Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Structural Basis of Substrate Recognition by the Multidrug Resistance Protein MRP1.

The multidrug resistance protein MRP1 is an ATP-binding cassette (ABC) transporter that confers resistance to many anticancer drugs and plays a role in the disposition and efficacy of several opiates, antidepressants, statins, and antibiotics. In addition, MRP1 regulates redox homeostasis, inflammation, and hormone secretion. Using electron cryomicroscopy, we determined the molecular structures of bovine MRP1 in two conformations: an apo form at 3.5 Å without any added substrate and a complex form at 3.3 Å with one of its physiological substrates, leukotriene C4. These structures show that by forming a single bipartite binding site, MRP1 can recognize a spectrum of substrates with different chemical structures. We also observed large conformational changes induced by leukotriene C4, explaining how substrate binding primes the transporter for ATP hydrolysis. Structural comparison of MRP1 and P-glycoprotein advances our understanding of the common and unique properties of these two important molecules in multidrug resistance to chemotherapy.

Hydrophobic moment drives penetration of bacterial membranes by transmembrane peptides.

With antimicrobial resistance (AMR) remaining a persistent and growing threat to human health worldwide, membrane-active peptides are gaining traction as an alternative strategy to overcome the issue. Membrane-embedded multi-drug resistant (MDR) efflux pumps are a prime target for membrane-active peptides, as they are a well-established contributor to clinically relevant AMR infections. Here, we describe a series of transmembrane peptides (TMs) to target the oligomerization motif of the AcrB component of the AcrAB-TolC MDR efflux pump from Escherichia coli. These peptides contain an N-terminal acetyl-A-(Sar)3 (sarcosine; N-methylglycine) tag and a C-terminal lysine tag-a design strategy our lab has utilized to improve the solubility and specificity of targeting for TMs previously. While these peptides have proven useful in preventing AcrB-mediated substrate efflux, the mechanisms by which these peptides associate with and penetrate the bacterial membrane remained unknown. In this study, we have shown peptide hydrophobic moment (µH)-the measure of concentrated hydrophobicity on one face of a lipopathic α-helix-drives bacterial membrane permeabilization and depolarization, likely through lateral-phase separation of negatively-charged POPG lipids and the disruption of lipid packing. Our results show peptide µH is an important consideration when designing membrane-active peptides and may be the determining factor in whether a TM will function in a permeabilizing or non-permeabilizing manner when embedded in the bacterial membrane.

Discovering targeted inhibitors for Escherichia coli efflux pump fusion proteins using computational and structure-guided approaches.

Multidrug resistance pathogens causing infections and illness remain largely untreated clinically. Efflux pumps are one of the primary processes through which bacteria develop resistance by transferring antibiotics from the interior of their cells to the outside environment. Inhibiting these pumps by developing efficient derivatives appears to be a promising strategy for restoring antibiotic potency. This investigation explores literature-reported inhibitors of E. coli efflux pump fusion proteins AcrB-AcrA and identify potential chemical derivatives of these inhibitors to overcome the limitations. Using computational and structure-guided approaches, a study was conducted with the selected inhibitors (AcrA:25-AcrB:59) obtained by data mining and their derivatives (AcrA:857-AcrB:3891) to identify their inhibitory effect on efflux pump using virtual screening, molecular docking and density functional theory (DFT) calculations. The finding indicates that Compound 2 (ZINC000072136376) has shown better binding and a significant inhibitory effect on AcrA, while Compound 3 (ZINC000072266819) has shown stronger binding and substantial inhibition effect on both non-mutant and mutated AcrB subunits. The identified derivatives could exhibit a better inhibitor and provide a potential approach for restoring the actions of resistant antibiotics.

Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

Insights into Allosteric Inhibition of the AcrB Efflux Pump: Role of Distinct Binding Pockets, Protomer Preferences, and Crosstalk Disruption.

