Multidrug Resistance in Mammals and Fungi-From MDR to PDR: A Rocky Road from Atomic Structures to Transport Mechanisms.

Multidrug resistance (MDR) can be a serious complication for the treatment of cancer as well as for microbial and parasitic infections. Dysregulated overexpression of several members of the ATP-binding cassette transporter families have been intimately linked to MDR phenomena. Three paradigm ABC transporter members, ABCB1 (P-gp), ABCC1 (MRP1) and ABCG2 (BCRP) appear to act as brothers in arms in promoting or causing MDR in a variety of therapeutic cancer settings. However, their molecular mechanisms of action, the basis for their broad and overlapping substrate selectivity, remains ill-posed. The rapidly increasing numbers of high-resolution atomic structures from X-ray crystallography or cryo-EM of mammalian ABC multidrug transporters initiated a new era towards a better understanding of structure-function relationships, and for the dynamics and mechanisms driving their transport cycles. In addition, the atomic structures offered new evolutionary perspectives in cases where transport systems have been structurally conserved from bacteria to humans, including the pleiotropic drug resistance (PDR) family in fungal pathogens for which high resolution structures are as yet unavailable. In this review, we will focus the discussion on comparative mechanisms of mammalian ABCG and fungal PDR transporters, owing to their close evolutionary relationships. In fact, the atomic structures of ABCG2 offer excellent models for a better understanding of fungal PDR transporters. Based on comparative structural models of ABCG transporters and fungal PDRs, we propose closely related or even conserved catalytic cycles, thus offering new therapeutic perspectives for preventing MDR in infectious disease settings.

Flagging Drugs That Inhibit the Bile Salt Export Pump.

The bile salt export pump (BSEP) is an ABC-transporter expressed at the canalicular membrane of hepatocytes. Its physiological role is to expel bile salts into the canaliculi from where they drain into the bile duct. Inhibition of this transporter may lead to intrahepatic cholestasis. Predictive computational models of BSEP inhibition may allow for fast identification of potentially harmful compounds in large databases. This article presents a predictive in silico model based on physicochemical descriptors that is able to flag compounds as potential BSEP inhibitors. This model was built using a training set of 670 compounds with available BSEP inhibition potencies. It successfully predicted BSEP inhibition for two independent test sets and was in a further step used for a virtual screening experiment. After in vitro testing of selected candidates, a marketed drug, bromocriptin, was identified for the first time as BSEP inhibitor. This demonstrates the usefulness of the model to identify new BSEP inhibitors and therefore potential cholestasis perpetrators.

Pore-exposed tyrosine residues of P-glycoprotein are important hydrogen-bonding partners for drugs.

The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein.

Influence of cell isolation method on the optimization of CD4+ T cell expansion using anti-CD3/CD28 coated beads.

BACKGROUNDS: Activation of CD4+ T lymphocytes with anti-CD3/CD28 coated magnetic beads promotes intrinsic resistance to HIV as well as cell expansion. The propose of this study is to define the optimal cell isolation protocol for expansion of CD4+ T lymphocytes by using anti-CD3/CD28 coated bead stimulation with an ultimate goal of using these cells for adoptive immunotherapy. METHODS: CD4+ T cells were isolated from healthy donor blood samples using three different methods including immunorosette formation, negative selection and CD8 depletion using immunomagnetic beads. These cells were activated with anti-CD3/CD28 coated beads at a bead to cell ratio of 1:1 and cell expansion was carried for 3 weeks. Cell numbers, cell viability and phenotypic characterization were determined by trypan blue exclusion and flow cytometry. RESULTS: Purified CD4+ T lymphocytes which were isolated via immunorosette formation can be expanded up to 1000-fold within 3 weeks with high viability (90%and high purity of CD4+ T lymphocytes (>95%). However, cell expansion from purified CD4+ T lymphocytes which were isolated by negative selection and CD8-depletion provided approximately 300-fold expansion. CONCLUSIONS: The results demonstrate that purified CD4+ T lymphocytes from immunorosette formation provided the highest CD4+ T lymphocyte expansion when stimulated with anti-CD3/CD28 coated beads. This method can be used to obtain a large number of expanded CD4+ T cells for adoptive immunotherapy.

Establishment of stable cell lines with high expression of heterodimers of human 4F2hc and human amino acid transporter LAT1 or LAT2 and delineation of their differential interaction with α-alkyl moieties.

System L is a major transport system for cellular uptake of neutral amino acids. Among system L transporters, L-type amino acid transporter 1 (LAT1) is responsible for the nutrient uptake in cancer cells, whereas L-type amino acid transporter 2 (LAT2) is a transporter for non-cancer cells. In this study, we have established HEK293 cell lines stably expressing high levels of human LAT1 and LAT2 forming heterodimers with native human 4F2hc of the cells. We have found that L-[(14)C]alanine is an appropriate substrate to examine the function of LAT2, whereas L-[(14)C]leucine is used for LAT1. By using L-[(14)C]alanine on LAT2, we have for the first time directly evaluated the function of human LAT2 expressed in mammalian cells and obtained its reliable kinetics. Using α-alkyl amino acids including α-methyl-alanine and α-ethyl-L-alanine, we have demonstrated that α-alkyl groups interfere with the interaction with LAT2. These cell lines with higher practical advantages would be useful for screening and analyzing compounds to develop LAT1-specific drugs that can be used for cancer diagnosis and therapeutics. The strategy that we took to establish the cell lines would also be applicable to the other heterodimeric transporters with important therapeutic implications.

The first intracellular loop is essential for the catalytic cycle of the human ABCG2 multidrug resistance transporter.

