Basal ATPase Research Articles

Myosin Isoform-Dependent Effect of Omecamtiv Mecarbil on the Regulation of Force Generation in Human Cardiac Muscle.

Omecamtiv mecarbil (OM) is a small molecule that has been shown to improve the function of the slow human ventricular myosin (MyHC) motor through a complex perturbation of the thin/thick filament regulatory state of the sarcomere mediated by binding to myosin allosteric sites coupled to inorganic phosphate (Pi) release. Here, myofibrils from samples of human left ventricle (β-slow MyHC-7) and left atrium (α-fast MyHC-6) from healthy donors were used to study the differential effects of μmolar [OM] on isometric force in relaxing conditions (pCa 9.0) and at maximal (pCa 4.5) or half-maximal (pCa 5.75) calcium activation, both under control conditions (15 °C; equimolar DMSO; contaminant inorganic phosphate [Pi] ~170 μM) and in the presence of 5 mM [Pi]. The activation state and OM concentration within the contractile lattice were rapidly altered by fast solution switching, demonstrating that the effect of OM was rapid and fully reversible with dose-dependent and myosin isoform-dependent features. In MyHC-7 ventricular myofibrils, OM increased submaximal and maximal Ca2+-activated isometric force with a complex dose-dependent effect peaking (40% increase) at 0.5 μM, whereas in MyHC-6 atrial myofibrils, it had no effect or-at concentrations above 5 µM-decreased the maximum Ca2+-activated force. In both ventricular and atrial myofibrils, OM strongly depressed the kinetics of force development and relaxation up to 90% at 10 μM [OM] and reduced the inhibition of force by inorganic phosphate. Interestingly, in the ventricle, but not in the atrium, OM induced a large dose-dependent Ca2+-independent force development and an increase in basal ATPase that were abolished by the presence of millimolar inorganic phosphate, consistent with the hypothesis that the widely reported Ca2+-sensitising effect of OM may be coupled to a change in the state of the thick filaments that resembles the on-off regulation of thin filaments by Ca2+. The complexity of this scenario may help to understand the disappointing results of clinical trials testing OM as inotropic support in systolic heart failure compared with currently available inotropic drugs that alter the calcium signalling cascade.

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 intra-cytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL. 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 while 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 wildtype TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions while the two residues in the cytosolic extension of TMH3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward facing conformation.

Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit fromfunctional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (kcat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Structure-function analysis of the ATPase domain of African swine fever virus topoisomerase.

The ATPase domain of type II topoisomerase provides energy by hydrolyzing ATP and coordinates with the DNA-binding/cleavage domain to drive and control DNA transport. The precise molecular mechanisms of how these domains respond to DNA binding and ATP hydrolysis signals and communicate with each other remain elusive. We determine the first high-resolution crystal structure of the ATPase domain of African swine fever virus (ASFV) topo II in complex with AMPPNP and biochemically investigate its function in ATPase and DNA relaxation activities. Importantly, we find that mutations at three characteristic regions of the ASFV ATPase domain produce parallel effects on the basal/DNA-stimulated ATPase and relaxation activities, implying the tight coupling of the ATP hydrolysis and strand passage process. Therefore, our data provide important implications for understanding the strand passage mechanism of the type II topoisomerase and the structural basis for developing ATPase domain-targeting antivirals against ASFV.

Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

ABCB5 is a member of the ABC transporter superfamily composed of 48 transporters, which have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma, and limbal stem cells. It has also been associated with multidrug resistance in several cancers. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half transporter (ABCB5β). Several studies have shown that the ABCB5β homodimer does not confer multidrug resistance, in contrast to ABCB5FL. In this study, using three complementary techniques, (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in melanoma: ABCB5β/B6 and ABCB5β/B9. Both heterodimers could be expressed in High-Five insect cells and ATPase assays revealed that both functional nucleotide-binding domains of homodimers and heterodimers are required for their basal ATPase activity. These results are an important step toward elucidating the functional role of ABCB5β in melanocytes and melanoma.

