Identifying novel inhibitors for hepatic organic anion transporting polypeptides by machine learning-based virtual screening

Integration of statistical learning methods with structure-based modeling approaches is a contemporary strategy to identify novel lead compounds in drug discovery. Hepatic organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1) are classical off-targets, and it is well recognized that their ability to interfere with a wide range of chemically unrelated drugs, environmental chemicals, or food additives can lead to unwanted adverse effects like liver toxicity and drug-drug or drug-food interactions. Therefore, the identification of novel (tool) compounds for hepatic OATPs by virtual screening approaches and subsequent experimental validation is a major asset for elucidating structure-function relationships of (related) transporters: they enhance our understanding about molecular determinants and structural aspects of hepatic OATPs driving ligand binding and selectivity. In the present study, we performed a consensus virtual screening approach by using different types of machine learning models (proteochemometric models, conformal prediction models, and XGBoost models for hepatic OATPs), followed by molecular docking of preselected hits using previously established structural models for hepatic OATPs. Screening the diverse REAL drug-like set (Enamine) shows a comparable hit rate for OATP1B1 (36% actives) and OATP1B3 (32% actives), while the hit rate for OATP2B1 was even higher (66% actives). Percentage inhibition values for 44 selected compounds were determined using dedicated in vitro assays and guided the prioritization of several highly potent novel hepatic OATP inhibitors: six (strong) OATP2B1 inhibitors (IC50 values ranging from 0.04 to 6 μM), three OATP1B1 inhibitors (2.69 to 10 μM), and five OATP1B3 inhibitors (1.53 to 10 μM) were identified. Strikingly, two novel OATP2B1 inhibitors were uncovered (C7 and H5) which show high affinity (IC50 values: 40 nM and 390 nM) comparable to the recently described estrone-based inhibitor (IC50 = 41 nM). A molecularly detailed explanation for the observed differences in ligand binding to the three transporters is given by means of structural comparison of the detected binding sites and docking poses.Medicinal Chemistr

The link between lithospheric scale deformations and deep fluid emanations: Inferences from the Southeastern Carpathians, Romania

Understanding the formation, migration and emanation of deep CO2, H2O and noble gases (He–Ne) in deep-seated deformation settings is crucial to understand the complex relationship between deep-originated fluids and lithospheric deformation. To gain a better insight into these phenomena, we studied the origin of H2O, CO2 and noble gases of gas-rich springs found in the Târgu Secuiesc Basin located in the southeasternmost part of the Carpathian-Pannonian region of Europe. This study area is one of the best natural examples to understand the connection between the deep sources of gas emanations and deep-seated deformation zones, providing an excellent analogue for regions worldwide with similar tectonic settings and fluid emanation properties. We studied the δ2H and δ18O stable isotopic ratios of the spring waters, and the δ13C, He and Ne stable isotopic ratio of the emanating CO2-rich gases dissolved in the mineral spring waters in Covasna town and its vicinity. Based on the δ2H, δ13C, δ18O stable isotopic ratios, the spring waters and the majority of the gases are released through two consecutive fluid infiltration events. The preservation of the metamorphic signal of the upwelling H2O is linked to the local groundwater flow and fault abundancy. Furthermore, the noble gas isotopic ratios show a high degree of atmospheric contamination in the dissolved water gasses that is most likely related to the local hydrogeology. Nevertheless, the elevated corrected helium stable isotopic ratios (Rc/Ra) of our filtered data suggest that part of the emanating gases have a potential upper mantle source component. Beneath the Southeastern Carpathians, mantle fluids can have multiple origin including the dehydration of the sinking slab hosting the Vrancea seismogenic zone, the local asthenospheric upwelling and the lithospheric mantle itself. The flux of the mantle fluids is enhanced by lithospheric scale deformation zones that also support the fluid inflow from the upper mantle into the lower crust. The upwelling CO2–H2O mantle fluids may induce the release of crustal fluids by shifting the pore fluid composition (X(CO2)) and, consequently, initiating decarbonisation and devolatilization metamorphic reactions as a result of carbonate and hydrous mineral destabilisation in the crust. Based on the p-T-X(CO2) conditions of calc-silicates and the local low geotherm, we emphasise the importance of the upwelling fluids in the release and upward migration of further H2O and CO2 in the shallower lower and upper crust. Our observations in the Southeastern Carpathians show a strong similarity to other deep-seated deformation zones worldwide (e.g., Himalayas, Alps, San Andreas Fault). We infer that migration of deep fluids may also play an important role in addition to temperature control on the generation of crustal fluids in deep-seated deformation zones

The Evolution of Single Cell-derived Colorectal Cancer Cell Lines is Dominated by the Continued Selection of Tumor Specific Genomic Imbalances, Despite Random Chromosomal Instability

