Defying Multidrug resistance! Modulation of Related Transporters by

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Defying Multidrug resistance! Modulation of Related Transporters by Flavonoids and Flavonolignans Christopher Steven Chambers, Jitka Viktorová, Kate#ina #eho#ová, David Biedermann, Lucie Turková, T. Macek, Vladimir Kren, and Kate#ina Valentová J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Journal of Agricultural and Food Chemistry

Defying Multidrug Resistance! Modulation of Related Transporters by Flavonoids and Flavonolignans Christopher S. Chambers$,†, Jitka Viktorová$,‡, Kateřina Řehořová‡, David Biedermann†, Lucie Turková†, Tomáš Macek‡, Vladimír Křen†, Kateřina Valentová*,†

†Laboratory

of Biotransformation, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ 142 20 Prague, Czech Republic. ‡Department

of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 5, CZ 166 28, Prague, Czech Republic. *Tel.: 420-296-442-509. E-mail: [email protected]. $Both

authors contributed equally to this manuscript

1 ACS Paragon Plus Environment


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Abstract

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Multidrug resistance (MDR) is a major challenge for the 21th century in both cancer

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chemotherapy and antibiotic treatment of bacterial infections. Efflux pumps and transport

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proteins play an important role in MDR. Compounds displaying inhibitory activity toward

5

these proteins are prospective for adjuvant treatment of such conditions. Natural low-cost

6

and non-toxic flavonoids, thanks to their vast structural diversity, offer a great pool of lead

7

structures with broad possibility of chemical derivatizations. Various flavonoids were found to

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reverse both antineoplastic and bacterial multidrug resistance by inhibiting Adenosine

9

triphosphate Binding Cassette (ABC)-transporters (human P-glycoprotein, multidrug

10

resistance-associated protein MRP-1, breast cancer resistance protein and bacterial ABC

11

transporters), other bacterial drug efflux pumps: major facilitator superfamily (MFS),

12

multidrug and toxic compound extrusion (MATE), small multidrug resistance (SMR) and

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resistance-nodulation-cell-division (RND) transporters, and glucose transporters. Flavonoids

14

and particularly flavonolignans are therefore highly prospective compounds for defying

15

multidrug resistance.

16 17

Keywords:

Multidrug

resistance

(MDR);

ABC

transporters;

glucose

transporters,

18

flavonolignans; flavonoids; cancer; methicillin resistant Staphylococcus aureus (MRSA).

19


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Introduction

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The 68th World Health Assembly1 in May 2015 (Geneva, CH) endorsed a global action plan to

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tackle antimicrobial resistance and one of the major objectives was to optimize the use of

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antimicrobial medicines, including combating the antibiotic resistance.2 The discovery and

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mainly implementation of novel systemic antibiotics has a stagnant trend (1983-7, 16 new

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antibiotics; 2003-7, only four and 2010-2015, only eight3 new antibiotics were approved by

26

FDA). There has been little investment into antibiotic discovery by the pharmaceutical

27

industry, mostly because financial returns are likely to be limited and due to strict

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governmental regulations. Therefore, identification of efficient and non-toxic compounds

29

with inhibitory activity towards multi-drug resistance (MDR) associated proteins seems to be

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an effective and feasible way to tackle antibiotic resistance.

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A rich source of such efficient non-toxic biologically active molecules comes from the plant

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kingdom,4 which is producing secondary metabolites5 of various natural compounds, such as

33

the (poly)phenols. These compounds exhibit UV-protectant and radical scavenging activity in

34

the plants; some of the phenolics act as toxins or anti-feedants and general antioxidant

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protectants in case of plant injury.6 Among a range of different plant phenols,

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phenylchromanes (derivatives of flavan, or flavonoids) play important roles in plant

37

organisms, e.g. as allelochemicals (7,8-benzoflavone), germination stimulators (isovitexin),

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phytoalexins and others. In the broadest sense, flavonoids consist of a common structural

39

motif called C6-C3-C6 containing 6-membered rings attached to a 3-carbon chain

40

(phenylpropanoids) and to another 6-membered ring. This motif also include some flavonoid

41

precursors, such as chalcones (chalconoids). In a more strict sense, the flavonoid family is

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characterized by 2, 3 or 4-phenylchroman moiety also called flavane, isoflavane and

43

neoflavane, respectively (see Figure 1 for basic structural motifs).5 3 ACS Paragon Plus Environment


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Flavonoids found in species from the plantae kingdom are highly important in the human diet,

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as they are relatively potent antioxidants and chemoprotectants in vitro with generally low or

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negligible toxicity. At present, the concept of “antioxidants” is often questioned in the

47

scientific community, due to the fact that concentrations necessary for direct antioxidant

48

(redox) activity are hardly achievable in vivo. Instead, low molecular antioxidants seem to act

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as pro-oxidants, which induce intrinsic chemoprotective pathways.7 The compounds having

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antioxidant activity and proven biological effects are now being further investigated for their

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structural issues, intracellular signalization and other effects dictated by their fine structure

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and stereo- and regioisomerism. One of the up and coming topically important biological

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effects of flavonoids are their inhibitory activities towards MDR associated proteins, both in

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somatic (typically cancer) cells and in parasites or in microorganisms.8

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The first studies on the ability of some flavonoids (genistein and quercetin) to cause partial

56

reversal of MDR resistance in cancer cells appeared at the end of the previous century9, 10 and

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specific flavonoids (Figure 2) have attracted attention for their MDR interactions. The

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flavonoids, which so far were found to exhibit MDR inhibiting activities, originate from a wide

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variety of plants and their parts, ranging from flowers of chamomile plant (apigenin) up to the

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pollen of Eucalyptus globulus Labill. (tricetin). Many of them are commonly consumed dietary

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flavonoids, contained in many evolutionary distinct plant species such as quercetin (oak bark,

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onion peel, radish, cranberry and many others); apigenin (often glycosylated, found in celery,

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parsley or lemon); chrysin (found in flowers and subsequently in honey); or kaempferol (often

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glycosylated and omnipresent in diverse plant families).

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A distinct class of MDR modulating flavonoids belong among flavonolignans, formed by the

66

oxidative coupling of a flavonoid e.g. taxifolin, quercetin and luteolin with a phenylpropanoid


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(lignan) such as coniferyl alcohol or sinapyl alcohol (Figure 3), which results in etheric or C-C

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bond formation.11 One of the main sources of flavonolignans is silymarin - an extract of

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Silybum marianum (L.) Gaertn. (Asteraceae) fruits.12 Silymarin, often wrongly presented as a

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single compound,13 is a complex extract consisting of a plethora of constituents. The

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flavonolignan composition also varies substantially due to the plant variety, environmental

72

conditions, and extraction and processing methods used.14

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The major silymarin flavonolignans are derived from the flavonoid taxifolin, coupled with the

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coniferyl alcohol in a radical manner. The radical coupling, by its nature, is not stereoselective

75

giving rise to a range of diastereomers or enantiomers as summarized in Figure 4. In most

76

preparations silybin (silibinin) A and silybin B are dominant. Depending on the chemo-variety,

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silychristin A or silydianin are also abundant. Isosilybins are always minor components as is

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flavonoid taxifolin. 2,3-Dehydroflavonolignans,15 formally derived from quercetin, were

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earlier considered to be negligent minor components probably arisen by mere oxidation of

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respective flavonolignans. Nevertheless, we have demonstrated recently that these

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compounds have notable biological activities,15 often surpassing their parent flavonolignans.16

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Other types of flavonolignans, “non-taxifolin derived”, are isolated from the white variety of

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the milk thistle (from naringenin and eriodictyol) or the tropical tree Hydnocarpus wightiana

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Blume (from luteolin, called hydnocarpins,17 see Figure 4). MDR inhibition by hydnocarpins

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has been serendipitously employed in traditional medicine in the treatment of leprosy with

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chaulmoogra oil, containing hydnocarpin in combination with cyclopentenoic fatty acids. The

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combination of these antibiotics, which inhibit the multiplication of mycobacteria, together

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with hydnocarpin enabled the treatment of such a persistent and debilitating disease as

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leprosy caused by Mycobacterium leprae.17 The aim of the present review is to summarize



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published knowledge about the ability of flavonoids and flavonolignans that contain features

91

that inhibit multidrug resistance in both cancer and bacterial cells.

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Flavonoids and flavonolignans involved in oncological therapy

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Nowadays, an increasing number of tumor types exhibit resistance to current anticancer

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drugs.18 Drug resistance (antineoplastic resistance) is usually defined, as the decrease in the

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efficiency of drugs to achieve therapeutic doses in the target site. Such resistance represents

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a major challenge in the treatment and overall in the patient survival.19 Numerous

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mechanisms of drug resistance in cancer therapy have been reported: e.g. drug inactivation,

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drug target alteration, DNA damage repair, cell death inhibition, epigenetic effects and

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metastases; drug efflux is nevertheless the most studied mechanism of cancer drug

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resistance.18 The drug efflux is mostly mediated by three transmembrane transporters that

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belong to the ATP Binding Cassette (ABC) protein family. ABC proteins use the hydrolysis of

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ATP as the source of energy for the transport of various molecules outside the cell against a

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concentration gradient.20 This type of resistance extensively limits the ADMET (absorption,

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distribution, metabolism, elimination and toxicity) properties of commercial drugs.21 The ABC

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superfamily consists of ca 49 major transporters divided into seven sub-families denoted by

106

letters (ABCA-ABCG).20 Transporters P-glycoprotein (P-gp, ABCB1), multidrug resistance-

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associated protein (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2) have

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the most significant role in clinical practice (Figure 5).

