HuR-miRNA complex activates RAS GTPase RalA to facilitate endosome targeting and extracellular export of miRNAs.

Extracellular vesicles-mediated exchange of miRNA cargos between diverse types of mammalian cells is a major mechanism of controlling cellular miRNA levels and activity, thus regulating the expression of miRNA-target genes in both donor and recipient cells. Despite tremendous excitement related to extracellular vesicles-associated miRNAs as biomarkers or having therapeutic potential, the mechanism of selective packaging of miRNAs into endosomes and multivesicular bodies for subsequent extracellular export is poorly studied due to the lack of an in vitro assay system. Here, we have developed an in vitro assay with endosomes isolated from mammalian macrophage cells to follow miRNA packaging into endocytic organelles. The synthetic miRNAs, used in the assay, get imported inside the isolated endosomes during the in vitro reaction and become protected from RNase in a time- and concentration-dependent manner. The selective miRNA accumulation inside endosomes requires both ATP and GTP hydrolysis and the miRNA-binding protein HuR. The HuR-miRNA complex binds and stimulates the endosomal RalA GTPase to facilitate the import of miRNAs into endosomes and their subsequent export as part of the extracellular vesicles. The endosomal targeting of miRNAs is also very much dependent on the endosome maturation process that is controlled by Rab5 protein and ATP. In summary, we provide an in vitro method to aid in the investigation of the mechanism of miRNA packaging process for its export from mammalian macrophage cells.

Human antigen R transfers miRNA to Syntaxin 5 to synergize miRNA export from activated macrophages.

Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from the endoplasmic reticulum to endosomes is a rate-determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin 5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 acts as an immunostimulant, we have identified the de novo RNA-binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAVL1 protein, Human antigen R. This activity ensures the export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.

Accelerated export of Dicer1 from lipid-challenged hepatocytes buffers cellular miRNA-122 levels and prevents cell death.

Hepatocytes on exposure to high levels of lipids reorganize the metabolic program while fighting against the toxicity associated with elevated cellular lipids. The mechanism of this metabolic reorientation and stress management in lipid-challenged hepatocytes has not been well explored. We have noted the lowering of miR-122, a liver-specific miRNA, in the liver of mice fed with either a high-fat diet or a methionine-choline-deficient diet that is associated with increased fat accumulation in mice liver. Interestingly, low miR-122 levels are attributed to the enhanced extracellular export of miRNA processor enzyme Dicer1 from hepatocytes in the presence of high lipids. Export of Dicer1 can also account for the increased cellular levels of pre-miR-122-the substrate of Dicer1. Interestingly, restoration of Dicer1 levels in the mouse liver resulted in a strong inflammatory response and cell death in the presence of high lipids. Increasing death of hepatocytes was found to be caused by increased miR-122 levels in hepatocytes restored for Dicer1. Thus, the Dicer1 export by hepatocytes seems to be a key mechanism to combat lipotoxic stress by shunting out miR-122 from stressed hepatocytes. Finally, as part of this stress management, we determined that the Ago2-interacting pool of Dicer1, responsible for mature microribonucleoprotein formation in mammalian cells, gets depleted. miRNA-binder and exporter protein HuR is found to accelerate Ago2-Dicer1 uncoupling to ensure export of Dicer1 via extracellular vesicles in lipid-loaded hepatocytes.

Probing the molecular mechanism of aggressive infection by antimony resistant Leishmania donovani.

The disease visceral leishmaniasis (VL) or kala azar is caused by the protozoan parasite, Leishmania donovani (LD). For many decades the pentavalent antimonial drugs countered the successive epidemics of the disease in the Indian sub-continent and elsewhere. With time, antimony resistant LD (LDR) developed and the drug in turn lost its efficacy. Infection of mammals with LDR gives rise to aggressive infection as compared to its sensitive counterpart (LDS) coupled with higher surge of IL-10 and TGF-ß. The IL-10 causes upregulation of multidrug resistant protein-1 which causes efflux of antimonials from LDR infected cells. This is believed to be a key mechanism of antimony resistance. MicroRNAs (miRNAs) are tiny post-transcriptional regulators of gene expression in mammalian cells and in macrophage play a pivotal role in controlling the expression of cytokines involved in infection process. Therefore, a change in miRNA profiles of macrophages infected with LDS or LDR could explain the differential cytokine response observed. Interestingly, the outcome of LD infection is also governed by the critical balance of pro- and anti-inflammatory cytokines which is inturn regulated by miRNA-Ago2 or miRNP complex and its antagonist RNA binding protein HuR. Here Ago2 plays the fulcrum whose phosphorylation and de-phosphorylation dictates the process; which in turn is controlled by PP2A and HuR. LDS and LDR upregulate PP2A and downregulate HuR at different magnitude leading to various levels of anti-inflammatory to proinflammatory cytokine production and resulting pathology in the host. While ectopic HuR expression alone is sufficient to clear LDS infection, simultaneous upregulation of HuR and inhibition of PP2A is required to inhibit LDR mediated infection. Therefore, tampering with miRNA pathway could be a new strategy to control infection caused by LDR parasite.

