Identification of isothiazolones analogues as potent bactericidal agents against antibiotic resistant CRE and MRSA strains.

Carbapenem-resistant Enterobacterales (CRE) has emerged as a worldwide spread nosocomial superbug exhibiting antimicrobial resistance (AMR) to all current antibiotics, leaving limited options for treating its infection. To discovery novel antibiotics against CRE, we designed and synthesized a series of 14 isothiazol-3(2H)-one analogues subjected to antibacterial activity evaluation against Escherichia coli (E. coli) BL21 (NDM-1) and clinical strain E. coli HN88 for investigating their structure-activity relationships (SAR). The results suggested that 5-chloroisothiazolone core with an N-(4-chlorophenyl) substitution 5a was the most potent antibacterial activity against the E. coli BL21 (NDM-1) with MIC value of less than 0.032 µg/mL, which was at least 8000-fold higher than the positive control Meropenem (MRM). It also displayed 2048-fold potent than the positive control MRM against E. coli HN88. Additionally, SAR analysis supported the conclusion that compounds with a chloro-group substituted on the 5-position of the heterocyclic ring was much more potent than other positions. The board spectrum analysis suggested that compound 5a showed a promising antimicrobial activity on MRSA and CRE pathogens. Meanwhile, cytotoxicity study of compound 5a suggested that it had a therapeutic index value of 875, suggesting future therapeutic potential. In vivo efficacy study declared that compound 5a could also protect the BALB/c mice against American type culture collection (ATCC) 43,300. Further screening of our compounds against a collection of CRE strains isolated from patients indicated that compound 5 g displayed much stronger antibacterial activity compared with MRM. In conclusion, our studies indicated that isothiazolones analogues could be potent bactericidal agents against CRE and MRSA pathogens.

A novel ESKAPE-sensitive peptide with enhanced stability and its application in controlling multiple bacterial contaminations in chilled fresh pork.

The co-existence of various pathogenic bacteria on the surface of pork products exacerbates difficulties in food safety control. Developing broad-spectrum and stable antibacterial agents that are not antibiotics is an unmet need. To address this issue, all l-arginine residues of a reported peptide (IIRR)4-NH2 (zp80) were substituted with the corresponding D enantiomers. This novel peptide (IIrr)4-NH2 (zp80r) was expected to maintain favourable bioactivity against ESKAPE strains and have enhanced proteolytic stability compared with zp80. In a series of experiments, zp80r maintained favourable bioactivities against starvation-induced persisters. Electron microscopy and fluorescent dye assays were used to verify the antibacterial mechanism of zp80r. Importantly, zp80r reduced bacterial colonies in chilled fresh pork contaminated with multiple bacterial species. This newly designed peptide is a potential antibacterial candidate to combat problematic foodborne pathogens during storage of pork.

C-terminal modification of a de novo designed antimicrobial peptide via capping of macrolactam rings.

In this work, by capping a macrolactam ring at the C-terminus of a de novo-designed peptide, namely zp80, we have constructed a small peptide library via the solid phase peptide synthesis for screening. Eight peptides bearing different aspartic acid-rich macrolactam rings but the same linear (IIRR)4 unit exhibited improved antibacterial activities, hemolytic activity, and selectivity index. Mechanistic studies revealed that they could destroy the integrity of bacterial envelope, leading to cytoplasm leakage and rapid dissipation of membrane potential. One of these peptides, zp90 with a macrolactam ring of (KaDGD), demonstrated preferential interaction with calcium ions at a stoichiometric ratio of 1:1, promoting the affinity of designed peptides to bacterial membrane. Overall, this work provides a feasible strategy for medicinal chemists to further develop potent, selective, and multifunctional de novo-designed antimicrobial peptides.

Flavonoid Monomers as Potent, Nontoxic, and Selective Modulators of the Breast Cancer Resistance Protein (ABCG2).