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P-gp was originally discovered in 1976 in the ovary cell mutants from Chinese hamster.22 Its

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presence has now been reported in membranes of polarized cells (such as liver, colon,

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jejunum, kidney and adrenal gland)20 with secretory function and also in cells with the barrier

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function like blood-brain barrier,8 where its physiological function is to protect the body


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against xenobiotics, to transport steroid hormones, ions and secrete cytokines. P-gp has broad

114

structural and functional substrate specificity.

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MRP1 was first discovered in 1992 in pulmonary carcinoma cells 23 and later in other polarized

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cells (e.g. skin, colon, cardiac and skeletal muscles).24 It shares a similar structure to P-gp, as it

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is composed of 12 transmembrane domains, several loops and two cytosolic nucleotide-

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binding domains (NBD). The main physiological function of this transporter is to be able to

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export both hydrophilic and hydrophobic xenobiotics, the transport of glutathione (in both

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oxidized and reduced form) and also its conjugates.25 MRP1 is the main transporter

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responsible for maintaining the GSSG:GSH cytosolic balance.24

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BCRP was discovered in 1998 in the human placenta,26 and was later also found in the

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intestine, brain endothelium, prostate and the central nervous system. BCRP is unlike the

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other transporters, as the structure consists of a half-transporter with only one ATP-binding

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site and half number of transmembrane domains. However, the crystal structure has not been

126

published yet and it is predicted to at least dimerize.27 BCRP represents the first barrier for

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drug absorption in the gut and in the maternal-fetus barrier, blood-brain barrier and other

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barrier systems. Its physiological function is therefore associated with the prevention of

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spreading of xenobiotics.27

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Despite the beneficial physiological functions of these transporters, that are responsible for

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efflux of a wide range of structurally dissimilar xenobiotics, their overproduction was

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described in cancer cells as the main mechanism for drug efflux. This leads to the resistance

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to all drugs transported by the same transporter. All three pumps transport many antitumor

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drugs (doxorubicin, mitoxantrone, etoposide); moreover, some of them transport other

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anticancer drugs such as vincristine and vinblastine (P-gp, MRP1), paclitaxel and colchicine (P7 ACS Paragon Plus Environment


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gp), cisplatin (MRP1) and others.28 In order to inhibit such efflux, transporter modulators have

137

been examined. These modulators are used as co-drugs in chemosensitization, which involves

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co-administration of an anticancer drug and the drug-efflux inhibitor.29 Several modes of

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actions are supposed to be: competitive and non-competitive binding of modulators,

140

physiological changes in the lipid bilayer affecting the transport or inhibition of transporter

141

expression.20 The first generation of modulators introduced (e.g. verapamil, doxorubicin,

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cyclosporine A) were found to have great toxicity, the second generation (R verapamil,

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emopamil) was more tolerable; however, they had a low transporter-selectivity. The third

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generation (valspodar, biricodar, laniquidar, zosuquidar, elacridar, and tariquidar) has failed

145

in clinical trials showing undesirable side effects21 (for the structures see Figure 6).

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The development of the fourth generation was based on natural products, as they have

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generally lower toxicity and higher selectivity. Such compounds include the flavonoids and

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their derivatives, which were previously shown to modulate ABC transporters activity.

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Nowadays, there are several approaches for studying the effect of flavonoids/flavonolignans

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on MDR: direct cytotoxicity of flavonoid/flavonolignan on MDR cell lines (without drugs),24, 30,

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31

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of flavonoid/flavonolignan to the domain of transporter (usually NBD domain) ,34-36 computer

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modeling,37 inhibition of transcription of transporter´s gene,9, 38 or modulation of transporter

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expression on protein level38-41 Moreover, cell lines usually used for in vitro studies are often

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normal sensitive cancer cell lines transfected by transporter-cDNA resulting in drug resistance

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(e.g. U-2 OS/MRP1,42 BHK21/MRP1,36 MCF7/GSTP1-1,43 MDCKII/MRP1 and MDCKII/MRP2,43

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H69AR/Bcl-2 and HeLa/ABCC1,39 Hek-293/ABCG2,37, 44 Hek-293/ABCB1,37 Hek-293/ABCC1,37

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HOC/MRP1,44 MDCK/BCRP38, MDCK/MDR138). Only few studies have used immortalized cell

inhibition of transporters using isolated membrane fractions (out of the cells),31-33 affinity


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lines of cancer cells that physiologically express MDR transporters.9, 40, 41, 45, 46 However, this

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expression is usually induced by long co-cultivation of the cancer cell line with the respective

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drug in low concentrations to select a drug-resistant sub-line.47,

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commercial drugs with/without addition of a single dose of flavonoids is compared. This

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approach was used for several commercial drugs: doxorubicin,40,

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daunorubicin,9, 45 mitoxantrone38, 50 and vincristine51. Finally, only a few papers dealt with

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dose-dependent drug sensitization (vinblastine,52 paclitaxel,52 daunomycin44) and there is very

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limited evidence from in vivo experiments.53 The studies are therefore very heterogeneous

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and any comparison is hard to compile.

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P-glycoprotein

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Several flavonoids (genistein, epicatechin gallate, catechin gallate, epigallocatechin gallate

170

and silymarin), are able to directly bind to the P-gp substrate binding site.20 Silybin and its

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semisynthetic derivatives were also shown to modulate P-gp and to act as its efflux

172

inhibitors.54 Moreover, silymarin was reported to bind to both substrate and ATP binding sites

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of P-gp in vitro.55 However, as this is a mixture of many compounds, this evaluation is

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inappropriate and scientifically incorrect. Quercetin, chrysin, kaempferol, naringenin,

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genistein and rutin are capable of direct interaction with ATP-binding site.8 The mechanism of

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epicatechin inhibition is described as a heterotropic allosteric activation.8 On the other hand,

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isoflavones and flavanone glycosides were inactive for P-gp inhibition.8,

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previously mentioned flavonoids, i.e. epigallocatechin gallate, biochanine A and quercetin

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showed a biphasic effect when applied to resistant cells: in low concentrations (roughly 10

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µM), they stimulated the transport by P-gp pump; however, in higher doses (50-100 µM), they

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acted as typical inhibitors.8 The dose-dependent manner of P-gp inhibition was published for

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the set of flavonoids: diosmetin, fisetin, naringin and tangeritin.8 A comprehensive study was

48

Often, the IC50 of

42, 49

daunomycin,32

21, 56

Some of



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realized by Mohana et al,21 who tested 40 flavonoids for their P-gp inhibition activity based on

184

the results of SAR (structure-activity relationship) analysis. In one concentration (10 µM)

185

measurement, several flavonoids totally failed and some of them showed low potency for

186

inhibition. Moderate inhibitory activity was demonstrated by epicatechin 3-O-gallate,

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tamarixetin, naringin, silybin, myricetin, pelargonidin and high inhibitory activity was

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demonstrated by quercetin (IC50 = 7 µM), theaflavin (IC50 = 20 µM) and rutin (IC50 = 8 µM).21

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Both quercetin and rutin were able to reverse the P-gp-based resistance at concentration level

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of 10 µM21 similarly as baicalein, a flavonoid isolated e.g. from Scutellaria radix.57 Moreover,

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quercetin demonstrated its ability to prevent doxorubicin resistance development by reducing

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P-gp expression.40 Similarly, icaritin, kaempferol and naringenin demonstrated the

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downregulation of P-gp expression at transcriptional level.