AcrB, a key component in bacterial efflux processes, exhibits distinct binding pockets that influence inhibitor interactions. In addition to the well-known distal binding pocket within the periplasmic domain, a noteworthy pocket amidst the transmembrane (TM) helices serves as an alternate binding site for inhibitors. The bacterial efflux mechanism involves a pivotal functional rotation of the TM protein, inducing conformational changes in each protomer and propelling drugs toward the outer membrane domain. Surprisingly, inhibitors binding to the TM domain display a preference for L protomers over T protomers. Metadynamics simulations elucidate that Lys940 in the TM domain of AcrB can adopt two conformations in L protomers, whereas the energy barrier for such transitions is higher in T protomers. This phenomenon results in stable inhibitor binding in l protomers. Upon a detailed analysis of unbinding pathways using random accelerated molecular dynamics and umbrella sampling, we have identified three distinct routes for ligand exit from the allosteric site, specifically involving regions within the TM domains─TM4, TM5, and TM10. To explore allosteric crosstalk, we focused on the following key residues: Val452 from the TM domain and Ala831 from the porter domain. Surprisingly, our findings reveal that inhibitor binding disrupts this communication. The shortest path connecting Val452 and Ala831 increases upon inhibitor binding, suggesting sabotage of the natural interdomain communication dynamics. This result highlights the intricate interplay between inhibitor binding and allosteric signaling within our studied system.

Assessment In vitro and In silico of the Activity of Thiadiazines as NorA Efflux Pump Inhibitors.

Antimicrobials fight microorganisms, preventing and treating infectious diseases. However, antimicrobial resistance (AMR) is a growing concern due to the inappropriate and excessive use of these drugs. Several mechanisms can lead to resistance, including efflux pumps such as the NorA pump in Staphylococcus aureus, which reduces the effectiveness of fluoroquinolones. Thiadiazines are heterocyclic compounds whose chemical structure resembles that of cephalosporins. Therefore, these compounds and their derivatives have been studied for their potential in combating increased bacterial resistance. To analyze this hypothesis, direct activity assays, antibiotic action-modifying activity, fluorescence assays to evaluate the retention of ethidium bromide inside bacteria, and molecular docking were carried out. These experiments involved serial dilutions in microplates against Staphylococcus aureus strain 1199B under the influence of six thiadiazine derivatives (IJ10, IJ11, IJ21, IJ22, IJ23, and IJ25). The tests revealed that, despite not showing effective direct activity, some thiadiazine derivatives (IJ11, IJ21, and IJ22) inhibited the function of the bromide pump both in microdilution tests and in fluorescence and docking assays. Particularly, the IJ11 compound stood out for its activity similar to efflux inhibitors, as well as its inhibition of the norfloxacin pump of this bacterium. Among the results of this study, it deserves to be highlighted for anchoring future experiments, as it represents the first investigation of this group of thiadiazine derivatives against the NorA pump.

Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters.

ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K+ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.

Still rocking in the structural era: A molecular overview of the small multidrug resistance (SMR) transporter family.

The small multidrug resistance (SMR) family is composed of widespread microbial membrane proteins that fulfill different transport functions. Four functional SMR subtypes have been identified, which variously transport the small, charged metabolite guanidinium, bulky hydrophobic drugs and antiseptics, polyamines, and glycolipids across the membrane bilayer. The transporters possess a minimalist architecture, with ∼100-residue subunits that require assembly into homodimers or heterodimers for transport. In part because of their simple construction, the SMRs are a tractable system for biochemical and biophysical analysis. Studies of SMR transporters over the last 25 years have yielded deep insights for diverse fields, including membrane protein topology and evolution, mechanisms of membrane transport, and bacterial multidrug resistance. Here, we review recent advances in understanding the structures and functions of SMR transporters. New molecular structures of SMRs representing two of the four functional subtypes reveal the conserved structural features that have permitted the emergence of disparate substrate transport functions in the SMR family and illuminate structural similarities with a distantly related membrane transporter family, SLC35/DMT.

Methodology for rigorous modeling of protein conformational changes by Rosetta using DEER distance restraints.

We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes.

Recognition of quinolone antibiotics by the multidrug efflux transporter MexB of Pseudomonas aeruginosa.

