九州大学学術情報リポジトリ

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九州大学学術情報リポジトリ

Kyushu University Institutional Repository

標的タンパク質特異的ラベル化のための反応基の探索とコバレントドラッグ開発への応用

渕田, 大和

http://hdl.handle.net/2324/1931853

出版情報：Kyushu University, 2017, 博士（創薬科学）, 課程博士バージョン：

権利関係：Public access to the fulltext file is restricted for unavoidable reason (3)

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Figure 1. Fluorescence assay system for evaluation ofreactivity using peptide-tag/ Zn¹¹-complex pair'.

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FF H CF2A inactive (< X 0.1) Figure 2. Relative reactivity toward a Cys residue.

ffi= iitft EGFR-TKI (tyrosine kinase inhibitor) 0) lffl~

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~fl£}t0)~.:h,t::~J:1.'.§fi5tt~1fTo NS-058 CT)!ffl~l=/Jx;/J L-t::

(Table. l)o

Table I. Summary of anti-proliferative activity (IC50 µM) against EGFR-dependent cell lines.

~ F

r

^(Linker) ^H ^HN

.v--

^Cl

Cl ~N d " N

o o I"' .:J

N 2fld generation EGFR-TKI

none cell line afatlnlb erlotlnlb

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H1975 0.19 4.7 1.0

H HN

nF

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I O I"' .:J

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a

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(irreversible EGFR inhibitor)

r9)

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.,.o.__.,...o)l)...i

e~otinib (reversible EGFR inhibitor)

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) y

₀

· \

/ , N y \ _H ₀

VY\ Z::-\r\

.J.. 0

.J.. ⁰

NS-016 NS-022 NS-023 NS-066 NS-058

0.024 0.029 <0.01 <0.01 < 0.01

> 10 8.8 3.7 >10 0.30

HI 975 *-lllJl~ll=M L-ts~n '~J:l.'.§fi5tt ~;,rs L-t;: t: v Ht-fr~ (NS-058) 0) 7 iv=\=-:,,?' ::'.7" ~ifT o ~~{;$:

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~!llm;O) 7 :/ F-7 Jv=\=-/ f lftft:IJDiitf.t (Cu-catalyzed azide alkyne cycloaddition; CuAAC) I= J:: ~ 7° ^P^- f O){t:/JD L. f;: ?' / /'\ :7 'lt~1ib't:J:~rn& L. f;:o tO)ff, SDS-PAGE ~fr,:;, t~:J:R.f1:~7.;-(;v{t:t;/J$~ J;~~

L-t::.o "'7,1,;r;v7:7t7°?1- ~1fTo afatinib ~w.1215:1:t, -1 :,,;:\=-.::z..;-(- H\!fr1e19 • ~1t1=1R1rL-t~k ft ?1 :,, /'\ :7 't'r c il=¥f ~ i¥J l=Iitr.t L-t:: - :n-r:', CF A ~ ifT o NS-058 ~~{215: /:t~ a\!f r1e19 ib o t, '/:trflirl lt-1

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-

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Figure 3. (a) Schematic illustration of gel-based ABPP. (b) Structures of afatinib and NS-058 derivatives. (c) Time-dependent reaction profiles in A431 cells ([Probe] = 3 µM, 0-8 hr, 37 °C). (d) Concentration-dependent reaction profiles in A431 cells ([Probe]= 0-10 µM, 3 hr, 37 °C).

~~. CTA ~~~~~MfflT0= c ~ ~ T k ~. ~ ~offiaQ~tt~ff~ T0= c c L k .

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Figure 4. (a) The structure of NS-062. (b) Western blot analysis ofEGFR signaling pathway in Hl 975 cells treated with NS-062. (c) Cocrystal structure ofEGFRLsssRff⁷⁹⁰M kinase domain bound to NS-062.

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NS-062 ~J!l3il!IJ:l~4Lt:::.c = ~. NS-062 /:l:control cJ;t~L-Cff;e':l::.~fQ(J)15t*~;JfpffilJL, afatinib c !Rlf~Jj'.O){f.ht:::.mijfWjn§"tt~~ Lt::. (Fig. 5a). E GI::., afatinib ~4ff-C/:l:"? r'J 7. O)~:m;~yn;'!ii

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b

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i

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5

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5 10 15

Days

20 25 Control afatinib NS-062

Figure 5. (a) In vivo anti-tumor activity of quinazoline compounds (20 mg/kg, oral administration twice daily). Each plot represents an average ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 versus control (two-tailed Student's t-test).

(b) Effect of quinazoline compounds on mice body weight after the 25-day continuous oral administration. Each bar represents an average ± SE. ** P < 0.01 versus afatinib (two-tailed Student's t-test).

ffi .=.iitft

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MT 0 tfH~~!l.~Ti5·11: 7i:ii¥fiffi L, t:.o -t O)~*' ~f?O) EGFR-TKI 0) osimertinib c 115'J;j:~Jj'.O) HI 975 *-IBIJ§

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I J... I Gl₅₀(50% growth inhibition) I J... I Gl₅₀

N N "' H1975 0.019µM N N "' H1975 0.016µM

H O, H292 1.4 µM H O, H292 0.22 µM

Figure 6. Structures of NSP-037, osimertinib and their anti-proliferative activity (GJ₅₀µM) against EGFR-dependent cell lines.

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九州大学学術情報リポジトリ