Overexpression of the polyspecific efflux transporter, P-glycoprotein (P-gp, MDR1, ABCB1 ), is a major mechanism by which cancer cells acquire multidrug resistance (MDR), the resistance to diverse chemotherapeutic drugs. Inhibiting drug transport by P-gp can resensitize cancer cells to chemotherapy, but there are no P-gp inhibitors available to patients. Clinically unsuccessful P-gp inhibitors tend to bind at the pump's transmembrane drug binding domains and are often P-gp transport substrates, resulting in lowered intracellular concentration of the drug and altered pharmacokinetics. In prior work, we used computationally accelerated drug discovery to identify novel P-gp inhibitors that target the pump's cytoplasmic nucleotide binding domains. Our first-draft study provided conclusive evidence that the nucleotide binding domains of P-gp are viable targets for drug discovery. Here we develop an enhanced, computationally accelerated drug discovery pipeline that expands upon our prior work by iteratively screening compounds against multiple conformations of P-gp with molecular docking. Targeted molecular dynamics simulations with our homology model of human P-gp were used to generate docking receptors in conformations mimicking a putative drug transport cycle. We offset the increased computational complexity using custom Tanimoto chemical datasets, which maximize the chemical diversity of ligands screened by docking. Using our expanded, virtual-assisted pipeline, we identified nine novel P-gp inhibitors that reverse MDR in two types of P-gp overexpressing human cancer cell lines, reflecting a 13.4% hit rate. Of these inhibitors, all were non-toxic to non-cancerous human cells, and six were not likely to be transport substrates of P-gp. Our novel P-gp inhibitors are chemically diverse and are good candidates for lead optimization. Our results demonstrate that the nucleotide binding domains of P-gp are an underappreciated target in the effort to reverse P-gp-mediated multidrug resistance in cancer.

Overexpression of the polyspecific efflux transporter, P-glycoprotein (P-gp, MDR1, ABCB1 ), is a major mechanism by which cancer cells acquire multidrug resistance (MDR), the resistance to diverse chemotherapeutic drugs. Inhibiting drug transport by P-gp can resensitize cancer cells to chemotherapy. However, there are no P-gp inhibitors available to patients. Clinically unsuccessful P-gp inhibitors tend to bind at the pump’s transmembrane drug binding domains and are often P-gp transport substrates, resulting in lowered intracellular concentration of the drug and altered pharmacokinetics. In prior work, we used virtual-assisted drug discovery to identify novel P-gp inhibitors that target the pump’s cytoplasmic nucleotide binding domains and showed that these domains are viable drug discovery targets. Here we develop an enhanced virtual-assisted pipeline that expands upon prior work by iteratively screening compounds against multiple regions and conformations of P-gp. This increased computational complexity is offset by custom Tanimoto chemical datasets which compress the initial docking library while maximizing chemical diversity. Molecules that were similar to resultant hits were subsequently retrieved and screened. We identified nine novel P-gp inhibitors that reverse MDR in two types of P-gp overexpressing human cancer cell lines, reflecting a 14% hit rate. Of these inhibitors, all were non-toxic to non-cancerous human cells, and six were not transport substrates of P-gp. Our novel P-gp inhibitors are chemically diverse and are good candidates for lead optimization. Our results demonstrate that the nucleotide binding domains of P-gp are an underused target in the effort to reverse P-gp-mediated multidrug resistance. ### Competing Interest Statement The authors have declared no competing interest.

The gall wasp, Hemadas nubilipennis Ashmead, is a pest of highbush and lowbush blueberry and can pose a challenge to control with foliar sprays due to adult activity being during bloom and because larval development is within plant tissues. We hypothesized that systemic insecticides that move within the blueberry vascular system would reach areas where H. nubilipennis eggs are laid, causing larval mortality. Three application methods, crown injection, soil drench, and foliar spray were applied to potted 'Jersey' blueberry bushes at 50% and 100% rates to quantify systemic residue concentrations in shoots and leaves. Additionally, systemic insecticides were evaluated for control of gall wasps using single-shoot bioassays and measuring larval mortality at 0.01%, 0.1%, 1%, and 10% of field rate provided within a floral pick. Systemic insecticides tested in both studies included imidacloprid, flupyradifurone, and spirotetramat. The potted bush residue study determined that insecticides moved from three tested sites of entry: the roots, crown cavity, and foliage. Results from the shoot bioassays found that the mean percent larval survival of H. nubilipennis was negatively correlated with the concentration of AI detected in galls. Imidacloprid and spirotetramat were found to have the greatest potential for control of H. nubilipennis due to mortality in the shoot bioassays and similar residue concentrations in the potted bush studies to shoot bioassays. Future research should evaluate systemic insecticides applied in highbush blueberry plantings for control of H. nubilipennis using the bioassay mortality assessment method developed in this study.

