靶向蛋白降解技術(shù)

分子膠

PROTAC

CRBN靶點(diǎn)藥物研發(fā)現(xiàn)狀

盡管人和小鼠CRBN蛋白具有高度同源性（94%），但V388和E377氨基酸殘基的人鼠差異足以破壞沙利度胺在小鼠體內(nèi)的抗腫瘤療效和致畸毒性。因此，缺乏合適的小鼠模型嚴(yán)重限制了相關(guān)基礎(chǔ)研究的開展以及新療法的研發(fā)。

南模生物長(zhǎng)期致力于藥物靶點(diǎn)人源化模型研究領(lǐng)域，自主開發(fā)了人源化CRBN敲入小鼠模型（hCRBN，目錄號(hào)：NM-HU-225071），該模型在靶細(xì)胞中表達(dá)全長(zhǎng)人CRBN蛋白，為評(píng)估CRBN靶向蛋白降解劑的體內(nèi)療效和毒性提供了強(qiáng)有力的工具。

Fig.1 Detection of CRBN expression in spleen, brain, liver, intestine and muscle tissues by RT-PCR.

Wild type: only one band at 185 bp with primers F1/R1 (mCrbn);

Homozygous: one band at 262 bp with primers F2/R2 (hCRBN) and one band at 374 bp with primers F3/R3 (hCRBN);

Abbr. M, DNA marker; HO, homozygous; WT, wild type.

Fig.2 Expression characterization of hCRBN in homozygous hCRBN KI mice by Western blot.

CC-885作為是一種CRBN調(diào)節(jié)劑，在多種腫瘤細(xì)胞中具有抗增殖活性。CC-885可以誘導(dǎo)IKZF1降解，還可以促進(jìn)CRBN與新型底物GSPT1的結(jié)合，靶向其降解。

Fig. 3 The degradation of GSPT1, CK1a and Myc by Lenalidomide and CC-885 in hCRBN knockin mice were determined by western blot.

The splenocytes derived from wild-type C57BL/6 (WT) and homozygous hCRBN KI (HO) mice were treated with 1% DMSO, Lenalidomide (10 nM, 100 nM, 1000 nM) and CC-885 (10 nM, 100 nM, 1000 nM) ,respectively, in RPMI-1640 cell culture medium for 18 hours ex vivo.

Fig.4 Blood concentration curves of Lenalidomidein in C57BL/6 and HO hCRBN mice (female, 10wks, n=3).（In cooperation with a client）

Fig.5 Blood concentration of CC-885 in C57BL/6 and hCRBN mice 1h after first administration (female, 10wks). (In cooperation with a client）

Fig.6 Mean body weight (A) and survival curve (B) of C57BL/6 and HO hCRBN KI mice treated with CC-885 or vehicle (n=8/group).?

All hCRBN mice treated with CC-885 dead within 2 days, while mice from other groups survived. The result indicates that CC-885 exhibits significant toxicity only in hCRBN humanized mice (In cooperation with a client).

Fig.7 The degradation of GSPT1, CK1a and Myc by CC-885 in C57BL/6 and hom hCRBN knockin mice in vivo (In cooperation with a client).

The wild-type C57BL/6 (WT, group 1&2) and homozygous hCRBN KI (HO, group 3&4) mice were treated with vehicle (group 1&3)? and 5 mg/kg CC-885 (group 2&4) , respectively. The mice were anesthetized, perfused, and tissues were harvested at 6 hr post the 2nd administration.