogen bonding between the amide nitrogen of NVPBYL719 and the amide carbonyl and the side chain amide group of Gln859 are especially important for attaining α-isoform selectivity because glutamine at this position is not conserved in the other isoforms (Figure 21) . Biochemical assays showed that NVP-BYL719 selectively inhibited the PI3Kα isoform (IC50 = 5 nM), exhibiting much higher IC50 values (0.25–1.2 μM) against the other isoforms. In addition, NVP-BYL719 potently inhibited Akt phosphorylation in Rat1-myr-



Future perspective The PI3K pathway is among the most frequently activated pathways in many diseases, and has emerged as an attractive target for drug development, especially for cancer treatment. This review has highlighted the current landscape of PI3K-selective inhibitor development and recent advances in achieving selectivity for PI3Ks over other protein kinases. Simultaneously inhibiting several kinases can result in target-related toxicity


N

N H

N

N

N

NH

CAL-101

12000 10000 8000 6000 4000

PIP5Kβ

PIP5Kα

mTOR

DNA-PK

hVPS34

Cllbeta

P110γ

0

P110δ

2000 P110β

CAL-101 (Calistoga Pharmaceuticals; WA, USA), a PI3Kδ-selective inhibitor, has been developed for the treatment of diseases that are associated with cellular autoimmune disorders [14,15] . PI3Kδ is also an attractive therapeutic target in B-cell-mediated hematological malignancies, such as CLL and NHL. Intriguingly, CAL-101 inhibits PI3Kδ 40- to 300-times more potently than other PI3K isoforms (IC50 : PI3Kδ = 2.5 nM; PI3Kα = 820 nM; PI3Kβ = 565 nM; PI3Kγ = 89 nM) (Figure 22) [15] . Even greater selectivity (400to 4000-fold) was observed relative to other related kinases in screens of CAL-101 against a panel of 402 diverse kinases (KinomeScan). The co-crystal structure of PI3Kδ with a derivative of CAL-101 verified that a hydrogen bond is formed between the nitrogen atom at the 1-position of adenine and the backbone of Val828. The 6-amino group of adenine forms an additional hydrogen bond with Glu826. However, it is not clear why CAL-101 is selective for the δ-isoform form from a structural standpoint. Because CAL-101 is selective for the δ isoform and is orally available, it is currently under investigation as a potentially less toxic drug candidate [17] . PhNVPe I/ II clinical trials of CAL-101 as a single agent or in combination with other agents are currently ongoing based on the early efficacy of CAL-101 in relapsed/refractory CLL and indolent NHL [61] . In addition, studies are in progress to test of the use of CAL-101 in plasma cell myeloma, acute myeloid leukemia, CLL, Hodgkin’s lymphoma and aggressive types of NHL.

F

P110α

CAL-101: quinazolinone scaffold, PI3Kδ-selective inhibitor

N O

IC50 (nM)

p110α cells [18] . The potency and selectivity of NVPBYL719 with respect to blocking the PI3K/Akt signaling pathway in cellular assays closely paralleled those observed in biochemical assays. The oral bioavailability of NVP-BYL719 was excellent in rats, mice and dogs, as well as other pharmacological properties, cellularlevel potency and selectivity profiles, were all within a range that satisfied criteria for progressing to Phase I clinical trials [18,60] . NVP-BYL719 is currently in Phase I/II clinical trials as a potential therapeutic agent to treat various cancers [18,54,55,60] .

Review

Figure 22. Structure and selectivity profiles of CAL-101. (A) PI3Kδ-selective inhibitor CAL-101 and (B) selectivity profile of CAL-101 [14] .

because many individual kinase pathways are involved in important cellular functions. By implementing successful structure-based drug approaches for designing PI3K-selective chemotypes and scrutinizing binding modes of PI3K–inhibitor complex structures, it is possible to provide general methodological guidelines for developing new PI3K inhibitors with desirable selectivity. For example, the introduction of some groups (e.g., Me, Cl, OMe) in the proper position of small molecules may achieve high selectivity by accessing PI3K-specific spaces of the gatekeeper region. As noted above, these inhibitors can be invaluable research tools for elucidating biological mechanisms as well as efficient therapeutic agents for combination therapy in cancer. Despite some success stories highlighted here, current kinase profiling strategies are still limited because selectivity assessment is largely restricted to biochemical selectivity assays. Eventually, exploring selectivity in the cell as a whole may be required to understand the phenotypes associated with selective inhibition. Financial & competing interest disclosure This research was supported by National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-2010-0022179, 2011-0016436) and the Institute for BasicScience (IBS) in Korea. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.


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Review Jeong, Kwon & Hong 15

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16

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17

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18

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19

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20

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21

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22

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23

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24

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25

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•

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26

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27

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28

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•

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29

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Review

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Identification of potent and selective MTH1 inhibitors.

SAH derived potent and selective EZH2 inhibitors.

Polyoxometalates--potent and selective ecto-nucleotidase inhibitors.

Synthesis and SAR study of potent and selective PI3Kδ inhibitors.

Design and synthesis of potent, selective inhibitors of matriptase.

Selective and potent proteomimetic inhibitors of intracellular protein-protein interactions.

Discovery of potent and selective 8-fluorotriazolopyridine c-Met inhibitors.

Ethynylflavones, highly potent, and selective inhibitors of cytochrome P450 1A1.

Structure-Based Design of Potent and Selective CK1γ Inhibitors.

Design and synthesis of potent, selective phenylimidazole-based FVIIa inhibitors.

Tetrahydroquinoline derivatives as potent and selective factor XIa inhibitors.

Functionalized N,N-Diphenylamines as Potent and Selective EPAC2 Inhibitors.

Anilino-monoindolylmaleimides as potent and selective JAK3 inhibitors.

9H-Carbazole-1-carboxamides as potent and selective JAK2 inhibitors.

A new series of synthetic thrombin-inhibitors (OM-inhibitors) having extremely potent and selective action.

Aliphatic propargylamines: potent, selective, irreversible monoamine oxidase B inhibitors.

Design and Development of a Series of Potent and Selective Type II Inhibitors of CDK8.

Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L.

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

The design and biological properties of potent and selective inhibitors of protein kinase C.

Analysis of subpocket selectivity and identification of potent selective inhibitors for matriptase and matriptase-2.

Design and Discovery of 2-Arylquinazolin-4-ones as Potent and Selective Inhibitors of Tankyrases.

Discovery and development of potent and selective inhibitors of histone methyltransferase g9a.

Selectivity Profiling and Biological Activity of Novel β-Carbolines as Potent and Selective DYRK1 Kinase Inhibitors.