Use of Traditional and Proteomic Methods in the Assessment of a Preclinical Model of Preeclampsia.

Recent studies have demonstrated downregulation of breast cancer resistance protein (BCRP/ABCG2) in placenta obtained from women with preeclampsia (PE). BCRP is highly expressed in placenta and plays an important role in preventing xenobiotics from entering the fetal compartment. Although PE is often therapeutically managed with drugs that are substrates of BCRP, there are limited studies on the impact of PE on fetal drug exposure. Due to ethical concerns, use of preclinical models is an important approach. Thus, by using proteomic and traditional methods, we characterized transporter changes in an immunologic rat model of PE to determine its utility and predictive value for future drug disposition studies. PE was induced by daily administration of low-dose endotoxin (0.01-0.04 mg/kg) to rats on gestational days (GD) 13-16, urine was collected, and rats were sacrificed on GD17 or GD18. PE rats shared similar phenotype to PE patients, including proteinuria, and increased levels of tumor necrosis factor α and interleukin 6. Transcript and protein levels of Bcrp were significantly downregulated in placenta of PE rats on GD18. multidrug resistance 1a, multidrug resistance 1b, and organic anion transporting polypeptide 2B1 mRNA were also decreased in PE. Proteomics revealed activation of various hallmarks of PE including immune activation, oxidative stress, endoplasmic reticulum stress and apoptosis. Overall, our results demonstrated that the immunologic PE rat model exhibits numerous similarities to human PE along with dysregulation of placental transporters. Therefore, this model may be useful in examining the impact of PE on the maternal and fetal disposition of BCRP substrates. SIGNIFICANCE STATEMENT: Fully characterizing preclinical models of disease is necessary to determine their validity to human conditions. Combining traditional and proteomic methods of model characterization, we identified numerous phenotypic similarities between our model of preeclampsia and human disease. The alignment with human pathophysiological changes allows for more confident use of this preclinical model.

Preclinical assessment of drug-drug interaction of fb-PMT, a novel anti-cancer thyrointegrin αvß3 antagonist.

The objective of the current study was to identify potential drug-drug interactions (DDIs) with the drug candidate fb-PMT, a novel anticancer thyrointegrin αvß3 antagonist. This was accomplished by using several in vitro assays to study interactions of fb-PMT with both cytochrome P450 (CYP) enzymes and drug transporters, two common mechanisms leading to adverse drug effects. In vitro experiments showed that fb-PMT exhibited weak reversible inhibition of CYP2C19 and CYP3A4. In addition, fb-PMT did not show time-dependent inhibition with any of the seven CYP isoforms tested, including 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. Human liver microsomal incubations demonstrated that fb-PMT is stable. Potential transporter-mediated DDIs with fb-PMT were assessed with two ATP binding cassette (ABC) family transporters (P-glycoprotein and breast cancer resistance protein) using Caco2 cells and seven solute carrier family (SLC) transporters (organic cation transporter OCT2, organic anion transporters OAT1 and OAT3, organic anion transporter peptides OATP1B1 and OATP1B3, and the multidrug and toxic extrusion proteins MATE1 and MATE2-K using transfected HEK293 cells). Fb-PMT was not a substrate for any of the nine transporters tested in this study, nor did it inhibit the activity of seven of the transporters tested. However, fb-PMT inhibited the uptake of rosuvastatin by both OATP1B1 and OATP1B3 with half-maximal inhibitory concentrations greater than 3 and less than 10 µM. In summary, data suggest that the systemic administration of fb-PMT is unlikely to lead to DDIs through CYP enzymes or ABC and SLC transporters in humans.

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.

Assessment of HBEC-5i endothelial cell line cultivated in astrocyte conditioned medium as a human blood-brain barrier model for ABC drug transport studies.

