Biochemistry. 2015 Oct 13;54(40):6195-206.

Allocrite Sensing and Binding by the Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1).

 

Yanyan Xu1,2*, Estefanía Egido1,3,4*, Xiaochun Li-Blatter1, Rita Müller1, Gracia Merino3,4, Simon Bernèche1,2, and Anna Seelig1

*Both authors contributed equally

1University of Basel, Biozentrum, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland

2SIB Swiss Institute of Bioinformatics, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland

3INDEGSAL, Campus Vegazana s/n, University of Leon, 24071 Leon, Spain

4Department of Biomedical Sciences – Physiology, Veterinary Faculty, Campus Vegazana s/n, University of Leon, 24071 Leon, Spain

 

Abstract:

The ATP binding cassette (ABC) transporters ABCG2 and ABCB1 perform ATP hydrolysis-dependent efflux of structurally highly diverse compounds, collectively called allocrites. Whereas much is known on allocrite-ABCB1 interactions, the chemical nature and strength of ABCG2-allocrite interactions have not yet been assessed. We quantified and characterized allocrite interactions with ABCG2 and ABCB1 using a set of 39 diverse compounds. We also investigated potential allocrite binding sites based on available transporter structures and structural models. We demonstrate that ABCG2 binds its allocrites from the lipid membrane, despite their hydrophilicity. Hence, allocrite binding to both transporters is a two-step process, starting with a lipid-water partitioning step, driven by hydrophobic interactions, followed by a transporter binding step in the lipid membrane. We show that allocrite binding to both transporters increases with the number of hydrogen bond acceptors in allocrites. Scrutinizing the transporter translocation pathways revealed ample hydrogen bond donors for allocrite binding. Importantly, the hydrogen bond donor strength is, on average, higher in ABCG2 than in ABCB1, which explains the higher measured allocrite affinity to ABCG2. π – π stacking and π – cation interactions play additional roles in allocrite binding to ABCG2 and ABCB1. With the present analysis we demonstrate that these membrane-mediated weak electrostatic interactions between transporters and allocrites allow for transporter promiscuity towards allocrites. In addition, we show that the different hydrogen bond donor strengths in the two transporters allow for affinity tuning. The different sensitivities of the transporters to allocrites’ charge and amphiphilicity provide transporter specificity.

PubMed ID: 26381710

 

Supplement:

Multidrug Resistance Caused by ABCB1 and ABCG2. Multidrug resistance (MDR) is defined as “simultaneous resistance to several structurally unrelated drugs that do not have a common mechanism of action” (5). MDR can arise from impaired drug delivery to cancer cells resulting from (i) poor absorption of orally administered drugs, (ii) increased drug metabolism and (iii) increased excretion, yielding lower levels of drug in the blood and thus reduced diffusion of drugs from the blood into the tumor mass. Moreover, resistance can arise in cancer cells themselves, due to genetic and epigenetic alterations that affect drug sensitivity (5).

MDR is strongly dependent on ATP binding cassette (ABC) transporters. Here we discuss the interaction of the two ABC transporters, ABCB1 (P-glycoprotein, MDR1) and ABCG2 (breast cancer resistance protein, BCRP) with their allocrites. The transporters are present at high concentrations in cell membranes of critical absorptive barriers such as the intestinal and the blood-brain barrier, as well as in excretory barriers in the liver and kidney. Both transporters capture drugs in the cytosolic leaflet of the plasma membrane and move them back to the extracellular leaflet, using the energy of ATP hydrolysis. Whereas drugs approach absorptive plasma membrane barriers from the extracellular side, they access excretory plasma membrane barriers from the cytosolic side. Active efflux thus counteracts passive influx and reduces or even inhibits drug uptake in the former, whereas it enhances drug excretion in the latter. Understanding the mechanisms underlying drug recognition by the two transporters is therefore of prime importance.

 

 FIG1AND 2

Figure 1A is reprinted in part with permission from Biochemistry 2015, 54, 6195-6206.  Copyright 2015 American Chemical Society

 

 

Drug-Transporter Interactions are Promiscuous – Both transporters capture drugs when they cross the lipid bilayer, and in particular when they reach the cytosolic membrane leaflet. Drug capturing works essentially via weak electrostatic interactions such as hydrogen bond formation between hydrogen bond donors (HBDs) in TMDs of the transporters (Fig. 1A) and hydrogen bond acceptors (HBAs) in drugs and/or π – π stacking interactions between π-electron systems in TMDs (Fig. 1B) and π-electron systems in drugs (Fig. 2A, B) (4,6).

The numerous HBDs and π-electron systems in the TMDs of the two transporters provide a large variety of transient binding possibilities for drugs (Fig. 1A, B). One transporter can thus capture drugs of different size and nature, provided they exhibit HBAs (Fig. 2A, B), which leads to its promiscuity towards drugs. As the two transporters capture drugs by the same basic mechanisms there is also promiscuity between the different transporters, or to put it differently there is “overlapping substrate specificity”.

