Identification of Binding Sites in the Nucleotide-Binding Domain of P-Glycoprotein for a Potent and Nontoxic Modulator, the Amine-Containing Monomeric Flavonoid FM04.

We have previously discovered an amine-containing flavonoid monomer FM04 as a potent P-glycoprotein (P-gp) inhibitor (EC50 = 83 nM). Here, a series of photoactive FM04 analogues were synthesized and used together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the FM04-binding sites on P-gp. Point mutations around the photo-crosslinked sites were made for verification. Together with the results from mutational studies, molecular docking, and molecular dynamics simulations, it was found that FM04 can interact with Q1193 and I1115 in the nucleotide-binding domain 2 (NBD2) of human P-gp. It was proposed that FM04 can inhibit P-gp in 2 novel mechanisms. FM04 can either bind to (1) Q1193, followed by interacting with the functionally critical residues H1195 and T1226 or (2) I1115 (a functionally critical residue itself), disrupting the R262-Q1081-Q1118 interaction pocket and uncoupling ICL2-NBD2 interaction and thereby inhibiting P-gp. Q1118 would subsequently be pushed to the ATP-binding site and stimulate ATPase.

Discovery of a Flavonoid FM04 as a Potent Inhibitor to Reverse P-Glycoprotein-Mediated Drug Resistance in Xenografts and Improve Oral Bioavailability of Paclitaxel.

Biotransformation of flavonoid dimer FD18 resulted in an active metabolite FM04. It was more druggable because of its improved physicochemical properties. FM04 (EC50 = 83 nM) was 1.8-fold more potent than FD18 in reversing P-glycoprotein (P-gp)-mediated paclitaxel (PTX) resistance in vitro. Similar to FD18, FM04 chemosensitized LCC6MDR cells towards multiple anticancer drugs by inhibiting the transport activity of P-gp and restoring intracellular drug levels. It stimulated the P-gp ATPase by 3.3-fold at 100 µM. Different from FD18, FM04 itself was not a transport substrate of P-gp and presumably, it cannot work as a competitive inhibitor. In the human melanoma MDA435/LCC6MDR xenograft, the co-administration of FM04 (28 mg/kg, I.P.) with PTX (12 mg/kg, I.V.) directly modulated P-gp-mediated PTX resistance and caused a 56% (*, p < 0.05) reduction in tumor volume without toxicity or animal death. When FM04 was administered orally at 45 mg/kg as a dual inhibitor of P-gp/CYP2C8 or 3A4 enzymes in the intestine, it increased the intestinal absorption of PTX from 0.2% to 14% in mice and caused about 57- to 66-fold improvement of AUC as compared to a single oral dose of PTX. Oral co-administration of FM04 (45 mg/kg) with PTX (40, 60 or 70 mg/kg) suppressed the human melanoma MDA435/LCC6 tumor growth with at least a 73% (***, p < 0.001) reduction in tumor volume without serious toxicity. Therefore, FM04 can be developed into a novel combination chemotherapy to treat cancer by directly targeting the P-gp overexpressed tumors or potentiating the oral bioavailability of P-gp substrate drugs.

Characterization of a Potent, Selective, and Safe Inhibitor, Ac15(Az8)2, in Reversing Multidrug Resistance Mediated by Breast Cancer Resistance Protein (BCRP/ABCG2).

Overexpression of breast cancer resistance transporter (BCRP/ABCG2) in cancers has been explained for the failure of chemotherapy in clinic. Inhibition of the transport activity of BCRP during chemotherapy should reverse multidrug resistance. In this study, a triazole-bridged flavonoid dimer Ac15(Az8)2 was identified as a potent, nontoxic, and selective BCRP inhibitor. Using BCRP-overexpressing cell lines, its EC50 for reversing BCRP-mediated topotecan resistance was 3 nM in MCF7/MX100 and 72 nM in S1M180 in vitro. Mechanistic studies revealed that Ac15(Az8)2 restored intracellular drug accumulation by inhibiting BCRP-ATPase activity and drug efflux. It did not down-regulate the cell surface BCRP level to enhance drug retention. It was not a transport substrate of BCRP and showed a non-competitive relationship with DOX in binding to BCRP. A pharmacokinetic study revealed that I.P. administration of 45 mg/kg of Ac15(Az8)2 resulted in plasma concentration above its EC50 (72 nM) for longer than 24 h. It increased the AUC of topotecan by 2-fold. In an in vivo model of BCRP-overexpressing S1M180 xenograft in Balb/c nude mice, it significantly reversed BCRP-mediated topotecan resistance and inhibited tumor growth by 40% with no serious body weight loss or death incidence. Moreover, it also increased the topotecan level in the S1M180 xenograft by 2-fold. Our results suggest that Ac15(Az8)2 is a promising candidate for further investigation into combination therapy for treating BCRP-overexpressing cancers.

