BX-795 | Sglt Signal

TBK1 inhibitors: a review of Review patent literature (2011 — 2014)
Tao Yu, Yanyan Yang, De Qing Yin, Sungyoul Hong, Young-Jin Son*, Jong-Hoon Kim* & Jae Youl Cho†
†Sungkyunkwan University, Department of Genetic Engineering, Suwon, Korea

Introduction: TANK-binding kinase 1 (TBK1) is a noncanonical IkB kinase family member that regulates the innate immune response. Misregulation of TBK1 activity can promote inflammatory disorders and oncogenesis; therefore, TBK1 inhibitors are considered a promising therapy for inflamma- tion and cancer.
Areas covered: In this review, the authors provide information on the role of TBK1 in human health and on recently developed inhibitors from patents granted from 2011 to 2014. The reader will gain an understanding of the mechanisms of TBK1 function as well as the structure and biological activity of recently developed TBK1 inhibitors. Google and NCBI search engines were used to find relevant patents and clinical information using “TBK1 inhibitor” as the search term.
Expert opinion: The role of TBK1 in various diseases has prompted the further investigation of significant targets. Although research on TBK1 inhibitors has increased over the last few years, only a few inhibitors of this kinase have been identified. In addition, almost all of the chemical inhibitors are modified from different scaffolds and/or chemotypes of pyrimidine. Specifically, com- pound BX795 is the representative one, which was first patented as a potent TBK1 inhibitor. Even though some compounds have displayed interesting potential inhibition and selectivity of TBK1 in vitro and in in vivo trials, the development of more efficient and selective TBK1 inhibitors is still required.

Keywords: BX795, cancer therapy, inflammation, inhibitory selectivity, pyrimidine derivatives, TBK1 inhibitor

Expert Opin. Ther. Patents [Early Online]

1. Introduction

TANK-binding kinase 1 (TBK1) (NAK or T2K) is a ubiquitously expressed member of the noncanonical IkB kinase (IKK) family of serine/threonine protein kinases that mediates the innate immune response against bacterial and viral challenges. A complex of TBK1 and its close homolog IKK epsilon (IKK” or IKKi) serves as a significant regulator in the induction of type I IFN expression [1]. TBK1 participates in the signaling of Toll-like receptors (TLRs), RIG-I like receptors (RLRs) and the STING (stimulator of IFN genes)-mediated sensing of cytosolic DNA, while induction of these receptors results in the activation of TBK1, which phosphorylates IFN regulatory factor IRF-3 [2]. Phosphorylated IRF-3 translocates to the nucleus to initiate transcription of the IFN-b gene. There are two main pathways that activate TBK1/IKK” signaling. The first includes the TLR3/TLR4 regulatory proteins, TIR domain-containing adapter molecule 1 (TICAM-1 or TRIF), and TNF receptor-associated factor 3 (TRAF3), and the second is through the retinoid-inducible gene 1 RLRs, IFN-b promoter stimulator protein 1, mediator of IRF3 activation (MITA or STING), and TRAF3 [3,4]. Upon activation, TBK1/IKK” phosphorylates IFN regulatory factors IRF3 and IRF7 (IRF3/7), which stimulate their translocation into the nucleus leading to the

reported a hydrophobic patch of the TBK1 ULD that con- tains Leu316, Ile353 and Val382 residues that are present in an intramolecular binding surface between the ULD and the C-terminal elongated helices, which contributes to its specific- ity for specific function [13]. Interestingly, the Lys63-linked polyubiquitination on Lys30 and Lys401 residues is thought to be critical for TBK1 activity [14]. In addition, a residue located in the C-terminal coiled-coil domain, Lys694, is responsible for the association with adaptor molecules NAP1, SINTBAD and TANK and has been shown to regulate the antiviral function of TBK1 [15].