We synthesize various substituted triazole-containing flavonoids and identify potent, nontoxic, and highly selective BCRP inhibitors. Ac18Az8, Ac32Az19, and Ac36Az9 possess m-methoxycarbonylbenzyloxy substitution at C-3 of the flavone moiety and substituted triazole at C-4' of the B-ring. They show low toxicity (IC50 toward L929 > 100 µM), potent BCRP-inhibitory activity (EC50 = 1-15 nM), and high BCRP selectivity (BCRP selectivity over MRP1 and P-gp > 67-714). They inhibit the efflux activity of BCRP, elevate the intracellular drug accumulation, and restore the drug sensitivity of BCRP-overexpressing cells. Like Ko143, Ac32Az19 remarkably exhibits a 100% 5D3 shift, indicating that it can bind and cause a conformational change of BCRP. Moreover, it significantly reduces the abundance of functional BCRP dimers/oligomers by half to retain more mitoxantrone in the BCRP-overexpressing cell line and that may account for its inhibitory activity. They are promising candidates to be developed into combination therapy to overcome MDR cancers with BCRP overexpression.

An ornithine-rich dodecapeptide with improved proteolytic stability selectively kills gram-negative food-borne pathogens and its action mode on Escherichia coli O157:H7.

Food-borne pathogenic bacteria are dispersed throughout the entire chain of the food industry. However, many food preservatives are limited by poor biocompatibility such as cumulative poisoning. The antimicrobial peptide is increasingly regarded as a promising preservative in food research due to its high bioactivity and low cytotoxicity. In this study, thirteen peptides were designed, synthesized, and screened for application as food preservatives. One of them, termed zp65, whose sequence is GIOAOIIIOIOO-NH2, demonstrated potent bactericidal effect against common Gram-negative strains including enterohemorrhagic Escherichia coli, Salmonella, and Citrobacter freundii. Encouragingly, zp65 showed negligible cytotoxicity to both mammalian cells and Galleria mellonella larvae. Peptide zp65 was prone to form α-helix structure in amphiphilic environments, facilitating its affinity with bacterial membrane. Furthermore, the proteolytic stability of zp65 was much higher than its derivatives consisting of totally natural amino acids. Isothermal titration calorimetry indicated that zp65 has a strong binding affinity to lipopolysaccharide with Kd = 1.3 µM, suggesting its possible action target on the bacterial envelope. Mechanistic studies revealed that this peptide also influenced the membrane potential of E.coli O157:H7 (O157) in a dose-dependent manner. Surprisingly, peptide zp65 did not induce disruption of membrane permeability even at a higher concentration of 4-fold minimal inhibitory concentration. By employing confocal microscopy, peptide zp65 labeled by fluorescein isothiocyanate mainly aggregated on the bacterial membrane. These results suggested that the bactericidal mode of action of zp65 is likely attributed to depolarization of the cell membrane. The minced lean beef experiment indicated that the maximum reduction of O157 reached 1.46 log colony-forming unit (CFU) per gram on day 1 after zp65 treatment at the dosage of 40 µg/g. Compared with the untreated cooked beef sample, the CFU of the zp65-treated group remained at a much lower level after 10-day storage. Subsequently, treatment with zp65 at concentrations above 32 µM also significantly reduced O157 viable counts in fresh tomato juice. And the zp65 treatment could rescue about 40% of Galleria mellonella larvae injected with O157-contaminated tomato juice. The peptide zp65 exhibits great potential and deserves further study as a candidate for food preservative.

Investigation of antibiofilm activity, antibacterial activity, and mechanistic studies of an amphiphilic peptide against Acinetobacter baumannii.

Biofilm-producing pathogens, such as Acinetobacter baumannii, have aroused escalating attention. Because these bacteria could secrete mixture with close-knit architecture and complicated components to resist traditional antibiotics. Here, we reported an amphiphilic peptide denoted as zp3 (GIIAGIIIKIKK-NH2), which showed favorable bioactivity against Acinetobacter baumannii ATCC 19606 (minimal inhibitory concentration, MIC = 4 µM) and low cytotoxicity to mammalian cells Vero (half maximal inhibitory concentration, IC50 > 100 µM). Importantly, zp3 could inhibit the formation of biofilm at micromole level and eliminate around 50% preformed biofilm at 32 µM after 6 h treatment. This peptide was able to bind with biofilm while maintaining a helical structure in a mimic biofilm-rich environment. In vivo test demonstrated that zp3 rescued 33.3% of larvae after 48 h infection and reduced 1 log live bacteria inside the animal body after 6 h treatment. The bactericidal mode for zp3 was attributed to the combination of influencing ions balance at low concentration and inducing permeability alteration and pore formation on the Acinetobacter baumannii membrane at high concentration. Application on medical textiles also proved that zp3 could perform a good antibacterial activity in practice.