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer’s disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer’s associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.

The MtrCDE system confers multidrug resistance to Neisseria gonorrheae, the causative agent of gonorrhea. Using free and directed molecular dynamics (MD) simulations, we analyzed the interactions between MtrD and azithromycin, a transport substrate of MtrD, and a last-resort clinical treatment for multidrug-resistant gonorrhea. We then simulated the interactions between MtrD and streptomycin, an apparent nonsubstrate of MtrD. Using known conformations of MtrD homologues, we simulated a potential dynamic transport cycle of MtrD using targeted MD techniques (TMD), and we noted that forces were not applied to ligands of interest. In these TMD simulations, we observed the transport of azithromycin and the rejection of streptomycin. In an unbiased, long-time scale simulation of AZY-bound MtrD, we observed the spontaneous diffusion of azithromycin through the periplasmic cleft. Our simulations show how the peristaltic motions of the periplasmic cleft facilitate the transport of substrates by MtrD. Our data also suggest that multiple transport pathways for macrolides may exist within the periplasmic cleft of MtrD.

Antibiotic-resistant gonorrheal infections are an urgent health concern in the United States. The MtrCDE system confers multidrug resistance to Neisseria gonorrhoeae, the causative agent of gonorrhea. Specifically, the inner membrane pump MtrD effluxes a variety of structurally diverse hydrophobic and amphiphilic substrates and thus confers resistance to a multitude of antibiotics. Using computational methods, we analyzed the interactions of MtrD with Azithromycin, an MtrD substrate, and one of the last remaining courses of treatment for multidrug resistant gonorrhea. Using targeted Molecular Dynamics (MD) simulations and known conformations of MtrD homologues, we guided MtrD through the conformational changes of a putative transport pathway by applying small forces to α-carbons of the protein backbone; forces were not applied to the ligands of interest. In these short timescale, targeted MD simulations, we demonstrate the transport of Azithromycin (in three possible protonation states) and the rejection of Streptomycin, which is not an MtrD substrate. To supplement our findings, we then demonstrate the spontaneous diffusion of Azithromycin through the periplasmic cleft in long time-scale, unbiased MD simulations. Our findings support the hypothesis that the transition from ‘Binding’ to ‘Extrusion’ is the energy requiring step in the transport process. To our knowledge, this is the first computational demonstration of substrate transport, and non-substrate rejection, by MtrD.

Fall armyworm is one of the main pests of conventional and Bacillus thuringiensis (Bt) corn in many countries in the Americas, Africa, Asia and in Australia. We conducted diet-overlay bioassays to determine the status of susceptibility to four Bt proteins (Cry1A.105, Cry2Ab2, Cry1F and Cry1Ac) in three different populations of fall armyworm from Mexico, and one population from Puerto Rico. Bioassays showed that fall armyworms from Puerto Rico were resistant to Cry1F with a resistance ratio 50 (RR50) higher than 10,000 ng/cm2 and to Cry1Ac with a RR50 = 12.2 ng/cm2, displaying the highest median lethal concentration (LC50) values to all Bt proteins tested. The effective concentration 50 (EC50) values further confirmed the loss of susceptibility to Cry1F and Cry1Ac in this population. However, LC50 and EC50 results with Cry1A.105 and Cry2Ab2 revealed that fall armyworm from Puerto Rico remained largely susceptible to these two proteins. The Mexican populations were highly susceptible to all the Bt proteins tested and displayed the lowest LC50 and EC50 values to all Bt proteins. Our results suggest that Cry1F and Cry1Ac resistance is stable in fall armyworm from Puerto Rico. However, this population remains susceptible to Cry1A.105 and Cry2Ab2. Results with Mexican fall armyworms suggest that possible deployment of Bt corn in Mexico will not be immediately challenged by Bt-resistant genes in those regions.