Regulation of the cell division hydrolase RipC by the FtsEX system in Mycobacterium tuberculosis

The FtsEX complex regulates, directly or via a protein mediator depending on bacterial genera, peptidoglycan degradation for cell division. In mycobacteria and Gram-positive bacteria, the FtsEX system directly activates peptidoglycan-hydrolases by a mechanism that remains unclear. Here we report our investigation of Mycobacterium tuberculosis FtsEX as a non-canonical regulator with high basal ATPase activity. The cryo-EM structures of the FtsEX system alone and in complex with RipC, as well as the ATP-activated state, unveil detailed information on the signal transduction mechanism, leading to the activation of RipC. Our findings indicate that RipC is recognized through a “Match and Fit” mechanism, resulting in an asymmetric rearrangement of the extracellular domains of FtsX and a unique inclined binding mode of RipC. This study provides insights into the molecular mechanisms of FtsEX and RipC regulation in the context of a critical human pathogen, guiding the design of drugs targeting peptidoglycan remodeling.

Redefine the role of d-α-Tocopheryl polyethylene glycol 1000 succinate on P-glycoprotein, multidrug resistance protein 1, and breast cancer resistance protein mediated cancer multidrug resistance

Cancer drug resistance is an ever-changing problem that most patients need to face in their later stages of treatment, especially the multidrug resistant (MDR) type. The drug efflux transporters, including P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP), play the crucial roles in this sophisticated battle. In recent decades, researchers try to find potential inhibitors to impede the drug efflux function of above transporters. d-α-Tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) is a prevalently used excipient in the formulation design. In the present study, the modulatory effects and mechanisms of vitamin E TPGS on the efflux transporters were investigated. And the cancer MDR reversing ability of vitamin E TPGS was evaluated as well. Stable-cloned transporter over-expressed cell lines were used for mechanisms study, while several types of MDR cancer cell lines were adopted as reversing evaluation models. The results exhibited that vitamin E TPGS significantly inhibited the efflux function of P-gp, MRP1, and BCRP under non-cytotoxic concentrations, but not influencing the protein expression levels. Through efflux assay and molecular docking, vitamin E TPGS was found to be an uncompetitive, non-competitive, and competitive inhibitor on chemotherapeutic drug doxorubicin efflux in P-gp, MRP1, and BCRP over-expressing cell lines, respectively. Furthermore, the basal ATPase activity of three transporters were significantly inhibited by vitamin E TPGS at 10 μM. And the cell membrane fluidity of P-gp over-expressing cell line was enhanced by 22.58% with 5 μM vitamin E TPGS treatment, compared to the parental Flp-In™-293 cell line (without P-gp). The resistance reversing ability of vitamin E TPGS was prominent in MCF-7/DOX MDR breast cancer cell line, which over-expressed P-gp, MRP1, and BCRP. These significant results suggested that vitamin E TPGS is a promising modulator on transporters mediated cancer MDR. Vitamin E TPGS is not an inert excipient, but possesses MDR-reversing pharmacological effects, and deserves a re-purposing application on the future combinatorial regimen design for MDR cancer treatment.

Residues from Homologous Transmembrane Helices 4 and 10 Are Critical for P-Glycoprotein (ABCB1)-Mediated Drug Transport

