Transport Of Conjugates Research Articles

Novel Penicillin-Based Sulfone-Siderophore Conjugates for Restoring β-Lactam Antibiotic Efficacy.

Membrane permeability is a natural defense barrier that contributes to increased bacterial drug resistance, particularly for Gram-negative pathogens. As such, accurate delivery of the antibacterial agent to the target has become a growing research area in the infectious diseases field as a means of improving drug efficacy. Although the efficient transport of siderophore-antibiotic conjugates into the cytosol still remains challenging, great success has been achieved in the delivery of β-lactam antibiotics into the periplasmic space via bacterial iron uptake pathways. Cefiderocol, the first siderophore-cephalosporin conjugate approved by the US Food and Drug Administration, is a good example. These conjugation strategies have also been applied to the precise delivery of β-lactamase inhibitors, such as penicillin-based sulfone 1, to restore β-lactam antibiotic efficacy in multidrug-resistant bacteria. Herein, we have explored the impact on the bacterial activity of 1 by modifying its iron chelator moiety. A set of derivatives functionalized with diverse iron chelator groups and linkages to the scaffold (compounds 2-8) were synthesized and assayed in vitro. The results on the ability of derivatives 2-8 to recover β-lactam antibiotic efficacy in difficult-to-treat pathogens that produce various β-lactamase enzymes, along with kinetic studies with the isolated enzymes, allowed us to identify compound 2, a novel β-lactamase inhibitor with an expanded spectrum of activity. Molecular dynamics simulation studies provided us with further information regarding the molecular basis of the relative inhibitory properties of the most relevant compound described herein.

Current Expansion of Silver and Gold Nanomaterials towards Cancer Theranostics: Development of Therapeutics

Abstract: Nanomaterial-based therapeutics is an emerging tool for the treatment of numerous types of cancer. Various types of polymeric, lipid and inorganic nanoparticles (NPs) result in a wider series of applications in cancer diagnosis and therapeutics. The NPs properties are due to high surface area to volume ratio, surface plasmon resonance, absorption in the visible spectrum and light scattering. These unique characteristics of NPs arise due to their optical surface properties for conjugation/surface modification and smaller size. In cancer therapeutics, NPs based products are used as a biomarker for early detection/diagnosis of tumours, drug nano-conjugates for the delivery of chemotherapeutic drugs to the tumour-specific site, chemo-protective agents, etc. : Furthermore, other advantages of NPs are biocompatibility, lesser toxicity, enhanced permeability and retention effect, higher stability, and specific targeting with a selective accumulation of nano drugs in the tissue of the tumour. The selective targeting of NPs to tumour tissue is possible by adding surface-active targeting agents i.e., antibodies. The selective transport of drug NPs conjugates to the cancer cells is increased and extravagated due to permeable vasculature from endothelial cells gap while failing the transport of drug NPs conjugates in normal cells. This review emphasizes metallic NPs, including silver NPs (AgNPs) and gold NPs (AuNPs), which are extensively reconnoitered in various applications in cellular targeting, imaging, drug delivery, DNA-NPs conjugates for biosensor/point of care devices development, photothermal/photodynamic therapy, protein-protein interaction, etc. In addition, this review discussed different synthetic methods of AgNPs and AuNPs and characterization methods. Furthermore, it highlighted the different properties and applications of AgNPs and AuNPs in cancer theranostics.

The Active Glucuronide Metabolite of the Brain Protectant IMM-H004 with Poor Blood-Brain Barrier Permeability Demonstrates a High Partition in the Rat Brain via Multiple Mechanisms.

Glucuronidation is an essential metabolic pathway for a variety of drugs. IMM-H004 is a novel neuroprotective agent against ischemic stroke, and its glucuronide metabolite IMM-H004G exhibits similar pharmacological activity. Despite possessing a higher molecular weight and polarity, brain exposure of IMM-H004G is much higher than that of IMM-H004. This study aimed to investigate the brain metabolism and transport mechanisms of IMM-H004 and IMM-H004G. First, the possibility of IMM-H004 glucuronidation in the brain was evaluated in several human brain cell lines and rat homogenate. Subsequently, the blood-brain barrier carrier-mediated transport mechanism of IMM-H004 and IMM-H004G was studied using overexpression cell models. In addition, intracerebroventricular injection, in situ brain perfusion model, and microdialysis/microinjection techniques were performed to study the distribution profiles of IMM-H004 and IMM-H004G. IMM-H004 could be metabolized to IMM-H004G in both rat brain and HEB cells mediated by UGT1A7. However, IMM-H004G could not be hydrolyzed back into IMM-H004. Furthermore, the entry and efflux of IMM-H004 in the brain were mediated by the pyrilamine-sensitive H+/OC antiporter and P-gp, respectively, while the transport of IMM-H004G from the blood to the brain was facilitated by OATP1A2 and OATP2B1. Ultimately, stronger concentration gradients and OATP-mediated uptake played a critical role in promoting greater brain exposure of IMM-H004G. The active glucuronide metabolite of the brain protectant IMM-H004 with poor blood-brain barrier permeability demonstrates a high partition in the rat brain via multiple mechanisms, and our findings deepen the understanding of the mechanisms underlying the blood-brain barrier metabolism and transport of active glucuronide conjugates.

