Iodomycin and iodipine, a structural analogue of azidopine, bind to a common domain in hamster P‐glycoprotein

Abstract

Both the overexpression of P-glycoprotein and the broad range of substrates of this ATP-binding cassette (ABC) transporter induce the phenomenon of multidrug resistance, one major cause of the failure of cancer chemotherapy in humans. This study reports that [ 125 I]iodipine, a structural analogue of the 1,4-dihydropyridine azidopine, shares a common binding site with iodomycin, a Bolton±Hunter derivative of the anthracycline daunomycin. This binding site is different from that described for iodoarylazidoprazosin, which is presumed to share a common binding site with azidopine. Edman sequencing revealed that [ 125 I]iodipine had photolabelled the same peptide as iodomycin and spans the primary sequence of hamster isoform pgp1 from amino acid 230 to amino acid 312.Keywords: ABC transporter; P-glycoprotein; photoaffinity labeling.The overexpression of P-glycoprotein is thought to be one reason for the failure of human cancer chemotherapy. P-glycoprotein confers resistance to a broad spectrum of structurally and chemically unrelated compounds by reducing the intracellular concentration of drugs [1]. This phenomenon of multidrug resistance (MDR) can be overcome by so-called modulators such as verapamil, cyclosporins and dihydropyridine calcium channel blockers, which resensitize the tumour cells by blocking the drug exclusion function of P-glycoprotein [2]. P-glycoprotein belongs to the protein superfamily of ATP-binding cassette (ABC) transporters. Predicted by hydropathy plot analysis, the 170-kDa large plasma membrane protein is made up of 12 transmembrane domains and two cytosolic nucleotide-binding folds [3]. A central issue of P-glycoprotein research is the question of how this protein is able to recognize and transport so many unrelated compounds. [14]. Azidopine photolabels two sites on P-glycoprotein, one in the N-terminal half between amino acid residues 198 and 440 and the other in the C-terminal half [10]. The binding site for azidopine was not analysed further (Fig. 5), but it is presumed that azidopine and [ 125 I]iodoarylazidoprazosin share a common binding site on P-glycoprotein [15].Another photoreactive substrate of P-glycoprotein is iodomycin, a photoreactive Bolton±Hunter derivative of the anthracycline daunomycin [16]. Recently we were able to localize by direct amino acid sequence analysis the [ 125 I]iodomycin-binding site of hamster isoform pgp1 [13] N-terminal in a segment of the primary sequence between amino acid 230 (TM4) and amino acid 312 (TM5).The 1,4-dihydropyridine derivative iodipine, known to be a modulator of MDR [17], was used at an early stage to characterize L-type Ca 2+ channels [18]. The photobinding of the inherently photoreactive iodipine to P-glycoprotein from Chinese hamster ovary (CHO) B30 plasma-membrane vesicles was demonstrated by . Iodipine shares the Bolton±Hunter group with iodomycin but otherwise has a structure very similar to that of azidopine (Fig. 1), for which reason the latter has been extensively employed as a reference compound in studies of drug binding ...

Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P‐glycoprotein

“…This sequence forms the distal part of TM4, the second cytoplasmic loop, and the proximal part of TM5 in the N‐terminal half of pgp1 [27]. The same BNPS‐skatol fragment has previously been reported by us to contain the binding sites for iodomycin and iodipine also [10,11]. The [ 125 I]iodoarylazidoprazosin‐labeled peptides obtained by BNPS‐skatol cleavage with a molecular mass of about 16 and 12 kDa were not accessible by this protocol.…”

“…Whereas for [ 125 I]iodomycin, the Bolton‐Hunter derivative of the anthracycline daunomycin [10], and for [ 125 I]iodipine, a 1,4‐dihydropyridine derivative [11], only one major photobinding site could be identified in P‐glycoprotein by Edman sequencing of photolabeled peptides, this study uncovered three photobinding sites for iodoarylazidoprazosin in pgp1. By applying techniques other than direct sequencing, multiple binding sites have been mapped in P‐glycoprotein for prazosin [15], azidopine [12], and 3′‐ p ‐benzoyldihydrocinnamoyltaxol [13] (Fig.…”

P-glycoprotein: from genomics to mechanism

“…The sophistication and accuracy of the methods used to identify regions of P-gp interacting with drug substrates vary considerably, and includes the use of antibodies raised against specific regions of P-gp (Bruggemann et al, 1989(Bruggemann et al, , 1992Greenberger, 1993;Morris et al, 1994;Demeule et al, 1998;Wu et al, 1998), HPLC in conjugation with Edman-MS (Demmer et al, 1997(Demmer et al, , 1999Isenberg et al, 2001), and more recently MALDI-MS (Borchers et al, 2002;Ecker et al, 2002). The earliest data were obtained by the immunological analysis of P-gp proteolytic fragments after photoaffinity labeling with [ 3 H]-azidopine, the 125 Iforskolin derivative, AIPPF, and the 125 I-prazosin derivative, IAAP (Bruggemann et al, 1989(Bruggemann et al, , 1992Greenberger, 1993;Morris et al, 1994).…”

Role of Transmembrane Domain/Transmembrane Domain Interfaces of PGlycoprotein (ABCB1) in Solute Transport. Convergent Information from Photoaffinity Labeling, Site Directed Mutagenesis and in Silico Importance Prediction

“…A variety of different substrates, including the prazosine analog iodoaryl-azidoprazosine (IAAP) [25,26] a benzoyldihydrocinnamoyl derivative of paclitaxel [27][28][29], the dihydropyridines azidopine and iodipine [30] and an azido-analogue of dexniguldipine [31] as well as the anthracycline iodomycin [30] were used. The majority of labeled component peptide fragments were large thus limiting refinement of binding regions.…”