P-glycoprotein (P-gp, ABCB1) transports structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs, thus contributing to multidrug-resistant cancer. Cryo-EM structures of human P-gp revealed that TMHs 4 and 10 contribute to the formation of the drug-binding cavity and undergo conformational changes during drug transport. To assess the role of the conformational changes in TMH4 and TMH10 during drug transport, we generated two mutants (TMH4-7A and TMH10-7A), each containing seven alanine substitutions. Analysis of the drug efflux function of these mutants using 15 fluorescent substrates revealed that most of the substrates were transported, indicating that even seven mutations in an individual helix have no significant effect on transport function. We then designed the TMH4,10-14A mutant combining seven mutations in both TMHs 4 and 10. Interestingly, when the TMH4,10-14A mutant was tested with 15 substrates, there was no efflux observed for fourteen. The basal ATPase activity of the TMH4,10-14A mutant, similar to that of the WT protein, was inhibited by zosuquidar but was not stimulated by verapamil or rhodamine 6G. Molecular dynamics simulations indicated that the mutations cause TMHs 4 and 10 to pack tighter to their proximal helices, reducing their independent mobility. In aggregate, our findings demonstrate the critical role of the residues of homologous TMHs 4 and 10 for substrate transport, consistent with conformational changes observed in the structure of P-gp.

Waste or die: The price to pay to stay alive.

Microorganisms need to constantly exchange with their habitat to capture nutrients and expel toxic compounds. The ATP-binding cassette (ABC) transporters, a family of membrane proteins especially abundant in microorganisms, are at the core of these processes. Due to their extraordinary ability to expel structurally unrelated compounds, some transporters play a protective role in different organisms. Yet, the downside of these multidrug transporters is their entanglement in the resistance to therapeutic treatments. Intriguingly, some multidrug ABC transporters show a high level of ATPase activity, even in the absence of transported substrates. Although this basal ATPase activity might seem a waste, we surmise that this inherent capacity allows multidrug transporters to promptly translocate any bound drug before it penetrates into the cell.

Finding inhibitors and new activators for the biliverdin transporter ABCB10.

The absence of the mitochondrial ATP binding cassette (ABC)-transporter ABCB10 in mice is embryonic lethal, with fetuses presenting severe anemia and oxidative damage. Initially, ABCB10 was thought to be a heme transporter, but recent evidence from our group and collaborators suggests instead that ABCB10 transports biliverdin, a heme degradation product with antioxidant properties. Since some ABC transporters are promiscuous, interacting and/or transporting a large variety of drugs, it is important to determine the specificity of ABCB10 for its substrate. Commonly, ABC transporters present a basal ATPase activity that is increased by substrate binding. As expected, the substrate biliverdin significantly increases the basal ATPase activity of the transporter. Now, we are studying the effect of related compounds on the ATPase activity of purified ABCB10 reconstituted in lipid nanodiscs. We have found inhibition with pyrroles closely related to biliverdin, suggesting that specific substrate-protein interactions are required for the biliverdin-induced activation. We have also found additional compounds (pyrroles and porphyrins) that stimulate the ATPase activity of the transporter, although at a lower level than biliverdin does. Competition assays are underway to determine if these compounds bind to a single site or if there is evidence of the coexistence of multiple drug binding sites on the transporter. Finding compounds that can modulate or inhibit the activity of this essential transporter brings additional tools for needed biochemical and structural studies and opens the door for possible therapeutic purposes. Funding: 1R01GM145938-01 to MEZ.

Inhibitors of ABCG2-mediated multidrug resistance: Lead generation through computer-aided drug design

Human breast cancer resistance protein (BCRP), known also as ABCG2, plays a major role in multiple drug resistance (MDR) in tumor cells. Through this ABC transporter, cancer cells acquire the ability of resistance to structurally and functionally unrelated anticancer drugs. Nowadays, the design of ABCG2 inhibitors as potential agents to enhance the chemotherapy efficacy is an interesting strategy. In this context, we have used computer-aided drug design (CADD) based on available data of a large series of potent inhibitors from our groups as an approach in guiding the design of effective ABCG2 inhibitors. We report therein the results on the use of the FLAPpharm method to elucidate the pharmacophoric features of one of the ABCG2 binding sites involved in the regulation of the basal ATPase activity of the transporter. The predictivity of the model was evaluated by testing three predicted compounds which were found to induce high inhibitory activity of BCRP, in the nanomolar range for the best of them.