The drug/proton antiporter MexB is the engine of the major efflux pump MexAB-OprM in Pseudomonas aeruginosa. This protein is known to transport a large variety of compounds, including antibiotics, thus conferring a multi-drug resistance phenotype. Due to the difficulty of producing co-crystals, only two X-ray structures of MexB in a complex with ligands are available to date, and mechanistic aspects are largely hypothesized based on the body of data collected for the homologous protein AcrB of Escherichia coli. In particular, a recent study (Ornik-Cha, Wilhelm, Kobylka et al., Nat. Commun., 2021, 12, 6919) reported a co-crystal structure of AcrB in a complex with levofloxacin, an antibiotic belonging to the important class of (fluoro)-quinolones. In this work, we performed a systematic ensemble docking campaign coupled to the cluster analysis and molecular-mechanics optimization of docking poses to study the interaction between 36 quinolone antibiotics and MexB. We additionally investigated surface complementarity between each molecule and the transporter and thoroughly assessed the computational protocol adopted against the known experimental data. Our study reveals different binding preferences of the investigated compounds towards the sub-sites of the large deep binding pocket of MexB, supporting the hypothesis that MexB substrates oscillate between different binding modes with similar affinity. Interestingly, small changes in the molecular structure translate into significant differences in MexB-quinolone interactions. All the predicted binding modes are available for download and visualization at the following link: https://www.dsf.unica.it/dock/mexb/quinolones.

Studying dynamics without explicit dynamics: A structure-based study of the export mechanism by AcrB.

Resistance-nodulation-cell division family proteins are transmembrane proteins identified as large spectrum drug transporters involved in multidrug resistance. A prototypical case in this superfamily, responsible for antibiotic resistance in selected gram-negative bacteria, is AcrB. AcrB forms a trimer using the proton motive force to efflux drugs, implementing a functional rotation mechanism. Unfortunately, the size of the system (1049 amino acid per monomer and membrane) has prevented a systematic dynamical exploration, so that the mild understanding of this coupled transport jeopardizes our ability to counter it. The large number of crystal structures of AcrB prompts studies to further our understanding of the mechanism. To this end, we present a novel strategy based on two key ingredients, which are to study dynamics by exploiting information embodied in the numerous crystal structures obtained to date, and to systematically consider subdomains, their dynamics, and their interactions. Along the way, we identify the subdomains responsible for dynamic events, refine the states (A, B, E) of the functional rotation mechanism, and analyze the evolution of intramonomer and intermonomer interfaces along the functional cycle. Our analysis shows the relevance of AcrB's efflux mechanism as a template within the HAE1 family but not beyond. It also paves the way to targeted simulations exploiting the most relevant degrees of freedom at certain steps, and to a targeting of specific interfaces to block the drug efflux. Our work shows that complex dynamics can be unveiled from static snapshots, a strategy that may be used on a variety of molecular machines of large size.

Study on functional sites in human multidrug resistance protein 1 (hMRP1).

Human multidrug resistance protein 1 (hMRP1) is an important member of the ATP-binding cassette (ABC) transporter superfamily. It can extrude a variety of anticancer drugs and physiological organic anions across the plasma membrane, which is activated by substrate binding, and is accompanied by large-scale cooperative movements between different domains. Currently, it remains unclear completely about how the specific interactions between hMRP1 and its substrate are and which critical residues are responsible for allosteric signal transduction. To the end, we first construct an inward-facing state of hMRP1 using homology modeling method, and then dock substrate proinflammatory agent leukotriene C4 (LTC4) to hMRP1 pocket. The result manifests LTC4 interacts with two parts of hMRP1 pocket, namely the positively charged pocket (P pocket) and hydrophobic pocket (H pocket), similar to its binding mode with bMRP1 (bovine MRP1). Additionally, we use the Gaussian network model (GNM)-based thermodynamic method proposed by us to identify the key residues whose perturbations markedly alter their binding free energy. Here the conventional GNM is improved with covalent/non-covalent interactions and secondary structure information considered (denoted as sscGNM). In the result, sscGNM improves the flexibility prediction, especially for the nucleotide binding domains with rich kinds of secondary structures. The 46 key residue clusters located in different subdomains are identified which are highly consistent with experimental observations. Furtherly, we explore the long-range cooperation within the transporter. This study is helpful for strengthening the understanding of the work mechanism in ABC exporters and can provide important information to scientists in drug design studies.