Bioisosteric investigation of ebselen: Synthesis and in vitro characterization of 1,2-benzisothiazol-3(2H)-one derivatives as potent New Delhi metallo-ß-lactamase inhibitors.

Carbapenem-resistant Enterobacteriaceae (CRE) producing New Delhi metallo-ß-lactamase (NDM-1) cause untreatable bacterial infections, posing a significant threat to human health. In the present study, by employing the concept of bioisosteric replacement of the selenium moiety of ebselen, we have designed, synthesized and characterized a small compound library of 2-substituted 1,2-benzisothiazol-3(2H)-one derivatives and related compounds for evaluating their cytotoxicity and synergistic activity in combination with meropenem against the E. coli Tg1 (NDM-1) strain. The most promising compound 3a demonstrated potent synergistic activity against a panel of clinically isolated NDM-1 positive CRE strains with FICI as low as 0.09. Moreover, its IC50 value and inhibition mechanism were also confirmed by using the enzyme inhibition assay and the ESI-MS analysis respectively. Importantly, compound 3a has acceptable toxicity and is not a PAINS. Because of its structural simplicity and potent synergistic activity in combination with meropenem, we propose that compound 3a may be a promising meropenem adjuvant and a new series of such compounds may worth further investigations.

Hydrophobic substituents on isatin derivatives enhance their inhibition against bacterial peptidoglycan glycosyltransferase activity.

Moenomycin A, the well-known natural product inhibitor of peptidoglycan glycosyltransferase (PGT), is a large amphiphilic molecule of molecular mass of 1583 g/mol and its bioavailablity as a drug is relatively poor. In searching for small-molecule ligands with high inhibition ability targeting the enzyme, we found that the addition of hydrophobic groups to an isatin-based inhibitor of bacterial PGT significantly improves its inhibition against the enzyme, as well as its antibacterial activity. The improvement in enzymatic inhibition can be attributed to a better binding of the small molecule inhibitor to the hydrophobic region of the membrane-bound bacterial cell wall synthesis enzyme and the plasma membrane. In the present study, a total of 20 new amphiphilic compounds were systematically designed and the relationship between molecular hydrophobicity and the antibacterial activity by targeting at PGT was demonstrated. The in vitro lipid II transglycosylation inhibitory effects (IC50) against E. coli PBP1b and MICs of the compounds were investigated. Optimized results including MIC values of 6 µg/mL for MSSA, MRSA, B. subtilis and 12 µg/mL for E. coli were obtained with an isatin derivative 5m which has a molecular mass of 335 g/mol.

De Novo Designed Hexadecapeptides Synergize Glycopeptide Antibiotics Vancomycin and Teicoplanin against Pathogenic Klebsiella pneumoniae via Disruption of Cell Permeability and Potential.

Given the worldwide prevalence of pathogenic drug-resistant bacteria and the slow pace of new antibacterial development, discovering new uses for approved drugs that are outside the scope of the original indication is increasingly becoming an attractive proposition. In this work, seven linear cationic hexadecapeptides were designed, synthesized, and characterized. These amphiphilic peptides are able to transform from the random coil structure in water to α-helix in SDS solution and have only modest bioactivity to limited bacterial strains when used alone. Surprisingly, one of them, namely, zp16, was found to demonstrate significant synergy with vancomycin and teicoplanin against highly pathogenic Klebsiella pneumoniae (KP) with FIC index as low as 0.03. Checkerboard assay indicated that, in the presence of 8 µM zp16, the minimum inhibitory concentration (MIC) of vancomycin greatly was reduced from >128 to 1 µM to clinically isolated carbapenem-resistant KP94. Additionally, the vancomycin-zp16 combination exhibited neglectable toxicity in vitro and in vivo. Further efficacy studies confirmed that the survival rate of a combination therapy at 100 mg/kg of zp16 and vancomycin was 30% higher than that of the single-drug treatment. More importantly, drug resistance has not developed to the combination even at the 20th serial passage of KP1088. Mechanistic studies revealed that zp16 could strengthen the vancomycin's influence on cell permeability and potential, leading to markedly reduced biofilm formation and rapid bactericidal effect. This new combination strategy expands the antibacterial spectrum of glycopeptide antibiotics and opens a new research direction for their application to treat pathogenic KP infection.