The lysosomal transporter TAPL has a dual role as peptide translocator and phosphatidylserine floppase

TAPL is a lysosomal ATP-binding cassette transporter that translocates a broad spectrum of polypeptides from the cytoplasm into the lysosomal lumen. Here we report that, in addition to its well-known role as a peptide translocator, TAPL exhibits an ATP-dependent phosphatidylserine floppase activity that is the possible cause of its high basal ATPase activity and of the lack of coupling between ATP hydrolysis and peptide efflux. We also present the cryo-EM structures of mouse TAPL complexed with (i) phospholipid, (ii) cholesteryl hemisuccinate (CHS) and 9-mer peptide, and (iii) ADP·BeF3. The inward-facing structure reveals that F449 protrudes into the cylindrical transport pathway and divides it into a large hydrophilic central cavity and a sizable hydrophobic upper cavity. In the structure, the peptide binds to TAPL in horizontally-stretched fashion within the central cavity, while lipid molecules plug vertically into the upper cavity. Together, our results suggest that TAPL uses different mechanisms to function as a peptide translocase and a phosphatidylserine floppase.

Myosin Transducer Inter-Strand Communication Is Critical for Normal ATPase Activity and Myofibril Structure.

Simple SummaryMyosin is a molecular motor protein that is critical for using stored energy to yield contraction in both skeletal muscle and in the heart. Mutations in myosin can result in inherited human hypertrophic cardiomyopathy, wherein the heart walls become thickened. This results in inability to adequately pump enough oxygenated blood to meet bodily demands, and can lead to abnormal heart beating patterns and the possibility of sudden death. Typically, over-active myosin can be causative for this disease. Here, we used the common fruit fly to study one of these cardiomyopathy-causing mutations by expressing the mutant protein in the indirect flight muscle of the fly. Surprisingly, we found that the mutation and associated mutations decrease myosin and muscle function instead of enhancing it. We show that at least some of these defects result from improper interactions within the myosin protein. This suggests that appropriate communications within myosin are critical for its function and further indicates that certain aspects of indirect flight muscle myosin are functionally distinct from those of myosin in the human heart. By understanding the intricate interactions within the myosin motor protein that are important for its normal role, therapeutics for improving mutant myosin function can be developed.The R249Q mutation in human β-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a β-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent β-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the β-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture.

Exploring the Role of Msh2‐Msh6 in Processing Holliday Junction DNA

Msh2‐Msh6 is a eukaryotic ATP‐dependent mismatch repair (MMR) protein that plays a key role in maintaining the fidelity of the genetic code. The protein recognizes and initiates repair for single base pair mismatches and short insertion deletion loops (IDL). Msh2‐Msh6 functions via a molecular switch mechanism, whereby ATP hydrolysis and ADP/ATP exchange facilitates the binding and release of the protein from the mismatch. Previous research has revealed that the protein binds G:T mismatch DNA with the highest affinity. Interestingly, in‐solution binding assays have revealed that Msh2‐Msh6 binds Holliday Junction DNA, a key recombination intermediate, with 1:1 stoichiometry and a similarly high affinity. However, the functional importance and structural details of this interaction are unclear. Thus far, X‐ray crystallography and Molecular Dynamics (MD) simulations are limited to the protein’s interactions with mismatch DNA only. To address the lack of structural information, we modeled the Msh2‐Msh6‐HJ interaction using the human Msh2‐Msh6 crystal structure in which we have docked in junction DNA instead of the mismatched DNA. The junction DNA used in all experiments and simulations is J3, a non‐migrating junction with 17 base pair long arms. These MD simulations will help refine the structural model and assess significant interactions between the protein and DNA. Furthermore, ATPase assays and equilibrium binding assays are used to elucidate the protein’s ATP hydrolysis activity and binding affinity in the presence of G:T mismatch DNA, J3 junction DNA, and J3 junction DNA containing a G:T mismatch. Preliminary experiments show that relative to the apoprotein’s basal ATPase activity, ATP hydrolysis is weakly stimulated when Msh2‐Msh6 is bound to G:T mismatch and J3. Gel mobility shift assays (GMSA) reveal binding affinities in the range of 9.1±2.6 nM to 23.5±11.8 nM for G:T mismatch, J3 junction, and mismatched J3 junction. These data illustrate that Msh2‐Msh6 not only binds G:T mismatch and J3 with similarly high affinity, but also that introducing a mismatch in J3 does not modulate the protein’s affinity for the junction. In addition, the GMSA data suggest that Msh2‐Msh6‐HJ binding may take on multiple conformations, consistent with previous Förster resonance energy transfer (FRET) calculations that show Msh2‐Msh6 opening the junction after binding.