Endothelial cells are main components of the Blood-Brain Barrier (BBB) and form a tight monolayer that regulates the passage of molecules, with the ATP-Binding Cassette (ABC) transporters efflux pumps. We have developed a human in vitro model of HBEC-5i endothelial cells cultivated alone or with human astrocytes conditioned medium on insert. HBEC-5i cells showed a tight monolayer within 14 days, expressing ZO-1 and claudin 5, a low apparent permeability to small molecules, with a TEER stability during five days. The P-gp, BCRP, MRPs transporters were well expressed and functional. Accumulation and efflux ratio measurement with different ABC transporters substrates (Rhodamine 123, BCECF AM, Hoechst 33342) and inhibitors (verapamil, Ko143, probenecid and cyclosporin A) were conducted. At barrier level, the functionality of ABC transporters was three-fold enhanced in astrocyte conditioned medium. We validated our model by the transport of pharmacological substrates: caffeine, rivaroxaban, and methotrexate. The rivaroxaban and methotrexate were released with an efflux ratio >3 and were decreased by more than half with inhibitors. HBEC-5i model could be used as relevant tool in preclinical studies for assessing the permeability of therapeutic molecules to cross human BBB.

In Vitro Assessment of the Effect of Antiepileptic Drugs on Expression and Function of ABC Transporters and Their Interactions with ABCC2.

ABC transporters have a significant role in drug disposition and response and various studies have implicated their involvement in epilepsy pharmacoresistance. Since genetic studies till now are inconclusive, we thought of investigating the role of xenobiotics as transcriptional modulators of ABC transporters. Here, we investigated the effect of six antiepileptic drugs (AEDs) viz. phenytoin, carbamazepine, valproate, lamotrigine, topiramate and levetiracetam, on the expression and function of ABCB1, ABCC1, ABCC2 and ABCG2 in Caco2 and HepG2 cell lines through real time PCR, western blot and functional activity assays. Further, the interaction of AEDs with maximally induced ABCC2 was studied. Carbamazepine caused a significant induction in expression of ABCB1 and ABCC2 in HepG2 and Caco2 cells, both at the transcript and protein level, together with increased functional activity. Valproate caused a significant increase in the expression and functional activity of ABCB1 in HepG2 only. No significant effect of phenytoin, lamotrigine, topiramate and levetiracetam on the transporters under study was observed in either of the cell lines. We demonstrated the interaction of carbamazepine and valproate with ABCC2 with ATPase and 5,6-carboxyfluorescein inhibition assays. Thus, altered functionality of ABCB1 and ABCC2 can affect the disposition and bioavailability of administered drugs, interfering with AED therapy.

Pre-Hepatectomy Assessment of Bile Transporter Expression by Gadoxetic Acid-Enhanced MRI in a Rat Model of Liver Cirrhosis.

BACKGROUND: Gadoxetic acid is a liver-specific intravenous T1 magnetic resonance (MR) contrast agent that is excreted via the hepatobiliary system. We hypothesize that hepatocyte expressions of bile transporters (OATP1 and MRP2) correlate with dynamic profile of Gadoxetic acid enhanced (GE)-MR imaging (MRI). METHODS: Two groups of rats, control (n = 6) and cirrhosis (n = 12), received gadoxetic acid enhanced MRI followed by 70% hepatectomy. The change in MR signal intensity from the baseline before the contrast injection (ΔSI) was analyzed every minute for 30 min. Dynamic signal intensity retention ratio (DSR) was defined as the mean ΔSI of the third 10-minmin period divided by the first 10-minmin period. Real-time PCR was utilized to quantify mRNA expressions. RESULTS: Compared to the control, cirrhosis group demonstrated lower mRNA levels of OATP1 (0.038 ± 0.020 vs. 0.232 ± 0.0979; p = 0.004), MRP2 (0.201 ± 0.084 vs. 0.7567 ± 0.254; p = 0.002), and OATP1/MRP2 mRNA ratio (0.193 ± 0.065 vs. 0.342 ± 0.206; p = 0.032). DSR was higher in the cirrhosis group (0.678 ± 0.554 vs -0.125 ± 0.839; p = 0.033). In the cirrhosis group, there was an inverse correlation between the ratios of OATP1/MRP2 mRNA and DSR (R = -0.709, p = 0.01). CONCLUSION: Bile transporters OATP1/MRP2 mRNA expression ratio in rat liver tissue decreased with DMN-induced liver injury. The expressions of bile transporters correlated with GE-MRI DSR. The GE-MRI DSR has potential utility in qualifying OATP1/MRP2 mRNA expression.