The human multidrug transporter ABCG2 is required for physiological detoxification and mediates anticancer drug resistance. Here, we identify pivotal residues in the first intracellular loop (ICL1), constituting an intrinsic part of the transmission interface. The architecture includes a triple helical bundle formed by the hot spot helix of the nucleotide-binding domain, the elbow helix, and ICL1. We show here that the highly conserved ICL1 residues G462, Y463, and Y464 are essential for the proper cross talk of the closed nucleotide-binding domain dimer with the transmembrane domains. Hence, ICL1 acts as a molecular spring, triggering the conformational switch of ABCG2 before substrate extrusion. These data suggest that the ABCG2 transmission interface may offer therapeutic options for the treatment of drug-resistant malignancies.

Picky ABCG5/G8 and promiscuous ABCG2 - a tale of fatty diets and drug toxicity.

Structural data on ABCG5/G8 and ABCG2 reveal a unique molecular architecture for subfamily G ATP-binding cassette (ABCG) transporters and disclose putative substrate-binding sites. ABCG5/G8 and ABCG2 appear to use several unique structural motifs to execute transport, including the triple helical bundles, the membrane-embedded polar relay, the re-entry helices, and a hydrophobic valve. Interestingly, ABCG2 shows extreme substrate promiscuity, whereas ABCG5/G8 transports only sterol molecules. ABCG2 structures suggest a large internal cavity, serving as a binding region for substrates and inhibitors, while mutational and pharmacological analyses support the notion of multiple binding sites. By contrast, ABCG5/G8 shows a collapsed cavity of insufficient size to hold substrates. Indeed, mutational analyses indicate a sterol-binding site at the hydrophobic interface between the transporter and the lipid bilayer. In this review, we highlight key differences and similarities between ABCG2 and ABCG5/G8 structures. We further discuss the relevance of distinct and shared structural features in the context of their physiological functions. Finally, we elaborate on how ABCG2 and ABCG5/G8 could pave the way for studies on other ABCG transporters.

The ABCG2 multidrug transporter is a pump gated by a valve and an extracellular lid.

The human ATP-binding cassette transporter ABCG2 is a key to anticancer resistance and physiological detoxification. However, the molecular mechanism of substrate transport remains enigmatic. A hydrophobic di-leucine motif in the ABCG2 core separates a large intracellular cavity from a smaller upper cavity. We show that the di-leucine motif acts as a valve that controls drug extrusion. Moreover, the extracellular structure engages the re-entry helix and all extracellular loops to form a roof architecture on top of the upper cavity. Disulfide bridges and a salt bridge limit roof flexibility, but provide a lid-like function to control drug release. We propose that drug translocation from the central to the upper cavities through the valve is driven by a squeezing motion, suggesting that ABCG2 operates similar to a peristaltic pump. Finally, the roof contains essential residues, offering therapeutic options to block ABCG2 by either targeting the valve or essential residues in the roof.

The structure of the human ABC transporter ABCG2 reveals a novel mechanism for drug extrusion.

The human ABC transporter ABCG2 (Breast Cancer Resistance Protein, BCRP) is implicated in anticancer resistance, in detoxification across barriers and linked to gout. Here, we generate a novel atomic model of ABCG2 using the crystal structure of ABCG5/G8. Extensive mutagenesis verifies the structure, disclosing hitherto unrecognized essential residues and domains in the homodimeric ABCG2 transporter. The elbow helix, the first intracellular loop (ICL1) and the nucleotide-binding domain (NBD) constitute pivotal elements of the architecture building the transmission interface that borders a central cavity which acts as a drug trap. The transmission interface is stabilized by salt-bridge interactions between the elbow helix and ICL1, as well as within ICL1, which is essential to control the conformational switch of ABCG2 to the outward-open drug-releasing conformation. Importantly, we propose that ICL1 operates like a molecular spring that holds the NBD dimer close to the membrane, thereby enabling efficient coupling of ATP hydrolysis during the catalytic cycle. These novel mechanistic data open new opportunities to therapeutically target ABCG2 in the context of related diseases.

L-glutamate enhances methylmercury toxicity by synergistically increasing oxidative stress.

Methylmercury (MeHg) is a well-known environmental toxicant. With its lipophilic nature and high reactivity to sulfhydryl groups, it is widely distributed and accumulated in the body to damage cells. Oxidative stress is proposed as a major mechanism underlying the cytotoxic action of MeHg. In the present study, we found that L-glutamate (L-Glu) concentration-dependently increased MeHg cytotoxicity in HeLa S3 cells. The enhancement of the toxicity was accompanied by enhanced apoptosis, increased production of reactive oxygen species, and decreased glutathione level. An anti-oxidant N-acetylcysteine largely alleviated the cytotoxicity, suggesting enhanced oxidative stress behind L-Glu-elicited increase of MeHg toxicity. The effect was specific to L-Glu and L-alpha-aminoadipate, whereas D-Glu, L-aspartate, and D-aspartate were not effective. In addition, the cystine uptake by the cells was mostly mediated by a L-Glu/L-alpha-aminoadipate-sensitive amino acid transport system x(-)(C). All these results suggest that the inhibition of system x(-)(C) by L-Glu underlies the enhancement of MeHg cytotoxicity. The enhancement was highly synergistic because MeHg and L-Glu alone had little toxic effect in the conditions used. This synergism was confirmed in neural cells (neuroblastoma cell lines). It is proposed that similar mechanisms may underlie the neural toxicity of MeHg, particularly in the locality of lesions characteristic of MeHg toxicity.