Substrate Efflux Research Articles

Microglia drive diurnal variation in susceptibility to inflammatory blood-brain barrier breakdown.

The blood-brain barrier (BBB) is critical for maintaining brain homeostasis but is susceptible to inflammatory dysfunction. While transporter-dependent efflux of some lipophilic substrates across the BBB shows circadian variation due to rhythmic transporter expression, basal transporter-independent permeability and leakage is nonrhythmic. Whether daily timing influences BBB permeability in response to inflammation is unknown. Here, we induced systemic inflammation through repeated LPS injections either in the morning (ZT1) or evening (ZT13) under standard lighting conditions; we then examined BBB permeability to a polar molecule that is not a transporter substrate, sodium fluorescein. We observed clear diurnal variation in inflammatory BBB permeability, with a striking increase in paracellular leak across the BBB specifically following evening LPS injection. Evening LPS led to persisting glia activation as well as inflammation in the brain that was not observed in the periphery. The exaggerated evening neuroinflammation and BBB disruption were suppressed by microglial depletion or through keeping mice in constant darkness. Our data show that diurnal rhythms in microglial inflammatory responses to LPS drive daily variability in BBB breakdown and reveal time of day as a key regulator of inflammatory BBB disruption.

Soloxolone N-3-(Dimethylamino)propylamide Restores Drug Sensitivity of Tumor Cells with Multidrug-Resistant Phenotype via Inhibition of P-Glycoprotein Efflux Function.

Multidrug resistance (MDR) remains a significant challenge in cancer therapy, primarily due to the overexpression of transmembrane drug transporters, with P-glycoprotein (P-gp) being a central focus. Consequently, the development of P-gp inhibitors has emerged as a promising strategy to combat MDR. Given the P-gp targeting potential of soloxolone amides previously predicted by us by an absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, the aim of the current study was to experimentally verify their P-gp inhibitory and MDR reversing activities in vitro. Screening of soloxolone amides as modulators of P-gp using molecular docking and cellular P-gp substrate efflux assays revealed the ability of compound 4 bearing a N-3-(dimethylamino)propylamide group to interact with the active site of P-gp and inhibit its transport function. Blind and site-specific molecular docking accompanied by a kinetic assay showed that 4 directly binds to the P-gp transmembrane domain with a binding energy similar to that of zosuquidar, a third-generation P-gp inhibitor (ΔG = -10.3 kcal/mol). In vitro assays confirmed that compound 4 enhanced the uptake of Rhodamine 123 (Rho123) and doxorubicin (DOX) by the P-gp-overexpressing human cervical carcinoma KB-8-5 (by 10.2- and 1.5-fold, respectively (p < 0.05, unpaired t-test)) and murine lymphosarcoma RLS40 (by 15.6- and 1.75-fold, respectively (p < 0.05, unpaired t-test)) cells at non-toxic concentrations. In these cell models, 4 showed comparable or slightly higher activity than the reference inhibitor verapamil (VPM), with the most pronounced effect of the hit compound in Rho123-loaded RLS40 cells, where 4 was 2-fold more effective than VPM. Moreover, 4 synergistically restored the sensitivity of KB-8-5 cells to the cytotoxic effect of DOX, demonstrating MDR reversal activity. Based on the data obtained, 4 can be considered as a drug candidate to combat the P-gp-mediated MDR of tumor cells and semisynthetic triterpenoids, with amide moieties in general representing a promising scaffold for the development of novel therapeutics for tumors with low susceptibility to antineoplastic agents.

Changes in Ergosterol Biosynthesis Alter the Response to Cycloheximide, 4-Nitroquinoline-N-Oxide, Weak Organic Acids, and Virulence in Candida glabrata.

The ERG6 gene encodes the sterol C24-methyltransferase converting zymosterol to fecosterol in the ergosterol biosynthetic pathway. Here, we extend the results of functional analysis of the CgERG6 gene, which was previously shown to modulate drug susceptibility in Candida glabrata mutant cells, by demonstrating that its deletion leads to increased susceptibility to cycloheximide, 4-nitroquinoline-N-oxide and weak organic acids, and such effects are associated with attenuated virulence. Together with abrogated efflux of drug substrates by CgCdr1p and CgPdr12p, the Cgerg6Δ mutation leads to reduced cell surface hydrophobicity and decreased virulence of the mutant cells of C. glabrata. The absence of CgErg6p impacts the lipid organization and function of the plasma membrane, resulting in non-specific permeability and abrogation of normal function of membrane-bound proteins accompanied by decreased virulence in Cgerg6Δ cells. Galleria mellonella larvae were used as a non-vertebrate animal host model to determine differences in the virulence potential of C. glabrata strains (parental strain and the Cgerg6Δ deletion mutant). We found that Cgerg6Δ mutant strain attenuated in virulence caused 25-30% survival of larvae compared with parental strain.

