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Page 8 of 17 Matsushita et al. Hepatoma Res 2018;4:61 I http://dx.doi.org/10.20517/2394-5079.2018.81

FIR and U2AF65 are also expected to interact with SAP155 through the domains with the similar amino
sequences. FBW7 has many WD motifs and most of the motifs are involved in the conformational
stabilization of the WD-repeated domain. Although those WD motifs are not related to the ligand
recognition of FBW7, there is an extra pair of W425 and D399 at the center of the WD-repeated domain.
Most of the ligands of FBW7 are the amino peptides that include phosphorylated Thr or Ser, because three
Arg residues are located at the center of the WD-repeated domain and hold the negatively charged peptides
by phosphorylation [Figure 3C]. The extra pair of W and D at the WD-repeated domain will not be involved
in the ligand recognition of the phosphorylated peptides, but the WD pair can interact with the peptide with
the above-mentioned conserved sequence from the structural viewpoint. Hence, FIR may be bound to the
WD-repeated domain and block the function of FBW7 [Figure 3C]. Together, latent disturbance of FBW7 by
FIR/FIRΔexon2/PUF60 in cancers inhibit degradation of substrate proteins.

[46]
FBW7 expression was decreased significantly in esophageal squamous cell carcinoma (ESCC) . Conversely,
FIR and FIRΔexon2 were overexpressed in ESCC. Especially, the knockdown of SAP155 (SF3B1), a splicing
[46]
factor required for proper AS of FIR pre-mRNA, decreased cyclin E . Therefore, disturbed AS of FIR
generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA
[46]
potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2 . A novel low molecular
[46]
weight chemical, BK697, with WD-like domain structure that inhibits FIR/FIRΔexon2 [Figures 2B and 3] ,
indicating simultaneous downregulation of FBW7 and E-cadherin accompanied with disturbed splicing of FIR
is required for migration [or epithelial-mesenchymal transition (EMT)] in cancers.

Cell growth inhibition by in silico-screened compounds against FIRΔexon2 protein
A small molecular weight chemical that has WD-like motif was identified by NPDepo screening [Figure 1-top,
Figure 4-(A), (C)]. From computer screening to search synthesized chemicals that mimicking the structure
of the identified compound using Namiki database (Namiki Shoji Co., Ltd., Tokyo, Japan) that was composed
of commercially available chemicals [Figure 4-(B), (B’)]. Recently, FIRΔexon2 was suggested to be potentially
bound to the substrate-binding degron pocket of FBW7. Since the substrate-binding degron pocket of FBW7
contains a unique structure of Trp (W) and Asp (D) combination (WD motif) and the WD motif is expected
to interact with FIRΔexon2 [Figure 3]. Actually, chemical skeleton of the two synthesized compounds were
[46]
regarded as a WD mimicking form [Figure 4 (A)-(D)] . All of the compounds bear a chemical skeleton
of aromatic ring connected to carboxyl group with a short linker. Hence, these compounds are analogues
of WD motif of FBW7 [Figure 4]. From these chemical structural findings of WD mimicking form, several
compounds were selected from the chemicals that have been synthesized in our previous studies [Figure 4] [46-49] .
Synthesized compounds were intended to inhibit FIRΔexon2 protein function.

Low molecular weight artificial chemical, BK697, that inhibits FIRΔexon2 protein function
suppressed tumor cell growth
Affiliated small molecular weight chemicals that have WD-like motif screened by NPDepo [Figure 5A, square].
Based on the computer screening, lots of similar chemicals were designed and seven compounds were selected
for treating with HLE and HLF cells to examine cell growth inhibition [Figure 5A, arrows]. Expectedly, BK697
effectively suppressed hepatoblastoma cells, HLE and HLF cells [Figure 5B]. BK697 suppressed FIR/FIRΔexon2
expression on dose-dependent manner in NUGC4 cells [Figure 5C, left] and HeLa cells [Figure 5C, right].
[34]
Particularly, FIR/PUF60 is required for HBV cccDNA replication , BK697 is a promising candidate for
hepatoma treatment by suppressing HBV cccDNA. Previously, FIR has been revealed to contribute to the
splicing of PKM1 to PKM2 in mice thymic lymphoma using six-plex tandem mass tag quantitative proteomic
analysis in mice model [Table 1] [50,51] . SAP155 (SF3B1) and FIR/PUF60 are required for E-cadherin expression
[52]
through engaging in its mRNA editing that is pivotal for cell-cell adhesion or EMT . Together, BK697
suppressed cell growth through interfering FIRΔexon2 with binding to analogues of WD-like motif of FBW7