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

BACKGROUND: Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism. METHODS: The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively. RESULTS: SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of JC-1 aggregates in such SP and MP cells became comparable. CONCLUSION: Inhibiting ABC transporters and depriving Hoechst 33342 dye are required for the accurate assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

Drug interaction profile of the HIV integrase inhibitor cabotegravir: assessment from in vitro studies and a clinical investigation with midazolam.

1. Cabotegravir (CAB; GSK1265744) is a potent HIV integrase inhibitor in clinical development as an oral lead-in tablet and long-acting injectable for the treatment and prevention of HIV infection. 2. This work investigated if CAB was a substrate for efflux transporters, the potential for CAB to interact with drug-metabolizing enzymes and transporters to cause clinical drug interactions, and the effect of CAB on the pharmacokinetics of midazolam, a CYP3A4 probe substrate, in humans. 3. CAB is a substrate for Pgp and BCRP; however, its high intrinsic membrane permeability limits the impact of these transporters on its intestinal absorption. 4. At clinically relevant concentrations, CAB did not inhibit or induce any of the CYP or UGT enzymes evaluated in vitro and had no effect on the clinical pharmacokinetics of midazolam. 5. CAB is an inhibitor of OAT1 (IC50 0.81 µM) and OAT3 (IC50 0.41 µM) but did not or only weakly inhibited Pgp, BCRP, MRP2, MRP4, MATE1, MATE2-K, OATP1B1, OATP1B3, OCT1, OCT2 or BSEP. 6. Based on regulatory guidelines and quantitative extrapolations, CAB has a low propensity to cause clinically significant drug interactions, except for coadministration with OAT1 or OAT3 substrates.

Identification of prognostic subgroups among acute myeloid leukemia patients with intermediate risk cytogenetics using a flow-cytometry-based assessment of ABC-transporter function.

BACKGROUND: A median fluorescence intensity ratio (MFIR) which measures the efflux of mitoxantrone (an ATP Binding Cassette (ABC) transporter substrate) with and without ABC transporter inhibition correlates with expression of MDR1 and BCRP in acute myeloid leukemia (AML) blasts. METHODS: This study evaluates the impacts of the MFIR on AML outcomes and its interaction with detection of the FLT3 ITD. RESULTS: Among 200 newly diagnosed AML patients, an MFIR of ≥ 1.9 (MFIR+) was detected in 60 (30%) leukemic blast samples. In multivariate analysis, MFIR was an independent prognostic factor for response to induction chemotherapy (OR=7.2, P<0.00001), DFS (HR=2.3, P=0.004) and OS (HR=2.2, P=0.0005) with the main effect being in the 141 patients with intermediate risk cytogenetics. Among intermediate risk cytogenetics patients: MFIR+ outcomes were similar to unfavorable cytogenetic risk (CR, 53% vs. 52%, P=1.0; OS, 11 vs. 9 months, P=0.79). MFIR status can further stratify the prognostic risk for patients with or without FLT3 ITD mutation. CONCLUSIONS: MFIR has value in predicting outcomes including DFS and OS as well as induction failure. This is particularly true for patients with intermediate risk cytogenetics and when combined with assessment for the FLT3-ITD mutation.

The Use of Transporter Probe Drug Cocktails for the Assessment of Transporter-Based Drug-Drug Interactions in a Clinical Setting-Proposal of a Four Component Transporter Cocktail.