Notably, the weak electrostatic interactions between drugs and the TMDs are particularly strong in the lipid environment, and weak in the aqueous environment. In contrast, water-soluble proteins such as serum albumin, which has been compared with ABCB1 because of its promiscuity (7), bind drugs in the aqueous phase by strong electrostatic or hydrophobic interactions.

Affinity Tuning – We demonstrated experimentally that the low affinity of typical ABCG2 allocrites for the lipid membrane is compensated by a high affinity to the transporter. The higher affinity of drugs to ABCG2 than to ABCB1 can be explained by the different HBDs in the TMDs of the two transporters. As seen in Fig. 3, ABCG2 exhibits more amino acids with strong hydrogen bond donor strength, decreasing in the order Cys > Tyr > Trp >Asn > Gln > Ser > Thr, than ABCB1. The abundance of phenyl groups in TMDs is also displayed in Fig. 3. In addition to π – π stacking interactions, phenyl groups can also engage in π – cation interactions, which is particularly relevant for ABCB1 with its many cationic allocrites.

 

 FIG3

Fig. 3. The abundance of the different amino acid residues with HBD side groups and phenyl residues in the TMDs of ABCB1 (human) (1) and ABCG2 (human) (2,3) are compared. Amino acid residues in x-axis are listed in the order of decreasing HBD strength. Amino acids of ABCB1 and ABCG2 (dimer) are indicated in blue and red, respectively. Reprinted with permission from Biochemistry 2015, 54, 6195-6206.  Copyright 2015 American Chemical Society

 

 

Specificity – Molecules in the cytosolic membrane leaflet are oriented with their polar part towards the interfacial membrane region. By diffusing laterally they may encounter charged residues in the protein’s interfacial region. With 15 cationic and 7 anionic residues, ABCB1 exhibits a high interfacial charge density (Fig. 4). The access routes to transmembrane helices TM 6 and TM 12, assumed to be most relevant for flopping or transport by ABCB1, are gated by a combination of anionic and cationic residues. This may explain why compounds with lower pKa values (i.e. stronger negative charge) are excluded from the transporter, unless they carry sufficient HBAs which can over-compensate the repulsive forces by hydrogen bond formation. Compared to ABCB1, ABCG2 with only two cationic residues (not shown) exhibits a distinctly lower putative interfacial charge density, which may explain the higher promiscuity of ABCG2 towards highly charged allocrites, including zwitterionic, double cationic, double anionic drugs (8). The present analysis suggests that charge-charge interactions play a particular role in sensing and gating, and to a lower extent also in allocrite binding. The network of possible attractive and repulsive electrostatic interactions differs in the two proteins, and gives rise to allocrite specificity.

 

FIG4Fig. 4. Charged residues of ABCB1 at the cytosolic membrane interface (NBDs are truncated). Representation of membrane position, as well as coloring of helices is the same as in Fig. 1A. Cationic amino acid residues are shown in blue, whereas anionic amino acid residues are in red. Reprinted in part with permission from Biochemistry 2015, 54, 6195-6206.  Copyright 2015 American Chemical Society

 

Energetic Cost for Drug Efflux by ABCB1 and ABCG2 – The efflux of one drug molecule by ABCB1 or ABCG2 requires the energy of hydrolysis of at least one ATP molecule (9). The question arises, why the cell spends so much energy to prevent entry of compounds carrying hydrogen bond acceptor patterns (Fig. 2A, B) into the cytosol? A likely answer is that preventing access of compounds that could interfere with DNA is worth the cost (4). We therefore propose that “multidrug resistance (MDR) defined as simultaneous resistance to a large number of structurally and functionally unrelated drugs” (5), is due to their common HBA patterns that could interfere with the genetic information of the cell (4). Variants of ABC transporters such as ABCB1 and ABCG2 evolved to capture these compounds in the lipid environment by attracting them by weak electrostatic interactions, and move them to the outer membrane leaflet. Variants of the metabolic enzyme cytochrome P450 capture drugs by similar mechanisms and further support MDR.

Overcoming or Circumventing MDR – Compounds carrying type II units (Fig 2B) are frequent among anticancer drugs and antibiotics and boost the expression of ABC transporters (4) at the epigenetic level (inducers of ABC tranporters). By this mechanism MDR can become intractable, particularly, if drugs have to cross several membrane barriers with upregulated ABC transporter levels, before reaching their target.

To overcome MDR due to ABC transporters, inhibitors have been used. Although, transporter inhibition generally works well in experimental settings with single cells, it often fails in more complex systems. A major difficulty in complex systems is reaching local drug concentrations that are sufficiently high for transporter inhibition (8). Local drug applications have been discussed and may be a future solution to the problem. So far the most successful strategy is synthesizing small hydrophobic, amphiphilic molecules which cross membranes by rapid diffusion and hence can escape the transporters (10).

 

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