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discovered as the first ABC transporter with clinical importance and the animal trials with

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flavonoids as P-gp inhibitors have been performed. It was demonstrated that rather than co-

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administration of the drug together with morin, quercetin, or silymarin, the pre-treatment

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with these compounds provides better results.8, 59 Despite the appropriate results of in vitro

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and in vivo studies focused on quercetin anticancer activity, many disadvantages of quercetin

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structure (such as low bioavailability, poor solubility, fast metabolism etc.) still persist. A

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recent comprehensive review of structure modification leading to overcoming such

201

disadvantages has shown an increased aqueous solubility for quercetin amino (e.g. glutamic)

202

acid conjugates and enhanced solubility in Dulbecco's Modified Eagle Medium (DMEM) when

203

C-7 hydroxy group was O-alkylated with methoxybutyl, pivaloylxymethyl (POM),

204

isopropyloxycarbonylmethyl (POC) groups. The stability was increased for derivatives

205

containing either mono or bis POC to over 96 hours whilst bis O-POM conjugate was stable

206

only over 24 hours. However, the 3,7-di-O-POM derivative was more stable in complete

29, 58

The P-gp transporter was


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DMEM (cDMEM), whilst 3-O-POC quercetin was the most stable derivative in both phosphate

208

buffered saline (over 96 h) and cDMEM (54 h, Figure 8).60

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Multidrug resistance-associated protein

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An increased doxorubicin accumulation in MRP1-transfected U-2 osteosarcoma cells was

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found for three flavonostilbenes (alopecurone A, B and D) isolated from Sophora

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alopecuroides (L.).42 At the non-toxic concentration (20 µM), these compounds decreased IC50

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of doxorubicin on these cells 12, 5 and 8 times, respectively. According to their qPCR analysis,

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the flavonostilbenes significantly affected the MRP1 expression.42 However, other tested

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flavonostilbenes, namely alopecurone F, sophoraflavanone G, lehmannin, liquiritin and

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luteolin did not show this expression. The transportation mediated by MRP1 was also inhibited

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by other flavonoids; namely apigenin, biochanin A, genistein, chalcone, silymarin, phloretin,

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morin, quercetin, naringenin, myricetin, chrysin and kaempferol.24, 29 The higher potency was

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detected in flavonoid dimers (e.g. apigenin dimer). The known mechanisms of action include

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the modulation of ATPase activity demonstrated by 2,3-dehydrosilybin and competition in

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substrate transport represented by flavopiridol.24 Inhibition of MRP1 was also demonstrated

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by 8-prenylnaringenin.29 Similarly, as in case of P-gp inhibition, quercetin was able to inhibit

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MRP1 expression and to reverse the resistance phenotype in gastric adenocarcinoma.61 The

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expression of both transporters was downregulated, also by kaempferol in promyelocytic

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leukemia cells62 and by icaritin in osteosarcoma cells.63 Finally, MRP1 expression and function

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was also suppressed by vitexin in colorectal cancer cells.64

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Breast cancer resistance protein

228

At present, several modulators of BCRP transporter with a flavonoid structure have been

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published based on data from cell cultures transfected with BCRP - apigenin; biochanin A;

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chrysin; chrysoeriol; daidzein; diosmetin; fisetin; genistein; hesperetin; kaempferol; laricitrin; 11 ACS Paragon Plus Environment


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luteolin; myricetin 3´,4´,5´-trimethylether; myricetin; naringenin; phloretin; quercetin; silybin;

232

tamarixetin; tricetin 3´,4´,5´-trimethylether; tricetin (reviewed in ref.27). From this long list, the

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following flavonoids are the most promising modulators: apigenin; chrysoeriol; diosmetin;

234

kaempferol; myricetin 3´,4´,5´-trimethylether; tamarixetin and tricetin 3´,4´,5´-trimethylether,

235

which have IC50 value below 0.1 µM similarly as the reference compound – Kol43 (selective

236

BCRP inhibitor, diketopiperazine structure).27

237

A prospective relatively new approach in the field of biologically active compounds represents

238

the concept of so-called hybrid compounds (hybrid molecules). These hybrids are composed

239

of two or more moieties with different modus operandi connected into a single structure. Such

240

hybrids are less sensitive to cancer cell resistance development.65 First attempts were

241

accomplished with an antioxidant and photoprotectant structure based on trans-resveratrol,

242

octyl methoxycinnamate and avobenzone subunits.66 Later, two flavonoids – genistein and

243

quercetin were used for the synthesis of a library of hybrid compounds and both showed a

244

higher anti-proliferative potency than the parent compounds upon human prostatic

245

carcinoma.30

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To conclude this chapter, the ideal structure of flavonoids with the best inhibition potential is

247

evaluated based on several SAR studies. Flavonoid structure-based modulators of P-gp

248

transporter should be hydrophobic molecules with a planar structure; with weak positive

249

charge at physiological pH; 2,3-unsaturated in the ring C and 5,3-hydroxylated.8, 21 The total

250

number of hydroxyl groups plays a vital role in the quality of inhibition, while triple

251

hydroxylated structures possess high inhibitory effect, molecules with four hydroxyl groups

252

exhibit weaker effect and pentahydroxylated structures enhance P-gp activity.8, 67


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SAR analysis suggests that the most prominent MRP-1 inhibitors should contain lavandulyl and

254

resveratrol moieties.42 For the interaction of flavonoid with NBD, C-5- and 7-hydroxy groups

255

on the A-ring as well as 2,3-double bond in ring C are important. Moreover, the number and

256

location of other hydroxy and methoxy groups significantly affect the inhibition activity.24

257

For inhibition of the BCRP transporter, the presence of a 2,3-double bond in ring C, attachment

258

of ring B to the position C-2, a hydroxyl group at the position C-5, a lack of the hydroxyl group

259

(however the presence of methoxy group is beneficial) at position C-3 and a hydrophobic

260

substituent at some of the positions C-6, C-7, C-8 or C-4′ are required (Figure 9).27

261

Glucose transporters

262

Another mechanism, by which flavonoids can affect multidrug resistant cancer cells, is their

263

effect on glucose transporters. Many types of cancer cells exhibit an increased glucose uptake

264

to satisfy the increased need for energy, which is necessary for the tumor growth

265

(approximately 30-fold higher glucose is demanded when compared with normal cells).68 One

266

new approach in cancer treatment, especially when a resistance to the standard

267

chemotherapy develops, is the active inhibition of glucose uptake as this leads to the cancer

268

cell starvation. It is during this process, that flavonoid testing and exploitation, can bring their

269

advantageous properties of which being naturally occurring compounds with no or little

270

negative side effects.

271

Glucose enters the cell via two types of glucose transport proteins (Figure 10): sodium–

272

glucose linked transporters (SGLTs) and facilitated diffusion glucose transporters (GLUTs). The

273

concentration of glucose in the cell depends on the levels of glucose transporters - the more

274

transport proteins cells express, the more glucose can be uptaken.69 The transport proteins

275

vary in kinetic and/or regulatory properties, which then enable to maintain the metabolic



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integrity at cellular, organ and consequently whole organism level.70 The expression of the

277

GLUT-1 plays a direct role in tumorigenesis, for example in the hepatocellular carcinoma.71

278

The downregulation of GLUT1 by apigenin, which is the most studied flavonoid in terms of

279

glucose transport inhibition, is thought to be responsible for its anticancer properties. This

280

flavonoid suppresses the overexpression of both GLUT1 and serine/threonine protein kinase

281

(p-Akt) in cisplatin-resistant laryngeal carcinoma cells. Consequently, it causes an increase in

282

the sensitivity to cisplatin.72 Apigenin and phloretin are the most efficient in reducing glucose

283

uptake and in modifying GLUT1 and GLUT4 levels. Genistein and daidzein were more efficient

284

in reducing glucose uptake in androgen-sensitive prostate cancer cells than in androgen-

285

insensitive cells, which was in agreement with the different GLUTs profiles in both types of

286

cells.73 Similarly, both quercetin and epigallocatechin gallate markedly decreased glucose

287

uptake in both estrogen receptor positive and negative breast carcinoma cells in a competitive

288

manner suggesting the inhibition of GLUT4.74

289

Glucose uptake by GLUT1-producing human lung cancer cells was suppressed by 30 µM

290

natural dihydrochalcone - (+)-cryptocaryone, isolated from Cryptocarya rubra. (+)-

291

Cryptocaryone also showed a cytotoxic effect towards a human colon cancer cell line with IC50

292

value

293

desmethylinfectocaryone was inactive. The (+)-desmethylinfectocaryone lacks the five-

294

membered lactone ring connected to the reduced A-ring of a flavanone unit, which seems to

295

be necessary for the cytotoxic effect.75 GLUT2 was inhibited by the flavonols myricetin, fisetin,

296

quercetin, and isoquercitrin.76 A potent inhibitor of SGLT is phloridzin,77 and its various

297

derivatives.78,

298

kushenol N, sophoraflavanone G, and kuraridin (Figure 11), as well as the methanolic extract

of

0.32

79

µM,

while

an

analogue

of

(+)-cryptocaryone,

known

as

(+)-

Some lavandulyl flavanones from Sophora flavescens, i.e. (−)-kurarinone,


Page 15 of 51


299

from the plant roots, also strongly inhibited both SGLT1 and SGLT2.80 Interestingly, SGLT1 is

300

often considered to be involved in the absorption of flavonoid glucosides in the small

301

intestine, although the efficiency of SGLT1 mediated transport is dramatically lowered by

302

subsequent efflux by MRP-2.77, 81, 82

303

In the case of flavonolignans, basal and insulin-dependent glucose uptake by 3T3-L1

304

adipocytes was dose-dependently reduced by both silybin and 2,3-dehydrosilybin, which were

305

then shown to act as competitive inhibitors of GLUT4 with Ki = 60 and 116 µM, respectively.83

306

Interestingly, 2,3-dehydrosilybin (stereomer A was somehow stronger than racemic mixture)

307

also exhibited pro-longevity properties in Caenorhabditis elegans dependent on the

308

expression of the Facilitative Glucose Transporter FGT-1, the homolog of mammalian GLUT2.84

309

Moreover, 2,3-dehydrosilybin and to a lesser extent also silybin and silychristin also decreased

310

glucose accumulation as glycogen in Mesocestoides vogae larvae.85 The potential of the

311

flavonolignans in influencing the glucose uptake by cancer cells is therefore obvious; however,

312

needs to be further explored.

