A chemically cross-linked knottin dimer binds integrins with picomolar affinity and inhibits tumor cell migration and proliferation.
A chemically cross-linked knottin dimer binds integrins with picomolar affinity and inhibits tumor cell migration and proliferation.
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DOI：
10.1021/ja508416e
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发表时间：
2015-01-14
影响因子：
15
通讯作者：
Cochran, Jennifer R.
中科院分区：
文献类型：
--
作者：
Kim, Jun W.;Cochran, Frank V.;Cochran, Jennifer R.
靶向和抑制αvβ3、αvβ5和α5β1整联蛋白的分子由于这些受体在介导血管生成和转移中的作用而引起了极大的兴趣。通过二聚化增加结合亲和力并因此增加整联蛋白抑制剂的功效的尝试已经是勉强有效的。在目前的工作中，我们实现了这一目标，通过使用肟为基础的化学共轭合成二聚体的整合素结合胱氨酸结（knottin）miniproteins与低皮摩尔结合亲和力的肿瘤细胞。将含有氨基氧基侧链的非天然氨基酸引入knottin单体内的不同位置，并与不同长度的含二聚体的交联剂反应，以产生具有不同分子拓扑结构的knottin二聚体。与通过靠近C-末端的氨基氧基交联的二聚体相比，通过位于蛋白质中间附近的氨基氧基官能团交联的二聚体对表达整合素的肿瘤细胞表现出更高的表观结合亲和力。相反，交联剂长度对整合素结合亲和力没有影响。基于化学的二聚化策略是至关重要的，因为通过与二价抗体结构域的基因融合产生的结蛋白二聚体相对于结蛋白单体在肿瘤细胞结合方面仅表现出适度的改善（小于5倍）。最好的肟共轭结蛋白二聚体实现了前所未有的150倍的明显结合亲和力增加结蛋白单体。此外，这种二聚体结合强3650倍，抑制肿瘤细胞的迁移和增殖相比，西仑吉肽，整合素靶向肽模拟物，在最近的临床试验中表现不佳，这表明进一步的治疗发展的承诺。
Molecules that target and inhibit αvβ3, αvβ5, and α5β1 integrins have generated great interest because of the role of these receptors in mediating angiogenesis and metastasis. Attempts to increase the binding affinity and hence the efficacy of integrin inhibitors by dimerization have been marginally effective. In the present work, we achieved this goal by using oxime-based chemical conjugation to synthesize dimers of integrin-binding cystine knot (knottin) miniproteins with low-picomolar binding affinity to tumor cells. A non-natural amino acid containing an aminooxy side chain was introduced at different locations within a knottin monomer and reacted with dialdehyde-containing cross-linkers of different lengths to create knottin dimers with varying molecular topologies. Dimers cross-linked through an aminooxy functional group located near the middle of the protein exhibited higher apparent binding affinity to integrin-expressing tumor cells compared with dimers cross-linked through an aminooxy group near the C-terminus. In contrast, the cross-linker length had no effect on the integrin binding affinity. A chemical-based dimerization strategy was critical, as knottin dimers created through genetic fusion to a bivalent antibody domain exhibited only modest improvement (less than 5-fold) in tumor cell binding relative to the knottin monomer. The best oxime-conjugated knottin dimer achieved an unprecedented 150-fold increase in apparent binding affinity over the knottin monomer. Also, this dimer bound 3650-fold stronger and inhibited tumor cell migration and proliferation compared with cilengitide, an integrin-targeting peptidomimetic that performed poorly in recent clinical trials, suggesting promise for further therapeutic development.
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影响因子：
2.9
作者：
Kimura RH;Levin AM;Cochran FV;Cochran JR
通讯作者：
Cochran JR
影响因子：
56.9
作者：
BROOKS, PC;CLARK, RAF;CHERESH, DA
通讯作者：
CHERESH, DA
影响因子：
11.2
作者：
Galli, R;Binda, E;Vescovi, A
通讯作者：
Vescovi, A
影响因子：
15
作者：
CANNE, LE;FERREDAMARE, AR;KENT, SBH
通讯作者：
KENT, SBH
影响因子：
3.4
作者：
Janssen, M;Oyen, WJG;Boerman, OC
通讯作者：
Boerman, OC