| Author: | Liu, Zhen |
| Title: | Reversal of P-gp-mediated paclitaxel resistance : identification of modulator-binding site on P-gp and rational design of next generation P-gp modulators |
| Advisors: | Chow, Ming-cheung Larry (ABCT) |
| Degree: | Ph.D. |
| Year: | 2022 |
| Subject: | Drug resistance in cancer cells Glycoproteins -- Analysis Biological response modifiers Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | ii, xix, 237 pages : color illustrations |
| Language: | English |
| Abstract: | One of the critical mechanisms for multidrug resistance (MDR) in cancer cells was caused by permeability-glycoprotein (P-gp) mediated drug efflux. Previously in our lab, we have developed a family of flavonoid analogs that showed an inhibition effect on P-gp mediated MDR both in vitro and in vivo. FM04 was one of the most potent flavonoid compounds with in vitro P-gp modulating activity of EC50 = 90.0 ± 15.0 nM. However, the exact binding site and binding mechanism of FM04 remained unknown. In this study, photo-crosslinking method was used to locate two binding sites for FM04 on the nucleotide-binding domain 2 (NBD2) of the P-gp. First, seven FM04 photo-affinity derivatives were designed and synthesized. Out of these derivatives, FM04X1, FM04H1, and FM04X4 showed similar activity (EC50 = 110 ± 10.1, 107 ± 15.7, and 66 ± 8.5) as FM04 in reversing P-gp mediated PTX resistance in LCC6MDR cell line that overexpressed P-gp. When applied to photo-crosslinking studies, FM04X4 showed the most potent photo-crosslinking efficiency. Further photo-crosslinking studies showed FM04X4 can photo-label human P-gp in LCC6MDR cell line and recombinant mouse P-gp expressed in Pichia Pastoris in a dose-dependent manner. Moreover, this photo-labeling of P-gp by FM04X4 can be inhibited by excess FM04, indicating that they were binding to the same binding site. Furthermore, the photo-crosslinked P-gp can be enriched by biotin azide and streptavidin agarose. Three photo-crosslinked peptides, namely Peptide A (Q1189-K1216), Peptide B (T1222-K1246), and Peptide C (G1110-F1119), were identified by analyzing the LC-MS/MS of the endoproteinase-digested mouse P-gp photo-labeled by FM04X4 and FM04H1. Peptide A and A partial contained mass adduction of FM04X4 and biotin-PEG4-azide clicked FM04X4 on residue Q1189. Peptide B and B partial contained mass adduction of FM04X4 and N-(3-Azidopropyl)biotin amide clicked FM04X4 on residue T1222. The FM04X4 and FM04H1 labeled Peptide C narrowed the photo-crosslinking position into G1110 or I1111. By analyzing the positions of the photo-crosslinking residues in mouse P-gp 3-D structure, residues Q1189 and T1222 were located on the same cavity at the surface of NBD2 while G1110 or I1111 were located at the interface of ICL2-NBD2. FM04 was docked into the Q1192-T1225 pocket of HM-P-gp (Q1189-T1222 in mouse P-gp), generating a model of FM04 having its "D ring" near the residue Q1192. The molecular dynamic (MD) simulation starting from the above model of FM04 was performed to test the stability of FM04 in this cavity. After 50 ns, the system stabilized with the "D ring" of FM04 close to the residue Q1192. However, residue R1137 showed the unfavorable interaction with FM04 at 100 ns, suggesting that P-gp modulation ability would be increased by adding an electron-rich group on the meta-position of the "D ring". With the goal of further improving the potency of FM04, twelve compounds were synthesized with different substitutions on the "D ring". Four compounds (FM04b, FM04d, FM04e, and FM04h) with methoxy groups on the meta- and para- position of "D ring" have lower EC50 values than FM04, at 69.3±4.7, 63.5±3.8, 66.0±7.9, 53.7±2.7, and 90 ± 15.0 nM, respectively. To further investigate how FM04 modulated P-gp, point mutations around the photo-crosslinked residues were performed. Point mutations on Q1193 and I1115 can retain P-gp activity while showing decreased FM04 modulation ability, suggesting that both residues may directly interact with FM04. Mutation of Q1193 showed different effects on the FM04 modulating ability. First, the small, hydrophobic, and negatively charged P-gp mutants of Q1193A, Q1193F, and Q1193E can no longer be modulated by FM04, indicating the loss of binding to FM04. Second, the Q1193K, Q1193C, and Q1193T mutants with positively charged or polar side chains showed decreased FM04 modulation ability on P-gp, indicating decreased binding with FM04. Thirdly, the Q1193N mutant with a similar side chain showed the same FM04 modulation ability as wild type, indicating unaltered interaction with FM04. Finally, the I1115F mutant with benzene ring in the side chain showed decreased FM04 modulation ability on P-gp activity, indicating decreased binding with FM04. In summary, using the photo-crosslinking approach, FM04 photo-crosslinked with Q1189, T1222, and G1110 or I1111 of the mouse P-gp. The mutational studies indicated that Q1189 and I1111 interacted with FM04. Using docking and MD simulation, two models of FM04 binding with P-gp were proposed. Model 1 indicated FM04 bound to Q1192-T1225 pocket of HM-P-gp (Q1189-T1222 in mouse P-gp), supported by the photo-crosslinking, MD simulation, new FM04 derivatives, and mutational studies. Model 2 demonstrated FM04 bound to the I1115 pocket of human P-gp (I1111 in mouse P-gp), causing P-gp modulation and ATPase stimulation. |
| Rights: | All rights reserved |
| Access: | open access |
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