Di Pietro A, Dayan G, Conseil G, Steinfels E, Krell T, Trompier D, Baubichon-Cortay H, Jault J
Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, Lyon, France.
Braz J Med Biol Res. 1999 Aug;32(8):925-39. doi: 10.1590/s0100-879x1999000800001.
Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.
癌细胞对化疗的耐药性主要由P-糖蛋白（Pgp）的过表达介导，P-糖蛋白是一种质膜ATP结合盒（ABC）转运蛋白，它以ATP水解为代价将细胞毒性药物排出细胞外。Pgp由两个同源部分组成，每个部分都包含一个跨膜结构域和一个胞质核苷酸结合结构域（NBD），该结构域包含两个参与ATP结合和水解的共有沃克基序A和B。该蛋白还包含ABC转运蛋白特有的S签名。Pgp介导的药物转运的分子机制尚不清楚。由于该转运蛋白具有极其广泛的底物特异性，其细胞功能被描述为“疏水真空吸尘器”。对Pgp机制的了解有限，部分原因是缺乏高分辨率结构，这在未能有效抑制其在癌细胞中的活性从而逆转多药耐药性（MDR）方面得到了很好的体现。与研究全长Pgp时遇到的困难相比，重组NBD可以大量获得为可溶性蛋白。本文展示了重组NBD的生化和生物物理特性，为建立结构-功能关系提供了一条合适的替代途径。研究表明，NBD在靠近ATP位点的区域结合ATP及其类似物以及MDR的有效调节剂，如疏水类固醇。有趣的是，黄酮类化合物也以高亲和力与NBD结合。它们的结合位点部分与ATP结合位点和类固醇相互作用区域重叠。因此，黄酮类化合物构成了一类新的有前途的Pgp双功能调节剂。