Lenacapavir Exhibits Atropisomerism-Mechanistic Pharmacokinetics and Disposition Studies of Lenacapavir Reveal Intestinal Excretion as a Major Clearance Pathway.

Lenacapavir (LEN), a long-acting injectable, is the first approved human immunodeficiency virus type 1 capsid inhibitor and one of a few Food and Drug Administration-approved drugs that exhibit atropisomerism. LEN exists as a mixture of two class 2 atropisomers that interconvert at a fast rate (half-life < 2 hours) with a ratio that is stable over time and unaffected by enzymes or binding to proteins in plasma. LEN exhibits low systemic clearance (CL) in nonclinical species and humans; however, in all species, the observed CL was higher than the in vitro predicted CL. The volume of distribution was moderate in nonclinical species and consistent with the tissue distribution observed by whole-body autoradiography in rats. LEN does not distribute to brain, consistent with being a P-glycoprotein (P-gp) substrate. Mechanistic drug disposition studies with [14C]LEN in intravenously dosed bile duct-cannulated rats and dogs showed a substantial amount of unchanged LEN (31%-60% of dose) excreted in feces, indicating that intestinal excretion (IE) was a major clearance pathway for LEN in both species. Coadministration of oral elacridar, a P-gp inhibitor, in rats decreased CL and IE of LEN. Renal excretion was < 1% of dose in both species. In plasma, almost all radioactivity was unchanged LEN. Low levels of metabolites in excreta included LEN conjugates with glutathione, pentose, and glucuronic acid, which were consistent with metabolites formed in vitro in Hμrel hepatocyte cocultures and those observed in human. Our studies highlight the importance of IE for efflux substrates that are highly metabolically stable compounds with slow elimination rates. SIGNIFICANCE STATEMENT: LEN is a long-acting injectable that exists as conformationally stable atropisomers. Due to an atropisomeric interconversion rate that significantly exceeds the in vivo elimination rate, the atropisomer ratio of LEN remains constant in circulation. The disposition of LEN highlights that intestinal excretion has a substantial part in the elimination of compounds that are metabolically highly stable and efflux transporter substrates.

Lansoprazole (LPZ) reverses multidrug resistance (MDR) in cancer through impeding ATP-binding cassette (ABC) transporter-mediated chemotherapeutic drug efflux and lysosomal sequestration

AimsLansoprazole is one of the many proton pump inhibitors (PPIs) that acts more strongly with ABCB1 and ABCG2. The present study is to investigate the potential of lansoprazole on reversal of ABCB1/G2-mediated MDR in cancer, in vitro and in vivo. MethodsReversal studies and combination evaluation were conducted to determine the synergistic anti-MDR effects on lansoprazole. Lysosomal staining was used to determination of lansoprazole on ABCB1-mediated lysosomal sequestration. Substrate accumulation and efflux assays, ATPase activity, and molecular docking were conducted to evaluate lansoprazole on ABCB1/G2 functions. Western blot and immunofluorescence were used to detect lansoprazole on ABCB1/G2 expression and subcellular localization. MDR nude mice models were established to evaluate the effects of lansoprazole on MDR in vivo. ResultsLansoprazole attenuated ABCB1/G2-mediated MDR and exhibited synergistic effects with substrate drugs in MDR cells. In vivo experiments demonstrated that lansoprazole attenuated ABCB1/G2-mediated MDR and exhibited synergistic effects that augmented the sensitivity of substrate anticancer drugs in ABCB1/G2-mediated settings without obvious toxicity. Lansoprazole impeded lysosomal sequestration mediated by ABCB1, leading to a substantial increase in intracellular accumulation of substrate drugs. The effects of lansoprazole were not attributable to downregulation or alterations in subcellular localization of ABCB1/G2. Lansoprazole promoted the ATPase activity of ABCB1/G2 and competitively bound to the substrate-binding region of ABCB1/G2. ConclusionsThese findings present novel therapeutic avenues whereby the combination of lansoprazole and chemotherapeutic agents mitigates MDR mediated by ABCB1/G2 overexpression.