Boosting the efficacy of anti-MRSA ß-lactam antibiotics via an easily accessible, non-cytotoxic and orally bioavailable FtsZ inhibitor.

The rapid emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains has undermined the therapeutic efficacy of existing ß-lactam antibiotics (BLAs), prompting an urgent need to discover novel BLAs adjuvants that can potentiate their anti-MRSA activities. In this study, cytotoxicity and antibacterial screening of a focused compound library enabled us to identify a compound, namely 28, which exhibited low cytotoxicity against normal cells and robust in vitro bactericidal synergy with different classes of BLAs against a panel of multidrug-resistant clinical MRSA isolates. A series of biochemical assays and microscopic studies have revealed that compound 28 is likely to interact with the S. aureus FtsZ protein at the T7-loop binding pocket and inhibit polymerization of FtsZ protein without interfering with its GTPase activity, resulting in extensive delocalization of Z-ring and morphological changes characterized by significant enlargement of the bacterial cell. Animal studies demonstrated that compound 28 had a favorable pharmacokinetic profile and exhibited potent synergistic efficacy with cefuroxime antibiotic in a murine systemic infection model of MRSA. Overall, compound 28 represents a promising lead of FtsZ inhibitor for further development of efficacious BLAs adjuvants to treat the staphylococcal infection.

A New Class of Safe, Potent, and Specific P-gp Modulator: Flavonoid Dimer FD18 Reverses P-gp-Mediated Multidrug Resistance in Human Breast Xenograft in Vivo.

Flavonoid dimer FD18 is a new class of dimeric P-gp modulator that can reverse cancer drug resistance. FD18 is a potent (EC50 = 148 nM for paclitaxel), safe (selective index = 574), and selective P-glycoprotein (P-gp) modulator. FD18 can modulate multidrug resistance toward paclitaxel, vinblastine, vincristine, doxorubicin, daunorubicin, and mitoxantrone in human breast cancer LCC6MDR in vitro. FD18 (1 µM) can revert chemosensitivity of LCC6MDR back to parental LCC6 level. FD18 was 11- to 46-fold more potent than verapamil. FD18 (1 µM) can increase accumulation of doxorubicin by 2.7-fold, daunorubicin (2.1-fold), and rhodamine 123 (5.2-fold) in LCC6MDR. FD18 inhibited P-gp-mediated doxorubicin efflux and has no effect on influx. FD18 at 1 µM did not affect the protein expression level of P-gp. Pharmacokinetics studies indicated that intraperitoneal administration of 45 mg/kg FD18 was enough to maintain a plasma level above EC50 (148 nM) for more than 600 min. Toxicity studies with FD18 (90 mg/kg, i.p. for 12 times in 22 days) with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) revealed no obvious toxicity or death in mice. In vivo efficacy studies indicated that FD18 (45 mg/kg, i.p. for 12 times in 22 days) together with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) resulted in a 46% reduction in LCC6MDR xenograft volume (n = 11; 648 ± 84 mm(3)) compared to paclitaxel control (n = 8; 1201 ± 118 mm(3)). There were no animal deaths or significant drop in body weight and vital organ wet weight. FD18 can increase paclitaxel accumulation in LCC6MDR xenograft by 1.8- to 2.2-fold. The present study suggests that FD18 represents a new class of safe and potent P-gp modulator in vivo.

Amine linked flavonoid dimers as modulators for P-glycoprotein-based multidrug resistance: structure-activity relationship and mechanism of modulation.

Here we report a great improvement in reversal potency of cancer drug resistance when flavonoid dimers possess a functionally substituted aminopolyethylene glycol linker. The most potent compound, 18, contains a N-benzyl group at the linker. It has many advantages including (1) high potencies in reversing P-glycoprotein (P-gp) mediated resistance in LCC6MDR cells to various anticancer drugs with EC(50) in the nanomolar range, (2) low toxicity and high therapeutic index, and (3) preferential inhibition of P-gp over multidrug resistance protein 1 and breast cancer resistance protein. Compound 18 stimulates P-gp-ATPase activity by 2.7-fold and mediates a dose-dependent inhibition of doxorubicin (DOX) transport activity. Lineweaver-Burk and Dixon plots suggest that 18 is a competitive inhibitor to DOX in binding to P-gp with a K(i) of 0.28-0.34 µM and a Hill coefficient of 1.17. Moreover, the LCC6MDR cell displays about 2.1-fold lower intracellular accumulation of 18 compared to the wild type, suggesting that 18 is a P-gp substrate as well.