To investigate the mechanism of action of compound 2i,
namely its potent inhibition of kynurenine production in A431
cells without rhIDO inhibition, the IDO expression level of the
cells was measured by Western blotting. IFN-γ induced
upregulation of the IDO protein level, whereas compound 2i
suppressed IDO expression in a concentration-dependent manner
(Fig. 2). This phenomenon was only observed for the cyclic
analogue 2i, that is, we have previously reported that the parent
S-benzylisothiourea 1 does not affect the IDO expression level in
IFN-γ-treated A431 cells.17 Therefore, we next explored the
effect on Janus kinase/signal transducer and activator of
transcription 1 (JAK/STAT1) signalling, because IFN-γ induces
IDO expression through this pathway.19 As shown in Fig. 2,
compound 2i reduced STAT1 expression, which was upregulated
by IFN-γ treatment, in a concentration-dependent manner. Taken
together, these results indicate that compound 2i suppressed IDO
expression by inhibiting STAT1 expression in IFN-γ-treated
A431 cells.
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In summary, we synthesised cyclic analogues of our IDO
inhibitor S-benzylisothiourea in an effort to circumvent its
possible toxicity. The 5-Cl-benzimidazole derivative 2b-6 was a
moderate rhIDO inhibitor with cellular inhibition of kynurenine
production. Compound 2i was found to exhibit potent inhibition
of cellular kynurenine production despite its unexpectedly minor
effect on the enzymatic activity of rhIDO. Analysis of the
mechanism of action revealed that compound 2i suppresses IDO
expression at the protein level by inhibiting STAT1 expression in
IFN-γ-treated A431 cells. With its distinct mechanism of action,
the kynurenine-production inhibitor 2i is expected to be a novel
lead compound for immunological cancer treatment.
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Acknowledgements
The authors thank Ms. Masumi Namba, Mr. Takeo Makino,
Ms. Hiromi Yamamoto and Mr. Kanta Nakayama for their
technical assistance for compound synthesis and analysis. This
study was supported by the Japan Society for the Promotion of
Science (JSPS) Grants-in-Aid for Scientific Research (C) (grant
number JP25460149 to Kenji Matsuno) and by a grant from the
Strategic Research Foundation Grant-aided Project for Private
Universities (grant number S1411005 to Yasutada Imamura and
Kenji Matsuno) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
Conflict of Interest (COI)
The authors declare no conflict of interest.
Supplementary Material
The online version of this article contains supplementary
material.
References