3. Potential clinical applications of TBK1 inhibitors

activation of type I IFN expression [5]. Eventually, the IFNs and their regulated downstream gene products regulate the suppression of viral replication, the clearance of virus-infected cells, and improvement of the immune response [6]. A simplified overview of the pathway is presented in Figure 1. A series of novel regulators are known to be involved in TBK1 signaling. STING and TBK1 reciprocally regulate each other to elicit antiviral signaling. The PPM1A (protein phosphatase, Mg2+/Mn2+-dependent, 1A) phosphatase func- tions to target the dephosphorylation of both STING and TBK1 [7]. Upon cytoplasmic DNA stimulation, autocrine motility factor receptor and INSIG1 (insulin-induced gene 1) interact to form an E3 ubiquitin ligase complex and catalyze K27-linked polyubiquitination of STING, which indirectly mediates TBK1 activity [8]. Furthermore, the down- stream of kinase (DOK)3 plays a positive and critical role in TLR3 signaling through formation of the TRAF3/TBK1 complex and activation of TBK1 and IFN regulatory factor 3 [9]. In regard to negative regulation, USP2b [ubiquitin-spe- cific protease (USP) 2b] serves as a deubiquitinating enzyme that can cleave K63-linked polyubiquitin chains from TBK1 to inhibit TBK1 kinase activity, and negatively regulate IFN signaling and the antiviral immune response [10]. In addition, NLRC3 (the nucleotide-binding, leucine-rich- repeat-containing protein) associates with both STING and TBK1 to impede STING–TBK1 interaction and reduce
downstream IFN production (Figure 1) [11].

2. Structural biology

TBK1 is a 729–amino acid (84-kDa) protein that consists of an N-terminal kinase domain, a middle ubiquitin-like domain (ULD) and an a-helical scaffold dimerization domain [12,13]. As reported previously, the ULD domain reg- ulates the kinase activity of TBK1 and also plays a critical role in signaling the mediating interactions with other mole- cules in the IFN signaling pathway. Novel research on this pathway has been published over the past few years. The Liang research team at the Chinese Academy of Sciences

The apparent role of TBK1 in immune response has highlighted its potential as a therapeutic target for the treatment of autoimmune diseases. Besides its role in the medi- ation of antiviral responses, TBK1 has also been proposed to be a target for cancer, inflammation and other diseases. There are an increasing number of reports demonstrating that TBK1 mediates cell apoptosis and proliferation in cancer cell lines, especially those that depend on oncogenic KRAS expression [16-18]. It was shown that TBK1 plays a key role in maintaining drug resistance in prostate cancer (PCa) cells [19]. TBK1 can interact with mammalian target of rapamycin and inhibit its function, which can induce cell-cycle arrest in PCa cells. Moreover, several reports have suggested that TBK1 is involved in breast cancer regulation [20-22]. Inhibition of TBK1 activity resulted in the suppression of growth in human HER2-positive breast cancer cells and the induction of cellular senescence, while TBK1 silencing decreased the expression of epithelial markers and increased the expression of mesenchymal markers in ERa (estrogen receptor)-positive breast cancer cells. Furthermore, TBK1 plays a significant role in lung cancer radiotherapy. TBK1 signaling mediates radiation-induced epithelial–mesenchymal transition by controlling glycogen syn- thase kinase 3b phosphorylation and zinc finger E-box binding protein 1 expression [23,24]. Meanwhile, increasing evidence sug- gests that TBK1 signaling plays an important role in inflamma- tory diseases. TBK1 is involved in regulating rheumatoid synovitis by controlling TLR3-mediated activation of IRF7 and subsequent transcription of the IP-10 gene, which is closely related to the pathogenesis of rheumatoid arthritis [25]. TBK1 also acts as a key regulator in neuroinflammation, micro- vascular inflammation and gastritis [26-28]. Interestingly, TBK1 contributes to obesity regulation through the repression of energy expenditure. TBK1 co-opts insulin targets to conserve energy during obesity, indicating that TBK1 could be a new therapeutic target for the treatment of obesity and type 2 diabetes [29]. TBK1 signaling has also been shown to be involved in mediating normal tension glaucoma, familial amyotrophic lateral sclerosis (ALS) and frontotemporal demen- tia diseases [30]. Additionally, TBK1-dependent mechanism for

Figure 1. TBK1 is an important protein for multiple signaling pathways.
IRF: Interferon regulatory factor; ISRE: Interferon-stimulated response element; NLRC5: NOD-like receptor family CARD domain containing 5; PPM1A: Protein phos- phatase, Mg2+/Mn2+-dependent, 1A; STING: Stimulator of interferon genes; TBK1: TANK-binding kinase1; TLR: Toll-like receptor; TRAF: TNF receptor-associated factors; TRIF: TIR-domain-containing adapter-inducing interferon-b; Ub: Ubiquitination; USP2b: Ubiquitin-specific protease (USP) 2b.

NF-kB signaling pathway contributes to autophagy addiction in K-Ras-driven non-small-cell lung cancer cells [31].
In summary, the structure and function of TBK1 and its role in various diseases has been studied extensively. As such, there is significant evidence to suggest that TBK1 is an excellent focus target for the treatment of multiple disease indications, making it an ideal target for the development of TBK1 inhibitors.