TLK1 Phosphorylates RAD54 To Promote Homology Driven DSB Repair

BackgroundEnvironmental agents like Ionizing radiation (I.R) can cause severe damage to DNA often causing double‐strand break (DSB). If unrepaired, DSB elicits chromosomal rearrangements, tumorigenesis, or apoptosis. The main mechanism to maintain the fidelity of the genetic information after DSBs is homologous recombination repair (HRR). The regulation of this process is unclear. Tousled‐like kinase 1 (TLK1), a Ser/Thr kinase that regulates DNA damage checkpoint and DSB repair has been found to interact with RAD54, a central DNA translocase enzyme in HRR. Our work aims to study how TLK1 regulates RAD54 activities during HRR? We hypothesize that phosphorylation of RAD54 at both NTD and CTD by TLK1 promotes HRR through RAD51‐nucleofilament interaction or during Branch Migration through RAD54 translocase activity.Material and methodsImmunoprecipitation and pull‐down assays show that TLK1 interacts with RAD54 in cells. We performed in‐vitro kinase assays with recombinant TLK1 to phosphorylate purified human RAD54 followed by mass spectrometry (MS). To understand the effect of TLK1 and mutation on RAD54 at the novel sites in HR efficiency, we use DR‐GFP assay in HeLa cells.ResultsWe find with pull‐downs and IP that TLK1 and RAD54 interact in vitro and ex vivo. Invitro TLK1 kinase assay with RAD54 followed by LC‐MS/MS reveals three novel sites of phosphorylation on RAD54 at T41, T59, and T700. The T41 and T59 lie in the unstructured domain (USD) region of N‐terminus (NTD). T700 lies in the Zn2+ finger motif in the C‐terminus (CTD) which contacts the DNA backbone for the translocase (t) function. HRR activity is monitored through the DR‐GFP assay which results in GFP positive cells after an efficient HR event. Our results with TLK1 specific inhibitor (J54‐10uM) or knockdown using TLK1 shRNA confirm that inhibition or depletion of TLK1 suppresses HRR activity in cells. To mimic the de‐phosphorylated or phosphorylated state of RAD54, we did a T to A/D site‐directed mutagenesis and generated DR‐GFP integrated HeLa‐RAD54 mutant cell lines in which RAD54 was genetically deleted. RAD54‐T700A/D, T2A/D (NTD double mutant‐T41, 59A/D), T3A/D (triple point) mutants expressed in HeLa‐RAD54K.O cell line were used to test the DNA repair proficiency in MMC sensitivity assay. We find T3A/D survival is lower than WT at the lowest concentration of MMC (0.1uM). All the D‐mutants are sensitive at 0.9uM MMC. Our data shows that compared to WT, T700D protein has 3‐fold higher basal ATPase activity in presence of dsDNA (‐ RAD51) than T2D and T3D. With RAD51, the activity of T700D was 2‐fold lower than T2D and T3D and 4‐fold lower than WT. RAD51 binding affinity was determined for all the mutant proteins and T700D had 20‐30% higher binding affinity than WT, T2D & T3D. This implies that 1) interaction of phosphorylated RAD54‐T700 with RAD51 can downregulate the ATPase activity during HR completion or, 2) pRAD54‐T700 may have higher translocase activity and greater RAD51 affinity that needs to be turned off in later HRR steps.ConclusionWe expect that pRAD54 by TLK1 will impact gene conversion events and shed light on HRR regulation at the post‐translational level of RAD54