Deciphering the functional role of clinical mutations in ABCB1, ABCC1, and ABCG2 ABC transporters in endometrial cancer.

ATP-binding cassette transporters represent a superfamily of dynamic membrane-based proteins with diverse yet common functions such as use of ATP hydrolysis to efflux substrates across cellular membranes. Three major transporters-P-glycoprotein (P-gp or ABCB1), multidrug resistance protein 1 (MRP1 or ABCC1), and breast cancer resistance protein (BCRP or ABCG2) are notoriously involved in therapy resistance in cancer patients. Despite exhaustive individual characterizations of each of these transporters, there is a lack of understanding in terms of the functional role of mutations in substrate binding and efflux, leading to drug resistance. We analyzed clinical variations reported in endometrial cancers for these transporters. For ABCB1, the majority of key mutations were present in the membrane-facing region, followed by the drug transport channel and ATP-binding regions. Similarly, for ABCG2, the majority of key mutations were located in the membrane-facing region, followed by the ATP-binding region and drug transport channel, thus highlighting the importance of membrane-mediated drug recruitment and efflux in ABCB1 and ABCG2. On the other hand, for ABCC1, the majority of key mutations were present in the inactive nucleotide-binding domain, followed by the drug transport channel and membrane-facing regions, highlighting the importance of the inactive nucleotide-binding domain in facilitating indirect drug efflux in ABCC1. The identified key mutations in endometrial cancer and mapped common mutations present across different types of cancers in ABCB1, ABCC1, and ABCG2 will facilitate the design and discovery of inhibitors targeting unexplored structural regions of these transporters and re-engineering of these transporters to tackle chemoresistance.

Model of P-Glycoprotein Ligand Binding and Validation with Efflux Substrate Matched Pairs.

The blood-brain barrier (BBB) poses a significant obstacle in developing therapeutics for neurodegenerative diseases and central nervous system (CNS) disorders. P-glycoprotein (P-gp), a multidrug resistance protein, is a critical gatekeeper in the BBB and plays a role in cancer chemoresistance. This paper uses cryo-EM P-gp structures as starting points with an induced fit docking (IFD) model to evaluate 19 pairs of compounds with known P-gp efflux data. The study reveals significant differences in binding energy and sheds light on structural modifications' impact on efflux properties. In the cases examined, fluorine incorporation influences the efflux by altering the molecular conformation rather than proximal heteroatom basicity. Although there are limitations in addressing covalent interactions or when binding extends into the more flexible vestibule region of the protein, the results provide valuable insights and potential strategies to overcome P-gp efflux, contributing to the advancement of drug development for both CNS disorders and cancer therapies.

Abstract 3102: A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University

Abstract Background: Drug development is important for cancer therapy to improve patient outcomes. In most cases, drug development starts in discovering candidate compounds from a chemical library in high-throughput screening (HTS). Tohoku University Graduate School of Pharmaceutical Science owns the original chemical library consisting of thousands of original chemical compounds. P-glycoprotein (P-gp), a major drug efflux transporter mediates multi-drug resistance (MDR) in cancer cells. Therefore, P-gp modulators could revere MDR and increase chemosensitivity. The aim of this study is to reveal an adequate method of HTS for discovering potent P-gp modulators from the chemical library in Tohoku University. Methods: The chemical library in Tohoku University had 5861 compounds in the present study, and the collection contains over 7 thousand compounds in 2023. P-gp overexpressing KB-V1, human epidermal carcinoma cell lines were obtained, and calcein AM, P-gp substrate was employed for fluorescent substrate efflux assays. A plate reader-based calcein AM efflux assay for HTS was established to select candidate compounds as P-gp modulators. After the primary HTS, structures of the candidate compounds were analyzed, then several analogous compounds were extracted from the chemical library. For the validation of the hit compounds in HTS and the analogs, a flow cytometry-based calcein AM efflux assay was conducted. In addition, reversal effects of P-gp modulators on the cytotoxicity of paclitaxel were measured in a cell proliferation assay. Results: Of 5861 compounds in the chemical library, 53 compounds showed 2-fold or higher fluorescence intensity, and then 13 compounds showed 3-fold or higher fluorescence intensity in a plate reader-based assay for the HTS. Especially 2 candidate compounds showed over 5-fold fluorescence intensity. In the analysis for structures of the candidate compounds, 20 analogous compounds were extracted from the chemical library. To verify the result of the plate reader-based assay, a flow cytometry-based assay was conducted for 53 hits and 20 analogs. As a result, the 2 candidate compounds that selected in the plate reader-based assay showed 60-fold, the highest fluorescence intensity in a flow cytometry-based assay. The other compounds showed less fluorescence intensity than these 2 candidates. In addition, both candidates enhanced the paclitaxel-induced cytotoxicity in a cell proliferation assay. Conclusions: A plate reader-based calcein AM efflux assay in HTS was an adequate method for discovering potent P-gp modulators from the chemical library in Tohoku University. Citation Format: Norihiko Sugisawa, Shinobu Ohnuma, Takayuki Doi, Michiaki Unno. A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3102.