Triazole Bridged Flavonoid Dimers as Potent, Nontoxic, and Highly Selective Breast Cancer Resistance Protein (BCRP/ABCG2) Inhibitors.

The present work describes the syntheses of diverse triazole bridged flavonoid dimers and identifies potent, nontoxic, and highly selective BCRP inhibitors. A homodimer, Ac22(Az8)2, with m-methoxycarbonylbenzyloxy substitution at C-3 of the flavone moieties and a bis-triazole-containing linker (21 atoms between the two flavones) showed low toxicity (IC50 toward L929, 3T3, and HFF-1 > 100 µM), potent BCRP-inhibitory activity (EC50 = 1-2 nM), and high BCRP selectivity (BCRP selectivity over MRP1 and P-gp > 455-909). Ac22(Az8)2 inhibits BCRP-ATPase activity, blocks the drug efflux activity of BCRP, elevates the intracellular drug accumulation, and finally restores the drug sensitivity of BCRP-overexpressing cells. It does not down-regulate the surface BCRP protein expression to enhance the drug retention. Therefore, Ac22(Az8)2 and similar flavonoid dimers appear to be promising candidates for further development into combination therapy to overcome MDR cancers with BCRP overexpression.

Phenol-Soluble-Modulin-Inspired Amphipathic Peptides Have Bactericidal Activity against Multidrug-Resistant Bacteria.

Phenol-soluble modulins (PSMs) are a large family of cytolytic peptide toxins produced by Staphylococcus aureus. Based on their amino acid sequences, we have constructed a small library of cationic isoleucine-rich peptides for antimicrobial evaluation. Relative to the parent PSMs, peptide zp3 (GIIAGIIIKIKK-NH2 ) was found to possess greatly improved physicochemical properties (soluble in water) and antibacterial activity (MIC=8 µm for E. coli, B. subtilis, and C. freundii) while maintaining low hemolytic activity (<5 % at 256 µm) and cytotoxicity (HEK293 cells IC50 >80 µm). We reasoned that the selective activity of zp3 toward bacterial cells is due to its amphiphilic nature and positive net charge. Moreover, it is difficult for bacteria to develop resistance against zp3. Through microscopic studies of E. coli, we demonstrated that zp3 can penetrate the bacterial membrane, thereby causing leakage of the bacterial cytoplasm. Our findings present a promising antimicrobial peptide lead, which has great potential for further chemical modification.

Antibacterial activity and mechanism of action of a thiophenyl substituted pyrimidine derivative.

The issue of multidrug resistant bacteria is a worldwide health threat. To develop new antibacterial agents with new mechanisms of action is thus an urgent request to address this antibiotic resistance crisis. In the present study, a new thiophenyl-pyrimidine derivative was prepared and utilized as an effective antibacterial agent against Gram-positive strains. In the tests against MRSA and VREs, the compound showed higher antibacterial potency than that of vancomycin and methicillin. The mode of action is probably attributed to the effective inhibition of FtsZ polymerization, GTPase activity, and bacterial cell division, which cause bactericidal effects. The compound could be a potential candidate for further development as an effective antibiotic to combat drug-resistant bacteria.

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.

Design, synthesis and antibacterial evaluation of 2,4-disubstituted-6-thiophenyl-pyrimidines.