Intratumor heterogeneity is a major challenge in cancer treatment. To decipher patterns of chromosomal heterogeneity, we analyzed six colorectal cancer cell lines by multiplex interphase FISH (miFISH). The mismatch repair deficient cell lines DLD-1 and HCT116 had the most stable copy numbers, whereas aneuploid cell lines (HT-29, SW480, SW620 and H508) displayed a higher degree of instability. We subsequently assessed the clonal evolution of single cells in two CRC cell lines, SW480 and HT-29, which both have aneuploid karyotypes but different degrees of chromosomal instability. The clonal compositions of the single cell-derived daughter lines, as assessed by miFISH, differed for HT-29 and SW480. Daughters of HT-29 were stable, clonal, with little heterogeneity. Daughters of SW480 were more heterogeneous, with the single cell-derived daughter lines separating into two distinct populations with different ploidy (hyper-diploid and near-triploid), morphology, gene expression and tumorigenicity. To better understand the evolutionary trajectory for the two SW480 populations, we constructed phylogenetic trees which showed ongoing instability in the daughter lines. When analyzing the evolutionary development over time, most single cell-derived daughter lines maintained their major clonal pattern, with the exception of one daughter line that showed a switch involving a loss of APC. Our meticulous analysis of the clonal evolution and composition of these colorectal cancer models shows that all chromosomes are subject to segregation errors, however, specific net genomic imbalances are maintained. Karyotype evolution is driven by the necessity to arrive at and maintain a specific plateau of chromosomal copy numbers as the drivers of carcinogenesis

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality

Clinically relevant mutations in the ABCG2 transporter uncovered by genetic analysis linked to erythrocyte membrane protein expression

The ABCG2 membrane protein is a key xeno- and endobiotic transporter, modulating the absorption and metabolism of pharmacological agents and causing multidrug resistance in cancer. ABCG2 is also involved in uric acid elimination and its impaired function is causative in gout. Analysis of ABCG2 expression in the erythrocyte membranes of healthy volunteers and gout patients showed an enrichment of lower expression levels in the patients. By genetic screening based on protein expression, we found a relatively frequent, novel ABCG2 mutation (ABCG2-M71V), which, according to cellular expression studies, causes reduced protein expression, although with preserved transporter capability. Molecular dynamics simulations indicated a stumbled dynamics of the mutant protein, while ABCG2-M71V expression in vitro could be corrected by therapeutically relevant small molecules. These results suggest that personalized medicine should consider this newly discovered ABCG2 mutation, and genetic analysis linked to protein expression provides a new tool to uncover clinically important mutations in membrane proteins. © 2018 The Author(s)

El manto litosférico en el sureste de España: aportaciones de los xenolitos englobados en rocas alcalinas del mioceno-plioceno

Pliocene-Quaternary nephelinitic and alkali basaltic lavas and pyroclastic deposits from Cofrentes and Tallante and Miocene lamproitic lavas from Aljorra host xenoliths and megacrysts of lithospheric mantle origino The xenolith suite consists of spinel Iherzolites and harzburgites and with protogranular, porphyroclastic and equigranular or transitional (protogranular to porphyroclastic) textures. The textural features provide evidence of ductile-brittle deformation of Tallante xenoliths, whose mineralogy and mineral chemistry data (presence of kaersutite and phlogopite) also bear evidence of fluid-induced metasomatism of the lithospheric mantle. P-T estima tes suggests that mostly protogranular xenoliths are reliable to define a palaeogeotherm, which is from 950-1185°C at 2,5-3.0 epa, 794-805°C and 1,1-1,3 epa for Cofrentes to 936-958°C, at 1,1-1,2 epa for Tallantes. The Aljorra peridotite suggests a temperature of -880 oC and a pressure of -1,8 epa. The picture of equilibrium P-T conditions indicates that the differences in the mantle lithosp-here composition is related to (1) its deformation state and (2) metasomatic events, but also to (3) the location of entrapment during magma ascent in the lithosphere generated by diapiric uprising of asthenospheric mantle. The location of the entrainment is either close to the magma generation area or is situated at the mantle-crust boundary

Lithospheric mantle below southeastern Spain: Evidence from mantle xenoliths in miocene-pliocene alkali rocks

Pliocene-Quaternary nephelinitic and alkali basaltic lavas and pyroclastic deposits from Cofrentes and Tallante and Miocene lamproitic lavas from Aljorra host xenoliths and megacrysts of lithospheric mantle origin. The xenolith suite consists of spinel Iherzolites and harzburgites and with protogranular, porphyroclastic and equigranular or transitional (protogranular to porphyroclastic) textures. The textural features provide evidence of ductile-brittle deformation of Tallante xenoliths, whose mineralogy and mineral chemistry data (presence of kaersutite and phlogopite) also bear evidence of fluid-induced metasomatism of the lithospheric mantle. P-T estimates suggests that mostly protogranular xenoliths are reliable to define a palaeogeotherm, which is from 950-1185°C at 2,5-3.0 CPa, 794-805°C and 1,7-7,3 CPa for Cofrentes to 936-958°C, at 1,1 -1,2 CPa for Tallantes. The Aljorra peridotite suggests a temperature of ~880°C and a pressure of ~1,8 GPa. The picture of equilibrium P-T conditions indicates that the differences in the mantle lithosphere composition is related to (1) its deformation state and (2) metasomatic events, but also to (3) the location of entrapment during magma ascent in the lithosphere generated by diapiric uprising of asthenospheric mantle. The location of the entrainment is either close to the magma generation area or is situated at the mantle-crust boundar