**1. Introduction**

#### **1.1 Multidrug resistance (MDR)**

Bacterial evolution tends to respond to the selection constraint of reckless antibiotic use, which has led to the emergence of drug-resistant strains mediated by

varied defense mechanisms. The main mechanisms whereby infectious agents develop resistance to antimicrobial chemotherapy include enzymatic inactivation, modification of the drug target(s), and reduction of intracellular drug concentration by changes in membrane permeability or by the overexpression of efflux pumps [1]. Multidrug resistance efflux pumps are recognized as an important component of resistance in both Gram-positive and Gram-negative bacteria [2]. Some bacterial efflux pumps may be selective for one substrate or transport antibiotics of different classes, conferring a multidrug resistance phenotype. With respect to efflux pumps, they provide a self-defense mechanism whereby antibiotics are extruded from the cell interior to the external environment. This results in sublethal drug concentrations at the active site that in turn may predispose the organism to the development of high-level target-based resistance [3]. Therefore, efflux pumps are viable antibacterial targets and the development of potent efflux pump inhibitors is a promising and valid strategy to rejuvenate the activity of antibiotics that are no longer effective against bacterial pathogens. The world is searching for new tools to combat multidrug resistance.

#### **1.2 P-glycoprotein (P-gp)**

ATP-binding cassette (ABC) transporters are found in all phyla and constitute one of the largest protein superfamilies. ABC transporters such as ABCB1 (P-glycoprotein/ P-gp), ABCG2, and ABCC1 are well known for their association with multidrug resistance, effluxing structurally diverse compounds, powered by the hydrolysis of ATP [4]. P-gp also plays an important role in the pharmacokinetics of many drugs, altering their absorption, distribution, and excretion. P-gp has been extensively studied since 1976, when it was identified as the multidrug efflux pump in Chinese hamster ovary cells that had been selected for resistance to colchicine [3].

In eukaryotes, it takes the form of a single polypeptide chain consisting of two transmembrane domains (TMDs) that are usually arranged into six transmembrane-spanning α-helices that form the pathway through which substrate crosses the membrane. These domains also form the substrate-binding site (or sites) which contribute to transport specificity. The two nucleotide-binding domains (NBDs) couple the energy of ATP catalysis to transport [5]. In some prokaryotes, however, the P-gp structure comprises a monomeric assembly, namely, a single TMD and a single NTD. The various domains can comprise one, two, or four polypeptide chains, encoded by the same or different genes, which assemble into monomers, homo- or heterodimers, or tetramers.

Prokaryotes harbor both importers for nutrient uptake (including amino acids, sugars, and metal ions) and exporters (drugs, toxins, polysaccharides, lipids, and proteins), whereas eukaryotes harbor only exporters [6]. It is believed that this transporter functions through an alternate access mechanism involving two different conformations. Drug binding occurs to the inward-facing from the cytoplasm or the inner leaflet of the bilayer. After binding two molecules of MgATP, the nucleotide-binding domains (NBDs) dimerize and switch the transmembrane domain (TMDs) from the inward- to the outward-facing conformation, followed by the release of the drug to the extracellular milieu. ATP hydrolysis, ADP/Pi release, and NBD dissociation reset the transporter to the inward-facing conformation. The switch from inward to outward form certainly requires a highly flexible structure [4, 7, 8].

Substrate "promiscuity" or polyspecificity is a well-known characteristic of P-gp and the subject of much research. Attempts have been made to understand the ability of P-gp to recognize various chemically and structurally diverse substrates

through biochemical investigations and structural studies. Despite all these studies, the molecular basis of this unusual property still remains poorly understood and is a matter of intense debate [9].
