Mechanism of CFTR correction by type I folding correctors.

Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.

Structure of the Human Lipid Exporter ABCA1.

ABCA1, an ATP-binding cassette (ABC) subfamily A exporter, mediates the cellular efflux of phospholipids and cholesterol to the extracellular acceptor apolipoprotein A-I (apoA-I) for generation of nascent high-density lipoprotein (HDL). Mutations of human ABCA1 are associated with Tangier disease and familial HDL deficiency. Here, we report the cryo-EM structure of human ABCA1 with nominal resolutions of 4.1 Å for the overall structure and 3.9 Å for the massive extracellular domain. The nucleotide-binding domains (NBDs) display a nucleotide-free state, while the two transmembrane domains (TMDs) contact each other through a narrow interface in the intracellular leaflet of the membrane. In addition to TMDs and NBDs, two extracellular domains of ABCA1 enclose an elongated hydrophobic tunnel. Structural mapping of dozens of disease-related mutations allows potential interpretation of their diverse pathogenic mechanisms. Structural-based analysis suggests a plausible "lateral access" mechanism for ABCA1-mediated lipid export that may be distinct from the conventional alternating-access paradigm.

Frozen motion: how cryo-EM changes the way we look at ABC transporters.

ATP-binding cassette (ABC) transporters are widely present molecular machines that transfer substrates across the cell membrane. ABC transporters are involved in numerous physiological processes and are often clinical targets. Structural biology is fundamental to obtain the molecular details underlying ABC transporter function and suggest approaches to modulate it. Until recently, X-ray crystallography has been the only method capable of providing high-resolution structures of ABC transporters. However, modern cryo-electron microscopy (cryo-EM) opens entirely new ways of studying these dynamic membrane proteins. Cryo-EM enables analyses of targets that resist X-ray crystallography, challenging multicomponent complexes, and the exploration of conformational dynamics. These unique capacities have turned cryo-EM into the dominant technique for structural studies of membrane proteins, including ABC transporters.

Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.

Differential dynamics and direct interaction of bound ligands with lipids in multidrug transporter ABCG2.

ABCG2 is an ATP-binding cassette (ABC) transporter that extrudes a wide range of xenobiotics and drugs from the cell and contributes to multidrug resistance in cancer cells. Following our recent structural characterization of topotecan-bound ABCG2, here, we present cryo-EM structures of ABCG2 under turnover conditions in complex with a special modulator and slow substrate, tariquidar, in nanodiscs. The structures reveal that similar to topotecan, tariquidar induces two distinct ABCG2 conformations under turnover conditions (turnover-1 and turnover-2). µs-scale molecular dynamics simulations of drug-bound and apo ABCG2 in native-like lipid bilayers, in both topotecan- and tariquidar-bound states, characterize the ligand size as a major determinant of its binding stability. The simulations highlight direct lipid-drug interactions for the smaller topotecan, which exhibits a highly dynamic binding mode. In contrast, the larger tariquidar occupies most of the available volume in the binding pocket, thus leaving little space for lipids to enter the cavity and interact with it. Similarly, when simulating ABCG2 in the apo inward-open state, we also observe spontaneous penetration of phospholipids into the binding cavity. The captured phospholipid diffusion pathway into ABCG2 offers a putative general path to recruit any hydrophobic/amphiphilic substrates directly from the membrane. Our simulations also reveal that ABCG2 rejects cholesterol as a substrate, which is omnipresent in plasma membranes that contain ABCG2. At the same time, cholesterol is found to prohibit the penetration of phospholipids into ABCG2. These molecular findings have direct functional ramifications on ABCG2's function as a transporter.

Interdomain communication in a homodimeric ABC transporter.

ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance. In type IV ABC transporters, each of the two nucleotide-binding domains is connected to a transmembrane domain by two coupling helices, which are part of cytosolic loops. Although there are many structural snapshots of different conformations, the interdomain communication is still enigmatic. Therefore, we analyzed the function of three conserved charged residues in the intracytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL (transporter associated with antigen processing-like). Substitution of D278 in coupling helix 1 by alanine interrupted peptide transport by impeding ATP hydrolysis. Alanine substitution of R288 and D292, both localized next to the coupling helix 1 extending to transmembrane helix 3, reduced peptide transport but increased basal ATPase activity. Surprisingly, the ATPase activity of the R288A variant dropped in a peptide-dependent manner, whereas ATPase activity of wildtype and D292A was unaffected. Interestingly, R288A and D292A mutants did not differentiate between ATP and GTP in respect of hydrolysis. However, in contrast to wildtye TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions, whereas the two residues in the cytosolic extension of transmembrane helix 3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward-facing conformation.

Structural and functional characterization of the nucleotide-binding domains of ABCA4 and their role in Stargardt disease.

ABCA4 is an ATP-binding cassette (ABC) transporter that prevents the buildup of toxic retinoid compounds by facilitating the transport of N-retinylidene-phosphatidylethanolamine across membranes of rod and cone photoreceptor cells. Over 1500 missense mutations in ABCA4, many in the nucleotide-binding domains (NBDs), have been genetically linked to Stargardt disease. Here, we show by cryo-EM that ABCA4 is converted from an open outward conformation to a closed conformation upon the binding of adenylyl-imidodiphosphate. Structural information and biochemical studies were used to further define the role of the NBDs in the functional properties of ABCA4 and the mechanisms by which mutations lead to the loss in activity. We show that ATPase activity in both NBDs is required for the functional activity of ABCA4. Mutations in Walker A asparagine residues cause a severe reduction in substrate-activated ATPase activity due to the loss in polar interactions with residues within the D-loops of the opposing NBD. The structural basis for how disease mutations in other NBD residues, including the R1108C, R2077W, R2107H, and L2027F, affect the structure and function of ABCA4 is described. Collectively, our studies provide insight into the structure and function of ABCA4 and mechanisms underlying Stargardt disease.

Structures of the peptidase-containing ABC transporter PCAT1 under equilibrium and nonequilibrium conditions.

ATP-binding cassette (ABC) transporters are ubiquitous molecular pumps that transport a broad range of substrates across biological membranes. Although the structure and function of ABC transporters has been studied extensively, our understanding of their energetics and dynamics remains limited. Here, we present studies of the peptidase-containing ABC transporter 1 (PCAT1), a polypeptide processing and secretion ABC transporter that functions via the classic alternating access mechanism. PCAT1 is a homodimer containing two peptidase (PEP) domains, two transmembrane domains, and two nucleotide-binding domains (NBDs). Using cryo-electron microscopy, we analyzed the structures of wild-type PCAT1 under conditions that either prevent or permit ATP hydrolysis and observed two completely different conformational distributions. In the presence of ATP but absence of Mg2+, PCAT1 adopts an NBD-dimerized, outward-facing conformation. The two PEP domains are dissociated from the transporter core, preventing uncoupled substrate cleavage. The addition of Mg2+ to promote ATP hydrolysis shifts the majority of the particles into NBD-separated, inward-facing conformations. Under this ATP turnover condition, only a small fraction of PCAT1 adopts the NBD-dimerized conformation. These data give rise to two mechanistic conclusions: 1) the ATP-bound, NBD-dimerized conformation is the lowest energy state, and 2) the rate-limiting step in the PCAT1 transport cycle is the formation of the NBD dimer. The thermodynamic conclusion is likely a general property shared by many ABC transporters. The kinetic bottleneck, however, varies from transporter to transporter.

Linker Domains: Why ABC Transporters 'Live in Fragments no Longer'.

ATP-binding cassette (ABC) transporters are membrane proteins present in all kingdoms of life. We have considered the disordered region that connects the N- and C-terminal halves in many eukaryotic ABC transporters, allowing all four consensus functional domains to be linked. The recent availability of structures of ABC transporters containing linker regions has allowed us to identify the start and end points of the connectors as well as hinting at their localisation. We address questions such as: Where did the linker regions come from? Why do some ABC transporters have connectors and others not? What are the rules and roles of the linker regions? What are the consequences of mutations in these connector regions for disease in humans?

