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.

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.

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.

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.

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.

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.

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.

Modulation of multidrug resistance protein 1 (MRP1/ABCC1)-mediated multidrug resistance by bivalent apigenin homodimers and their derivatives.

Here we showed that bivalency approach is effective in modulating multidrug resistance protein 1 (MRP1/ABCC1)-mediated doxorubicin (DOX) and etoposide (VP16) resistance in human 2008/MRP1 ovarian carcinoma cells. Flavonoid dimers bearing five or six ethylene glycol (EG) units with 6-methyl (4e, 4f) or 7-methyl (5e, 5f) substitution on the ring A of flavonoid dimers have the highest modulating activity for DOX against MRP1 with an EC(50) ranging from 73 to 133 nM. At 0.5 microM, the flavonoid dimer 4e was sufficient to restore DOX accumulation in 2008/MRP1 to parental 2008/P level. Lineweaver-Burk and Dixon plot suggested that it is likely a competitive inhibitor of DOX transport with a K(i) = 0.2 microM. Our data suggest that flavonoid dimers have a high affinity toward binding to DOX recognition site of MRP1. This results in inhibiting DOX transport, increasing intracellular DOX retention, and finally resensitizing 2008/MRP1 to DOX. The present study demonstrates that flavonoid dimers can be employed as an effective modulator of MRP1-mediated drug resistance in cancer cells.

Quinacrine and a novel apigenin dimer can synergistically increase the pentamidine susceptibility of the protozoan parasite Leishmania.

OBJECTIVES: The aim of this study was to investigate the synergistic effect of quinacrine and a novel apigenin dimer (compound 9d) on reversing pentamidine resistance of Leishmania parasites. METHODS: Pentamidine-resistant cell lines, LePentR50 and LdAG83PentR50, were generated by gradually increasing pentamidine pressure on wild-type promastigotes. We tested the effects of different combinations of quinacrine and an apigenin dimer on modulating the pentamidine resistance levels of LePentR50 and LdAG83PentR50 using an MTS proliferation assay. We then measured the accumulation level of pentamidine using HPLC. The fractional inhibitory concentration index (FICI) method was used to evaluate the interaction between quinacrine and the apigenin dimer on reversing pentamidine resistance in Leishmania. RESULTS: LePentR50 and LdAG83PentR50 promastigotes were approximately 8.6- and 4.6-fold more resistant to pentamidine than their wild-type parents. Amastigotes derived from LePentR50 and LdAG83PentR50 were also pentamidine-resistant. We found that quinacrine can increase the susceptibility of Leishmania to pentamidine. Quinacrine, when used at 6 microM, can increase the IC(50) of pentamidine by 3.8-, 3.4-, 3.5- and 6.3-fold in wild-type Leishmania enriettii Le, LePentR50, wild-type Leishmania donovani LdAG83 and LdAG83PentR50, respectively. Quinine, quinidine and verapamil did not show any sensitizing effect. The sensitizing effect of quinacrine was: (i) dose-dependent; (ii) not associated with an increase in pentamidine accumulation; and (iii) only observed in pentamidine-resistant but not sodium stibogluconate-resistant or vinblastine-resistant parasites. Other than quinacrine, we also found that an apigenin dimer (compound 9d), previously shown to be able to inhibit ABCB1-mediated cancer drug resistance in mammalian cells, can also increase the pentamidine susceptibility of Leishmania. 9d, when used at 6 microM, can increase the IC(50) of pentamidine by 2.5-, 4.2-, 1.6- and 1.9-fold in Le, LePentR50, LdAG83 and LdAG83PentR50, respectively. Unlike quinacrine, sensitization by 9d was accompanied by an increase in pentamidine accumulation, presumably due to the inhibition of an ABC transporter. Using the FICI method, we found that quinacrine and 9d can act synergistically. When they are used in a 1:1 ratio, they sensitize LePentR50 to pentamidine by 19-fold, with an FICI of 0.48 (P < 0.005), indicating that they might act synergistically. CONCLUSIONS: Our findings support the notion that the pentamidine susceptibility of Leishmania is mediated by multiple targets. Quinacrine and apigenin dimer 9d, each inhibiting its own target, can have a synergistic effect when used together to sensitize Leishmania to pentamidine.

Flavonoid dimers as bivalent modulators for p-glycoprotein-based multidrug resistance: structure-activity relationships.