The breast cancer resistance protein (BCRP) is an ABC transporter that uses energy derived from ATP hydrolysis to export toxins from the cell. In cancer cells, these transporters are overexpressed and remove chemotherapeutic drugs from the cell. This confers multidrug resistance. We have previously conducted high‐throughput in silico ligand docking studies to identify potential inhibitors of BCRP. These drug‐like molecules inhibit ATP hydrolysis by targeting the nucleotide binding domain. Biochemical and biophysical analyses of these inhibitors are necessary to study their inhibition mechanisms. Large amounts of purified protein are required to carry out these analyses. Our lab previously generated a gene for human BCRP that was codon optimized for protein expression in the yeast, PichiaPinkTM. Here we report our attempts to purify BCRP from this strain as well as from a second, BCRP over‐expressing Pichia Pastoris strain. Growth conditions were previously optimized using methanol to induce BCRP expression. We also report our efforts to purify BCRP into detergent mixed micelles using Ni‐NTA affinity and ion exchange chromatography. Attempts to assemble BCRP containing nativelike membrane nanodiscs to achieve maximum ATP hydrolysis are also reported. Obtaining purified BCRP in a native‐like environment with high activity is crucial for the assessment of BCRP inhibition mechanisms of our newly found drug‐like compounds.Support or Funding InformationThis work is supported by the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D., of Naples, FL.

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer cells due to its role in the transport of chemotherapeutic drugs out of the cells in an ATP‐dependent process. To search for P‐gp inhibitors as co‐therapeutics to combat MDR, we previously used high‐throughput in silico ligand docking studies to identify drug‐like compounds. To assess functionality of the hit compounds we measured their effects on P‐gp ATPase hydrolysis activity. Some of the compounds inhibited ATP hydrolysis of purified P‐gp and successfully reversed the MDR in prostate and ovarian cancer cell lines. We subsequently used computational approaches and structure‐based design to produce variants of one of the hit compounds with predicted higher affinity to P‐gp. The efficacy of these variants in reversing MDR in cell culture as well as the biochemical evaluation of inhibition was assessed. High expression of human P‐gp in the yeast Pichia pastoris in a biologically active form was previously reported and the purified P‐gp was used on our recent assays. Currently, reconstitution of P‐gp into nanodiscs allowed for human P‐gp to be solubilized in its active form with ATPase activity enhanced by 2–3 fold as well as increased stability. Here we report the evaluation of the potential of our previously discovered drug‐like compounds to serve as inhibitors of human P‐gp ATP hydrolysis activity.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D. of Naples Florida. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.

P‐glycoprotein (P‐gp) belongs to the ATP‐Binding Cassette (ABC) transporter family of proteins and is strongly linked with multidrug resistance (MDR) in tumors where overexpression often abolishes the effects of chemotherapeutics by removing the therapeutics from the cells. In hopes of creating co‐therapeutics to re‐sensitize tumors to chemotherapeutics, we previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. To evaluate the mechanism of inhibition in human P‐gp, we have isolated human P‐gp from the yeast Pichia pastoris using mixed detergent micelles. In order to effectively assess the potential inhibitors, we must be able to sufficiently stimulate our protein and have a high enough activity to see the inhibitory effects of the potential inhibitors. In previous work, we found that the activity of P‐gp assembled into membrane nanodiscs had significantly higher basal activity in the absence of transport substrate than P‐gp in detergent micelles. However, significant increase in activity in the presence of the substrate verapamil was not observed in micelles or nanodiscs. Here, we report methods of protein purification and nanodisc assembly modified to have optimally functional human P‐gp. The resulting purified P‐gp will be used to evaluate the inhibition mechanism of lead compounds that were shown to reverse MDR in cancer cell culture.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D. of Naples, Florida. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.