The Resistance of Cancer Cells to Palbociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor, is Mediated by the ABCB1 Transporter

Palbociclib was approved by the United States Food and Drug Administration for use, in combination with letrozole, as a first-line treatment for estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER+/HER2-) postmenopausal metastatic breast cancer. However, recent studies show that palbociclib may be an inhibitor of the ABCB1 transporter, although this remains to be elucidated. Therefore, we conducted experiments to determine the interaction of palbociclib with the ABCB1 transporter. Our in vitro results indicated that the efficacy of palbociclib was significantly decreased in the ABCB1-overexpressing cell lines. Furthermore, the resistance of ABCB1-overexpressing cells to palbociclib was reversed by 3 μM of the ABCB1 inhibitor, verapamil. Moreover, the incubation of ABCB1-overexpressing KB-C2 and SW620/Ad300 cells with up to 5 μM of palbociclib for 72 h, significantly upregulated the protein expression of ABCB1. The incubation with 3 µM of palbociclib for 2h significantly increased the intracellular accumulation of [3H]-paclitaxel, a substrate of ABCB1, in ABCB1 overexpressing KB-C2 cells but not in the corresponding non-resistant parental KB-3-1 cell line. However, the incubation of KB-C2 cells with 3 μM of palbociclib for 72 h decreased the intracellular accumulation of [3H]-paclitaxel due to an increase in the expression of the ABCB1 protein. Palbociclib produced a concentration-dependent increase in the basal ATPase activity of the ABCB1 transporter (EC50 = 4.73 μM). Molecular docking data indicated that palbociclib had a high binding affinity for the ABCB1 transporter at the substrate binding site, suggesting that palbociclib may compete with other ABCB1 substrates for the substrate binding site of the ABCB1. Overall, our results indicate that palbociclib is a substrate for the ABCB1 transporter and that its in vitro anticancer efficacy is significantly decreased in cancer cells overexpressing the ABCB1.

Interaction of A3 adenosine receptor ligands with the human multidrug transporter ABCG2

Various adenosine receptor nucleoside-like ligands were found to modulate ATP hydrolysis by the multidrug transporter ABCG2. Both ribose-containing and rigidified (N)-methanocarba nucleosides (C2-, N6- and 5′-modified), as well as adenines (C2-, N6-, and deaza modified), were included. 57 compounds out of 63 tested either stimulated (50) or inhibited (7) basal ATPase activity. Structure-activity analysis showed a separation of adenosine receptor and ABCG2 activities. The 7-deaza modification had favorable effects in both (N)-methanocarba nucleosides and adenines. Adenine 37c (MRS7608) and (N)-methanocarba 7-deaza-5′-ethyl ester 60 (MRS7343) were found to be potent stimulators of ABCG2 ATPase activity with EC50 values of 13.2 ± 1.7 and 13.2 ± 2.2 nM, respectively. Both had affinity in the micromolar range for A3 adenosine receptor and lacked the 5′-amide agonist-enabling group (37c was reported as a weak A3 antagonist, Ki 6.82 μM). Compound 60 significantly inhibited ABCG2 substrate transport (IC50 0.44 μM). Docking simulations predicted the interaction of 60 with 21 residues in the drug-binding pocket of ABCG2.

Modeling the stimulation by glutathione of the steady state kinetics of an adenosine triphosphate binding cassette transporter.

We report the steady state ATPase activities of the ATP Binding Cassette (ABC) exporter NaAtm1 in the absence and presence of a transported substrate, oxidized glutathione (GSSG), in detergent, nanodiscs, and proteoliposomes. The steady state kinetic data were fit to the “nonessential activator model” where the basal ATPase rate of the transporter is stimulated by GSSG. The detailed kinetic parameters varied between the different reconstitution conditions, highlighting the importance of the lipid environment for NaAtm1 function. The increased ATPase rates in the presence of GSSG more than compensate for the modest negative cooperativity observed between MgATP and GSSG in lipid environments. These studies highlight the central role of the elusive ternary complex in accelerating the ATPase rate that is at the heart of coupling mechanism between substrate transport and ATP hydrolysis.