Molecular determinant deciphering of MIC-guided RND efflux substrates in E. coli

Antimicrobial resistance poses an urgent and formidable global public health threat. The escalation of bacterial multidrug resistance to antibiotics has the potential to become a leading cause of global mortality if there is no substantial improvement in antimicrobial development and therapy protocols. In light of this, it is imperative to identify the molecular determinants responsible for the reduced antibiotic activity associated with RND efflux pumps. This comprehensive study meticulously examines Minimum Inhibitory Concentration (MIC) data obtained from in vitro tests for various antibiotic families and non-active dye compounds, sourced from diverse literature references. The primary focus of this study is to assess the susceptibility of these agents to efflux-resistant Escherichia coli strains, integrating both MIC data and relevant physicochemical properties. The central objective is to unveil the specific substituents that significantly influence the uptake process mediated by the AcrAB-TolC efflux system. This exploration seeks to reveal the consequences of these substituents on pharmacodynamic responses, providing valuable insights into Structure-Activity Relationships. It is noteworthy that this analysis represents a pioneering effort, with prospective implications for RND efflux pump-producing strains. Ultimately, deciphering efflux markers is crucial to effectively mitigate the emergence of specific resistance and to better monitor the role of this primary resistance mechanism in Gram-negative bacteria, particularly as observed in clinical antibiotic therapy practice.

Maternal nitric oxide homeostasis impacts female gametophyte development under optimal and stress conditions.

In adverse environments, the number of fertilizable female gametophytes (FGs) in plants is reduced, leading to increased survival of the remaining offspring. How the maternal plant perceives internal growth cues and external stress conditions to alter FG development remains largely unknown. We report that homeostasis of the stress signaling molecule nitric oxide (NO) plays a key role in controlling FG development under both optimal and stress conditions. NO homeostasis is precisely regulated by S-nitrosoglutathione reductase (GSNOR). Prior to fertilization, GSNOR protein is exclusively accumulated in sporophytic tissues and indirectly controls FG development in Arabidopsis (Arabidopsis thaliana). In GSNOR null mutants, NO species accumulated in the degenerating sporophytic nucellus, and auxin efflux into the developing FG was restricted, which inhibited FG development, resulting in reduced fertility. Importantly, restoring GSNOR expression in maternal, but not gametophytic tissues, or increasing auxin efflux substrate significantly increased the proportion of normal FGs and fertility. Furthermore, GSNOR overexpression or added auxin efflux substrate increased fertility under drought and salt stress. These data indicate that NO homeostasis is critical to normal auxin transport and maternal control of FG development, which in turn determine seed yield. Understanding this aspect of fertility control could contribute to mediating yield loss under adverse conditions.

ABCC4 suppresses glioblastoma progression and recurrence by restraining cGMP-PKG signalling.

Cyclic nucleotides are critical mediators of cellular signalling in glioblastoma. However, the clinical relevance and mechanisms of regulating cyclic nucleotides in glioblastoma progression and recurrence have yet to be thoroughly explored. In silico, mRNA, and protein level analyses identified the primary regulator of cyclic nucleotides in recurrent human glioblastoma. Lentiviral and pharmacological manipulations examined the functional impact of cyclic nucleotide signalling in human glioma cell lines and primary glioblastoma cells. An orthotopic xenograft mice model coupled with aspirin hydrogels verified the in vivo outcome of targeting cyclic nucleotide signalling. Elevated intracellular levels of cGMP, instead of cAMP, due to a lower substrate efflux from ATP-binding cassette sub-family C member 4 (ABCC4) is engaged in the recurrence of glioblastoma. ABCC4 gene expression is negatively associated with recurrence and overall survival outcomes in glioblastoma specimens. ABCC4 loss-of-function activates cGMP-PKG signalling, promoting malignancy in glioblastoma cells and xenografts. Hydrogels loaded with aspirin, inhibiting glioblastoma progression partly by upregulating ABCC4 expressions, augment the efficacy of standard-of-care therapies in orthotopic glioblastoma xenografts. ABCC4, repressing the cGMP-PKG signalling pathway, is a tumour suppressor in glioblastoma progression and recurrence. Aspirin hydrogels impede glioblastoma progression through ABCC4 restoration and constitute a viable translational approach.