Antibiotic resistance by bacterial pathogens is a persistent problem in medicine. One method of resistance is through RND efflux transporters, which can move antibiotics out of the cells and the periplasm, thus preventing cell death. Many of these transporters can move several very different antibiotics out of the cells, thus conferring multidrug resistance to the pathogens. This project focuses on one such RND transporter found in Neisseria gonorrhoeae known as mtrCDE. Our goal is to identify potential inhibitors of mtrCDE by observing their effects on the minimum inhibitory concentrations (MICs) of antibiotics. Ultimately, we aim to develop inhibitors of mtrCDE with the goal of restoring antibiotic sensitivity to antibiotic‐resistant N. gonorrhoeae. Potential inhibitors have been identified through computational means. In the system described here, the genes for mtrCDE have been transformed into an E. coli strain that has had the homologous E. coli RND transporter deleted for facilitating testing. We have attempted to optimize MIC assays to determine the minimum concentration of antibiotic required to inhibit the growth of cells expressing the mtrCDE genes. To date, the largest effects in these assays have been observed with the detergent Triton X‐100. In addition, the plasmid encoding mtrCDE included a gene conferring chloramphenicol resistance for selection. The media for initial growth of cells contained chloramphenicol to maintain this selection. Initial studies showed that inclusion of chloramphenicol with other antibiotics overwhelms even resistant bacteria. The effects of inclusion of dimethyl sulfoxide (DMSO), a commonly used carrier vehicle for experimental compounds, was tested in the assays since our compound libraries use this solvent. Experiments suggested that inclusion of low concentrations of DMSO does not affect the MIC values observed, but that the cells do reflect a dose response to varying concentrations of DMSO that needs to be carefully controlled. Continuing optimization of these assays with various antibiotics is being performed to determine MIC values, compare them with reported MIC values, and identify antibiotics that are effluxed by mtrCDE that have no published MIC values for N. gonorrhoeae or E. coli. We report here these and other results of our MIC assay optimization for multidrug resistant, gram‐negative bacterial pathogens.Support or Funding InformationDepartment of Biological Sciences, the Center for Drug Discovery, Design and Delivery (CD4), the Center for Scientific Computation, Engaged Learning, and the Hamilton Scholars Program, Southern Methodist University, Dallas, TX

Multi‐drug resistance (MDR) occurs when cancer cells become resistant to a diverse array of chemotherapeutics and xenobiotics. Among the many mechanisms of MDR, one of the most prominent is the overexpression of ATP‐binding cassette (ABC) transporters. ABC transporters harness the energy from ATP hydrolysis to transport xenobiotics, drugs, and other toxic compounds out of the cell. When ABC transporters are overexpressed in cancer cells, their efflux activity can lower the intracellular concentration of medicinal compounds to sub‐therapeutic levels. One member of the ABC transporter family, the Breast Cancer Resistance Protein (BCRP, or ABCG2), confers MDR to a variety of cancers. Transient inhibition of BCRP should therefore restore sensitivity of resistant cells to chemotherapeutics. In previous work by our lab, several potential BCRP inhibitors were identified via computational methods. Using cell viability assays, we tested the potential BCRP inhibitors using a BCRP overexpressing cell line. Here we compare the effectiveness of the inhibitors at re‐sensitizing the cells to Mitoxantrone relative to the parental cell line which does not overexpress BCRP. The potential toxicities of the experimental compounds were also assessed using the non‐cancerous HFL1 cell line.

Antibiotic-resistant gonorrheal infections are a rising health concern in the United States. The MtrCDE efflux system confers antibiotic resistance to N. gonorrhoeae, the causative agent of gonorrheal disease. The system consists of the MtrD pump, the periplasmic adaptor MtrC, and the outer membrane channel MtrE. MtrD exports hydrophobic and amphiphilic substrates in a wide range of sizes, and thus mediates resistance to a variety of antibiotics. Using computational methods, we focused on interactions of MtrD with a substrate, a non-substrate, and a suspected substrate of MtrD. To generate a plausible starting point for our simulations, we docked ligands to the Access and Deep pocket of the MtrD Drug Binding Domain. We then used free and directed Molecular Dynamics simulations with NAMD to guide MtrD along six known conformations of MtrD homologues to explore a putative drug transport pathway. We describe potential paths through the Drug Binding Domain for each ligand, and our simulations showed that ligand interactions with the gate loop can affect the outcome of the simulation. We investigate the transport mechanism of MtrD to aid future efforts to target the pump itself, and thereby restore antibiotic sensitivity to resistant N. gonorrhoeae.

Multi‐drug resistance (MDR) occurs when cancer cells become resistant to multiple chemotherapeutic drugs that are functionally and structurally different. MDR usually leads to failure of chemotherapy treatment and subsequently to poor patient prognosis. Cancers can become resistant to antineoplastic drugs by over‐expressing one or more ATP‐binding cassette (ABC) transporters that act as drug efflux pumps and expel xenobiotics from cells. One such protein is the Breast Cancer Resistance Protein (BCRP, or ABCG2), which is believed to confer MDR to several types of cancer. Inhibition of BCRP cellular activity is therefore thought as a way to restore sensitivity to chemotherapeutics. In previous work, we identified several potential inhibitors of BCRP using high‐throughput in silico screens. Using cell viability and cellular accumulation assays of BCRP substrates, we assessed these compounds for toxicity to cells and the ability to reverse MDR in the BCRP‐overexpressing breast cancer cell line, MCF7‐M100. Here we expand our studies of the potential BCRP inhibitors to two other BCRP‐overexpressing cell lines, the BCRP overexpressing colon cancer and non‐small cell lung cancer cell lines, S1M1‐80 and MX20, respectively. For each cell line, the level of BCRP overexpression was assessed at RNA and protein level and the MDR phenotype was confirmed using cell viability assays. The effects of our newly discovered inhibitors on reversing MDR in these two cell lines is reported.Support or Funding InformationThis work is supported by the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D., of Naples, FL.