Probe drug cocktails are used clinically to assess the potential for drug-drug interactions (DDIs), and in particular, DDIs resulting from coadministration of substrates and inhibitors of cytochrome P450 enzymes. However, a probe drug cocktail has not been identified to assess DDIs involving inhibition of drug transporters. We propose a cocktail consisting of the following substrates to explore the potential for DDIs caused by inhibition of key transporters: digoxin (P-glycoprotein, P-gp), rosuvastatin (breast cancer resistance protein, BCRP; organic anion transporting polypeptides, OATP), metformin (organic cation transporter, OCT; multidrug and toxin extrusion transporters, MATE), and furosemide (organic anion transporter, OAT). Furosemide was evaluated in vitro, and is a substrate of OAT1 and OAT3, with Km values of 38.9 and 21.5 µM, respectively. Furosemide was also identified as a substrate of BCRP, OATP1B1, and OATP1B3. Furosemide inhibited BCRP (50% inhibition of drug transport: 170 µM), but did not inhibit OATP1B1, OATP1B3, OCT2, MATE1, and MATE2-K at concentrations below 300 µM, and P-gp at concentrations below 2000 µM. Conservative approaches for the estimation of the likelihood of in vivo DDIs indicate a remote chance of in vivo transporter inhibition by these probe drugs when administered at low single oral doses. This four component probe drug cocktail is therefore proposed for clinical evaluation.

Structural and genetic assessment of the ABCA4-associated optical gap phenotype.

PURPOSE: To investigate the developmental stages and genetic etiology of the optical gap phenotype in recessive Stargardt disease (STGD1). METHODS: Single and longitudinal data points from 15 patients, including four sibling pairs, exhibiting an optical gap phenotype on spectral-domain optical coherence tomography (SD-OCT) with confirmed disease-causing ABCA4 alleles were retrospectively analyzed. Fundus images with corresponding SD-OCT scans were collected with a confocal scanning laser ophthalmoscope. Structural phenotypes were assigned to three developmental stages according to SD-OCT. The ABCA4 gene was screened in all patients. RESULTS: At least two disease-causing ABCA4 variants where identified in each patient; all except one (91%) were compound heterozygous for the p.G1961E mutation. All patients exhibited structural findings on SD-OCT that grouped into three progressive developmental stages over several years. Stage 1 was characterized by mild disruptions of the ellipsoid zone (EZ) band over the fovea. Stage 2 was a progressive expansion of the EZ band loss resulting in an empty lesion devoid of photoreceptors. Stage 3 observed a structural collapse of the inner retinal layers into the optical gap space leading to involvement and atrophy of the RPE thereafter. CONCLUSIONS: The optical gap phenotype in STGD1 can be structurally divided into three progressive stages spanning several years. This particular phenotype also appears to be highly associated with the p.G1961E mutation of ABCA4. Taken together, it appears that a focal loss of photoreceptors sequentially precedes RPE dysfunction in the early development of ABCA4-associated optical gap lesions.

In vitro methods in drug transporter interaction assessment.

Drug transporter proteins recruit to pharmacological barrier tissues and profoundly affect the ADME properties of a large number of drugs. In vitro assays optimized for drug transporters have grown into routine tools in the determination of molecular level interactions as well as prediction of barrier penetration and system level pharmacokinetics. Regulatory position mandates increasing interest in the application of these assays during drug development.

Functional ATP-binding cassette drug efflux transporters in isolated human and rat hepatocytes significantly affect assessment of drug disposition.

Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-superfamily drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of hepatocytes. In the present study, the expression and activity of ABC transporters BCRP, BSEP, P-gp, MRP2, MRP3, and MRP4 in human and rat cryopreserved hepatocytes were investigated. In commercially available human cryopreserved hepatocytes, all drug efflux transporters except human BCRP (hBCRP) exhibited similar expression levels as in fresh liver biopsies. Expression levels of hBCRP were 60% lower in cryopreserved human hepatocytes than in liver tissue, which could lead to, at most, a 2.5-fold reduction in hBCRP-mediated efflux. Fresh rat hepatocytes showed significantly lower levels of rat BCRP compared with liver expression levels; expression levels of other ABC transporters were unchanged. ABC transporters in human cryopreserved cells were localized to the plasma membrane. Functional studies could demonstrate P-gp and BCRP activity in both human cryopreserved and fresh rat hepatocytes. Inhibiting P-gp-mediated efflux by elacridar in in vitro experiments significantly decreased fexofenadine efflux from hepatocytes, resulting in an increase in apparent fexofenadine uptake. The results from the present study clearly indicate that ABC transporter-mediated efflux in freshly isolated as well as cryopreserved rat and human hepatocytes should be taken into account in in vitro experiments used for modeling of drug metabolism and disposition.