The increasing incidences of multidrug resistant bacterial infections urge the development of novel antibacterial having a new mechanism of action. The small molecule-based inhibitors targeting at the cell division protein FtsZ has been recognized as a promising approach to search for new antibacterial with high potency. In the present study, a series of novel 2,4-disubstituted-6-thiophenyl-pyrimidine derivatives were synthesized and their antibacterial activities against clinically related pathogens were investigated. The compounds show strong antibacterial activities against MRSA and VREs. The antibacterial activity of compound Bb2 against MRSA and VREs (MIC values: 2 µg/mL) is stronger than that of methicillin and vancomycin. From the in vitro and in vivo results, Bb2 was found to inhibit GTPase activity and FtsZ polymerization. The compound is able to inhibit bacterial cell division through interacting with GTP binding site of FtsZ and thus causing cell death. In addition, S. aureus was found to develop resistance to methicillin but not for Bb2, which was proved in our resistance generation experiments.

Discovery of Novel Flavonoid Dimers To Reverse Multidrug Resistance Protein 1 (MRP1, ABCC1) Mediated Drug Resistance in Cancers Using a High Throughput Platform with "Click Chemistry".

A 300-member flavonoid dimer library of multidrug resistance-associated protein 1 (MRP1, ABCC1) modulators was rapidly assembled using "click chemistry". Subsequent high-throughput screening has led to the discovery of highly potent (EC50 ranging from 53 to 298 nM) and safe (selective indexes ranging from >190 to >1887) MRP1 modulators. Some dimers have potency about 6.5- to 36-fold and 64- to 358-fold higher than the well-known MRP1 inhibitors, verapamil, and MK571, respectively. They inhibited DOX efflux and restored intracellular DOX concentration. The most potent modulator, Ac3Az11, was predicted to bind to the bipartite substrate-binding site of MRP1 in a competitive manner. Moreover, it provided sufficient concentration to maintain its plasma level above its in vitro EC50 (53 nM for DOX) for about 90 min. Overall, we demonstrate that "click chemistry" coupled with high throughput screening is a rapid, reliable, and efficient tool in the discovery of compounds having potent MRP1-modualting activity.

Investigation of synergistic antimicrobial effects of the drug combinations of meropenem and 1,2-benzisoselenazol-3(2H)-one derivatives on carbapenem-resistant Enterobacteriaceae producing NDM-1.

The worldwide prevalence of NDM-1-producing bacteria has drastically undermined the clinical efficacy of the last line antibiotic of carbapenems, prompting a need to devise effective strategy to preserve their clinical value. Our previous studies have shown that ebselen can restore the efficacy of meropenem against a laboratory strain that produces NDM-1. Here we report the construction of a focused compound library of 1,2-benzisoselenazol-3(2H)-one derivatives which comprise a total of forty-six candidate compounds. The structure-activity relationship of these compounds and their potential to serve as an adjuvant to enhance the antimicrobial efficacy of meropenem against a collection of clinical NDM-1-producing carbapenem-resistant Enterobacteriaceae isolates was examined. Drug combination assays indicated that these derivatives exhibited synergistic antimicrobial activity when used along with meropenem, effectively restoring the activity of carbapenems against the resistant strains tested in a Galleria mellonella larvae in vivo infection model. The mode of inhibition of one compound, namely 11_a38, which was depicted when tested on the purified NDM-1 enzyme, indicated that it could covalently bind to the enzyme and displaced one zinc ion from the active site. Overall, this study provides a novel 1,2-benzisoselenazol-3(2H)-one scaffold that exhibits strong synergistic antimicrobial activity with carbapenems, and low cytotoxicity. The prospect of application of such compounds as carbapenem adjuvants warrants further evaluation.

Modified Penicillin Molecule with Carbapenem-Like Stereochemistry Specifically Inhibits Class C ß-Lactamases.