Repositioning application of polyoxyethylene (20) sorbitan monooleate on ocular drug resistance and cancer multi-drug resistance by inhibiting the ATPase activity of human multidrug resistance protein 1 and P-glycoprotein

Drug efflux transporters were highly related to the clinical drug resistance issues, such as cancer multi-drug resistance (MDR) and ocular drug resistance. In the present study, with the focus on human multi-drug resistance protein 1 (MRP1) and P-glycoprotein (P-gp), the inhibitory kinetics of polyoxyethylene (20) sorbitan monooleate (Tween 80) on both drug binding sites and ATPase were in-depth evaluated. We used the stable-cloned ABCB1/Flp-In™-293 and ABCC1/Flp-In™-293 cell lines, and inside-out membrane vesicles for underlying mechanisms investigation while used the drug induced cancer MDR cell line KB/VIN and human retinal pigmented epithelium cell line ARPE-19 for efficacy evaluation. Results showed that Tween 80 exhibited non-competitive inhibition on the doxorubicin efflux of P-gp and MRP1, with the inhibitory affinity 0.00195% (14.89 μM) and 0.00245% (18.7 μM), respectively. Tween 80 inhibited the basal ATPase activity of P-gp and MRP1 in a dose-dependent manner (0.0002–0.02%) and demonstrated significant reversing effects on the doxorubicin, paclitaxel, and vincristine resistance at the concentration of 0.001% (7.63 μM). This was the first thorough study revealing the interactions between Tween 80 and P-gp or MRP1 at a molecular level and these findings suggested that Tween 80 was a potential candidate for future combinatorial regimens applied in the “drug resistance” issue.

Effects of Polyphenols on P-Glycoprotein (ABCB1) Activity.

P-glycoprotein (Pgp, ABCB1) is a member of one of the largest families of active transporter proteins called ABC transporters. Thanks to its expression in tissues with barrier functions and its broad substrate spectrum, it is an important determinant of the absorption, metabolism and excretion of many drugs. Pgp and/or some other drug transporting ABC proteins (e.g., ABCG2, MRP1) are overexpressed in nearly all cancers and cancer stem cells by which cancer cells become resistant against many drugs. Thus, Pgp inhibition might be a strategy for fighting against drug-resistant cancer cells. Previous studies have shown that certain polyphenols interact with human Pgp. We tested the effect of 15 polyphenols of sour cherry origin on the basal and verapamil-stimulated ATPase activity of Pgp, calcein-AM and daunorubicin transport as well as on the conformation of Pgp using the conformation sensitive UIC2 mAb. We found that quercetin, quercetin-3-glucoside, narcissoside and ellagic acid inhibited the ATPase activity of Pgp and increased the accumulation of calcein and daunorubicin by Pgp-positive cells. Cyanidin-3O-sophoroside, catechin, naringenin, kuromanin and caffeic acid increased the ATPase activity of Pgp, while they had only a weaker effect on the intracellular accumulation of fluorescent Pgp substrates. Several tested polyphenols including epicatechin, trans-ferulic acid, oenin, malvin and chlorogenic acid were ineffective in all assays applied. Interestingly, catechin and epicatechin behave differently, although they are stereoisomers. We also investigated the effect of quercetin, naringenin and ellagic acid added in combination with verapamil on the transport activity of Pgp. In these experiments, we found that the transport inhibitory effect of the tested polyphenols and verapamil was additive or synergistic. Generally, our data demonstrate diverse interactions of the tested polyphenols with Pgp. Our results also call attention to the potential risks of drug–drug interactions (DDIs) associated with the consumption of dietary polyphenols concurrently with chemotherapy treatment involving Pgp substrate/inhibitor drugs.