Taurine Is Covalently Incorporated into Alpha-Tubulin.

Taurine is the most abundant free amino acid in the human body. It is found in relatively high concentrations (1-10 mM) in many animal tissues but not in plants. It has been studied since the early 1800s but has not been found to be covalently incorporated into proteins in any animal tissue. Taurine has been found in only one macromolecular complex as a post-transcriptional modification to mitochondrial tRNA. Tubulin is the subunit of microtubules found in all eukaryotic species and almost all eukaryotic cells and subject to numerous post-translational modifications (PTMs). An important PTM on α-tubulin is the removal and re-ligation of the final carboxyl residue, tyrosine. We here demonstrate that taurine can be covalently incorporated at the C-terminal end of alpha-tubulin in avian erythrocytes in a reaction that requires the de-tyrosination PTM and prevents the re-tyrosination PTM. Further, this is, to our knowledge, the first instance of taurine incorporation into a large protein.

Site-Specific Bioconjugation and Multi-Bioorthogonal Labeling via Rapid Formation of a Boron-Nitrogen Heterocycle.

Precise control of covalent bond formation in the presence of multiple functional groups is pertinent in the development of many next-generation bioconjugates and materials. Strategies derived from bioorthogonal chemistries are contributing greatly in that regard; however, the gain of chemoselectivity is often compromised by the slow rates of many of these existing chemistries. Recent work on a variation of the classical aldehyde/ketone condensation based on ortho-carbonylphenylboronic acids has uncovered markedly accelerated rates compared to those of the simple carbonyl counterparts. The products of these reactions are distinct, often in the form of boron-nitrogen heterocycles. In particular, we have shown that 2-formylphenylboronic acid (2fPBA), when coupled with an α-amino-hydrazide, produces a unique zwitterionic and stable 2,3,1-benzodiazaborine derivative. In this work, we apply this chemistry to generate chemically defined and functional bioconjugates, herein illustrated with immunoconjugates. We show that an antibody and a fluorophore (as payload) equipped with the relevant reactive handles undergo rapid conjugation at near-stoichiometric ratios, displaying a reaction half-life of only ∼5 min with 2 equiv of the linker payload. Importantly, the reaction can be extended to multicomponent labeling by partnering with the popular strain-promoted azide-alkyne cycloaddition and tetrazine- trans-cyclooctene (Tz-TCO) ligation. The mutual orthogonality to both of these chemistries allows simultaneous triple bioorthogonal conjugations, a rare feat thus far that will widen the scope of various multilabeling applications. Further collaboration with the Tz-TCO reaction enables rapid one-pot synthesis of a site-specific dual-payload antibody conjugate. Altogether, we envision that the 2fPBA-α-amino-hydrazide ligation will facilitate efficient assembly of diverse bioconjugates and materials, enabling access to more complex modalities via partnership with other orthogonal chemistries.

Reversible HuR-microRNA binding controls extracellular export of miR-122 and augments stress response.

microRNAs (miRNAs), the tiny but stable regulatory RNAs in metazoan cells, can undergo selective turnover in presence of specific internal and external cues to control cellular response against the changing environment. We have observed reduction in cellular miR-122 content, due to their accelerated extracellular export in human hepatic cells starved for small metabolites including amino acids. In this context, a new role of human ELAV protein HuR has been identified. HuR, a negative regulator of miRNA function, accelerates extracellular vesicle (EV)-mediated export of miRNAs in human cells. In stressed cells, HuR replaces miRNPs from target messages and is both necessary and sufficient for the extracellular export of corresponding miRNAs. HuR could reversibly bind miRNAs to replace them from Ago2 and subsequently itself gets freed from bound miRNAs upon ubiquitination. The ubiquitinated form of HuR is predominantly associated with multivesicular bodies (MVB) where HuR-unbound miRNAs also reside. These MVB-associated pool of miRNAs get exported out via EVs thereby delimiting cellular miR-122 level during starvation. Therefore, by modulating extracellular export of miR-122, HuR could control stress response in starved human hepatic cells.