4. Patent evaluation

Since TBK1 serves as a potential therapeutic target, an enormous amount of research has been conducted to identify and develop novel TBK1 inhibitors. Currently known com- pounds like BX795 and CYT387 are representative potent

TBK1 inhibitors and have provided the basic structure for molecule design using medicinal chemistry approaches (Figure 2). However, these compounds have been demon- strated to be nonselective, and they were originally prepared as inhibitors of other targets [32,33]. Therefore, researchers sought to design and screen a wide array of effective and specific compounds that target TBK1, and fortunately the development of TBK1 inhibitors has attracted increased attention over the past few years. In this review, we focus on recent patents and the application of TBK1 inhibitors published between 2011 and 2014.

4.1 University of Dundee
Based on the nonspecific mechanism of TBK1 inhibition by
BX795, the Clark group at the University of Dundee

selectivity against other kinase activity (Aurora B and
O O
CDK2). Fortunately, compound 9 displayed selectivity with

N N N N N
H H H H

BX795

N H
H
N N

N N
H H

MRT67307

Figure 2. Chemical structure of pharmacological TBK1 inhibitors developed by University of Dundee.

continued to explore TBK1 inhibitors [34]. They modified the structure of BX795 and generated the improved compound MRT67307 (Figure 2), which exhibited highly effective and selective TBK1 inhibition [35]. The IC50 values of MRT67307 were 160 and 19 nM against IKK” and TBK1, respectively, and this compound lacked inhibition of IKKa
and IKKb even at 10 µM. Different from BX795,
MRT67307 did not suppress the activation of JNK and p38 MAPK, implying that MRT67307 could be used as a useful probe for research determining TBK1 and IKK” function. Importantly, this evidence was consistent with testing in RAW264.7 macrophages.

4.2 AstraZeneca
Over the past 4 years, AstraZeneca has been very active in the
development of TBK1 inhibitors. In 2012, AstraZeneca reported a total of 44 compounds that were part of an azaben-
zimidazole derivative series and almost all of these compounds showed effective inhibition on TBK1 kinase activity at 1 µM.
The IC50 of several compounds in the series reached a concen- tration of 10 nM [35]. The structures of these compounds are shown in Figure 3. Compounds 4, 6 and 7 exhibited particu- larly excellent inhibition of TBK1 (IC50 = 4, 3 and 4 nM, respectively). However, compounds 4, 6 and 7 lacked

high efficacy. Moreover, compound 9 also showed distinct activity against TBK1 in HEK293 cells.
More recently, studies focusing on azabenzimidazole deriv- atives were published in 2014. Johannes et al. at AstraZeneca developed an additional 35 compounds with 6-aryl-azabenzi- midazole derivatives [35]. They focused on screening the 6-substituted azabenzimidazoles against TBK1 and identified a series of potential effective inhibitors, including compound 15 that pairs a C6 aryl group with a C7-nitrogen linkage via the introduction of a thiazole group. As a result, compound 15 showed interesting nanomolar inhibitory effects against TBK1 activity with selectivity over the CDK2 and Aurora B-cell–cycle kinases (Figure 4). However, no animal in vivo testing was conducted during the development process by AstraZeneca, which should be conducted in the future. In addition, the synergistic effect of TBK1 inhibitors in combination with MEK inhibitor was also demonstrated, which was tested as a combination therapy for the treatment of cancers. AZ13102909, developed by AstraZeneca, has an inhibition against TBK1 with IC50 of 5 nM. Vu et al. reported that treatment of AZ13102909 combined with MEK inhibitors (AZD6244) can clearly enhance the apopto- sis of cancer cells [36]. Zhu et al. presented similar results in their work as well [33]. These findings are quite useful for help- ing people to study therapeutic regiment of serious cancers with resistance capacity to apoptosis. The further clinic trials are expected to be operated.

4.3 Technical University of Dortmund
Researchers at the Technical University of Dortmund pat-
ented a new class of pyrimidine-based TBK1 inhibitors using activity-based screening approaches [37]. Particularly, 2,4,6-substituted pyrimidine scaffolds such as tozasertib and scaffold I were considered the main structure for further ratio- nal design to improve inhibitory activity. After consecutive validation and optimization, a focused library of derivatives with activities in the nanomolar range was synthesized and is represented by compounds 16 — 21 (Figure 5). These com- pounds were reported to possess selective inhibition of
TBK1 with IC50 values ranging from 0.06 to 0.38 µM.
Compound 16 was the most potent inhibitor, which has the advantageous effect of an electron-withdrawing functional group by the addition of a nitro group. Further studies in macrophages indicated that these derivatives were effective anti-inflammatory agents in various in vitro studies.