313

The role of flavonoids in bacterial multidrug resistance

314

Bacterial multidrug resistance is another example of MDR and one of the most challenging

315

problems in modern medicine. The first antibiotic resistance was discovered in the 1940s,

316

which was within a few years after introduction of penicillin and since that time numerous

317

types of bacterial resistance have been described.2 It is evident especially in the developing

318

countries, where more than 50,000 people die every year as a consequence of MDR infection,

319

due to the faster spread of infection that is caused by poor hygiene conditions and

320

inappropriate antibiotic use; their low price leads to their extensive and unqualified

321

application.86



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322

Plant extracts

323

Medicinal herbs have been commonly used as natural antibiotics in traditional medicine and

324

their role has also been investigated in the fight against MDR bacteria (Table 1). These are

325

frequently isolated as shoot extracts in a polar solvent and their efficiency is compared with

326

the synthetic antibiotics. The studied plants belong to a wide range of monocotyledonous and

327

dicotyledonous families and they are characterized by high levels of secondary metabolite

328

content (such as alkaloids, flavonoids, coumarins, triterpenes, sterols, saponins etc.). For

329

example, phytochemical screening of several Cameroon plants revealed the presence of the

330

most common secondary metabolites.87-95 Many of them have been reported to have

331

antibacterial activity towards MDR bacterial strains.96 In the case of medicinal herbs,

332

antimicrobial activity against MDR bacteria is usually presented as the effect of the crude plant

333

extract, which can be very misleading as such extracts will hardly reach the target cells (see

334

the section on the bioavailability of flavonoids). Furthermore, the exact composition of the

335

extracts, the compounds responsible for the effect, content of the active compounds and the

336

mechanism of action (synergistic effect of the components, etc.) has not been described in

337

these studies. Moreover, high supra-physiological inhibition concentrations (mostly units of

338

mg/mL up to 50 mg/mL)97 and weak characterization of extracts is very limiting for the further

339

use.

340

Isolated flavonoids

341

MRSA (methicillin resistant Staphylococcus aureus) and VRE (vancomycin resistant

342

Enterococcus faecalis) are the only MDR Gram-positive bacteria that are intensively studied in

343

relation with flavonoids (Table 1 and Table 2), as they cause the most challenging infections

344

in hospitals.98 Many flavonoids, especially isoflavones (apigenin, genistein, daidzein) have

345

been excluded for low degree of antimicrobial activity against MDR G+ bacteria.99 However, 16 ACS Paragon Plus Environment

Page 17 of 51


346

luteolin with minimal inhibitory concentration (MIC) up to 100 µM against MRSA strains is a

347

promising candidate for further research.100 Luteolin contains hydroxyl substituents at

348

positions C-5, C-7, C-3′, and C-4′, which are important structural features enhancing

349

antimicrobial activity of flavonoids.101 The analogous positive effect was observed for the OH-

350

group at C-3′ and 5′ in chalcones.102 Other auspicious compounds are phenylpropanoyl flavans

351

and hydroxycinnamoylated dihydrochalcones (balsacones) showing MIC < 10 µM against 10

352

tested MRSA isolates. The presence of cinnamoyl and p-hydroxycinnamoyl moieties at

353

position C-8 and C-3 led to enhanced activity while the methoxy group had a negative

354

impact.103 In contrast, the missing methoxy group in meta position to the C-3 OH in chalcones

355

reduces their MDR-antibacterial efficiency.102 Meanwhile a different structure activity

356

relationship study showed that substitution of hydrophilic sulfonic group in position 5′ in the

357

case of quercetin and morin significantly increased the anti-MRSA activity.104

358

The mechanism of flavonoid action against MRSA has been rarely described. A prenylated

359

flavonoid artonin I inhibited MRSA efflux pumps together with depolarization of cell

360

membrane, which resulted in the loss of cell integrity, as demonstrated by TEM microscopy.105

361

The special case is the inhibition of MRSA biofilm formation that was observed by co-

362

cultivation with flavonoid aglycones (myricetin, hesperetin, phloretin) and glycosides

363

(myricitrin, hesperidin, phloridzin), nevertheless the relationship between structure and

364

inhibition is still unknown.106

365

Gram-negative bacteria have a special structure of the cell wall, differing them from Gram-

366

positive bacteria. Lipopolysaccharide outer membrane, peptidoglycan cell wall and an inner

367

membrane makes the penetration of antibiotics harder, therefore identification of an

368

alternative to ineffective antibiotic is very difficult.107 As with the Gram-positive bacteria,



Page 18 of 51

369

chalcones have shown effective degrees of antibacterial activity against MDR Gram-negative

370

bacteria. 2′,4′-Dihydroxychalcone was active against MDR Proteus mirabilis strain at supra-

371

physiological MIC 260 µM. The efficiency can be linked to carbonyl region with the OH group

372

at

373

trihydroxychalcone) showed lower activity, which demonstrates that the presence of OH

374

group at C-4 reduced the effectiveness.108

375

Pseudomonas aeruginosa is an important Gram-negative pathogen, which is troublesome due

376

to its ability to adhere to the surfaces and form biofilms. This is why it is one of the most

377

frequently tested MDR Gram-negative bacteria (Table 1 and Table 2). For example, gliricidin-

378

7-O-hexoside, quercetin-7-O-rutinoside (rutin), keampferol-3-O-rutinoside and myricetin-3-O-

379

rhamnoside inhibited the growth of planktonic cells at MIC < 10 µM.109 Similarly, rutin showed

380

the ability to inhibit P. aeruginosa biofilm formation.110

381

Flavonoids as bacterial efflux pump inhibitors (EPI)

382

In contrast to previously discussed direct toxic effects of flavonoids on MDR bacteria, an

383

alternative can be found in the reversion of MDR phenotype, followed by toxic effect of

384

previously inactive antibiotics. Up to now, if we disregard gene mutations that leads to

385

resistance, five main molecular mechanisms of bacterial quenching of antibiotics have been

386

reported (Figure 12). The first described mechanism of resistance was the efflux of tetracycline

387

out of the cells of E. coli. Since that time, five major families of bacterial efflux pumps have

388

been reported with different substrate specificity. For Gram-positive bacteria, ATP-binding

389

cassette family (ABC) transporters, the major facilitator superfamily (MFS) transporters, the

390

multidrug and toxic compound extrusion family (MATE) transporters and the small multidrug

391

resistance family (SMR) transporters are common. Both ABC and MFS transporters could be

carbon

2′

and

4′.

Surprisingly,

another

chalcone,

isoliquiritigenin

(2′,4′,4-


Page 19 of 51


392

found in Gram-negative bacteria together with the resistance-nodulation-cell-division family

393

(RND) transporters.111

394

Several flavonoids have been used as enhancers for antibiotics because many flavonoids can

395

inhibit efflux pumps. For this purpose, flavonoids do not need to be toxic in low

396

concentrations, as in the case of direct antimicrobial activity, but they must be able to assure

397

antibiotic presence in the resistant cells (due to inhibition of efflux pumps, penicillin binding

398

protein - PBP2a, or increasing the permeability of membrane) and reverse the resistance.

399

Quercetin, as the most studied flavonoid and its isomer morin were tested against multidrug

400

resistant MRSA together with a spectrum of antibiotics (β-lactams, fluoroquinolones,

401

macrolides, and tetracycline) and it was shown that both of them can inhibit the mechanism

402

of bacterial resistance.112 The same results were observed with 100 clinical isolates of MRSA,

403

but only quercetin and some other flavonoids (rutin, morin, luteolin) were effective in

404

combination with the antibiotics. The fact that flavonoids influence only the cell wall was

405

tested via potassium leakage.113-115

406

Synergistic effect of silybin and antibiotics was also confirmed. In combination with

407

ciprofloxacin or benzalkonium chloride, silybin can clearly enhance the antibiotic efficiency

408

due to efflux pump inhibition of MRSA. It was proved that silybin can reduce the expression

409

of efflux pumps genes norA and qacA/B, and reverse the MDR phenotype.116 Also

410

hydnocarpins and its derivatives were shown to efficiently inhibit the MDR efflux pump norA

411

of Staphylococcus aureus, inhibit biofilm formation by this bacterium and increase its

412

susceptibility to enrofloxacin.117, 118 Very similar results were published in the case of apigenin

413

in combination with ampicillin and ceftriaxone.115 Rutin can inhibit MDR of P. aeruginosa at

414

supra-physiological MIC 1.31 mM, but at four times lower concentration, it can be used as an



Page 20 of 51

415

efficient modulator for gentamicin resistance and inhibitor of biofilm formation.110 This effect

416

of flavonoid applicability has not been vastly explored. From the above-mentioned facts, the

417

synergistic effect of “flavonoid-antibiotic” seems to be potentially the most promising for the

418

future anti-MDR studies.