Inverse pH Gradient-Assay for Facile Characterization of Proton-Antiporters in Xenopus Oocytes.

Xenopus oocytes represent one of the most versatile model systems for characterizing the properties of membrane transporters. However, for studying proton-coupled antiporters, the use of Xenopus oocytes has so far been limited to so-called injection-based transport assays. In such assays, where the compound is injected directly into the oocytes' cytosol and transport is detected by monitoring substrate efflux, poor control over internal diffusion and concentration are incompatible with mechanistic characterizations. In this study, we present an inverse pH-gradient transport assay. Herein, an outward-facing proton gradient enables the characterization of proton antiporters via facile import-based transport assays. We describe two approaches for establishing sustained outward-facing proton gradients across the oocyte membrane, namely by applying alkaline external conditions or through surprisingly stable carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)-mediated acidification of the cytosol. Previously, genetic evidence has shown that DTX18 from Arabidopsis thaliana is essential for the deposition of the hydroxycinnamic acid amide p-coumaroylagmatine (coumaroylagmatine) defence compound on the leaf surface. However, direct evidence for its ability to transport coumarol-agmatine has not been provided. Here, using Xenopus oocytes as expression hosts, we demonstrate DTX18's ability to transport coumaroyl-agmatine via both injection-based and inverse pH-gradient transport assays. Notably, by showing that DTX18 is capable of accumulating its substrate against its concentration gradient, we showcase the compatibility of the latter with mechanistic investigations.

ABCB1 overexpression through locus amplification represents an actionable target to combat paclitaxel resistance in pancreatic cancer cells

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and the chemotherapies such as gemcitabine/nab-paclitaxel are confronted with intrinsic or acquired resistance. The aim of this study was to investigate mechanisms underlying paclitaxel resistance in PDAC and explore strategies to overcome it.MethodsThree paclitaxel (PR) and gemcitabine resistant (GR) PDAC models were established. Transcriptomics and proteomics were used to identify conserved mechanisms of drug resistance. Genetic and pharmacological approaches were used to overcome paclitaxel resistance.ResultsUpregulation of ABCB1 through locus amplification was identified as a conserved feature unique to PR cells. ABCB1 was not affected in any of the GR models and no cross resistance was observed. The ABCB1 inhibitor verapamil or siRNA-mediated ABCB1 depletion sensitized PR cells to paclitaxel and prevented efflux of ABCB1 substrates in all models. ABCB1 expression was associated with a trend towards shorter survival in patients who had received gemcitabine/nab-paclitaxel treatment. A pharmacological screen identified known and novel kinase inhibitors that attenuate efflux of ABCB1 substrates and sensitize PR PDAC cells to paclitaxel.ConclusionUpregulation of ABCB1 through locus amplification represents a novel, conserved mechanism of PDAC paclitaxel resistance. Kinase inhibitors identified in this study can be further (pre) clinically explored as therapeutic strategies to overcome paclitaxel resistance in PDAC.

PH-Responsive Polypropylene Sulfide Magnetic Nanocarrier-Mediated Chemo-Hyperthermia Kills Breast Cancer Stem Cells by Long-Term Reversal of Multidrug Resistance and Chemotherapy Resensitization.