P‐glycoprotein (P‐gp) belongs to the ATP‐Binding Cassette (ABC) transporter family of protein. This class of membrane proteins is strongly linked with multidrug resistance (MDR) in tumors where overexpression of these proteins often nullifies the effects of chemotherapeutics by removing the therapeutics from the cells. In hopes of creating co‐therapeutics to re‐sensitize tumors to chemotherapeutics, we previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. The effects on P‐gp ATPase hydrolysis activity were used as an initial biochemical screen for inhibitor effectiveness followed by assessments of MDR‐reversal in various P‐gp overexpressing cancer cell lines. For the initial biochemical analyses, P‐glycoprotein (MDR3) from mouse was used, which is closely related to the human (MDR1) protein. To evaluate the mechanism of inhibition also in the human P‐protein, we have isolated human P‐gp from the yeast Pichia pastoris using mixed detergent micelles. Lipid activation of ABC transporters has been shown previously to be a vital step in ensuring best enzyme activity for the analysis of such proteins. In our pursuit to optimize the P‐gp ATPase activity, we have varied amounts and combinations of different lipids including phosphatidylcholine (PC), E. coli lipids and cholesterol and assessed the resulting ATPase hydrolysis activities in coupled enzyme assays. Preliminary results showed P‐gp activity in the presence of PC was significantly higher than when E. coli lipids were used. Here we report our efforts to achieve the best protein:lipid ratios as well as assess various techniques to achieve the high ATP hydrolysis activity of P‐gp to test our newly found drug‐like molecules in their effectiveness as P‐gp inhibitors.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Solid set canopy delivery systems (SSCDS) are a novel foliar agrochemical delivery system designed as an alternative for airblast sprayers in high density fruit production. This study tested the pest management potential, coverage, and chemical deposition of an SSCDS using commercially available microsprinkler components over the course of a growing season. Spray coverage and deposition for a representative airblast sprayer and SSCDS were evaluated using water sensitive paper and tartrazine dye, respectively. Foliar sprays for pest suppression were applied through both systems, and damage assessments were taken at the midpoint and end of the growing season. SSCDS sprays demonstrated similar levels of coverage on the adaxial leaf surface as airblast sprays, but significantly lower coverage on the abaxial surface. However, mean levels of foliar chemical deposition was generally higher in the SSCDS. Evaluations found minimal arthropod and fungal damage in both airblast and SSCDS treated plots compared to untreated trees. The SSCDS was shown to be a viable alternative to the airblast, with inherent advantages such as rapid application time and improved worker safety. Furthermore, higher deposition on SSCDS treated foliage supports the hypothesis that SSCDS provide a higher droplet capture rate in the canopy, with less off-target loss and drift than airblast sprayers.