4.4 MRC Technology
MRC Technology patented a class of BX795 pyrimidine
derivatives (compounds 22 — 29) as potent TBK1 selective inhibitors [35]. More than 40 different 5-substituted- 2,4-dichloropyrimidine intermediates were developed by replacing functional groups including 5-iodo, bromine, urea, aryl amides and N-methyl piperidine (Figure 6). Among these,

NH
Br N
N

NH NH

Br
N
O

NH NH

Br
N
O

N N

NH2

N N H
H

1 2 3

NH NH

Br
N
O

NH N

Br
N
O

NH NH

Br
N

O NH2

N N N
H H H

4 5 6

NH NH

HN
O
Br

H2NOC

NH NH

HN
O

H2NOC

NH NH

HN
O

N N N
O N
O N
N
N H N
H H

OMe

7 8 9

Figure 3. Structure of TBK1 inhibitors developed by AstraZeneca Company.

O O
O O
NH NH NH NH
O
HO

HN O
Br Br
N N

S O
H2N
N

S HN

O O

N N
H H

10 11

O O

N N
H N H

12 13

O
O NH
NH
O O
HO H2N

N HN

S
N N

O O

N N
H H
14 15 AZ13102909

Figure 4. Additional structure of TBK1 inhibitors patented by AstraZeneca Company.

H H
H N N
N N N
N

NH NH NH
Me
NO2 Pr

O H
O H N O H
H
H H

16 17 18

NH2 O

CF3

19 20 21

Figure 5. Pyrimidine derivatives patented as TBK1 inhibitors by Technical University of Dortmund.

compound 25 with a small cycloalkyl amide was found to show optimal potency for TBK1 activity (IC50 = 2 nM), and selectivity against PDK1 improved considerably. More- over, compound 29 with a 1-methyl-3-amino pyrazole group substitution showed high efficacy (IC50 = 8 nM) and selectiv- ity as well as reasonable cellular potency and stability in both human and mouse liver microsomes. Importantly, compound 29 inhibited the LPS-induced release of pro-inflammatory cytokines in mice, indicating it is a good candidate for TBK1 signaling in inflammation.

4.5 University of North Carolina
The research team at the University of North Carolina
recently developed a series of compounds that inhibit TBK1. They applied positional scanning peptide library technology to determine the optimal phosphorylation motif using GST-TBK1 purified from HEK293T cells, and then validated the TBK1 peptide substrate for high-throughput chemical screening. The screen resulted in 4727 kinase- focused compounds for in vitro inhibitors of TBK1 [38].
Among these, 227 compounds suppressed TBK1 kinase activity at a concentration of 10 µM. In particular, several compounds showed submicromolar concentrations that
inhibit TBK1, including compounds 29 — 33 (IC50 values of 0.4 — 0.8 µM) (Figure 7). Further studies examining the
ability of these compounds to inhibit TBK1 in cell-based assays are in progress. However, none of these compounds are among the most effective TBK1 inhibitors, and many

lack specificity implying that more experimentation is required to optimize the use of this principle.

4.6 The Scripps Research Institute
A structurally rigid 2-amino-4-(30-cyano-40-pyrrolidine)phe-
nyl-pyrimidine scaffold was patented by researchers at the Scripps Research Institute of United States as a potent TBK1 inhibitor [39]. As compound SR8185 was reported as a JNK inhibitor candidate, this group modified the structure of SR8185 and explored structure–activity relationship studies, and successfully generated compounds 34 and 35 with excellent inhibition and specificity for TBK1 (IC50 values were both lower than 1 nM) (Figure 8). Moreover, compounds 34 and 35 distinctly inhibited cell viability in human breast, prostate and oral cancer cell lines. Interestingly, these two compounds significantly impaired tumor development in xenograft and allograft mouse models. Most importantly, these two inhibitors had low molecular weight, low cytochrome P450 inhibition and high metabolic stability, which are key factors for development as anticancer drugs. This suggests that compounds 34 and 35 could be potential candidates for clinical trials in the near future.