419

Bioavailability of flavonoids

420

The effect of flavonoids on MDR-associated transporters is only possible if the compounds are

421

able to reach the transporter. In other words, this depends on their bioavailability and this

422

applies especially for plant extracts. Orally ingested polyphenols can be partially absorbed

423

from the small intestine. However, as most of them are consumed as esters, glycosides or

424

even polymers, these molecules must first be hydrolyzed by intestinal enzymes, or by the

425

colonic microbiota. The absorption itself is quite limited and by then, the flavonoids are rapidly

426

metabolized by Phase II metabolism enzymes yielding methylated, sulfated and/or

427

glucuronidated metabolites.119 Therefore, most parent flavonoids are found in body fluids

428

only in nM to low µM ranges and glucuronidated, sulfated, and methylated derivatives are

429

found in plasma in often higher concentrations.120, 121 On the other hand, many human (e.g.

430

endothelial) cells harbour enzymes enabling deconjugation of phase II metabolites and

431

releasing parent flavonoids in tissues when they can exert local activity. Furthermore, efficient

432

concentrations of polyphenols can be easily achieved in the gastrointestinal tract122 or

433

topically e.g. in the skin123 with great potential for local treatment of gastrointestinal and skin

434

malignancies. Moreover, large differences in bioavailability are documented and some

435

flavonoid classes seem to be absorbed sufficiently at least at distinct populations119-121, 124, 125

436

to exert MDR-modulating effects in vivo. An alternative option is the intravenous or

437

intramuscular application of flavonoids in order to deliver them to cells without excessive

438

metabolic transformation. 20 ACS Paragon Plus Environment

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439

In conclusion, both flavonoids and flavonolignans were shown to be prospective natural low-

440

cost and non-toxic compounds able to reverse both antineoplastic and bacterial multidrug

441

resistance by inhibiting ABC and other bacterial drug efflux pumps and glucose transporters.

442

In accordance with recent consensus on antioxidants7, 126 no direct antioxidant activity but

443

rather receptor interactions seem to play a major role in MDR inhibition by flavonoids.

444

Flavonoids, as a result of their vast structural diversity, offer a great pool of lead structures

445

with a broad possibility of various chemical derivatizations. Flavonoids and particularly

446

flavonolignans are therefore highly prospective group of compounds for defying multidrug

447

resistance.

448

Abbreviations used

449

ABC, Adenosine triphosphate Binding Cassette; ADMET, absorption, distribution, metabolism,

450

elimination and toxicity; ATP, Adenosine triphosphate; DMEM, Dulbecco's Modified Eagle

451

Medium; FDA, Food and Drug Administration; FGT-1, Facilitative Glucose Transporter; GLUTs,

452

facilitated diffusion glucose transporters; MATE, multidrug and toxic compound extrusion;

453

MDR, Multidrug resistance; MFS, major facilitator superfamily; MIC, minimal inhibitory

454

concentration; MRP-1, multidrug resistance-associated protein; MRSA, methicillin

455

resistant Staphylococcus aureus; NBD, nucleotide-binding domains; p-Akt, serine/threonine

456

protein

457

pivaloylxymethyl; qPCR, quantitative polymerase chain reaction; RND, resistance-nodulation-

458

cell-division; SAR, structure-activity relationship; SGLTs, sodium–glucose linked transporters;

459

SMR, small multidrug resistance; VRE, vancomycin resistant Enterococcus faecalis.

kinase;

P-gp,

P-glycoprotein;

POC,

isopropyloxycarbonylmethyl;

POM,

References 1. 2.

https://www.who.int/mediacentre/news/releases/2015/wha-25-may-2015/en/. World Health Organisation: Global antimicrobial resistance surveillance system (GLASS) report: early implementation 2016-2017. Geneva, Switzerland. https://www.who.int/antimicrobialresistance/en/. In.



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Cole, S. P.; Bhardwaj, G.; Gerlach, J. H.; Mackie, J. E.; Grant, C. E.; Almquist, K. C.; Stewart, A. J.; Kurz, E. U.; Duncan, A. M.; Deeley, R. G., Overexpression of a transporter gene in a multidrugresistant human lung cancer cell line. Science 1992, 258, 1650-1654. Lorendeau, D.; Dury, L.; Nasr, R.; Boumendjel, A.; Teodori, E.; Gutschow, M.; Falson, P.; Di Pietro, A.; Baubichon-Cortay, H., MRP1-dependent collateral sensitivity of multidrug-resistant cancer cells: Identifying selective modulators inducing cellular glutathione depletion. Curr. Med. Chem. 2017, 24, 1186 - 1213. Cole, S. P., Multidrug resistance protein 1 (MRP1, ABCC1), a "multitasking" ATP-binding cassette (ABC) transporter. J. Biol. Chem. 2014, 289, 30880-30888. Doyle, L. A.; Yang, W.; Abruzzo, L. V.; Krogmann, T.; Gao, Y.; Rishi, A. K.; Ross, D. D., A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl. Acad. Sci. U S A 1998, 95, 15665-15670. Peña-Solórzano, D.; Stark, S. A.; König, B.; Sierra, C. A.; Ochoa-Puentes, C., ABCG2/BCRP: Specific and nonspecific modulators. Med Res Rev 2017, 37, 987-1050. Stacy, A. E.; Jansson, P. J.; Richardson, D. R., Molecular pharmacology of ABCG2 and its role in chemoresistance. Mol. Pharmacol. 2013, 84, 655-669. Abdallah, H. M.; Al-Abd, A. M.; El-Dine, R. S.; El-Halawany, A. M., P-glycoprotein inhibitors of natural origin as potential tumor chemo-sensitizers: A review. J. Adv. Res. 2015, 6, 45-62. Chen, Q. H.; Yu, K.; Zhang, X.; Chen, G.; Hoover, A.; Leon, F.; Wang, R.; Subrahmanyam, N.; Addo Mekuria, E.; Harinantenaina Rakotondraibe, L., A new class of hybrid anticancer agents inspired by the synergistic effects of curcumin and genistein: Design, synthesis, and anti-proliferative evaluation. Bioorg. Med. Chem. Lett. 2015, 25, 4553-4556. Leslie, E. M.; Deeley, R. G.; Cole, S. P., Bioflavonoid stimulation of glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1). Drug Metab. Dispos. 2003, 31, 11-5. Comte, G.; Daskiewicz, J. B.; Bayet, C.; Conseil, G.; Viornery-Vanier, A.; Dumontet, C.; Di Pietro, A.; Barron, D., C-Isoprenylation of flavonoids enhances binding affinity toward P-glycoprotein and modulation of cancer cell chemoresistance. J. Med. Chem. 2001, 44, 763-8. Maitrejean, M.; Comte, G.; Barron, D.; El Kirat, K.; Conseil, G.; Di Pietro, A., The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of Pglycoprotein. Bioorg. Med. Chem. Lett. 2000, 10, 157-60. Bois, F.; Boumendjel, A.; Mariotte, A. M.; Conseil, G.; Di Petro, A., Synthesis and biological activity of 4-alkoxy chalcones: potential hydrophobic modulators of P-glycoprotein-mediated multidrug resistance. Bioorg. Med. Chem. 1999, 7, 2691-5. Boumendjel, A.; Beney, C.; Deka, N.; Mariotte, A. M.; Lawson, M. A.; Trompier, D.; BaubichonCortay, H.; Di Pietro, A., 4-Hydroxy-6-methoxyaurones with high-affinity binding to cytosolic domain of P-glycoprotein. Chem. Pharm. Bull. (Tokyo) 2002, 50, 854-6. Trompier, D.; Baubichon-Cortay, H.; Chang, X. B.; Maitrejean, M.; Barron, D.; Riordon, J. R.; Di Pietro, A., Multiple flavonoid-binding sites within multidrug resistance protein MRP1. Cell Mol. Life Sci. 2003, 60, 2164-77. Valdameri, G.; Gauthier, C.; Terreux, R.; Kachadourian, R.; Day, B. J.; Winnischofer, S. M. B.; Rocha, M. E. M.; Frachet, V.; Ronot, X.; Di Pietro, A.; Boumendjel, A., Investigation of chalcones as selective inhibitors of the breast cancer resistance protein: critical role of methoxylation in both inhibition potency and cytotoxicity. J. Med. Chem. 2012, 55, 3193-3200. Pick, A.; Muller, H.; Mayer, R.; Haenisch, B.; Pajeva, I. K.; Weigt, M.; Bonisch, H.; Muller, C. E.; Wiese, M., Structure-activity relationships of flavonoids as inhibitors of breast cancer resistance protein (BCRP). Bioorg. Med. Chem. 2011, 19, 2090-102. Laberge, R. M.; Karwatsky, J.; Lincoln, M. C.; Leimanis, M. L.; Georges, E., Modulation of GSH levels in ABCC1 expressing tumor cells triggers apoptosis through oxidative stress. Biochem. Pharmacol. 2007, 73, 1727-37. Desrini, S.; Mustofa; Sholikhah, E. N., The effect of quercetin and doxorubicin combination in inhibiting resistance in MCF-7 cell. Bangladesh J. Med. Sci. 2017, 16, 91.



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A. P.; Rousset, F.; Ruskovska, T.; Saraiva, N.; Sasson, S.; Schröder, K.; Semen, K.; Seredenina, T.; Shakirzyanova, A.; Smith, G. L.; Soldati, T.; Sousa, B. C.; Spickett, C. M.; Stancic, A.; Stasia, M. J.; Steinbrenner, H.; Stepanić, V.; Steven, S.; Tokatlidis, K.; Tuncay, E.; Turan, B.; Ursini, F.; Vacek, J.; Vajnerova, O.; Valentová, K.; Van Breusegem, F.; Varisli, L.; Veal, E. A.; Yalçın, A. S.; Yelisyeyeva, O.; Žarković, N.; Zatloukalová, M.; Zielonka, J.; Touyz, R. M.; Papapetropoulos, A.; Grune, T.; Lamas, S.; Schmidt, H. H. H. W.; Di Lisa, F.; Daiber, A., European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS). Redox Biol. 2017, 13, 94-162. Deng, D.; Yan, N., GLUT, SGLT, and SWEET: Structural and mechanistic investigations of the glucose transporters. Protein Sci. 2016, 25, 546-558. Wright, E. M.; Loo, D. D.; Hirayama, B. A.; Turk, E., Surprising versatility of Na+-glucose cotransporters: SLC5. Physiology 2004, 19, 370-376. Khan, U. A.; Rahman, H.; Qasim, M.; Hussain, A.; Azizllah, A.; Murad, W.; Khan, Z.; Anees, M.; Adnan, M., Alkanna tinctoria leaves extracts: A prospective remedy against multidrug resistant human pathogenic bacteria. BMC Complement. Altern. Med. 2015, 15, 127. Hamdi, A.; Jaramillo-Carmona, S.; Srairi Beji, R.; Tej, R.; Zaoui, S.; Rodríguez-Arcos, R.; JiménezAraujo, A.; Kasri, M.; Lachaal, M.; Karray Bouraoui, N.; Guillén-Bejarano, R., The phytochemical and bioactivity profiles of wild Asparagus albus (L.) plant. Food Res. Int. 2017, 99, 720-729. Haque, S. M.; Chakraborty, A.; Dey, D.; Mukherjee, S.; Nayak, S.; Ghosh, B., Improved micropropagation of Bacopa monnieri (L.) Wettst. (Plantaginaceae) and antimicrobial activity of in vitro and ex vitro raised plants against multidrug-resistant clinical isolates of urinary tract infecting (UTI) and respiratory tract infecting (RTI) bacteria. Clin. Phytosci. 2017, 3, 17. Rath, S.; Padhy, R. N., Monitoring in vitro antibacterial efficacy of 26 Indian spices against multidrug resistant urinary tract infecting bacteria. Integr. Med. Res. 2014, 3, 133-141. Rath, S.; Padhy, R. N., Monitoring in vitro antibacterial efficacy of Terminalia alata Heyne ex. Roth, against MDR enteropathogenic bacteria isolated from clinical samples. J. Acute Med. 2013, 3, 93-102. Swain, S. S.; Padhy, R. N., In vitro antibacterial efficacy of plants used by an Indian aboriginal tribe against pathogenic bacteria isolated from clinical samples. J. Taibah Univ. Med. Sci. 2015, 10, 379-390. Sahu, M. C.; Padhy, R. N., In vitro antibacterial potency of Butea monosperma Lam. against 12 clinically isolated multidrug resistant bacteria. Asian Pac. J. Trop. Dis. 2013, 3, 217-226. Khomarlou, N.; Aberoomand-Azar, P.; Lashgari, A. P.; Hakakian, A.; Ranjbar, R.; Ayatollahi, S. A., Evaluation of antibacterial activity against multidrug-resistance (MDR) bacteria and antioxidant effects of the ethanolic extract and fractions of Chenopodium album (sub sp Striatum). Int. J. Pharm. Sci. Res. 2017, 8, 3696-3708. Rahman, H.; Khan, U. A.; Qasim, M.; Muhammad, N.; Khan, M. D.; Asif, M.; Azizullah, A.; Adnan, M.; Murad, W., Ethnomedicinal Cichorium intybus seed extracts: An impending preparation against multidrug resistant bacterial pathogens. Jundishapur J Microbiol 2016, 9, e35436. Siddhartha, E., Sarojamma, V. and Ramakrishna, V. , Bioactive compound rich indian spices suppresses the growth of beta-lactamase produced multidrug resistant bacteria. JKIMSU 2017, 6, 10-24. Ahumada-Santos, Y. P.; Soto-Sotomayor, M. E.; Báez-Flores, M. E.; Díaz-Camacho, S. P.; LópezAngulo, G.; Eslava-Campos, C. A.; Delgado-Vargas, F., Antibacterial synergism of Echeveria subrigida (B. L. Rob & Seaton) and commercial antibiotics against multidrug resistant Escherichia coli and Staphylococcus aureus. Eur. J. Integr. Med. 2016, 8, 638-644. Dubey, D.; Padhy, R. N., Antibacterial activity of Lantana camara L. against multidrug resistant pathogens from ICU patients of a teaching hospital. J. Herb. Med. 2013, 3, 65-75. Nayak, N.; Rath, S.; Mishra, M. P.; Ghosh, G.; Padhy, R. N., Antibacterial activity of the terrestrial fern Lygodium flexuosum (L.) Sw. against multidrug resistant enteric- and uro-pathogenic bacteria. J. Acute Dis. 2013, 2, 270-276.



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142. Shaheen, A. Y.; Sheikh, A. A.; Rabbani, M.; Aslam, A.; Bibi, T.; Liaqat, F.; Muhammad, J.; Rehmani, S. F., Antibacterial activity of herbal extracts against multi-drug resistant Escherichia coli recovered from retail chicken meat. Pak. J. Pharm. Sci. 2015, 28, 1295-1300. 143. Vambe, M., Aremu, A. O., Chukwujekwu, J. C., Finnie, J. F. and Van Staden, J. , Antibacterial screening, synergy studies and phenolic content of seven South African medicinal plants against drug-sensitive and -resistant microbial strains. South Afr. J. Bot. 2018, 144, 250-259. 144. Bernaitis, A., M., Shenoy, R. P., Mathew, J. and Khan, D. M., Comparative evaluation of the antimicrobial activity of ethanol extract of Taxus baccata, Phyllanthus debilis, Plectranthus amboinicus against multi drug resistant bacteria. Int. J. Pharm. Sci. Res. 2013, 6, 10-24. 145. Akinjogunla, O. J. a. O., A. O., Thermostability and in-vitro antibacterial activity of aqueous extracts of Tetrapleura tetraptera pods on multidrug resistant clinical isolates. Br. J. Pharm. Res. 2016, 14, 1-16. 146. Lakshmipriya, T., Soumya, T., Jayasree, P. R. and Kumar, P. R. M. , Antioxidant, antimicrobial and antiproliferative activities of leaf extracts of the indian traditional medicinal plant Wrightia arborea. Int. J. Pharm. Sci. Res. 2017, 8, 1124-1133. 147. Mbaveng, A. T.; Sandjo, L. P.; Tankeo, S. B.; Ndifor, A. R.; Pantaleon, A.; Nagdjui, B. T.; Kuete, V., Antibacterial activity of nineteen selected natural products against multi-drug resistant Gramnegative phenotypes. SpringerPlus 2015, 4, 823. 148. Bhaskar, B. V.; Mohan, A. R.; Babu, T. M. C.; Rajesh, S. S.; Bhuvaneswar, C.; Sivaraman, T.; Gunasekar, D.; Rajendra, W., Antibacterial efficacy of fractions and compounds from Indigofera barberi: Identification of DNA gyrase B inhibitors through pharmacophore based virtual screening. Process Biochem. 2016, 51, 2208-2221.

Funding The work was supported by the Czech Science Foundation project 18-00150S, by the Operational

Program

Prague-Competitiveness

projects

CZ.2.16/3.1.00/21537

and

CZ.2.16/3.1.00/24503, and by the Czech National Program of Sustainability NPU I (LO) (MSMT43760/2015).