Cancer stem cells (CSCs) present a formidable challenge in cancer treatment due to their inherent resistance to chemotherapy, primarily driven by the overexpression of ABC transporters and multidrug resistance (MDR). Despite extensive research on pharmacological small-molecule inhibitors, effectively managing MDR and improving chemotherapeutic outcomes remain elusive. On the other hand, magnetic hyperthermia (MHT) holds great promise as a cancer therapeutic, but there is limited research on its potential to reverse MDR in breast CSCs and effectively eliminate CSCs through combined chemo-hyperthermia. To address these gaps, we developed tumor microenvironment-sensitive, drug-loaded poly(propylene sulfide) (PPS)-coated magnetic nanoparticles (PPS-MnFe). These nanoparticles were employed to investigate hyperthermia sensitivity and MDR reversion in breast CSCs, comparing their performance to that of small-molecule inhibitors. Additionally, we explored the efficacy of combined chemo-hyperthermia in killing CSCs. CSC-enriched breast cancer cells were subjected to low-dose MHT at 42 °C for 30 min and then treated with the chemical MDR inhibitor salinomycin (SAL). The effectiveness of each treatment in inhibiting MDR was assessed by measuring the efflux of the MDR substrate, rhodamine 123 (R123) dye. Notably, MHT induced a prolonged reversal of MDR activity compared with SAL treatment alone. After successfully inhibiting MDR, the breast CSCs were exposed to chemotherapy using paclitaxel to trigger synergistic cell death. The combination of MHT and chemotherapy demonstrated remarkable reductions in stemness properties, MDR reversal, and the effective eradication of breast CSCs in this innovative dual-modality approach.

Utilizing a Dual Human Transporter MDCKII-MDR1-BCRP Cell Line to Assess Efflux at the Blood Brain Barrier.

To facilitate the design of drugs readily able to cross the blood brain barrier (BBB), a Madin-Darby canine kidney (MDCK) cell line was established that over expresses both P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), the main human efflux transporters of the BBB. Proteomics analyses indicate BCRP is expressed at a higher level than Pgp in this cell line. This cell line shows good activity for both transporters [BCRP substrate dantrolene efflux ratio (ER) 16.3 ± 0.9, Pgp substrate quinidine ER 27.5 ± 1.2], and use of selective transporter inhibitors enables an assessment of the relative contributions to overall ERs. The MDCKII-MDR1-BCRP ER negatively correlates with rat unbound brain/unbound plasma ratio, Kpuu Highly brain penetrant compounds with rat Kpuu ≥ 0.3 show ERs ≤ 2 in the MDCKII-MDR1-BCRP assay while compounds predominantly excluded from the brain, Kpuu ≤ 0.05, demonstrate ERs ≥ 20. A subset of compounds with MDCKII-MDR1-BCRP ER < 2 and rat Kpuu < 0.3 were shown to be substrates of rat Pgp using a rat transfected cell line, MDCKII-rMdr1a. These compounds also showed ERs > 2 in the human National Institutes of Health (NIH) MDCKI-MDR1 (high Pgp expression) cell line, which suggests that they are weak human Pgp substrates. Characterization of 37 drugs targeting the central nervous system in the MDCKII-MDR1-BCRP efflux assay show 36 have ERs < 2. In drug discovery, use of the MDCKII-MDR1-BCRP in parallel with the NIH MDCKI-MDR1 cell line is useful for identification of compounds with high brain penetration. SIGNIFICANCE STATEMENT: A single cell line that includes both the major human efflux transporters of the blood brain barrier (MDCKII-MDR1-BCRP) has been established facilitating the rapid identification of efflux substrates and enabling the design of brain penetrant molecules. Efflux ratios using this cell line demonstrate a clear relationship with brain penetration as defined by rat brain Kpuu.

Probing the pH Sensitivity of OprM: Insights into Metastable States and Semi-Open Conformation.

Efflux pumps are specialized transport proteins that play a key role in the bacterial defense against a wide spectrum of antibiotics. Hence, understanding the biophysical mechanism associated with this complex system of drug expulsion becomes crucial. This work deals with some vital aspects of the outer membrane factor (OMF) of MexAB-OprM. After being passed through MexB and MexA, efflux substrates have to go through OprM for their final judgment. Thus, it is very important to understand the periplasmic pore opening mechanism and the associated biophysical changes during this process. Our study captures a detailed analysis of the pore opening mechanism involving OprM. With powerful molecular dynamics (MD) techniques such as well-tempered metadynamics, the presence of metastable states in between open and closed states was confirmed. Also, upon mutating R376, the energy barrier for the conversion of the close to open conformation decreases, indicating an important role played by the residue. Further, constant pH MD was performed to capture the effect of pH in both conformations. OprM exhibits distinct conformational states at pH values greater than 5.5 and lower than 5.5, suggesting its pH-responsive characteristics. Overall, our study elucidates a crucial undertaking toward discovering potential inhibitors for MexAB-OprM efflux pumps.