4.7 Domainex
Domainex is one of the most advanced drug discovery compa- nies studying TBK1/IKK” kinase inhibitors. It has identified that first-in-class small-molecule inhibitors of TBK1/IKK”. Domainex’s compounds exhibited high potency in animal

F F

O O

N N
H H
Br Br
Br

22 23 24

F F
HN F

O O
N N O

N N
N N H H
H H

25 26 27

N N
H H

28 29

Figure 6. Chemical structures of TBK1 inhibitors patented by MRC Technology.

disease models, and were orally bioavailable, which can be applied for treatment of TBK1-related diseases. During the past 3 years, Domainex developed more than 80 pyrimidinyl compounds that have specific substitution pattern, showing selective and high potency against TBK1 kinase (Figure 9) [40]. Encouragingly, a series of compounds exhibited nanomo- lar level inhibition. In particular, compounds 39 and 40 have shown the most potent suppressive activity toward TBK1 (IC50 = 1 — 2 nM). Moreover, these compounds exhib- ited higher selectivity, since the inhibition to IKKb, JNK-1 and JNK-3 was seen at 200 times higher concentra- tions like 200 nM or even 10000 nM. Furthermore, in vivo effects in animal inflammatory disease model were examined subsequently. Interestingly, the selective compounds could inhibit various pro-inflammatory cytokines including TNF-a, RANTES, IL-1b and IL-6 without displaying side effects. These results strongly suggest that these drugs can be

promising compounds available for treatment of inflamma- tory diseases. However, more investigations and investment should be continued for proceeding clinic trials in the future.

4.8 Myrexis
Myrexis is one of the lead companies in the research of
discovering, developing and commercializing TBK1 inhibi- tors for treatment of various diseases. As patented before, MPI-0485520 is one of the representative compounds, and has shown excellent inhibition and selectivity against TBK1 (IC50 = 500 pM) [41]. Moreover, subsequent in vitro and in vivo tests provided it to be a promising therapeutic compound for the treatment of autoimmune diseases and cancer, which are known to be caused by activated TBK1. In further investigations, Myrexis synthesized and examined more than 670 amino-pyrimidine compounds [42]. Among them, more than 280 compounds displayed lowered

30 31

OH N O

32 33

Figure 7. The potent TBK1 inhibitors patented by University of North Carolina.

HN
O

N N
H

SR8185

Amlexanox

N
N
N

N N
N N H
H

34 35

Figure 8. The potent TBK1 inhibitors patented by the Scripps Research Institute and University of Michigan.

N N
N

NH2
N N
H
N N
O H

H
NH2 N

O
N N
H

36 37 38

N N

CN CN

N N
H N N
H
39 40

OH F

N
N
CN
CN

H N
H N

N N
O H
N N
H

41 42

Figure 9. The potent TBK1 inhibitors patented by Domainex.

activities with IC50 values in a range of 10 nM, which are partially shown in Figure 10. More importantly, several com- pounds have consistently potent suppression at both in vitro and in vivo conditions. In particular, compounds 45, 46 and 47 inhibited the secretion of RANTES with IC50 values of

less than about 10 nM. Compound 44 suppressed produc- tion of RNATES, IP-10 and IFN-b with IC50 values of about 60 and 40 nM, which indicated their therapeutic potential in treating patients with rheumatoid arthritis and other related diseases.

H
H N N
N N

43 44

H H H
N N N N N N

H N

HO N
N

45 46 47

Figure 10. The potent TBK1 inhibitors patented by Myrexis.

4.9 Other institutions
Researchers at many institutions are developing novel and
potent TBK1 inhibitors. Lithium salt was reported to inhibit TBK1 activity by a research team from Shandong University in China [35]. They demonstrated that lithium chloride attenuates LPS-, polyinosinic-polycytidylic acid- and Sendai virus-induced IFN-b production and IFN regulatory factor
3 activation in macrophages, which was consistent with
in vivo testing through targeting TBK1. The inhibition was at IC50 values lower than 5 µM. Researchers at the University
of Michigan recently patented a repurposed compound named amlexanox that showed obvious inhibition of
TBK1 activity (IC50 = 1 — 2 µM) (Figure 8) [35]. Moreover,
treatment of obese mice with amlexanox clearly increased energy expenditure through improved thermogenesis that resulted in weight loss, insulin sensitivity improvement, and steatosis decrease in obese mice. Most importantly, amlexanox could be a potential candidate for clinical evaluation in the treatment of obesity and related disorders due to its demonstrated safety in patients.