Decreased prevalence of the Plasmodium falciparum Pfcrt K76T and Pfmdr1 and N86Y mutations post-chloroquine treatment withdrawal in Katete District, Eastern Zambia.

BACKGROUND: In 2002, Zambia withdrew chloroquine as first-line treatment for Plasmodium falciparum malaria due to increased treatment failure and worldwide spread of chloroquine resistance. The artemisinin combination regimen, artemether-lumefantrine, replaced chloroquine (CQ) as first choice malaria treatment. The present study determined the prevalence of CQ resistance molecular markers in the Pfcrt and Pfmdr1 genes in Eastern Zambia at 9 and 13 years after the removal of drug pressure. METHODS: Samples collected from Katete District during the drug therapeutic efficacy assessments conducted in 2012 and 2016 were assayed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) to determine the prevalence of genetic mutations, K76T on the Pfcrt gene and N86Y on the Pfmdr1 gene. A total of 204 P. falciparum-positive DBS samples collected at these two time points were further analysed. RESULTS: Among the samples analysed for Pfcrt K76T and Pfmdr1 N86Y in the present study, 112 (82.4%) P. falciparum-infected samples collected in 2012 were successfully amplified for Pfcrt and 94 (69.1%) for Pfmdr1, while 69 (65.7%) and 72 (68.6%) samples from 2016 were successfully amplified for Pfcrt and Pfmdr1, respectively. In 2012, the prevalence of Pfcrt 76K (sensitive) was 97.3%, 76T (resistant) was 1.8%, and 0.8% had both 76K and 76T codons (mixed). Similarly in 2012, the prevalence of Pfmdr1 86N (sensitive) was 97.9% and 86Y (resistant) was 2.1%. In the 2016 samples, the prevalence of the respective samples was 100% Pfcrt 76K and Pfmdr1 86N. CONCLUSION: This study shows that there was a complete recovery of chloroquine-sensitive parasites by 2016 in Katete District, Eastern Zambia, 13 years following the withdrawal of CQ in the country. These findings add to the body of evidence for a fitness cost in CQ-resistant P. falciparum in Zambia and elsewhere. Further studies are recommended to monitor resistance countrywide and explore the feasibility of integration of the former best anti-malarial in combination therapy in the future.

Lactobionamide-based fluorinated detergent for functional and structural stabilization of membrane proteins.

Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. Conventional detergents such as n-Dodecyl ß-D-maltoside have been used largely and efficiently to solubilize MPs with varying degrees of success concerning MPs functionality and stability. Fluorinated surfactants (FSs) have shown a great potential for the stabilization of various MPs. However, so far only a limited number of reports have demonstrated the ability of FSs to solubilize MPs from biological membranes. We report herein the use of a fluorinated lactobionamide-based detergent named FLAC6 for functional and structural stabilization of membrane proteins. We first demonstrated that FLAC6 efficiently solubilized three membrane proteins i.e. the native adenosine receptor A2AR, a G protein-coupled receptor, and two native transporters AcrB and BmrA. The resulting affinity purified MPs were highly pure, homogenous and aggregates free. Furthermore, the functionality of each MP was well maintained. Finally, striking overstabilization features were observed. Indeed, the Tm of native A2AR, AcrB and BmrA could be improved by 7, ~9 and ~ 23 °C, respectively when FLAC6 was used instead of the reference detergent. This work illustrates that FLAC6 is an efficient tool to maintain structural and functional integrities of different MPs belonging to different classes, providing a new avenue for functional stabilization of highly druggable and challenging membrane proteins involved in unmet medical needs.

Prediction model of human ABCC2/MRP2 efflux pump inhibitors: a QSAR study.