Subcutaneous Administration of Monoclonal Antibodies: Pharmacology, Delivery, Immunogenicity, and Learnings From Applications to Clinical Development.

Subcutaneous (s.c.) administration of monoclonal antibodies (mAbs) can reduce treatment burden for patients and healthcare systems compared with intravenous (i.v.) infusion through shorter administration times, made possible by convenient, patient-centric devices. A deeper understanding of clinical pharmacology principles related to efficacy and safety of s.c.-administered mAbs over the past decade has streamlined s.c. product development. This review presents learnings from key constituents of the s.c. mAb development pathway, including pharmacology, administration variables, immunogenicity, and delivery devices. Restricted mAb transportation through the hypodermis explains their incomplete absorption at a relatively slow rate (pharmacokinetic (PK)) and may impact mAb-cellular interactions and/or onset and magnitude of physiological responses (pharmacodynamic). Injection volumes, formulation, rate and site of injection, and needle attributes may affect PKs and the occurrence/severity of adverse events like injection-site reactions or pain, with important consequences for treatment adherence. A review of immunogenicity data for numerous compounds reveals that incidence of anti-drug antibodies (ADAs) is generally comparable across i.v. and s.c. routes, and complementary factors including response magnitude (ADA titer), persistence over time, and neutralizing antibody presence are needed to assess clinical impact. Finally, four case studies showcase how s.c. biologics have been clinically developed: (i) by implementation of i.v./s.c. bridging strategies to streamline PD-1/PD-L1 inhibitor development, (ii) through co-development with i.v. presentations for anti-severe acute respiratory syndrome-coronavirus 2 antibodies to support rapid deployment of both formulations, (iii) as the lead route for bispecific T cell engagers (BTCEs) to mitigate BTCE-mediated cytokine release syndrome, and (iv) for pediatric patients in the case of dupilumab.

The D2D3 form of uPAR acts as an immunotoxin and may cause diabetes and kidney disease.

Soluble urokinase plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. In addition to suPAR, proteolysis of membrane-bound uPAR results in circulating D1 and D2D3 proteins. We showed that when exposed to a high-fat diet, transgenic mice expressing D2D3 protein developed progressive kidney disease marked by microalbuminuria, elevated serum creatinine, and glomerular hypertrophy. D2D3 transgenic mice also exhibited insulin-dependent diabetes mellitus evidenced by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic ß cell mass, and high fasting blood glucose. Injection of anti-uPAR antibody restored ß cell mass and function in D2D3 transgenic mice. At the cellular level, the D2D3 protein impaired ß cell proliferation and inhibited the bioenergetics of ß cells, leading to dysregulated cytoskeletal dynamics and subsequent impairment in the maturation and trafficking of insulin granules. D2D3 protein was predominantly detected in the sera of patients with nephropathy and insulin-dependent diabetes mellitus. These sera inhibited glucose-stimulated insulin release from human islets in a D2D3-dependent manner. Our study showed that D2D3 injures the kidney and pancreas and suggests that targeting this protein could provide a therapy for kidney diseases and insulin-dependent diabetes mellitus.

Effectiveness of cyclooxygenase-2 inhibition in limiting abdominal aortic aneurysm progression in mice correlates with a differentiated smooth muscle cell phenotype.

Abdominal aortic aneurysms (AAAs) are a chronic condition that often progress over years to produce a weakened aorta with increased susceptibility for rupture, and currently, there are no pharmacological treatments available to slow disease progression. AAA development has been characterized by increased expression of cyclooxygenase-2 (COX-2), and inactivation of COX-2 before disease initiation reduces AAA incidence in a mouse model of the disease. The current study determined the effectiveness of COX-2 inhibition on AAA progression when treatment was begun after initiation of the disease. COX-2 inhibitor treatment with celecoxib was initiated after angiotensin II-induced AAA formation in a strain of nonhyperlipidemic mice that we have previously identified as highly susceptible to AAA development. When analyzed at different time points during progression of the disease, celecoxib treatment significantly reduced the incidence and severity of AAAs. The celecoxib treatment also protected the mice from aortic rupture and death. The aneurysmal lesion displayed an altered smooth muscle cell (SMC) phenotype, whereas celecoxib treatment was associated with increased expression of differentiated SMC markers and reduced dedifferentiation marker expression during AAA progression. Maintenance of a differentiated SMC phenotype is associated with the effectiveness of COX-2 inhibition for limiting AAA progression in nonhyperlipidemic mice.

