The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Structure, function, and inhibition of catalytically asymmetric ABC transporters: Lessons from the PDR subfamily.

ATP-binding cassette (ABC) superfamily comprises a large group of ubiquitous transmembrane proteins that play a crucial role in transporting a diverse spectrum of substrates across cellular membranes. They participate in a wide array of physiological and pathological processes including nutrient uptake, antigen presentation, toxin elimination, and drug resistance in cancer and microbial cells. ABC transporters couple ATP binding and hydrolysis to undergo conformational changes allowing substrate translocation. Within this superfamily, a set of ABC transporters has lost the capacity to hydrolyze ATP at one of their nucleotide-binding sites (NBS), called the non-catalytic NBS, whose importance became evident with extensive biochemistry carried out on yeast pleiotropic drug resistance (PDR) transporters. Recent single-particle cryogenic electron microscopy (cryo-EM) advances have further catapulted our understanding of the architecture of these pumps. We provide here a comprehensive overview of the structural and functional aspects of catalytically asymmetric ABC pumps with an emphasis on the PDR subfamily. Furthermore, given the increasing evidence of efflux-mediated antifungal resistance in clinical settings, we also discuss potential grounds to explore PDR transporters as therapeutic targets.

Synthesis and Anticancer Cytotoxicity of Azaaurones Overcoming Multidrug Resistance.

The resistance of tumors against anticancer drugs is a major impediment for chemotherapy. Tumors often develop multidrug resistance as a result of the cellular efflux of chemotherapeutic agents by ABC transporters such as P-glycoprotein (ABCB1/P-gp), Multidrug Resistance Protein 1 (ABCC1/MRP1), or Breast Cancer Resistance Protein (ABCG2/BCRP). By screening a chemolibrary comprising 140 compounds, we identified a set of naturally occurring aurones inducing higher cytotoxicity against P-gp-overexpressing multidrug-resistant (MDR) cells versus sensitive (parental, non-P-gp-overexpressing) cells. Follow-up studies conducted with the P-gp inhibitor tariquidar indicated that the MDR-selective toxicity of azaaurones is not mediated by P-gp. Azaaurone analogs possessing pronounced effects were then designed and synthesized. The knowledge gained from structure-activity relationships will pave the way for the design of a new class of anticancer drugs selectively targeting multidrug-resistant cancer cells.

5-Oxo-hexahydroquinoline derivatives as modulators of P-gp, MRP1 and BCRP transporters to overcome multidrug resistance in cancer cells.

Multidrug resistance (MDR) in cancer cells is often associated with overexpression of ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1) and breast cancer resistance protein (BCRP/ABCG2). Modulators of these transporters might be helpful in overcoming MDR. Moreover, exploiting collateral sensitivity (CS) could be another approach for efficient treatment of cancer. Twelve novel 5-oxo-hexahydroquinoline derivatives bearing different aromatic substitutions at C4, while having 2-pyridyl alkyl carboxylate substituents at the C3 were synthesized and evaluated for MDR reversal activity by flow cytometric determination of rhodamine 123, calcein and mitoxantrone accumulations in P-gp, MRP1 and BCRP-overexpressing cell lines, respectively. Furthermore, to confirm the P-gp inhibitory activity, the effect of compounds on the reduction of doxorubicin's IC50 of drug-resistant human uterine sarcoma cell line, MES-SA/DX5, was evaluated. Compounds D6, D5 and D3 (bearing 3-chlorophenyl, 2,3-dichlorophenyl and 4-chlorophenyl substituents at C4 position of 5-oxo-hexahydroquinoline core) were the most potent P-gp, MRP1 and BCRP inhibitors, respectively, causing significant MDR reversal at concentrations of 1-10 µM. Additionally, D4 (containing 3-flourophenyl) was the most effective MRP1-dependent CS inducing agent. Overall, chlorine containing compounds D6, C4 and D3 were capable of significant inhibition of all 3 important efflux pumps in cancer cells. Moreover, D6 also induced CS triggered by reducing glutathione efflux. In conclusion, some of the 5-oxo-hexahydroquinoline derivatives are effective efflux pump inhibitors capable of simultaneously blocking 3 important ABC transporters involved in MDR, and represent promising agents to overcome MDR in cancer cells.

Peroxisomal ATP-binding cassette transporters form mainly tetramers.

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane.

W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans.

Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Here, we report suppressor analysis on the drug-susceptible transmembrane domain mutant V532D that identified the suppressor mutation W1038S, close to the D-loop of the non-catalytic ATP-binding site. Introduction of the W1038S mutation in the background of V532D mutant conferred resistance for most of the substrates to the latter. Such restoration is accompanied by a severe reduction of ATPase activity, of about 85%, while that of the V532D mutant is half-reduced. Conversely, alanine substitution of the highly conserved aspartate D1033A in that D-loop rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity, suggesting altogether that ATP hydrolysis may not hold the key to restoration mechanism. Analysis of the ABCG5/ABCG8-based 3D-model of Cdr1p suggested that the W1038S substitution leads to the loss of hydrophobic interactions and H-bond with residues of the neighbor NBD1, in the non-catalytic ATP-binding site area. The compensatory effect within TMDs accounting for transport restoration in the V532D-W1038S variant may, therefore, be mainly due to an increase in NBDs mobility at the non-catalytic interface.

Methyl-cyclopentadienyl Ruthenium Compounds with 2,2'-Bipyridine Derivatives Display Strong Anticancer Activity and Multidrug Resistance Potential.

New ruthenium methyl-cyclopentadienyl compounds bearing bipyridine derivatives with the general formula [Ru(η5-MeCp)(PPh3)(4,4'-R-2,2'-bpy)]+ (Ru1, R = H; Ru2, R = CH3; and Ru3, R = CH2OH) have been synthesized and characterized by spectroscopic and analytical techniques. Ru1 crystallized in the monoclinic P21/ c, Ru2 in the triclinic P1̅, and Ru3 in the monoclinic P21/ n space group. In all molecular structures, the ruthenium center adopts a "piano stool" distribution. Density functional theory calculations were performed for all complexes, and the results support spectroscopic data. Ru1 and Ru3 were poor substrates of the main multidrug resistance human pumps, ABCB1, ABCG2, ABCC1, and ABCC2, while Ru2 displayed inhibitory properties of ABCC1 and ABCC2 pumps. Importantly, all compounds displayed a very high cytotoxic profile for ovarian cancer cells (sensitive and resistant) that was much more pronounced than that observed with cisplatin, making them very promising anticancer agents.

Monoterpene indole alkaloid azine derivatives as MDR reversal agents.

Aiming at generating a library of bioactive indole alkaloid derivatives as multidrug resistance (MDR) reversers, two epimeric indole alkaloids (1 and 2) were submitted to chemical transformations, giving rise to twenty-four derivatives (5-28), bearing new aromatic or aliphatic azine moieties. The structure of the compounds was established by 1D and 2D NMR (COSY, HMBC, HMQC and NOESY) experiments. Two different strategies were employed for assessing their anti-MDR potential, namely through the evaluation of their activity as inhibitors of typical MDR ABC transporters overexpressed by cell transfection, such as ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP), or by evaluating their ability as collateral sensitivity (CS) agents in cells overexpressing MRP1. A considerable MDR reversing activity was observed for compounds bearing the aromatic azine moiety. The strongest and most selective P-gp inhibition was found for the epimeric azines 5 and 6, bearing a para-methylbenzylidene moiety. Instead, compounds 17 and 18 that possess a di-substituted benzylidene portion with methoxy and hydroxyl groups, selectively inhibited MRP1 drug-efflux. None of these compounds inhibited BCRP. Compounds 5, 6 and 18 were further investigated in drug combination experiments, which corroborated their anti-MDR potential. Moreover, it was observed that compound 12, with an aromatic azine moiety, and compounds 23-26, sharing a new aliphatic substituent, displayed a CS activity, selectively killing MRP1-overexpressing cells. Among these last compounds, it could be established that addition of 19, 23 and 25 to MRP1-overexpressing cells led to glutathione depletion triggering cell death through apoptosis.

Glycosyl-Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins.

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl-substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane-cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b, 8 c, and 9 b preserved the ATPase function of BmrA, an ATP-binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a, 8 b, 8 f, 9 a, and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G-protein-coupled adenosine receptor A2A R. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.

Two different centered monoclinic crystals of the E. coli outer-membrane protein OmpF originate from the same building block.

Macromolecule crystal formation can be divided in two major steps: 1. the formation of a nucleus and 2. the growth of this nucleus into a full mature crystal. The latter is well described and understood, while the former remains elusive due to the difficulty to study it and is described by nucleation theories. Here we report the structure of the Escherichia coli outer membrane porin OmpF in two centered monoclinic space groups. Strikingly, the two crystals originate from the same building block, made of two trimers of OmpF interacting via their rough side. The different crystallization conditions trigger the formation of distinct arrangement of these building blocks, leading to the formation of translational non-crystallographic symmetry (tNCS) in one case, made possible by the loose lateral packing mediated by detergents. In light of nucleation theories, these results allow us to speculate that these two crystals originate from nuclei made of either clusters of building blocks, or already forming columns that later associate laterally using detergents as glue.