A longitudinal study of Stargardt disease: quantitative assessment of fundus autofluorescence, progression, and genotype correlations.

PURPOSE: We characterized subtypes of fundus autofluorescence (AF) and the progression of retinal atrophy, and correlated these findings with genotype in Stargardt disease. METHODS: Full clinical examination and AF imaging was undertaken in 68 patients with Stargardt disease. The baseline data were compared to those at follow-up. Patients were classified into three AF subtypes: type 1 had a localized low signal at the fovea surrounded by a homogeneous background, type 2 had a localized low signal at the macula surrounded by a heterogeneous background with numerous foci of abnormal signal, and type 3 had multiple low signal areas at the posterior pole with a heterogeneous background. At baseline, there were 19 patients with type 1, 41 with type 2, and 8 with type 3 disease. The areas of reduced AF signal were measured and rate of atrophy enlargement (RAE) was calculated as the difference of the atrophy size over time (mm²) divided by the follow-up interval (years). Molecular screening of ABCA4 was undertaken. RESULTS: The mean follow-up interval was 9.1 years. A total of 42% cases with type 1 disease progressed to type 2, and 12% with type 2 progressed to type 3. The RAE (mm²/y) based upon baseline AF subtypes was significantly different; 0.06 in type 1, 0.67 in type 2, and 4.37 in type 3. ABCA4 variants were identified in 57 patients. There was a significant association between AF subtype and genotype. CONCLUSIONS: The AF pattern at baseline influences the enlargement of atrophy over time and has genetic correlates. These data are likely to assist in the provision of counseling on prognosis in Stargardt disease and be valuable for future clinical trials.

Pharmacokinetic assessment of efflux transport in sunitinib distribution to the brain.

This study quantitatively assessed transport mechanisms that limit the brain distribution of sunitinib and investigated adjuvant strategies to improve its brain delivery for the treatment of glioblastoma multiforme (GBM). Sunitinib has not shown significant activity in GBM clinical trials, despite positive results seen in preclinical xenograft studies. We performed in vivo studies in transgenic Friend leukemia virus strain B mice: wild-type, Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) genotypes were examined. The brain-to-plasma area under the curve ratio after an oral dose (20 mg/kg) was similar to the steady-state tissue distribution coefficient, indicating linear distribution kinetics in mice over this concentration range. Furthermore, the distribution of sunitinib to the brain increased after administration of selective P-glycoprotein (P-gp) or breast cancer resistance protein (Bcrp) pharmacological inhibitors and a dual inhibitor, elacridar, comparable to that of the corresponding transgenic genotype. The brain-to-plasma ratio after coadministration of elacridar in wild-type mice was ≈ 12 compared with ≈ 17.3 in Mdr1a/b(-/-)Bcrp1(-/-) mice. Overall, these findings indicate that there is a cooperation at the blood-brain barrier (BBB) in restricting the brain penetration of sunitinib, and brain delivery can be enhanced by administration of a dual inhibitor. These data indicate that the presence of cooperative efflux transporters, P-gp and Bcrp, in an intact BBB can protect invasive glioma cells from chemotherapy. Thus, one may consider the use of transporter inhibition as a powerful adjuvant in the design of future clinical trials for the targeted delivery of sunitinib in GBM.

Assessment of the roles of P-glycoprotein and cytochrome P450 in triptolide-induced liver toxicity in sandwich-cultured rat hepatocyte model.