Synthesis, cytotoxic properties and tubulin polymerization inhibitory activity of novel 2-pyrazoline derivatives.

A series of novel 1-(3',4',5'-trimethoxybenzoyl)-3,5-diarylpyrazoline derivatives were synthesized and evaluated for their cytotoxic properties on different cancer cell lines and tubulin polymerization inhibitory activity. Compounds 6d and 6e exhibited remarkable cytotoxic activity against different cancer cell lines with good tubulin polymerization inhibitory activity. Compound 6d exhibited moderate selectivity toward renal cancer and breast cancer subpanels with selectivity ratios of 3.06 and 5.11, respectively, at the cytostatic activity (TGI) level. Compounds 6e and 6d achieved good tubulin polymerization inhibitory activity with IC(50) values of 17 and 40 µM, respectively. The photomicrographs made for compounds 6d and 6e on cellular microtubules indicated that the cytotoxicity of these compounds can be attributed to their ability to interfere with microtubule assembly. Molecular modeling studies involving compound 6e with the colchicine binding site of α,ß-tubulin revealed hydrogen-bonding and hydrophobic interactions with several amino acids in the colchicine binding site of ß-tubulin.

Leishmania survives by exporting miR-146a from infected to resident cells to subjugate inflammation.

Leishmania donovani, the causative agent of visceral leishmaniasis, infects and resides within tissue macrophage cells. It is not clear how the parasite infected cells crosstalk with the noninfected cells to regulate the infection process. During infection, Leishmania adopts a dual strategy for its survival by regulating the intercellular transport of host miRNAs to restrict inflammation. The parasite, by preventing mitochondrial function of host cells, restricts the entry of liver cell derived miR-122-containing extracellular vesicles in infected macrophages to curtail the inflammatory response associated with miR-122 entry. On contrary, the parasite up-regulates the export of miR-146a from the infected macrophages. The miR-146a, associated with the extracellular vesicles released by infected cells, restricts miR-122 production in hepatocytes while polarizing neighbouring naïve macrophages to the M2 state by affecting the cytokine expression. On entering the recipient macrophages, miR-146a dominates the miRNA antagonist RNA-binding protein HuR to inhibit the expression of proinflammatory cytokine mRNAs having HuR-interacting AU-rich elements whereas up-regulates anti-inflammatory IL-10 by exporting the miR-21 to polarize the recipient cells to M2 stage.

Simultaneous stabilization of actin cytoskeleton in multiple nephron-specific cells protects the kidney from diverse injury.

Chronic kidney diseases and acute kidney injury are mechanistically distinct kidney diseases. While chronic kidney diseases are associated with podocyte injury, acute kidney injury affects renal tubular epithelial cells. Despite these differences, a cardinal feature of both acute and chronic kidney diseases is dysregulated actin cytoskeleton. We have shown that pharmacological activation of GTPase dynamin ameliorates podocyte injury in murine models of chronic kidney diseases by promoting actin polymerization. Here we establish dynamin's role in modulating stiffness and polarity of renal tubular epithelial cells by crosslinking actin filaments into branched networks. Activation of dynamin's crosslinking capability by a small molecule agonist stabilizes the actomyosin cortex of the apical membrane against injury, which in turn preserves renal function in various murine models of acute kidney injury. Notably, a dynamin agonist simultaneously attenuates podocyte and tubular injury in the genetic murine model of Alport syndrome. Our study provides evidence for the feasibility and highlights the benefits of novel holistic nephron-protective therapies.

4-aminophenylalanine as a biocompatible nucleophilic catalyst for hydrazone ligations at low temperature and neutral pH.

