**1. Introduction**

Multidrug resistance (MDR) remains a problem for the chemotherapy of cancer patients [1,2]. Polyspecific efflux transporters of the plasma membrane that prevent the accumulation of a range of drugs to cytotoxic levels are a common cause of MDR [3,4]. The primary transporter associated with failure of chemotherapy is ABCB1 (previously known as P-glycoprotein and MDR1). Outside of the cancer clinic, ABCB1 is an important determinant of drug absorption, distribution, and excretion of many drugs including antibiotics, anti-epileptics and antiarrhythmics due to its native expression in a range of tissues including the apical membranes of gut epithelia and endothelial cells of the blood-brain barrier, and the canalicular membrane of the liver hepatocytes [5]. The polyspecificity of ABCB1 for many drugs of different structure and chemical class is a key feature of the transporter that needs to be understood to allow for drug designs which avoid recognition by the transporter and for the design of specific inhibitors. In this regard, the recent report by Alam et al. [6] of the structure of ABCB1 in complex with a transport substrate, the anticancer drug taxol, represents a milestone in its field. The complex was solved by single particle imaging using cryoelectron microscopy (cryoEM) with the taxol molecule occluded within the transmembrane domains of the transporter. Twelve amino acids were involved in drug coordination. These were primarily via weak Van der Waals interactions. However,

**Citation:** Sasitharan, K.; Iqbal, H.A.; Bifsa, F.; Olszewska, A.; Linton, K.J. ABCB1 Does Not Require the Side-Chain Hydrogen-Bond Donors Gln347, Gln725, Gln<sup>990</sup> to Confer Cellular Resistance to the Anticancer Drug Taxol. *Int. J. Mol. Sci.* **2021**, *22*, 8561. https://doi.org/10.3390/ ijms22168561

Academic Editor: Jose J.G. Marin

Received: 28 May 2021 Accepted: 28 July 2021 Published: 9 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

three glutamines, Gln347, Gln725, and Gln990, located respectively in transmembrane helix (TMH) 6, TMH7, and TMH12, were highlighted to form hydrogen bond contacts (Figure 1). The study was not without limitations. The medium resolution of 3.5 Å, the low level of functional activity of the transporter within the nanodisc particles and the trapping of the transporter conformation using an inhibitory antibody may impact the physiological significance of the findings. The affect these have on the veracity of structural detail remain unclear and the implications of the binding pocket were not tested further.

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**Figure 1.** The taxol binding pocket. Ribbon depiction of ABCB1 with taxol occluded by the transmembrane domains (TMD1, blue-turquoise spectrum; TMD2, green-orange spectrum). The nucleotide binding domains, NBD1 and NBD2, are shown in green and red, respectively (pdb: 6QEX). The right-hand panel shows a 12Å slice in the Z plane of the taxol binding site. The three glutamines Gln347, Gln725, and Gln<sup>990</sup> highlighted by Alam et al. [6] to hydrogen bond (dashed grey lines) with the taxol are show in single letter code and stick format with the bond lengths (N-O) indicated in black in Ångstroms. The combination of bond angle and length suggests that Gln<sup>725</sup> forms the strongest and only H-bond with the baccatin III tetracyclic ring, while Gln<sup>347</sup> and Gln<sup>990</sup> form weaker H-bonds with the carbonyl and hydroxyl, respectively, which link to the diphenolic tail of the drug.

Prior structure-activity studies, where "activity" refers to whether the one hundred chemicals that were tested are transport substrates of ABCB1 or not, had already suggested the importance of multiple free electron pairs (and their spatial pattern) in defining the transport substrates [7–9]. Taxol is particularly rich in these structural motifs with six pairs (or triplets) of acceptor sites separated by 2.5 or 4.6 Å, respectively, available for electrostatic interaction with the transporter. Two of these motifs were observed to form hydrogen bonds in the structure determined by Alam et al. (Gln<sup>347</sup> and Gln<sup>990</sup> coordinate the different oxygens within the same motif). Taken together, the simplest interpretation of these earlier structure-activity relationship data and the recent empirical structural data is that the three glutamines are likely key to drug recognition.

In the current study, we asked whether any or all of the glutamines are necessary for triggering the transport cycle and whether they have a role in the polyspecificity of the transporter. The glutamines were replaced with alanine residues to create single, paired and triple mutants. We then measured the effect on the transport efficiency of three different classes of drug in transiently-transfected cells and tested whether the mutants are able to confer resistance to taxol after stable expression in Flp-In cells.

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