Atomic modelling and systematic mutagenesis identify residues in multiple drug binding sites that are essential for drug resistance in the major Candida transporter Cdr1.

The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Candida albicans, shows promiscuity towards the substrate it exports and plays a major role in antifungal resistance. It has two transmembrane domains (TMDs) comprising of six transmembrane helices (TMH) that envisage and confer the substrate specificity and two nucleotide binding domains (NBDs), interconnected by extracellular loops (ECLs) and intracellular loops (ICLs) Cdr1p. This study explores the diverse substrate specificity spectrum to get a deeper insight into the structural and functional features of Cdr1p. By screening with the variety of compounds towards an in-house TMH 252 mutant library of Cdr1p, we establish new substrates of Cdr1p. The localization of substrate-susceptible mutants in an ABCG5/G8 homology model highlights the common and specific binding pockets inside the membrane domain, where rhodamines and tetrazoliums mainly engage the N-moiety of Cdr1p, binding between TMH 2, 11 and surrounded by TMH 1, 5. Whereas, tin chlorides involve both N and C moieties located at the interface of TMH 2, 11, 1 and 5. Further, screening of the in house TMH mutant library of Cdr1p displays the TMH12 interaction with tetrazolium chloride, trimethyltin chloride and a Ca2+ ionophore, A23187. In silico localization reveals a binding site at the TMH 12, 9 and 10 interface, which is widely exposed to the lipid interface. Together, for the first time, our study shows the molecular localization of Cdr1p substrates-binding sites and demonstrates the participation of TMH12 in a peripheral drug binding site.

Multidrug ABC transporter Cdr1 of Candida albicans harbors specific and overlapping binding sites for human steroid hormones transport.

The present study examines the kinetics of steroids efflux mediated by the Candida drug resistance protein 1 (Cdr1p) and evaluates their interaction with the protein. We exploited our in-house mutant library for targeting the 252 residues forming the twelve transmembrane helices (TMHs) of Cdr1p. The screening revealed 65 and 58 residues critical for ß-estradiol and corticosterone transport, respectively. Notably, up to 83% critical residues for corticosterone face the lipid interface compared to 54% for ß-estradiol. Molecular docking identified a possible peripheral corticosterone-binding site made of 8/14 critical/non-critical residues between TMHs 3, 4 and 6. ß-estradiol transport was severely hampered by alanine replacements of Cdr1p core residues involving TMHs 2, 5 and 8, in a binding site made of 10/14 critical residues mainly shared with rhodamine 6G with which it competes. By contrast, TMH11 was poorly impacted, although being part of the core domain. Finally, we observed the presence of several contiguous stretches of 3-5 critical residues in TMHs 2, 5 and 10 that points to a rotation motion of these helices during the substrate transport cycle. The selective structural arrangement of the steroid-binding pockets in the core region and at the lipid-TMD interface, which was never reported before, together with the possible rotation of some TMHs may be the structural basis of the drug-transport mechanism achieved by these type II ABC transporters.

Gradient reconstitution of membrane proteins for solid-state NMR studies.

We here adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. We established conditions under which quantitative reconstitution of active protein at low lipid-to-protein ratios can be obtained, and also how to upscale these conditions in order to produce adequate amounts for NMR. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution, and shall represent a valuable alternative in solid-state NMR membrane protein sample preparation.

pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport.

Multidrug resistance 1 (MDR1) is a member of the major facilitator superfamily that contributes to MDR of Candida albicans This antiporter belongs to the drug/H(+) antiporter 1 family, pairing the downhill gradient of protons to drug extrusion. Hence, drug efflux from cytosol to extracellular space and the parallel import of H(+) towards cytosol are inextricably linked processes. For monitoring the drug/H(+) antiporter activity of Mdr1p, we developed a new system, exploiting a GFP variant pHluorin, which changes its fluorescence properties with pH. This enabled us to measure the cytosolic pH correlated to drug efflux. Since protonation of charged residues is a key step in proton movement, we explored the role of all charged residues of the 12 transmembrane segments (TMSs) of Mdr1p in drug/H(+) transport by mutational analysis. This revealed that the conserved residue R(215), positioned close to the C-terminal end of TMS-4, is critical for drug/H(+) antiport, allowing protonation over a range of pH, in contrast with its H(215) or K(215) variants that failed to transport drugs at basic pH. Mutation of other residues of TMS-4 highlights the role of this TMS in drug transport, as confirmed by in silico modelling of Mdr1p and docking of drugs. The model points to the importance of R(215) in proton transport, suggesting that it may adopt two main conformations, one oriented towards the extracellular face and the other towards the centre of Mdr1p. Together, our results not only establish a new system for monitoring drug/H(+) transport, but also unveil a positively charged residue critical to Mdr1p function.

Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion.

UNLABELLED: In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE: HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain.

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.

The Hydrophilic C-terminus of Yeast Plasma-membrane Na+/H+ Antiporters Impacts Their Ability to Transport K.

Yeast plasma-membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkali-metal cations and protons in cells. They are predicted to consist of 13 transmembrane segments (TMSs) and a large hydrophilic C-terminal cytoplasmic part with seven conserved domains. The substrate specificity, specifically the ability to recognize and transport K+ cations in addition to Na+ and Li+, differs among homologs. In this work, we reveal that the composition of the C-terminus impacts the ability of antiporters to transport particular cations. In the osmotolerant yeast Zygosaccharomyces rouxii, the Sod2-22 antiporter only efficiently exports Na+ and Li+, but not K+. The introduction of a negative charge or removal of a positive charge in one of the C-terminal conserved regions (C3) enabled ZrSod2-22 to transport K+. The same mutations rescued the low level of activity and purely Li+ specificity of ZrSod2-22 with the A179T mutation in TMS6, suggesting a possible interaction between this TMS and the C-terminus. The truncation or replacement of the C-terminal part of ZrSod2-22 with the C-terminus of a K+-transporting Nha/Sod antiporter (Saccharomyces cerevisiae Nha1 or Z. rouxii Nha1) also resulted in an antiporter with the capacity to export K+. In addition, in ScNha1, the replacement of three positively charged arginine residues 539-541 in the C3 region with alanine caused its inability to provide cells with tolerance to Li+. All our results demonstrate that the physiological functions of yeast Nha/Sod antiporters, either in salt tolerance or in K+ homeostasis, depend on the composition of their C-terminal parts.

Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species.

Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.

Localization of putative binding sites for cyclic guanosine monophosphate and the anti-cancer drug 5-fluoro-2'-deoxyuridine-5'-monophosphate on ABCC11 in silico models.

BACKGROUND: The Multidrug Resistance Protein ABCC11/MRP8 is expressed in physiological barriers and tumor breast tissues in which it secretes various substrates including cGMP (cyclic guanosine monophosphate) and 5FdUMP (5-fluoro-2'-deoxyuridine-5'-monophosphate), the active metabolite of the anticancer drug 5-FluoroUracil (frequently included to anticancer therapy).Previously, we described that ABCC11 high levels are associated to the estrogen receptor (ER) expression level in breast tumors and in cell lines resistant to tamoxifen. Consequently, by lowering the intracellular concentration of anticancer drugs, ABCC11 likely promotes a multidrug resistance (MDR) phenotype and decreases efficiency of anticancer therapy of 5FdUMP. Since no experimental data about binding sites of ABCC11 substrate are available, we decided to in silico localize putative substrate interaction sites of the nucleotide derivatives. Taking advantage of molecular dynamics simulation, we also analysed their evolution under computational physiological conditions and during the time. RESULTS: Since ABCC11 crystal structure is not resolved yet, we used the X-ray structures of the mouse mdr3 (homologous to human ABCB1) and of the bacterial homolog Sav1866 to generate two independent ABCC11 homology models in inward- and outward-facing conformations. Based on docking analyses, two putative binding pockets, for cGMP and 5FdUMP, were localized in both inward- and outward-facing conformations. Furthermore, based on our 3D models, and available biochemical data from homologous transporters, we identified several residues, potentially critical in ABCC11 transport function. Additionally, molecular dynamics simulation on our inward-facing model revealed for the first time conformation changes assumed to occur during transport process. CONCLUSIONS: ABCC11 would present two binding sites for cGMP and for 5FdUMP. Substrates likely first bind at the intracellular side of the transmembrane segment while ABCC11 is open forward the cytoplasm (inward-facing conformation). Then, along with conformational changes, it would pass through ABCC11 and fix the second site (close to the extracellular side), until the protein open itself to the extracellular space and allow substrate release.

Modular construction of quaternary hemiaminal-based inhibitor candidates and their in cellulo assessment with HIV-1 protease.

Non-peptidomimetic drug-like protease inhibitors have potential for circumventing drug resistance. We developed a much-improved synthetic route to our previously reported inhibitor candidate displaying an unusual quaternized hemi-aminal. This functional group forms from a linear precursor upon passage into physiological media. Seven variants were prepared and tested in cellulo with our HIV-1 fusion-protein technology that result in an eGFP-based fluorescent readout. Three candidates showed inhibition potency above 20µM and toxicity at higher concentrations, making them attractive targets for further refinement. Importantly, our class of original inhibitor candidates is not recognized by two major multidrug resistance pumps, quite in contrast to most clinically applied HIV-1 protease inhibitors.