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and their Zebrafish Homologs at the Blood-Brain Barrier.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish where expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disrpution. We transfected HEK-293 cells with both NanoLuc and human ABCB1 or ABCG2, or their zebrafish homologs Abcb4 and Abcg2a, which are functionally homologous to human P-gp and ABCG2, respectively, and expressed at the zebrafish BBB. We evaluated the brightness of ten NanoLuc substrates, then screened the eight brightest for their ability to be effluxed by the ABC transporters. We identified one ABCB1 substrate, two Abcb4 substrates, six ABCG2 substrates, and four Abcg2a substrates. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. Significance Statement The ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

Integrative analysis of transcriptome and metabolome provides insights into the mechanisms of leaf variegation in Heliopsis helianthoides.

BACKGROUND: In the field of ornamental horticulture, phenotypic mutations, particularly in leaf color, are of great interest due to their potential in developing new plant varieties. The introduction of variegated leaf traits in plants like Heliopsis helianthoides, a perennial herbaceous species with ecological adaptability, provides a rich resource for molecular breeding and research on pigment metabolism and photosynthesis. We aimed to explore the mechanism of leaf variegation of Heliopsis helianthoides (using HY2021F1-0915 variegated mutant named HY, and green-leaf control check named CK in 2020 April, May and June) by analyzing the transcriptome and metabolome. RESULTS: Leaf color and physiological parameters were found to be significantly different between HY and CK types. Chlorophyll content of HY was lower than that of CK samples. Combined with the result of Weighted Gene Co-expression Network Analysis (WGCNA), 26 consistently downregulated differentially expressed genes (DEGs) were screened in HY compared to CK subtypes. Among the DEGs, 9 genes were verified to be downregulated in HY than CK by qRT-PCR. The reduction of chlorophyll content in HY might be due to the downregulation of FSD2. Low expression level of PFE2, annotated as ferritin-4, might also contribute to the interveinal chlorosis of HY. Based on metabolome data, differential metabolites (DEMs) between HY and CK samples were significantly enriched on ABC transporters in three months. By integrating DEGs and DEMs, they were enriched on carotenoids pathway. Downregulation of four carotenoid pigments might be one of the reasons for HY's light color. CONCLUSION: FSD2 and PFE2 (ferritin-4) were identified as key genes which likely contribute to the reduced chlorophyll content and interveinal chlorosis observed in HY. The differential metabolites were significantly enriched in ABC transporters. Carotenoid biosynthesis pathway was highlighted with decreased pigments in HY individuals. These findings not only enhance our understanding of leaf variegation mechanisms but also offer valuable insights for future plant breeding strategies aimed at preserving and enhancing variegated-leaf traits in ornamental plants.

lncRNAs: New players of cancer drug resistance via targeting ABC transporters.

Cancer drug resistance poses a significant obstacle to successful chemotherapy, primarily driven by the activity of ATP-binding cassette (ABC) transporters, which actively efflux chemotherapeutic agents from cancer cells, reducing their intracellular concentrations and therapeutic efficacy. Recent studies have highlighted the pivotal role of long noncoding RNAs (lncRNAs) in regulating this resistance, positioning them as crucial modulators of ABC transporter function. lncRNAs, once considered transcriptional noise, are now recognized for their complex regulatory capabilities at various cellular levels, including chromatin modification, transcription, and post-transcriptional processing. This review synthesizes current research demonstrating how lncRNAs influence cancer drug resistance by modulating the expression and activity of ABC transporters. lncRNAs can act as molecular sponges, sequestering microRNAs that would otherwise downregulate ABC transporter genes. Additionally, they can alter the epigenetic landscape of these genes, affecting their transcriptional activity. Mechanistic insights reveal that lncRNAs contribute to the activity of ABC transporters, thereby altering the efflux of chemotherapeutic drugs and promoting drug resistance. Understanding these interactions provides a new perspective on the molecular basis of chemoresistance, emphasizing the regulatory network of lncRNAs and ABC transporters. This knowledge not only deepens our understanding of the biological mechanisms underlying drug resistance but also suggests novel therapeutic strategies. In conclusion, the intricate interplay between lncRNAs and ABC transporters is crucial for developing innovative solutions to combat cancer drug resistance, underscoring the importance of continued research in this field.