5. Conclusion

In summary, significant progress has been made in the development and application of TBK1 inhibitors over the

past 4 years. Most of the patented compounds display potent inhibitory activity against TBK1 in enzymatic and cellular assays, and several interesting potential inhibitors have been examined in animal models. These inhibitors are modified from the basic structure of BX795, and can be divided mainly into two classes: the benzimidazole and pyrimidine derivatives. Even though it is relatively easy to generate effective TBK1 inhibitors, selectivity for TBK1 is less easily achieved. It has also been proven difficult to achieve useful clinical efficacy with TBK1 inhibitors in the therapeutic treatment of several diseases.

6. Expert opinion

Numerous studies demonstrate that TBK1 signaling plays a critical role in many different and interrelated physiological processes, including the immune response of virus infection, inflammation, cancer, diabetes and dementia diseases. Therefore, blocking TBK1 signaling could be of great help for the treatment of these diseases, making TBK1 an attractive target for the development of therapeutics. As such, the identification and development of novel inhibitors to mediate TBK1 activity becomes an important field of research.
Since the first TBK1 inhibitor BX795 was reported in 2009, extensive research and broad efforts have been devoted to the

development of TBK1 inhibitors. Over 35 applications describing novel TBK1 inhibitors have been awarded patents from 2011 to 2014. Almost all of the novel chemicals in these patents include a variety of different scaffolds that are modified from the structure of BX795, for example, 6-aryl-azabenzimi- dazole, 2,4,6-substituted pyrimidine, 5-substituted-2,4- dichloropyrimidine and 2-amino-4-(30-cyano-40-pyrroli- dine)phenyl-pyrimidine. Among these, compounds 34 and 35 showed the best inhibition of TBK1 activity. Most impor- tantly, the compounds displayed consistent effects in in vitro and in vivo testing, prioritizing them for further toxicological testing, drug metabolism testing and clinical trials.
During the process of drug development, the ability of the compound to penetrate the cell membrane is considered an important factor that can directly guarantee the quantity of drug absorption in vivo. As examined, different chemical structures might have different properties with the potential to interfere with cell penetration. Isopropyl groups can improve cell penetration compared to nitro- and fluorine groups [37], indicating that isopropyl residues should be highlighted as an optimal functional group in the process of drug design. Moreover, para-phenyl connected to a functional group can influence the property of electron donating due to its spatial arrangement, which has critical inhibitory effects. Researchers should pay special attention to these key factors. In addition, it is well known that the selectivity of inhibitors is considered a significant characteristic during the develop- ment of kinase inhibitors. As examined, the gatekeeper residue on the hinge that is variable in the off-target kinases has attracted more focus. For example, the 6-position of azabenzimidazole points to the gatekeeper, and therefore

modifications at this position could modulate selectivity against other kinases. Also, the modification at the solvent channel, which is different among kinases, can also increase selectivity. In summary, these important factors should be taken seriously for researchers developing effective and selec- tive TBK1 inhibitors. More advanced drug design approaches are expected to increase the improvement of small molecules against TBK1 kinase.
Conclusively, numerous studies focusing on TBK1 inhibi- tors have produced patents over the past 4 years; however, even though interest in the development of TBK1 inhibitors is growing, progress remains significantly low. Currently, no targeted TBK1 inhibitor has reached the market, even at the clinical phase. Therefore, more focus should be put toward the development of TBK1 inhibitors in the future. Hopefully, novel, safe, efficient and selective TBK1 inhibitors available for clinical use will be introduced to the market in the near future.

Declaration of interest

Writing assistance was utilized in the production of this manuscript from E-world editing company and funded by Sunkyunkwan University. The authors have no relevant affili- ations 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. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Affiliation
Tao Yu1, Yanyan Yang1, De Qing Yin2, Sungyoul Hong3, Young-Jin Son*4, Jong-Hoon Kim*5 & Jae Youl Cho†3
†,*Authors for correspondence
1Qingdao University, Medical College, Qingdao 266071, China
2Linyi Center for Disease Control and Prevention, Linyi 276000, China 3Sungkyunkwan University, Department of Genetic Engineering, 300 Chuncheon-Dong, Suwon 440-746, Korea
Tel: +82 312 907 868;
Fax: +82 312 907 870;
E-mail: [email protected]
4Sunchon National University, Department of Pharmacy, Suncheon 540-742, Republic of Korea Tel: +82 617 503 755;
Fax: +82 617 503 708;
E-mail: [email protected]
5Chonbuk National University, College of Veterinary Medicine, Biosafety Research Institute, Department of Veterinary Physiology, Jeonju 561-756, Republic of Korea
Tel: +82 632 702 563;
Fax: +82 632 703 780;
E-mail: [email protected] BX-795