The blood‐brain barrier (BBB) is a physical and dynamic barrier that separates the central nervous system from the circulatory system in order to protect the brain from harmful endogenous and exogenous chemicals. The capillary endothelial cells that form the BBB are firmly sealed via tight junctions and express relatively high amounts of ATP‐binding cassette efflux transporters like P‐glycoprotein (P‐gp) and the breast cancer resistant protein (BCRP) to protect the brain from entry of potentially harmful chemicals. While protective to the brain under normal circumstances, this high level of expression of these proteins at the BBB are problematic when attempting to treat diseases of the brain like neurodegenerative diseases or brain cancers since P‐gp and BCRP impede entry of therapeutic drugs into the brain. Previously, we have computationally identified several drug‐like molecules that inhibit or modulate P‐gp and/or BCRP activities. The cell line, hCMEC/d3, was used in our studies as a model for the BBB. Using qPCR, we confirmed a higher level of expression of both P‐gp and BCRP in hCMEC/d3 cells when compared to non‐cancerous HFL‐1 cells. When hCMEC/d3 cells were treated with the P‐gp and BCRP inhibitors, accumulation of the P‐gp substrate rhodamine‐123 as well as the P‐gp and BCRP substrates, mitoxantrone and daunorubicin, was observed. We conclude that our newly discovered P‐gp and BCRP inhibitors may make good leads for the development of co‐therapeutics to inhibit and/or modulate the activity of P‐gp and BCRP in the BBB and facilitate therapeutic drug uptake through the BBB and into the brain. Support or Funding Information This work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas as well as the Hamilton Undergraduate Research Scholars Program, the SMU Summer Research and Undergraduate Research Assistantship Programs as well as the SMU Engaged Learning Program. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The integral membrane efflux pump P-glycoprotein (P-gp, ABCB1) is a causative agent of multidrug resistance (MDR) to a wide variety of chemotherapeutics in human cancers. In many cancer subtypes, chemotherapy treatment can both induce and select for the overexpression of P-gp. When P-gp is overexpressed, the pump lowers the intracellular concentration of many drugs to sub-therapeutic levels, thereby conferring general MDR to the cancer, and rendering further chemotherapeutic treatment ineffective. Given the clinical importance of the enzyme, P-gp has long been a target for drug discovery and the focus of extensive mechanistic research. Until recently, the lack of known human P-gp structures has hindered efforts to understand the unusually polyspecific substrate profile and complex transport mechanism of the enzyme. Prior research by our laboratory has demonstrated the possibility of multiple transport pathways through the drug binding domains (DBDs) of the transporter, and of variable substrate binding sites using targeted molecular dynamics (TMD) simulations on homology models of human P-gp. Following the recent release of the cryo-EM structure of human P-gp, we performed novel simulations of the solved structure using four known, low energy conformations of P-gp homologues in various states along a putative ABC transporter catalytic efflux cycle. These simulations were done at long timescales using a combination of free and directed GPU-accelerated molecular dynamics with NAMD. These analyses included the new human P-gp structure and demonstrated the pumping of known P-gp substrates across the cell membrane. Parameterizations and simulations of molecules that are either suspected transport substrates or putative transport inhibitors of P-gp were performed. These computational studies support earlier observations, and explore the hypothetical mechanisms of P-gp-mediated substrate efflux and of P-gp transport inhibition. This work was supported by NIH NIGMS [R15GM094771-02].

Soil columns were collected from a blueberry field, and insecticide solutions were allowed to leach through these columns. Insecticides from four different chemical classes were applied at two different rates: the concentration at which the insecticides wash off blueberries under rainfall conditions and the labeled field rate at which they are sprayed. The soil columns were divided into thirds; top, middle and bottom. Soil bioassays using Eisenia foetida Savigny, as an indicator species, were set up to determine the toxicity of the insecticides at a top, middle and bottom layer of the soil column. The mass of E. foetida was also measured after the bioassay experiment was completed. The concentrations at which insecticides wash-off of blueberries from rainfall were not lethal to E. foetida. In order to support mortality data, insecticide residues were quantified in the soil layers for each insecticide. Under field rate leaching conditions, carbaryl showed the high levels of toxicity in the top and middle layers of soil suggesting that it has the highest risk to organisms from leaching. This study will help blueberry growers make informed decisions about insecticide use, which can help minimize contamination of the environment.

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Overexpression of ATP‐binding cassette (ABC) transporters, P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are two well‐studied drug transporters which are associated with MDR. These two transporters also act as a major functional unit of the blood brain barrier to protect the brain from xenobiotics and toxins. Lack of clinically approved P‐gp and BCRP inhibitors renders chemotherapy treatments of many MDR cancers ineffective and obstructs drug uptake into the brain. Using computational methods, we previously identified a novel class of P‐gp inhibitors that were not transport substrates of the pump. Here we describe the effects of chemical variants of the previously identified P‐gp inhibitor, SMU‐29. The variants were generated using computational approaches or by structure‐based design to improve protein binding interactions. All variants showed improved efficacy in reversing paclitaxel resistance in the P‐gp over‐expressing DU145 TXR prostate cancer cell line and their ability of increasing P‐gp substrate calcein in these cells. Five of these variants were not transported by P‐gp and three of them were substrates of the pump. Four variants only affected P‐gp, but not BCRP function, making them more selective than the parental compound, SMU 29. The three variants that affected BCRP reversed MDR in a BCRP over‐expressing breast cancer cell line, MCF‐7 M100, and increased accumulation of BCRP substrates such as Hoechst 33342, mitoxantrone and daunorubicin in these cells, indicating the potential to be used as BCRP inhibitors. The variants that affected both P‐gp and BCRP also showed higher accumulation of P‐gp and BCRP substrates in a blood brain barrier model cell line, hCMEC/D3, showing the potential to open the blood brain barrier for increased drug uptake into the brain. Support or Funding Information This work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .