Denis Barron

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Abstract. Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodium falciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro rational screening was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

Isoprenylated flavonoids—a survey

Abstract The structural variation of isoprenylated flavonoids, including the chalcones, flavones and flavonols, as well as their dihydro derivatives are reviewed. Emphasis is mainly directed to the modification of the side attachments arising from C 5 , C 10 or C 15 groups, and the frequency of their occurrence among the different classes of flavonoids. Some aspects related to their biosynthesis and enzymology, as well as their biological/pharmacological activities are also discussed. The natural occurrences of the various classes of isoprenylated flavonoids are tabulated as part of this review.

The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of P-glycoprotein.

A new series of potential flavonoidic modulators of P-glycoprotein activity has been prepared. The flavanolignan silybin was first oxidised to dehydrosilybin and then C-alkylated with either prenyl or geranyl bromide. The resulting isoprenoid dehydrosilybins were shown to display high in vitro affinities for direct binding to P-glycoprotein, which ranged them among the best flavonoids ever tested.

Multiple flavonoid-binding sites within multidrug resistance protein MRP1.

Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N3-[α-32P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C4 (LTC4) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N3-[α-32P]ADP trapping, weakened inhibition of LTC4 transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.

ORGANOLITHIUM MEDIATED SYNTHESIS OF PRENYLCHALCONES AS POTENTIAL INHIBITORS OF CHEMORESISTANCE

A number of substituted chalcones have been prepared by a novel LiHMDS-mediated aldol condensation, the first method consistent with the use of alkali-labile protecting groups such as tert-butyldiphenylsilyl or tert-butyldimethylsilyl. Chalcone substitution by prenylation increases their binding affinity to P-glycoprotein responsible for cancer cells chemoresistance.

High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

ABSTRACT In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters.

Combination of Suboptimal Doses of Inhibitors Targeting Different Domains of LtrMDR1 Efficiently Overcomes Resistance of Leishmania spp. to Miltefosine by Inhibiting Drug Efflux

ABSTRACT Miltefosine (hexadecylphosphocholine) is the first orally active drug approved for the treatment of leishmaniasis. We have previously shown the involvement of LtrMDR1, a P-glycoprotein-like transporter belonging to the ATP-binding cassette superfamily, in miltefosine resistance in Leishmania. Here we show that overexpression of LtrMDR1 increases miltefosine efflux, leading to a decrease in drug accumulation in the parasites. Although LtrMDR1 modulation might be an efficient way to overcome this resistance, a main drawback associated with the use of P-glycoprotein inhibitors is related to their intrinsic toxicity. In order to diminish possible side effects, we have combined suboptimal doses of modulators targeting both the cytosolic and transmembrane domains of LtrMDR1. Preliminary structure-activity relationships have allowed us to design a new and potent flavonoid derivative with high affinity for the cytosolic nucleotide-binding domains. As modulators directed to the transmembrane domains, we have selected one of the most potent dihydro-β-agarofuran sesquiterpenes described, and we have also studied the effects of two of the most promising, latest-developed modulators of human P-glycoprotein, zosuquidar (LY335979) and elacridar (GF120918). The results show that this combinatorial strategy efficiently overcomes P-glycoprotein-mediated parasite miltefosine resistance by increasing intracellular miltefosine accumulation without any side effect in the parental, sensitive, Leishmania line and in different mammalian cell lines.

Rearrangement of 5-O-prenyl flavones: a regioselective access to 6-C-(1,1-dimethylallyl)- and 8-C-(3,3-dimethylallyl)-flavones

Abstract Regioselective control of the Claisen rearrangement of 7-MEM-5-prenyl chrysin was achieved by microwave irradiation. The nature of the products was influenced by the irradiation power and the type of solvent. Irradiation at 750 W in N,N-diethylaniline specifically yielded the 8-(3,3-dimethylallyl) para-rearranged product while refluxing in N,N-diethylbutylamine gave selective access to the 6-(1,1-dimethylallyl) ortho-rearranged compound.

Isoprenoid flavonoids are new leads in the modulation of chemoresistance

Flavonoid compounds are able to bind to P-glycoprotein (P-gp), a transporter involved in chemoresistance of cancer cells. This interaction involves, at least in part, two overlapping sites in the cytosolic domains of P-gp, the ATP site and a hydrophobic steroid-binding site. We have studied the structure-activity relationships toward binding to P-gp. Modification of the substitution pattern of the flavonoid ring by hydroxylation, methoxylation or introduction of nitrogen-containing substituents had little effect. On the contrary, the presence of a 3-hydroxyl (flavonols), and especially of a C-isoprenoid chain increased the affinity of flavonoids towards P-gp. More detailed examination of the interaction with the ATP site was conducted through inhibition by flavonoids of the photolabeling by radioactive 8-azido-TNP-ATP. Only simple flavonols were demonstrated to bind to the ATP site. When position 3 was free (flavones) or when a hydrophobic C-prenyl substituent was present, interaction was rather directed to the hydrophobic site. A number of flavonoid compounds were tested for their ability to modulate multidrug resistance in resistant leukemic K562/R7 cells. Again, prenyl flavonoids were potent modulators. Simple flavonoids were ineffective in this model. The beneficial effect of prenylation was lower in polyhydroxylated compounds, suggesting a crucial role of hydrophobicity in P-gp modulation.

Regioselective syntheses of 6-(1,1-dimethylallyl)- and 8-(3,3-dimethylallyl) chrysins

Abstract The first regioselective syntheses of 6-(1,1-dimethylallyl)- and 8-(3,3-dimethylallyl) chrysins have been designed. Claisen rearrangement of protected 5- O -(3,3-dimethylallyl) chrysin in N , N -diethylaniline at 200–217°C gave selective access to the 8-(3,3-dimethylallyl) isomer. Similar rearrangement in N , N -diethylbutylamine at 140–160°C, or in cycloheptane/Eu(fod) 3 at 100°C, led to the formation of the 6-(1,1-dimethylallyl) isomer. Four different protecting groups for position 7 of chrysin have been compared, and found to follow the order of interest Bz>MOM>TBDPS>MEM.