Bacterial ß-lactamases readily inactivate most penicillins and cephalosporins by hydrolyzing and "opening" their signature ß-lactam ring. In contrast, carbapenems resist hydrolysis by many serine-based class A, C, and D ß-lactamases due to their unique stereochemical features. To improve the resistance profile of penicillins, we synthesized a modified penicillin molecule, MPC-1, by "grafting" carbapenem-like stereochemistry onto the penicillin core. Chemical modifications include the trans conformation of hydrogen atoms at C-5 and C-6 instead of cis, and a 6-α hydroxyethyl moiety to replace the original 6-ß aminoacyl group. MPC-1 selectively inhibits class C ß-lactamases, such as P99, by forming a nonhydrolyzable acyl adduct, and its inhibitory potency is ∼2 to 5 times higher than that for clinically used ß-lactamase inhibitors clavulanate and sulbactam. The crystal structure of MPC-1 forming the acyl adduct with P99 reveals a novel binding mode for MPC-1 that resembles carbapenem bound in the active site of class A ß-lactamases. Furthermore, in this novel binding mode, the carboxyl group of MPC-1 blocks the deacylation reaction by occluding the critical catalytic water molecule and renders the acyl adduct nonhydrolyzable. Our results suggest that by incorporating carbapenem-like stereochemistry, the current collection of over 100 penicillins and cephalosporins can be modified into candidate compounds for development of novel ß-lactamase inhibitors.

Efficient Synthesis of Amine-Linked 2,4,6-Trisubstituted Pyrimidines as a New Class of Bacterial FtsZ Inhibitors.

We have recently identified a new class of filamenting temperature-sensitive mutant Z (FtsZ)-interacting compounds that possess a 2,4,6-trisubstituted pyrimidine-quinuclidine scaffold with moderate antibacterial activity. Employing this scaffold as a molecular template, a compound library of amine-linked 2,4,6-trisubstituted pyrimidines with 99 candidates was successfully established by employing an efficient convergent synthesis designed to explore their structure-activity relationship. The results of minimum inhibitory concentration (MIC) assay against Staphylococcus aureus strains and cytotoxicity assay against the mouse L929 cell line identified those compounds with potent antistaphylococcal properties (MIC ranges from 3 to 8 µg/mL) and some extent of cytotoxicity against normal cells (IC50 ranges from 6 to 27 µM). Importantly, three compounds also exhibited potent antibacterial activities against nine clinically isolated methicillin-resistant S. aureus (MRSA) strains. One of the compounds, 14av_amine16, exhibited low spontaneous frequency of resistance, low toxicity against Galleria mellonella larvae, and the ability to rescue G. mellonella larvae (20% survival rate at a dosage of 100 mg/kg) infected with a lethal dose of MRSA ATCC 43300 strain. Biological characterization of compound 14av_amine16 by saturation transfer difference NMR, light scattering assay, and guanosine triphosphatase hydrolysis assay with purified S. aureus FtsZ protein verified that it interacted with the FtsZ protein. Such a property of FtsZ inhibitors was further confirmed by observing iconic filamentous cell phenotype and mislocalization of the Z-ring formation of Bacillus subtilis. Taken together, these 2,4,6-trisubstituted pyrimidine derivatives represent a novel scaffold of S. aureus FtsZ inhibitors.

Flavonoid dimers are highly potent killers of multidrug resistant cancer cells overexpressing MRP1.

MRP1 overexpression in multidrug-resistant cancer cells has been shown to be responsible for collateral sensitivity to some flavonoids that stimulate a huge MRP1-mediated GSH efflux. This massive GSH depletion triggers the death of these cancer cells. We describe here that bivalent flavonoid dimers strikingly stimulate such MRP1-mediated GSH efflux and trigger a 50-100 fold more potent cell death than their corresponding monomers. This selective and massive cell death of MRP1-overexpressing cells (both transfected and drug-selected cell lines) is no longer observed either upon catalytic inactivation of MRP1 or its knockdown by siRNA. The best flavonoid dimer, 4e, kills MRP1-overexpressing cells with a selective ratio higher than 1000 compared to control cells and an EC50 value of 0.1 µM, so far unequaled as a collateral sensitivity agent targeting ABC transporters. This result portends the flavonoid dimer 4e as a very promising compound to appraise in vivo the therapeutic potential of collateral sensitivity for eradication of MRP1-overexpressing chemoresistant cancer cells in tumors.

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.