Hydrazone formation and similar reactions are highly versatile and specific, but their application to biological systems has been limited by their characteristically slow reaction kinetics at neutral pH. Catalysis of these reactions through imine formation with aromatic amines such as aniline has broadened the applicability of these reactions to biomolecular labeling. High concentrations of the catalyst are necessary, which may be incompatible with the native structure of certain proteins. In this study, we investigated the utility of 4-aminophenylalanine (4a-Phe) as a catalyst for these reactions. We find that 4a-Phe is nearly as effective as aniline in catalyzing hydrazone formation between the reactive amino acid 3-formyltyrosine (3f-Tyr) and hydrazine-containing fluorophores, both free in solution and incorporated into the protein tubulin. The catalyst 4a-Phe maintains ∼70% of the catalytic efficacy of aniline and is less detrimental to the native structure of tubulin. Examination of the temperature dependence of imine formation between 3f-Tyr and 4a-Phe shows an increase in imine concentration accompanying a decrease in temperature, confirming the exothermic nature of the equilibrium reaction. Interestingly, decreasing the temperature of the 4a-Phe-catalyzed hydrazone reaction between 3f-Tyr and the fluorophore 7-hydrazinyl-4-methylcoumarin increases the overall rate of the reaction. This result indicates that the temperature dependence of the catalyst-aldehyde equilibrium is greater than the temperature dependence of the rate constant for hydrazone formation from this intermediate, and that the rate of hydrazone formation a direct function of the concentration of the intermediate imine. These results provide a platform for conducting nucleophilic catalysis under conditions that are more compatible with biomolecular targets than previously demonstrated, thereby expanding the utility of hydrazone ligations in biological systems.

Design and synthesis of simplified taxol analogs based on the T-Taxol bioactive conformation.

A series of compounds designed to adopt a conformation similar to the tubulin-binding T-Taxol conformation of the anticancer drug paclitaxel has been synthesized. Both the internally bridged analogs 37-39, 41 and the open-chain analogs 27-29 and 43 were prepared. The bridged analogs 37-39 and 41 were synthesized by Grubbs' metatheses of compounds 30-32 and 33, which, in turn, were prepared by coupling ß-lactams 24-26 with alcohols 22 and 23. Both the bridged and the open-chain analogs showed moderate to good cytotoxicity.

Aurones: small molecule visible range fluorescent probes suitable for biomacromolecules.

Aurones, derivatives of 2-benylidenebenzofuran-3(2H)-one, are natural products that serve as plant pigments. There have been reports that some of these substances fluoresce, but little information about their optical properties is in the literature. In this report, series of aurone derivatives were synthesized as possible fluorescent probes that can be excited by visible light. We found that an amine substituent shifted the lowest energy absorption band from the near-UV to the visible region of the electromagnetic spectrum. Four amine-substituted aurone derivatives were synthesized to explore the effect of this substituent on the absorption and emission properties of the aurone chromophore. The emission maxima and intensities of the molecules are strongly dependent on the nature of the substituent and the solvent polarity. Overall, the emission intensity increases and the maximum wavelength decreases in less polar solvents; thus, the aurones may be useful probes for hydrophobic sites on biological molecules. A limited investigation with model protein, nucleic acid and fixed cells supports this idea. It is known that the sulfur analog of aurone can undergo photo-induced E/Z isomerization. This possibility was investigated for one of the aminoaurones, which was observed to reversible photoisomerize. The two isomers have similar absorption spectra, but the emission properties are distinct. We conclude that appropriately substituted aurones are potentially useful as biological probes and photoswitches.

A Novel Fluorogenic Assay for the Detection of Nephrotoxin-Induced Oxidative Stress in Live Cells and Renal Tissue.

Drug-induced kidney injury frequently leads to aborted clinical trials and drug withdrawals. Sufficiently sensitive sensors capable of detecting mild signs of chemical insult in cell-based screening assays are critical to identifying and eliminating potential toxins in the preclinical stage. Oxidative stress is a common early manifestation of chemical toxicity, and biomolecule carbonylation is an irreversible repercussion of oxidative stress. Here, we present a novel fluorogenic assay using a sensor, TFCH, that responds to biomolecule carbonylation and efficiently detects modest forms of renal injury with much greater sensitivity than standard assays for nephrotoxins. We demonstrate that this sensor can be deployed in live kidney cells and in renal tissue. Our robust assay may help inform preclinical decisions to recall unsafe drug candidates. The application of this sensor in identifying and analyzing diverse pathologies is envisioned.

GW182 Proteins Restrict Extracellular Vesicle-Mediated Export of MicroRNAs in Mammalian Cancer Cells.

MicroRNAs (miRNAs) are small regulatory RNAs of relatively long half-life in non-proliferative human cells. However, in cancer cells the half-lives of miRNAs are comparatively short. To understand the mechanism of rapid miRNA turnover in cancer cells, we explored the effect of target mRNAs on the abundance of the miRNAs that repress them. We have noted an accelerated extracellular vesicle (EV)-mediated export of miRNAs in presence of their target mRNAs in mammalian cells, and this target-driven miRNA-export process is retarded by Ago2-interacting protein GW182B. The GW182 group of proteins are localized to GW182 bodies or RNA processing bodies in mammalian cells, and GW182B-dependent retardation of miRNA export depends on GW body integrity and is independent of the HuR protein-mediated auxiliary pathway of miRNA export. Our data thus support the existence of a HuR-independent pathway of miRNA export in human cells that can be targeted in MDA-MB-231 cancer cells, to increase the level of cellular let-7a, a known negative regulator of cancer growth.