The overexpression of ABCC2/MRP2, an ATP-binding cassette transporter, contributes to multidrug resistance in cancer cells. In this study, a quantitative structure-activity relationship (QSAR) analysis on ABCC2 inhibitors has been carried out, aiming to establish a computational prediction model for ABCC2 modulators. Seven classification models and two regression models were built by SONNIA 4.2, and two other regression models were built by MOE 2008.10 based on a data set comprising 372 compounds collected from 16 relevant publications. The CPG-C iABCC2 model for classifying ABCC2 inhibitors has total accuracy of 0.88 and Matthews correlation coefficient MCC = 0.75. The CPG-C iEG model for classifying ABCC2 inhibitors (substrate EG: ß-estradiol 17-ß-D-glucuronide) has total accuracy of 0.91 and MCC = 0.82. The regression model PLS EG-IC50 for predicting ABCC2 inhibitors (substrate EG) gave root-mean-square error RMSE = 0.26, Q2 = 0.73 and [Formula: see text]. The regression model PLS CDCF-IC50 for predicting ABCC2 inhibitors [substrate CDCF: 5(6)-carboxy-2',7'-dichlorofluorescein] gave RMSE = 0.31, Q2 = 0.74 and [Formula: see text]. Four 2D-QSAR models were applied to 1661 compounds, with results indicating 369 compounds having the ability to reverse the efflux of both EG and CDCF by ABCC2, 152 among them having IC50 < 100 µM.

Structure and activity of lipid bilayer within a membrane-protein transporter.

Membrane proteins function in native cell membranes, but extraction into isolated particles is needed for many biochemical and structural analyses. Commonly used detergent-extraction methods destroy naturally associated lipid bilayers. Here, we devised a detergent-free method for preparing cell-membrane nanoparticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Å resolution. We discovered a remarkably well-organized lipid-bilayer structure associated with transmembrane domains of the AcrB trimer. This bilayer patch comprises 24 lipid molecules; inner leaflet chains are packed in a hexagonal array, whereas the outer leaflet has highly irregular but ordered packing. Protein side chains interact with both leaflets and participate in the hexagonal pattern. We suggest that the lipid bilayer supports and harmonizes peristaltic motions through AcrB trimers. In AcrB D407A, a putative proton-relay mutant, lipid bilayer buttresses protein interactions lost in crystal structures after detergent-solubilization. Our detergent-free system preserves lipid-protein interactions for visualization and should be broadly applicable.

Mutagenic Analysis of the Putative ABCC6 Substrate-Binding Cavity Using a New Homology Model.

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.

The First Cytoplasmic Loop in the Core Structure of the ABCC1 (Multidrug Resistance Protein 1; MRP1) Transporter Contains Multiple Amino Acids Essential for Its Expression.

ABCC1 (human multidrug resistance protein 1 (hMRP1)) is an ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions and confers multidrug resistance through active drug efflux. The 17 transmembrane α-helices of hMRP1 are distributed among three membrane spanning domains (MSD0, 1, 2) with MSD1,2 each followed by a nucleotide binding domain to form the 4-domain core structure. Eight conserved residues in the first cytoplasmic loop (CL4) of MSD1 in the descending α-helix (Gly392, Tyr404, Arg405), the perpendicular coupling helix (Asn412, Arg415, Lys416), and the ascending α-helix (Glu422, Phe434) were targeted for mutagenesis. Mutants with both alanine and same charge substitutions of the coupling helix residues were expressed in HEK cells at wild-type hMRP1 levels and their transport activity was only moderately compromised. In contrast, mutants of the flanking amino acids (G392I, Y404A, R405A/K, E422A/D, and F434Y) were very poorly expressed although Y404F, E422D, and F434A were readily expressed and transport competent. Modeling analyses indicated that Glu422 and Arg615 could form an ion pair that might stabilize transporter expression. However, this was not supported by exchange mutations E422R/R615E which failed to improve hMRP1 levels. Additional structures accompanied by rigorous biochemical validations are needed to better understand the bonding interactions crucial for stable hMRP1 expression.

Novel Symmetrical Cage Compounds as Inhibitors of the Symmetrical MRP4-Efflux Pump for Anticancer Therapy.

Within the last decades cancer treatment improved by the availability of more specifically acting drugs that address molecular target structures in cancer cells. However, those target-sensitive drugs suffer from ongoing resistances resulting from mutations and moreover they are affected by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be treated with the common drugs, so that there have been long efforts to develop drugs to combat that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4 we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant anticancer drug as proof-of-principle to encourage for further preclinical studies.