Functional characterization of two TolC in the resistance to drugs and metals and in the virulence of Riemerella anatipestifer.

Riemerella anatipestifer (RA) is a notorious duck pathogen, characterized by a multitude of serotypes that exhibit no cross-reaction with one another. Moreover, RA is resistant to various antibacterial agents. Consequently, understanding the mechanisms behind resistance and identifying potential targets for drug development have become pressing needs. In this study, we show that the two TolC proteins play a role in the resistance to different drugs and metals and in the virulence. The results suggest that TolCA has a wider range of efflux substrates than TolCB. Except for gentamicin, neither TolCA nor TolCB was involved in the efflux of the other tested antibiotics. Strikingly, TolCA but not TolCB enhanced the frequency of resistance-conferring mutations. Moreover, TolCA was involved in RA virulence. Given its conservation in RA, TolCA has potential as a drug target for the development of therapeutics against RA infections.

Combined in silico and in vitro approaches to identify P-glycoprotein-inhibiting pesticides.

The P-glycoprotein (P-gp) efflux pump plays a major role in xenobiotic detoxification. The inhibition of its activity by environmental contaminants remains however rather little characterised. The present study was designed to develop a combination of different approaches to identify P-gp inhibitors among a large number of pesticides using in silico and in vitro models. First, the prediction performance of four web tools was evaluated alone or in combination using a set of recently marketed drugs. The best combination of web tools-AdmetSAR2.0/PgpRules/pkCSM-was next used to predict P-gp activity inhibition by 762 pesticides. Among the 187 pesticides predicted to be P-gp inhibitors, 11 were tested in vitro for their ability to inhibit the efflux of reference substrates (rhodamine 123 and Hoechst 33342) in P-gp overexpressing MCF7R cells and to inhibit the efflux of the reference substrate rhodamine 123 in the Caco-2 cell monolayer. In MCF7R cell assays, ivermectin B1a, emamectin B1 benzoate, spinosad, dimethomorph and tralkoxydim inhibited P-gp activity; ivermectin B1a, emamectin B1 benzoate and spinosad were determined to be stronger inhibitors (half-maximal inhibitory concentration [IC50 ] of 3 ± 1, 5 ± 1 and 7 ± 1 µM, respectively) than dimethomorph and tralkoxydim (IC50 of 102 ± 7 and 88 ± 7 µM, respectively). Ivermectin B1a, emamectin B1 benzoate, spinosad and dimethomorph also inhibited P-gp activity in Caco-2 cell monolayer assays, with dimethomorph being a weaker P-gp inhibitor. These combined approaches could be used to identify P-gp inhibitors among food contaminants, but need to be optimised and adapted for high-throughput screening.

Mosloflavone from Fissistigma petelotii ameliorates oncogenic multidrug resistance by STAT3 signaling modulation and P-glycoprotein blockade

BackgroundOncogenic multidrug resistance (MDR) is a tough question in cancer therapy. However, no effective medications targeting oncogenic MDR are currently available. Studies have demonstrated that mosloflavone exerts anti-inflammatory effects, yet, its potential to ameliorate MDR remains unclear. PurposeThis study aimed to access the capability and elucidate molecular mechanisms of mosloflavone as a MDR resensitizing candidate. MethodsWe investigated the ability of mosloflavone to reverse oncogenic MDR and investigated its underlying mechanisms through cytotoxicity assay, cell cycle assay, apoptosis assay, and zebrafish xenograft model. The modulatory interplay between mosloflavone and P-gp was investigated through analysis of calcein-AM uptake, substrate efflux, ATPase assays, and molecular docking simulation. ResultsMosloflavone inhibited P-gp efflux function in an uncompetitive manner without altering ABCB1 gene expression. In addition, it stimulated P-gp ATPase activity by binding to an active site distinct from that of verapamil. Regarding MDR reversal potential, mosloflavone resensitized MDR cancer cells to chemotherapies by arresting cell cycle and triggering apoptosis, possibly by enhancing reactive oxygen species accumulation and reducing phospho-STAT3. Moreover, in the zebrafish xenograft model, mosloflavone significantly potentiated the antitumor effect of paclitaxel. ConclusionOur findings underscore the potential of mosloflavone as a novel dual modulator of STAT3 and P-gp, indicating it is a promising candidate for overcoming MDR in cancer treatment.