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Figure captions Figure 1. Structures of benzylchromanes – the basic flavonoid skeletons and their precursor (chalcone). Figure 2. Selected flavonoids (flavonoid moieties in blue) with multidrug resistance modulating activity. Figure 3. Formation of flavonolignans exemplified on the reaction of coniferyl alcohol with taxifolin. N.b. only the silybins A and B are shown but the reaction yields also other products. Figure 4. Structures of selected flavonolignans (flavonoid moiety – blue; lignin part - black). Figure 5. Mechanisms of inhibition of main mammalian ABC transporters by flavonoid compounds. Mechanisms of P-gp inhibition (left): 1. Interference with binding and hydrolysis of ATP (e.g. quercetin, naringenin, genistein); 2. Blocking drug binding site/competitive substrates (e.g. genistein, silymarin); 3. Binding to the allosteric site (e.g. epicatechin). Mechanisms of MRP1 inhibition (center): 1. Competitive substrates (e.g. flavopiridol, apigenin); 2. Modulation of ATPase activity (e.g. dehydrosilybin). Mechanism of BCRP inhibition (right) is still unknown because of lack of knowledge on high-resolution crystal structure of this transporter. Figure 6. Former leads in MDR inhibition development, Abu = L-2-amino butyric acid, MeGly, MeVal, MeLeu = N-methyl amino acid. Figure 7. Ability of the flavonoid structure-based modulators to inhibit the main ABC transporters (P-gp, MRP1 and BCRP). *In contrast to other modulators, which are individual compounds, silymarin is a complex mixture of flavonoids and flavonolignans extracted from Silybum marianum (L.) Gaertn. fruits.



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Figure 8. Chemical modifications of quercetin to improve solubility and stability. Bu = butyl, POM = pivaloyloxymethyl, POC = isopropyloxycarbonylmethyl. Figure 9. Idealized minimal structures of MDR inhibitors. The role of C-4 keto group has not been conclusively clarified. In grey: optional OH group. Figure 10. Glucose transporters belonging to the solute carrier family (SLC) in humans. GLUTs - glucose transporters - facilitative diffusion mediated by up to 14 human transporters, are subdivided into three classes. Class I includes: GLUT1 - occurs in fetal tissues, erythrocytes and barrier tissues, and is responsible for basal glucose uptake required for respiration, usually upregulated during oncogenesis; GLUT2 - is expressed at a very high level in pancreatic β-cells and in the basolateral membranes of intestinal and kidney epithelial cells and of hepatocytes, and is responsible for equilibration of glucose between the extra/intracellular space and startup of insulin secretion; GLUT3 - the major neuronal glucose transporter, also expressed in placenta, lymphocytes, macrophages, and platelets; GLUT4 - adipocytes, skeletal muscles, is responsible for glucose homeostasis. SGLTs - six proteins are identified in human, among which SGLT1 and SGLT2 are the best characterized, SGLT1 is primarily expressed in intestine, while SGLT2 is highly expressed in the kidney where reabsorbs the glucose.70, 127, 128 Figure 11. Structures of SGLT inhibitory flavonoids from the roots of Sophora flavescens. Figure 12. Main mechanisms of bacterial drug resistance. 1. Bacterial multidrug efflux pumps: Transporters using the proton motive force to exclude antibiotics (ATB; inhibited e.g. by both silybin A and B). 2. Decreased uptake: Limiting the influx of ATB by changing the permeability of membrane. 3. Inactivating enzymes: ATB elimination by e.g. phosphorylation, acetylation, adenylation. 4. Target alterations: Modification of target sites (e.g. ribosomes) to avoid


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recognition by ATB. 5. Bypass pathway: The adjustment of essential synthesis pathway that is normally inhibited by ATB.



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Tables Table 1. Summary of the Published Data (2013-2018) on the Effect of Plant Extracts Containing Flavonoids on MDR Bacteria. Plant species

Alkanna tinctoria

Asparagus albus

Bacopa monnieri

Solvent extract

Secondary metabolites content

hexane

Alkaloids, Bufadienoloides, Flavonoids, Proteins, Pseudotannis, Resins, Saponins, Steroids, Tannins

ethanol acetone methanol

mixture of rutin, nicotiflorin, narcisin, q3-O-gluc, q-3,4′-digluc among other compounds

MDR bacteria (MIC) Gram-negative

Ref.

Gram-positive A. baumannii (25 mg/mL)

S. aureus (25 mg/mL)

E. coli (25 mg/mL)

129

P. aeruginosa (25 mg/mL) S. aureus (1.56 mg/mL) Str. agalactia mg/mL)

(0.78

E. coli (1.56 mg/mL)

E. coli (5-7.5 mg/mL)

Alkaloids, Flavonoids, Phenols

130

P. aeruginosa (1.56 mg/mL) 131

K. pneumoniae (2.5-15 mg/mL) A. baumannii (1.5-4.3 mg/mL) C. freundii (1.5-9.6 mg/mL)

Buchanania latifolia, Ocimum tenuiflorum, Senna xanthocarpum and Indian spices

methanol

Alkaloids, Glycosides, Resins, Terpenoids, Tannins, Flavonoids, Steroids

E. faecalis mg/mL)

(0.7-3.4

S. aureus (0.7-3.4 mg/mL)

E. aerogenes (3.4-4.3 mg/mL) E. coli (0.7-4.3 mg/mL)

132-134

K. pneumoniae (1.5-3.4 mg/mL) P. aeruginosa (0.7-9.6 mg/mL) P. mirabilis (3.4-9.6 mg/mL) P. vulgaris (3.4-9.6 mg/mL) Acinetobacter sp. (2.6 mg/mL)

Butea monosperma

methanol

Alkaloids, Flavonoids, Saponins, Tannins

E. faecalis (5.9 mg/mL)

Citrobacter sp. (2.6 mg/mL)


Chr. violaceum (5.9 mg/mL)

135

E. coli (5.9 mg/mL)


Page 35 of 51


K. pneumoniae (1.2 mg/mL) P. aeruginosa (1.2 mg/mL) Sal. typhi (0.5 mg/mL) E. aerogenes (0.1-1 mg/mL) E. cloacae (0.5-1 mg/mL)

Alkaloids, Anthocyanins, Cameroonian plants

methanol

Anthraquinones, Coumarins, Flavonoids Phenols, Saponins, Triterpenes

Tannins,

Sterols,

S. aureus (0.1-1 mg/mL)

E. coli (0.1-1 mg/mL)

87-95

K. pneumoniae (0.1-1 mg/mL) P. aeruginosa (0.1-1 mg/mL) P. stuartii (0.1-1 mg/mL) E. coli (0.3-2.5 mg/mL) Sal. enteritidis (0.6-2.5 mg/mL)

Chenopodium album

ethanol

Sal. typhimurium (0.3-2.5 mg/mL)

Flavonoids, Phenols


136

Sal. infantis (0.3-2.5 mg/mL) Sh. flexneri (0.6-2.5 mg/mL) Sh. dysenteriae (0.3-2.5 mg/mL) A. baumannii (6.3 mg/mL)

Alkaloids, Bufadienolides, Carbohydrates, Flavonoids, Gallotannins, Proteins, Resins, Saponins, Triterpenoids

S. aureus (6.5 mg/mL)

methanol

Alkaloids, Flavonoids, Glycosides, Phenols, Resins, Steroids, Tannins, Terpenoids

S. aureus mg/mL)

Echeveria subrigida

methanol

Flavonoids, Coumarins, Tannins

S. aureus (3.2 mg/mL)

Hibiscus sabdariffa

methanol

Alkaloids, Flavonoids, Phenols, Saponins

Cichorium intybus seeds

aqueous

culinary Indian

ethanol

spices

(0.02-0.5

E. coli (12.5 mg/mL)

137

P. aeruginosa (6.5 mg/mL) E. coli (0.02-0.5 mg/mL) K. pneumoniae (0.02-0.5 mg/mL) P. aeruginosa (0.02-0.5 mg/mL)

138

E. coli (0.01-0.5 mg/mL)

139

A. baumannii (25-50 mg/mL)

97



Page 36 of 51

A.baumannii (2-3 mg/mL) Lantana camara

dichloromethane a methanolb

Alkaloids,a,b, Flavonoids,ab, Glycosidesa, Saponnins,ab, Steroidsab, Tanninsa, a,b Terpenoids

E. faecalis (6 mg/mL) S. aureus (3-6 mg/mL) S. pyogenes (3-6 mg/mL)

C. freundii (3-6 mg/mL) P. aeruginosa (6-13 mg/mL)

140

P. mirabilis (3-6 mg/mL) P. vulgaris (3 mg/mL) E. aerogenes (3.1 mg/mL) E. coli (12.5 mg/mL) K. pneumoniae (6.3 mg/mL) P. aeruginosa (12.5 mg/mL) P. mirabilis (6.3 mg/mL)

141

142

Lygodium flexuosum

methanol

Glycosides, Terpenoids, Carbohydrates, Tannins, Flavonoids, Sterols

Mentha piperita

ethanol

Polyphenols, Flavonoids, Terpenoids

E. coli (1.4 mg/mL)

South African medicinal plants

methanol

Polyphenols, Flavonoids

E. coli (0.6-1 mg/mL)

E. faecalis (6.3 mg/mL) S. aureus (3.1 mg/mL)

dichloromethane

143

K. pneumoniae (0.6-1 mg/mL) A. baumannii (0.2-0.4 mg/mL)

Taxus baccata, Phyllanthus debilis,

ethanol

E. faecalis mg/mL)

Flavonoids, Lipids

(0.2-0.3


E. cloacae (0.3-0.4 mg/mL) E. coli (0.2-0.4 mg/mL)

144

K. pneumoniae (0.2-0.3 mg/mL) P. aeruginosa (0.2-0.4 mg/mL) P. rettgeri (0.25-0.3 mg/mL)

Tetrapleura tetraptera

aqueous

Alkaloids, Flavonoids, Steroids, Tannins, Terpenoids

Wrightia arborea

methanol

Polyphenols, Flavonoids

S. aureus (40 mg/mL)

E. coli (5-20 mg/mL)

145

Salmonella spp. (40 mg/mL) Klebsiella spp. (6.2 mg/mL)

146


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Table 2. List of articles (2013-2018) testing MDR bacteria and specific flavonoids/flavonolignans. Flavonoids

MDR bacteria (MIC) Gram-positive

Ref.