An induced mutation of ABC-transporter component VraF(K84E) contributes to vancomycin resistance and virulence in Staphylococcus aureus strain MW2.

Staphylococcus aureus is a notorious pathogen responsible for various severe diseases. Due to the emergence of drug-resistant strains, the prevention and treatment of S. aureus infections have become increasingly challenging. Vancomycin is considered to be one of the last-resort drugs for treating most methicillin-resistant S. aureus (MRSA), so it is of great significance to further reveal the mechanism of vancomycin resistance. VraFG is one of the few important ABC (ATP-binding cassette) transporters in S. aureus that can form TCS (two-component systems)/ABC transporter modules. ABC transporters can couple the energy released from ATP hydrolysis to translocate solutes across the cell membrane. In this study, we obtained a strain with decreased vancomycin susceptibility after serial passaging and selection. Subsequently, whole-genome sequencing was performed on this laboratory-derived strain MWA2 and a novel single point mutation was discovered in vraF gene, leading to decreased sensitivity to vancomycin and daptomycin. Furthermore, the mutation reduces autolysis of S. aureus and downregulates the expression of lytM, isaA, and atlA. Additionally, we observed that the mutant has a less net negative surface charge than wild-type strain. We also noted an increase in the expression of the dlt operon and mprF gene, which are associated with cell surface charge and serve to hinder the binding of cationic peptides by promoting electrostatic repulsion. Moreover, this mutation has been shown to enhance hemolytic activity, expand subcutaneous abscesses, reflecting an increased virulence. This study confirms the impact of a point mutation of VraF on S. aureus antibiotic resistance and virulence, contributing to a broader understanding of ABC transporter function and providing new targets for treating S. aureus infections.

Opposing genetic polymorphisms of two ABC transporters contribute to the variation of nukacin resistance in Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S. mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG, an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG, 19/108 strains (17.6%) carried only mukFEG, and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG. We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type ß). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type ß (MukFß). Then, we constructed a mukFEG-deletion mutant complemented with MukFαEG or MukFßEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes. IMPORTANCE: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans. This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution.

Differential Selectivity of Human and Mouse ABCC4/Abcc4 for Arsenic Metabolites.

Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring >100-fold higher doses for tumour induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette (ABC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the GSH conjugate of the highly toxic monomethylarsonous acid (MMAIII), MMA(GS)2, and the major human urinary arsenic metabolite dimethylarsinic acid (DMAV). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M4I-10 epitope was first mapped to an octapeptide (411HVQDFTA418F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in HEK293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMAIII, monomethylarsonic acid, dimethylarsinous acid or DMAV], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)2 or DMAV, despite a >3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17ß-estradiol-17-(ß-D-glucuronide). Abcc4(+/+) mouse embryonic fibroblasts (MEFs) were ~3-fold more resistant to arsenate than Abcc4(-/-) MEFs; however, further characterization indicated this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to differences in human and mouse arsenic toxicokinetics. Significance Statement Toxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. The human ATP-binding cassette transporter hABCC4 is a high affinity transporter of toxicologically important arsenic metabolites. Here we used multiple cell models to demonstrate that mouse Abcc4 does not protect cells against, or transport, any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.

Zinc finger proteins and ATP-binding cassette transporter-dependent multidrug resistance.