Mitochondrial Reprogramming By Bcl-2 Inhibitor Venetoclax Enhances αCD19 CAR-T Cell Fitness and Anti-Tumor Efficacy

Introduction Patients undergoing anti-CD19 chimeric antigen receptor T cell (CAR T) therapy for the treatment of aggressive B-cell non-Hodgkin lymphomas (B-NHL) can expect a complete response rate of merely 43-68%. Insubstantial clinical responses to CAR T therapy and a small window of survival for patients facing relapsed/refractory (R/R) disease highlights the critical need for improvement of CAR T therapy in aggressive B-NHL cases. Recent findings suggest that enriching for a CD8 + T Stem Cell Memory (T SCM) population within adoptive cell therapies translates to enhanced antitumor function and persistence in vivo, and thus produces favorable clinical outcomes. T SCM cells display heightened self-renewal, low mitochondrial membrane potential (∆Ψm), and are metabolically dependent upon oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO). Selection of CD8 + T SCM subsets is therefore necessary to ensure a robust antitumor response. Previously, we revealed synergy between the Bcl-2 inhibitor, Venetoclax, and CAR T cells in the context of B-NHL. Venetoclax exposure increased CD8 + T SCM cell frequency, decreased ∆Ψm, reduced T Regulatory (T REG) cell frequency, and bolstered effector cytokine production. Though the role of Bcl-2 inhibition within B-NHLs is well characterized (FDA-approved for frontline and relapsed/refractory chronic lymphocytic leukemia [CLL] treatment), further investigation regarding the impact of Venetoclax on the patient T cell compartment is needed. In this study, we demonstrate a role for Venetoclax as a qualitative enhancer of bulk CAR T cell product using both healthy-donor and B-NHL patient-derived T cells, and implicate modulation of mitochondrial pathways as a likely mechanism of action. Methods Whole peripheral blood mononuclear cells were collected from consenting healthy human donors or CLL patients undergoing ramp-up with Venetoclax (BDR 164122) at Roswell Park Comprehensive Cancer Center, and activated with αCD3/αCD28. CLL samples were collected at baseline, 1 day, 7 days, and 1-month timepoints. Transduction was performed with lentiviral vectors encoding CD28 or 4-1BB-based α-CD19 CAR constructs 24 hours post-activation. Transduced cells were cultured in RPMI with hIL-2 and hIL-7. In vivo studies were performed on 7-week-old SCID mice using Raji-Luc, Rec-1, and U2932. Venetoclax was given daily [100mg/kg] via oral gavage. CAR T cells were delivered by single infusion via tail-vein injection at a dose of either 1x10 6 or 3x10 6 cells total.Mice were imaged via IVIS spectrum. Western blot was performed on stimulated untransduced T cells or CAR T cells exposed to Venetoclax. Total protein was quantified, and membranes were probed for apoptotic and mitochondrial protein expression. Patient sample immunophenotyping was performed on CLL patient samples using panels interrogating memory differentiation, exhaustion, and T REG populations. Results Consistent with our hypothesis, a significant increase in mitochondrial proteins associated with FAO and OXPHOS - ACC, PDH, and AMPKα - was observed in exposed CAR T cells, as well increased expression of transcription factor FOXO1 - an essential driver of CD8 + memory differentiation. Voltage-dependent anion channel (VDAC) expression increased, suggesting heightened efflux of respiratory substrates into the tricarboxylic acid cycle. Hexokinase-I, a critical glycolytic enzyme, was significantly downregulated. We next investigated our combination therapy in vivo and observed extended survival outcome by 15 days. Further in vivo studies investigating preclinical models of mantle cell lymphoma and activated B-cell diffuse large B-cell lymphoma are currently ongoing. Within our CLL patient samples, 24-hour and 7-day timepoints reflected a favorable population increase in CD8 + T SCM cells as compared to baseline samples, and marginally restored CD8 + T cell frequencies. Conclusion Metabolic properties guide T cell memory differentiation and fitness. There exists potential to manipulate this mechanism and select the most metabolically fit T cell population for adoptive cell therapies. In addition to answering fundamental questions of CAR T cell biology, the combination of Venetoclax and CAR T cell therapy may provide a solution to the observed clinical gap, in which CAR T therapy as a single agent does not afford favorable response rates in the context of aggressive and R/R B-NHLs.