Gram-negative

2,4-dihydroxychalcone

P. mirabilis (0.26 mM)*

108

antalantoflavone

E. coli (0.38 mM)*

88

bidwillon

E. coli (1.26 mM)*

P. stuartii (0.63 mM)*

K. pneumonia (0.63 mM)*

88

neocyclomorusin

E. coli (0.29-0.59 mM)*



88

6α-hydroxyphaseollidin

E. coli (1.51 mM)*



88

E. coli (0.10 mM)

P. stuartii (0.02 mM)

K. pneumonia (0.02 mM)

E. cloacae (0.02 mM)

P. aeruginosa (0.79 mM)*

neobavaisoflavone

88

artonin I

S. aureus (0.04 mM)

105

balsacone

S. aureus (< 0.01 mM)

103

3′,5′-dihydroxy-1′-methoxychalcone

S. aureus (0.24-0.48 mM)*

102

1′,3′-dihydroxy-2′,5′dimethoxychalcone

S. aureus (0.05-0.42 mM)

1,5-diacetate-3′-methoxychalcone

S. aureus (0.36 mM)*

E. aerogenes (0.42 mM)*

102

102

gliricidin-7-O-hexoside

P. mirabilis (IC50=0.1 µM)

P. vulgaris (IC50=1 nM)

P. aeruginosa ( IC50=0.04 µM)

109

quercetin-7-O-rutinoside

P. mirabilis (IC50=0.01 mM)

P. vulgaris (IC50=8 nM)

P. aeruginosa ( IC50=0.01 mM)

109

luteolin

S. aureus (0.1-0.4 mM)*

quercetin

S. aureus (0.21 M)*

E. coli (0.21 M)*

P. aeruginosa (0.21 M)*

104

morin

S. aureus (0.10 M)*

E. coli (13 mM)*


104

quercetin-5′-sulfonic acid

S. aureus (0.08 M)*

E. coli (0.16 M)*


104

morin-5′-sulfonic acid

S. aureus (0.13 M)*

E. coli (0.13 M)*


104

100



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neocyclomorusin

E. coli (0.07 mM) P. stuartii (0.07 mM)

E. aerogenes (0.04-0.07 mM) E. cloacae (0.04 mM)

K. pneumoniae (0.02 mM) P. aeruginosa (0.15 mM)*

147

candidone

E. coli (0.02-0.73 mM) P. stuartii (0.18 mM)*

E. aerogenes (0.45-0.73 mM)* E. cloacae (0.72 mM)*


147

neobavaisoflavone

E. coli (0.05-0.79 mM) P. stuartii (0.02 mM)

E. aerogenes (0.10-0.79 mM) E. cloacae (0.79 mM)*


147

daidzein

E. coli (0.50 mM)* P. stuartii (0.50 mM)*

E. aerogenes (0.50-1.01 mM)* E. cloacae (1.01 mM)*

K. pneumoniae (0.50 mM)* P. aeruginosa (1.01 mM)*

147

isowighteone

E. coli (0.38 mM)*

quercetin

Citrobacter spp. (0.2 mM)*

147

E. coli (0.4 mM)*

K. pneumoniae (0.4 mM)*

148

P. aeruginosa (0.8 mM)* rutin

P. aeruginosa (1.3 mM)*

110

Gram-positive bacteria: Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes Gram-negative bacteria: Acinetobacter baumannii, Acinetobacter species, Citrobacter freundii, Citrobacter koseri, Citrobacter species, Chromobacterium violaceum, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, Salmonella enteritidis, Salmonella infantis, Salmonella species, Salmonella typhimurium, Shigella flexneri, Shigella dysenteriae *

Supra-physiological concentrations.


Page 39 of 51


Figure graphics O

O O

O flavane

isoflavane

neoflavane

chalcone

Figure 1.



Page 40 of 51

R3

R4 HO

O

R5 R2

R apigenin diosmetin fisetin hesperetin chrysin chrysoeriol kaempferol laricitrin luteolin myricetin myricetin 3',4',5'-trimethylether quercetin rutin tamarixetin tricetin tricetin 3',4',5'-trimethylеthеr

HO

R1 OH OH OH OH H OH OH OH OH OH OH OH OH OH OH OH

O R2 H OH H OH OH H OH OH OH OH OH OH α-rutinosyl OH H H

R3 H H H H H H H OH H OH OMe H H H OH OMe

R4 OH OMe OH OMe H OH OH OH OH OH OMe OH OH OH OH OMe

R5 H OH OH OH H OMe H OMe OH OH OMe OH OH OMe OH OMe OH

O

OH HO

R1

O

R R

R1 genistein biochanin A

R2

3

O

HO

O

OH

HO

O

OH O baicalein

2

R3

OH

OH

O

OH

OH H H OH H OMe

OH epigallocatechin gallate

OH

R1 HO

1

OH

O

OH

O chalcone

OH O naringenin 8-prenylnaringenin

R1 H 3-methylbut-2enyloxy

HO

OH

HO

OH

O

OH OH

HO

O

OH O

OH O phloretin

HO OH theaflavin

Figure 2.


Page 41 of 51


O HO

O

OH

O

OH O taxifolin

silybin B

OH

O

OMe trans-coniferyl alcohol

OH

OH O

plant peroxidase

OH OH

OMe

O OH

OH

HO

OH

HO

O

O OH

OH OMe OH

OH O silybin A

Figure 3.



Page 42 of 51

OH O HO

O

OH O

O OH

O

HO

O

O

OH OH O

OH OH O

isosilybin A

isosilybin B

O

OH

O O

HO

OH

O

HO

OH

OH

O

O O OH

O

HO

O

O

OH

OH O

OH O 2,3-dehydrosilychristin A

2,3-dehydrosilybin A

OH

O O

O

OH

OH

OH

OH

OH

silydianin

silychristin A

O

O

OH O

OH O

HO

OH O

O

OH

OH

HO

O OH

O

O HO

O

OH

O

OH O OH

OH O OH O

hydnocarpin

hydnocarpin D

Figure 4.


Page 43 of 51


Figure 5.



Page 44 of 51

NH2

1st Generation

O

OH

O

O

OH O

O

H

O

OH

OH O

OH

MeVal MeLeu

O

N

H

D-Ala

Val Me Leu

Ala

doxorubicine

Abu MeGly MeLeu

MeLeu

N

O

O

OH O

N

verapamil

cyclosporine A

2nd Generation

N

N

O

N

N

O

emopamil

O O

R verapamil

3rd Generation O

N N

O

O

O N

O

H

O

MeVal MeLeu

O

MeLeu

O

F O

H

N

O

Val MeGly MeLeu

Val Me Leu

valspodar N

O

OH N

H

Ala

H

O

D-Ala

O

biricodar F

N

N O N O

N H

N O O

O

O

HN

N H tariquidar

N

zosuquidar

N H

O

O

N

N

O N

elacridar

O laniquidar

O

Figure 6.


Page 45 of 51


Figure 7.



Page 46 of 51

OH Replacement with OBu, OPOM, OPOC, or OCO-NGlutamic acid

OH HO

O 7 3

OH

OH

Replacement with OPOM or OPOC

O

Figure 8.


Page 47 of 51


hydrophobic substituent site

4´

HO

8

7

A 6

O C

5

2

3

B

OH

HO

O

OH

OH

OH O idealized P-gp and MRP-1 inhibitor

OH O idealized BCRP inhibitor

Figure 9.



Page 48 of 51

Figure 10.


Page 49 of 51


(-)-kurarinone kushenol N sophoraflavanone G

R1 H CH3 H

R2 OH OCH3 OH

Figure 11.



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Figure 12.


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Graphic for table of contents


Defying Multidrug resistance! Modulation of Related Transporters by

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