Triptolide (TP), a main bioactive component of Tripterygium wilfordii Hook F., is a promising agent for treatment of autoimmune diseases. However, a high incidence of dose-limiting hepatotoxicity was observed in the clinic. Sandwich-cultured rat hepatocyte model was used in this study to identify the involvement of P-glycoprotein (P-gp) in TP disposition and to evaluate TP-induced hepatotoxicity after modulation of P-gp by the known inhibitors, ritonavir and tariquidar, and known inducers, phenobarbital, quercetin, and H(2)O(2). Our data showed that biliary clearance of TP reduced 73.7% and 84.2% upon treatment of ritonavir (25 µM) and tariquidar (5 µM), respectively. In contrast, increases of 346%, 280%, and 273% in biliary clearance of TP were observed with treatment of phenobarbital (1.0 mM), quercetin (20 µM), and H(2)O(2) (0.5 mM), respectively. The TP-induced hepatotoxicity increased by twofold when CYP activity was blocked by 1-aminobenzotriazole, suggesting that CYP and P-gp may both contribute to the detoxification of TP in the SCRH model. In addition, hepatotoxicity and the expression of apoptosis proteins Bax and Bcl-2 were correlated qualitatively with the TP exposure duration and its intracellular concentration, which, in turn, can be modulated by P-gp inhibitors or inducers. Our results for the first time demonstrated that in addition to CYP-mediated metabolism, P-gp also plays an important role in the disposition of TP and TP-induced hepatotoxicity. Thus, the modulation of canalicular P-gp has a potential to cause drug-drug interaction between TP and the coadministered P-gp inhibitors or inducers in the clinic.

Cost, effectiveness and environmental relevance of multidrug transporters in sea urchin embryos.

ATP-binding cassette transporters protect cells via efflux of xenobiotics and endogenous byproducts of detoxification. While the cost of this ATP-dependent extrusion is known at the molecular level, i.e. the ATP used for each efflux event, the overall cost to a cell or organism of operating this defense is unclear, especially as the cost of efflux changes depending on environmental conditions. During prolonged exposure to xenobiotics, multidrug transporter activity could be costly and ineffective because effluxed substrate molecules are not modified in the process and could thus undergo repeated cycles of efflux and re-entry. Here we use embryos of the purple sea urchin, Strongylocentrotus purpuratus, as a model to determine transport costs and benefits under environmentally relevant xenobiotic concentrations. Strikingly, our results show that efflux transporter activity costs less than 0.2% of total ATP usage, as a proportion of oxygen consumption. The benefits of transport, defined as the reduction in substrate accumulation due to transporter activity, depended largely, but not entirely, on the rate of passive flux of each substrate across the plasma membrane. One of the substrates tested exhibited rapid membrane permeation coupled with high rates of efflux, thus inducing rapid and futile cycles of efflux followed by re-entry of the substrate. This combination significantly reduced transporter effectiveness as a defense and increased costs even at relatively low substrate concentrations. Despite these effects with certain substrates, our results show that efflux transporters are a remarkably effective and low-cost first line of defense against exposure to environmentally relevant concentrations of xenobiotics.

Modular construction of quaternary hemiaminal-based inhibitor candidates and their in cellulo assessment with HIV-1 protease.

Non-peptidomimetic drug-like protease inhibitors have potential for circumventing drug resistance. We developed a much-improved synthetic route to our previously reported inhibitor candidate displaying an unusual quaternized hemi-aminal. This functional group forms from a linear precursor upon passage into physiological media. Seven variants were prepared and tested in cellulo with our HIV-1 fusion-protein technology that result in an eGFP-based fluorescent readout. Three candidates showed inhibition potency above 20µM and toxicity at higher concentrations, making them attractive targets for further refinement. Importantly, our class of original inhibitor candidates is not recognized by two major multidrug resistance pumps, quite in contrast to most clinically applied HIV-1 protease inhibitors.