BACKGROUND: Multidrug resistance (MDR) remains a significant challenge in cancer treatment, leading to poor clinical outcomes. Dysregulation of ATP-binding cassette (ABC) transporters has been identified as a key contributor to MDR. Zinc finger proteins (ZNPs) are key regulators of transcription and have emerged as potential contributors to cancer drug resistance. Bridging the knowledge gap between ZNPs and MDR is essential to understand a source of heterogeneity in cancer treatment. This review sought to elucidate how different ZNPs modulate the transcriptional regulation of ABC genes, contributing to resistance to cancer therapies. METHODS: The search was conducted using PubMed, Google Scholar, EMBASE and Web of Science. RESULTS: In addition to ABC-blockers, the transcriptional features regulated by ZNP are expected to play a role in reversing ABC-mediated MDR and predicting the efficacy of anticancer treatments. Among the ZNP-induced epithelial to mesenchymal transition, SNAIL, SLUG and Zebs have been identified as important factors in promoting MDR through activation of ATM, NFκB and PI3K/Akt pathways, exposing the metabolism to potential ZNP-MDR interactions. Additionally, nuclear receptors, such as VDR, ER and PXR have been found to modulate certain ABC regulations. Other C2H2-type zinc fingers, including Kruppel-like factors, Gli and Sp also have the potential to contribute to MDR. CONCLUSION: Besides reviewing evidence on the effects of ZNP dysregulation on ABC-related chemoresistance in malignancies, significant markers of ZNP functions are discussed to highlight the clinical implications of gene-to-gene and microenvironment-to-gene interactions on MDR prospects. Future research on ZNP-derived biomarkers is crucial for addressing heterogeneity in cancer therapy.

BI-2865, a pan-KRAS inhibitor, reverses the P-glycoprotein induced multidrug resistance in vitro and in vivo.

BACKGROUND: Multidrug resistance (MDR) limits successful cancer chemotherapy. P-glycoprotein (P-gp), BCRP and MRP1 are the key triggers of MDR. Unfortunately, no MDR modulator was approved by FDA to date. Here, we will investigate the effect of BI-2865, a pan-KRAS inhibitor, on reversing MDR induced by P-gp, BCRP and MRP1 in vitro and in vivo, and its reversal mechanisms will be explored. METHODS: The cytotoxicity of BI-2865 and its MDR removal effect in vitro were tested by MTT assays, and the corresponding reversal function in vivo was assessed through the P-gp mediated KBv200 xenografts in mice. BI-2865 induced alterations of drug discharge and reservation in cells were estimated by experiments of Flow cytometry with fluorescent doxorubicin, and the chemo-drug accumulation in xenografts' tumor were analyzed through LC-MS. Mechanisms of BI-2865 inhibiting P-gp substrate's efflux were analyzed through the vanadate-sensitive ATPase assay, [125I]-IAAP-photolabeling assay and computer molecular docking. The effects of BI-2865 on P-gp expression and KRAS-downstream signaling were detected via Western blotting, Flow cytometry and/or qRT-PCR. Subcellular localization of P-gp was visualized by Immunofluorescence. RESULTS: We found BI-2865 notably fortified response of P-gp-driven MDR cancer cells to the administration of chemo-drugs including paclitaxel, vincristine and doxorubicin, while such an effect was not observed in their parental sensitive cells and BCRP or MRP1-driven MDR cells. Importantly, the mice vivo combination study has verified that BI-2865 effectively improved the anti-tumor action of paclitaxel without toxic injury. In mechanism, BI-2865 prompted doxorubicin accumulating in carcinoma cells by directly blocking the efflux function of P-gp, which more specifically, was achieved by BI-2865 competitively binding to the drug-binding sites of P-gp. What's more, at the effective MDR reversal concentrations, BI-2865 neither varied the expression and location of P-gp nor reduced its downstream AKT or ERK1/2 signaling activity. CONCLUSIONS: This study uncovered a new application of BI-2865 as a MDR modulator, which might be used to effectively, safely and specifically improve chemotherapeutic efficacy in the clinical P-gp mediated MDR refractory cancers.

Natural products for combating multidrug resistance in cancer.

Cancer cells frequently develop resistance to chemotherapeutic therapies and targeted drugs, which has been a significant challenge in cancer management. With the growing advances in technologies in isolation and identification of natural products, the potential of natural products in combating cancer multidrug resistance has received substantial attention. Importantly, natural products can impact multiple targets, which can be valuable in overcoming drug resistance from different perspectives. In the current review, we will describe the well-established mechanisms underlying multidrug resistance, and introduce natural products that could target these multidrug resistant mechanisms. Specifically, we will discuss natural compounds such as curcumin, resveratrol, baicalein, chrysin and more, and their potential roles in combating multidrug resistance. This review article aims to provide a systematic summary of recent advances of natural products in combating cancer drug resistance, and will provide rationales for novel drug discovery.

Cadmium transport by mammalian ATP-binding cassette transporters.

Cellular responses to toxic metals depend on metal accessibility to intracellular targets, reaching interaction sites, and the intracellular metal concentration, which is mainly determined by uptake pathways, binding/sequestration and efflux pathways. ATP-binding cassette (ABC) transporters are ubiquitous in the human body-usually in epithelia-and are responsible for the transfer of indispensable physiological substrates (e.g. lipids and heme), protection against potentially toxic substances, maintenance of fluid composition, and excretion of metabolic waste products. Derailed regulation and gene variants of ABC transporters culminate in a wide array of pathophysiological disease states, such as oncogenic multidrug resistance or cystic fibrosis. Cadmium (Cd) has no known physiological role in mammalians and poses a health risk due to its release into the environment as a result of industrial activities, and eventually passes into the food chain. Epithelial cells, especially within the liver, lungs, gastrointestinal tract and kidneys, are particularly susceptible to the multifaceted effects of Cd because of the plethora of uptake pathways available. Pertinent to their broad substrate spectra, ABC transporters represent a major cellular efflux pathway for Cd and Cd complexes. In this review, we summarize current knowledge concerning transport of Cd and its complexes (mainly Cd bound to glutathione) by the ABC transporters ABCB1 (P-glycoprotein, MDR1), ABCB6, ABCC1 (multidrug resistance related protein 1, MRP1), ABCC7 (cystic fibrosis transmembrane regulator, CFTR), and ABCG2 (breast cancer related protein, BCRP). Potential detoxification strategies underlying ABC transporter-mediated efflux of Cd and Cd complexes are discussed.

Magnolol derivatives as specific and noncytotoxic inhibitors of breast cancer resistance protein (BCRP/ABCG2).

The breast cancer resistance protein (BCRP/ABCG2) transporter mediates the efflux of numerous antineoplastic drugs, playing a central role in multidrug resistance related to cancer. The absence of successful clinical trials using specific ABCG2 inhibitors reveals the urge to identify new compounds to attend this critical demand. In this work, a series of 13 magnolol derivatives was tested as ABCG2 inhibitors. Only two compounds, derivatives 10 and 11, showed partial and complete ABCG2 inhibitory effect, respectively. This inhibition was selective toward ABCG2, since none of the 13 compounds inhibited neither P-glycoprotein nor MRP1. Both inhibitors (10 and 11) were not transported by ABCG2 and demonstrated a low cytotoxic profile even at high concentrations (up to 100 µM). 11 emerged as the most promising compound of the series, considering the ratio between cytotoxicity (IG50) and ABCG2 inhibition potency (IC50), showing a therapeutic ratio (TR) higher than observed for 10 (10.5 versus 1.6, respectively). This derivative showed a substrate-independent and a mixed type of inhibition. The effect of compound 11 on the ABCG2 ATPase activity and thermostability revealed allosteric protein changes. This compound did not affect the expression levels of ABCG2 and increased the binding of the conformational-sensitive antibody 5D3. A docking study showed that 11 did not share the same binding site with ABCG2 substrate mitoxantrone. Finally, 11 could revert the chemoresistance to SN-38 mediated by ABCG2.