Testosterone Metabolite 6ß-Hydroxytestosterone Contributes to Angiotensin II-Induced Abdominal Aortic Aneurysms in Apoe-/- Male Mice.

Background Sex is a prominent risk factor for abdominal aortic aneurysms (AAAs), and angiotensin II (Ang II) induces AAA formation to a greater degree in male than in female mice. We previously reported that cytochrome P450 1B1 contributes to the development of hypertension, as well as AAAs, in male mice. We also found that a cytochrome P450 1B1-generated metabolite of testosterone, 6ß-hydroxytestosterone (6ß-OHT), contributes to Ang II-induced hypertension and associated cardiovascular and renal pathogenesis in male mice. The current study was conducted to determine the contribution of 6ß-OHT to Ang II-induced AAA development in Apoe-/- male mice. Methods and Results Intact or castrated Apoe-/-/Cyp1b1+/+ and Apoe-/-/Cyp1b1-/- male mice were infused with Ang II or its vehicle for 28 days, and administered 6ß-OHT every third day for the duration of the experiment. Abdominal aortas were then evaluated for development of AAAs. We observed a significant increase in the incidence and severity of AAAs in intact Ang II-infused Apoe-/-/Cyp1b1+/+ mice, compared with vehicle-treated mice, which were minimized in castrated Apoe-/-/Cyp1b1+/+ and intact Apoe-/-/Cyp1b1-/- mice infused with Ang II. Treatment with 6ß-OHT significantly restored the incidence and severity of AAAs in Ang II-infused castrated Apoe-/-/Cyp1b1+/+ and intact Apoe-/-/Cyp1b1-/- mice. However, administration of testosterone failed to increase AAA incidence and severity in Ang II-infused intact Apoe-/-/Cyp1b1-/- mice. Conclusions Our results indicate that the testosterone-cytochrome P450 1B1-generated metabolite 6ß-OHT contributes to Ang II-induced AAA development in Apoe-/- male mice.

Metabolomic and transcriptomic analysis reveals endogenous substrates and metabolic adaptation in rats lacking Abcg2 and Abcb1a transporters.

Abcg2/Bcrp and Abcb1a/Pgp are xenobiotic efflux transporters limiting substrate permeability in the gastrointestinal system and brain, and increasing renal and hepatic drug clearance. The systemic impact of Bcrp and Pgp ablation on metabolic homeostasis of endogenous substrates is incompletely understood. We performed untargeted metabolomics of cerebrospinal fluid (CSF) and plasma, transcriptomics of brain, liver and kidney from male Sprague Dawley rats (WT) and Bcrp/Pgp double knock-out (dKO) rats, and integrated metabolomic/transcriptomic analysis to identify putative substrates and perturbations in canonical metabolic pathways. A predictive Bayesian machine learning model was used to predict in silico those metabolites with greater substrate-like features for either transporters. The CSF and plasma levels of 169 metabolites, nutrients, signaling molecules, antioxidants and lipids were significantly altered in dKO rats, compared to WT rats. These metabolite changes suggested alterations in histidine, branched chain amino acid, purine and pyrimidine metabolism in the dKO rats. Levels of methylated and sulfated metabolites and some primary bile acids were increased in dKO CSF or plasma. Elevated uric acid levels appeared to be a primary driver of changes in purine and pyrimidine biosynthesis. Alterations in Bcrp/Pgp dKO CSF levels of antioxidants, precursors of neurotransmitters, and uric acid suggests the transporters may contribute to the regulation of a healthy central nervous system in rats. Microbiome-generated metabolites were found to be elevated in dKO rat plasma and CSF. The altered dKO metabolome appeared to cause compensatory transcriptional change in urate biosynthesis and response to lipopolysaccharide in brain, oxidation-reduction processes and response to oxidative stress and porphyrin biosynthesis in kidney, and circadian rhythm genes in liver. These findings present insight into endogenous functions of Bcrp and Pgp, the impact that transporter substrates, inhibitors or polymorphisms may have on metabolism, how transporter inhibition could rewire drug sensitivity indirectly through metabolic changes, and identify functional Bcrp biomarkers.