We recently described the modulatory activities of apigenin homodimers linked by ethylene glycol units in multidrug- resistant breast cancer and leukemic cells overexpressing ABCB1 (P-glycoprotein, P-gp). To further improve the potency of these dimers, a small library of flavonoid homodimers and heterodimers were synthesized, and their in vitro activity in reversing cellular resistance to paclitaxel, along with structure-activity relationships (SAR), were evaluated using a P-gp-expressing human breast cancer cell line. Among these synthesized homodimers, many showed more potent reversing activity than that of the parent compound and verapamil. Two compounds in particular showed promising reversing activity at sub-micromolar concentrations with no cytotoxic effects. Regarding SAR trends, flavonoid dimers with nonpolar and hydrophobic substituents (e.g., methyl and ethyl groups) generally showed more potent resistance-reversing activity than that of dimers with polar and hydrophilic substituents (e.g. hydroxy groups) at the C3, C6, and C7 positions, but not at C5. In terms of substituent steric bulk at C6, it was found that the flavonoid dimer with methyl groups was optimal, with bulkier substituents leading to lower reversing activity. Comparisons of flavonoid heterodimers with the corresponding homodimers revealed that the two binding sites on P-gp for flavonoid moieties are quite similar to each other. Besides paclitaxel, these new compounds also increased drug accumulation and enhanced the cytotoxicity of other cancer drugs such as doxorubicin, vincristine, and vinblastine by decreasing the IC(50) values 4-45-fold.

Flavonoid dimers as bivalent modulators for pentamidine and sodium stiboglucanate resistance in leishmania.

Drug resistance by overexpression of ATP-binding cassette (ABC) transporters is an impediment in the treatment of leishmaniasis. Flavonoids are known to reverse multidrug resistance (MDR) in Leishmania and mammalian cancers by inhibiting ABC transporters. Here, we found that synthetic flavonoid dimers with three (compound 9c) or four (compound 9d) ethylene glycol units exhibited a significantly higher reversing activity than other shorter or longer ethylene glycol-ligated dimers, with approximately 3-fold sensitization of pentamidine and sodium stibogluconate (SSG) resistance in Leishmania, respectively. This modulatory effect was dosage dependent and not observed in apigenin monomers with the linker, suggesting that the modulatory effect is due to its bivalent nature. The mechanism of reversal activity was due to increased intracellular accumulation of pentamidine and total antimony in Leishmania. Compared to other MDR modulators such as verapamil, reserpine, quinine, quinacrine, and quinidine, compounds 9c and 9d were the only agents that can reverse SSG resistance. In terms of reversing pentamidine resistance, 9c and 9d have activities comparable to those of reserpine and quinacrine. Modulators 9c and 9d exhibited reversal activity on pentamidine resistance among LeMDR1(-/-), LeMDR1(+/+), and LeMDR1-overexpressed mutants, suggesting that these modulators are specific to a non-LeMDR1 pentamidine transporter. The LeMDR1 copy number is inversely related to pentamidine resistance, suggesting that it might be involved in importing pentamidine into the mitochondria. In summary, bivalency could be a useful strategy for the development of more potent ABC transporter modulators and flavonoid dimers represent a promising reversal agent for overcoming pentamidine and SSG resistance in parasite Leishmania.

Flavonoid dimers as bivalent modulators for P-glycoprotein-based multidrug resistance: synthetic apigenin homodimers linked with defined-length poly(ethylene glycol) spacers increase drug retention and enhance chemosensitivity in resistant cancer cells.

Much effort has been spent on searching for better P-glycoprotein- (P-gp-) based multidrug resistance (MDR) modulators. Our approach was to target the binding sites of P-gp using dimers of dietary flavonoids. A series of apigenin-based flavonoid dimers, linked by poly(ethylene glycol) chains of various lengths, have been synthesized. These flavonoid dimers modulate drug chemosensitivity and retention in breast and leukemic MDR cells with the optimal number of ethylene glycol units equal to 2-4. Compound 9d bearing four ethylene glycol units increased drug accumulation in drug-resistant cells and enhanced cytotoxicity of paclitaxel, doxorubicin, daunomycin, vincristine, and vinblastine in drug-resistant breast cancer and leukemia cells in vitro, resulting in reduction of IC50 by 5-50 times. This compound also stimulated P-gp's ATPase activity by 3.3-fold. Its modulating activity was presumably by binding to the substrate binding sites of P-gp and disrupting drug efflux.