Is monolithic configuration superior to random packing in a fixed bed reactor for CLC like processes?

In light of the geometric deficiencies and diffusion constraints of conventional packed-bed reactors, the potential application of monolithic configurations in fixed-bed reactors was explored using the structure-resolved CFD approach. This work delves into how geometric features of fixed beds influence flow patterns and related transport phenomena. To assess the performance of mass transfer and heterogeneous reactions in different configurations, we conducted simulations of fixed-bed chemical looping combustion (CLC) processes, in which a simplified model of non-catalytic gas-solid reactions is implemented to reduce the computational costs. Our findings highlight the importance of face contact between reaction tube and the monolithic structure, which effectively eliminates the inherent wall effects of random packing and enhances the radial heat conduction. More uniform geometry in the monolithic configuration naturally suppresses violent interstitial channeling flow in the random packing. Notably, the honeycomb configuration demonstrates an exceptionally low pressure drop, about two orders of magnitude smaller than that of the Raschig ring packing, and the designable monolithic configurations offers the potential to further reduce the pressure drop with enlarged voidage. Open-cell foams exhibit excellent convective and radial heat transport properties, promote homogeneous flow fields, and demonstrate superior performance in conjugate transport and heterogeneous reactions.

Design, synthesis, insecticidal activities and translocation of amino acid-tralopyril conjugates as vectorizing agrochemicals.

Conjugating amino acid moieties to active ingredients has been recognized as an effective method for improving the precise targeting of the active form to the specific site. Based on the vectorization strategy, a series of amino acid-tralopyril conjugates were designed and synthesized as novel proinsecticide candidates, with the potential capability of root uptake and translocation to the foliage of crops. Bioassay results showed excellent insecticidal activities of some conjugates, in particular, the conjugates 6b, 6e, and 7e, against the diamondback moth (Plutella xylostella), with equivalent insecticidal activity to chlorfenapyr (CFP). Importantly, conjugate 6e exhibited significantly higher in vivo insecticidal activity against P. xylostella than CFP. Furthermore, the systemic test experiments with Brassica chinensis demonstrated that conjugates 6e and 7e could be transported to the leaves, in contrast to CFP, which remained in the root. This study demonstrated the feasibility of amino acid fragment conjugation as a vectorization strategy for transporting non-systemic insecticides into the leaves of B. chinensis while maintaining in vivo insecticidal activity. The findings also provide insights for subsequent mechanism studies on the uptake and transport of amino acid-insecticide conjugates in plants. © 2023 Society of Chemical Industry.

Sustained intra-cartilage delivery of interleukin-1 receptor antagonist using cationic peptide and protein-based carriers

Blocking the interleukin-1 (IL-1) catabolic cascade following joint trauma can be achieved using its receptor antagonist, IL-1Ra. However, its clinical translation for osteoarthritis therapy has been unsuccessful due to its rapid joint clearance and lack of targeting and penetration into deep cartilage layers at therapeutic concentrations. Here, we target the high negative charge of cartilage aggrecan-glycosaminoglycans (GAGs) by attaching cationic carriers to IL-1Ra. IL-1Ra was conjugated to the cartilage targeting glycoprotein, Avidin, and a short length optimally charged cationic peptide carrier (CPC+14). It is hypothesized that electro-diffusive transport and binding properties of IL-1Ra-Avidin and IL-1Ra-CPC+14 will create intra-cartilage depots of IL-1Ra, resulting in long-term suppression of IL-1 catabolism with only a single administration. IL-1Ra was conjugated to Avidin or CPC+14 using site specific maleimide linkers, and confirmed using gel electrophoresis, high-performance liquid chromatography (HPLC), and mass spectrometry. Intra-cartilage transport and retention of conjugates was compared with native IL-1Ra. Attenuation of IL-1 catabolic signaling with one-time dose of IL-1Ra-CPC+14 and IL-1Ra-Avidin was assessed over 16 days using IL-1α challenged bovine cartilage and compared with unmodified IL-1Ra. Positively charged IL-1Ra penetrated through the full-thickness of cartilage, creating a drug depot. A single dose of unmodified IL-1Ra was not sufficient to attenuate IL-1-induced cartilage deterioration over 16 days. However, when delivered using Avidin, and to a greater extent CPC+14, IL-1Ra significantly suppressed cytokine induced GAG loss and nitrite release while improving cell metabolism and viability. Charge-based cartilage targeting drug delivery systems hold promise as they can enable long-term therapeutic benefit with only a single dose.

Shrinkage during vacuum cooling of porous foods: Conjugate mechanistic modelling and experimental validation

A conjugate modelling of momentum, heat and mass transport, and structural deformation during steamed bread vacuum cooling (VC) was developed and validated with nuclear magnetic resonance/magnetic resonance imaging. The moisture variation was correlated with shrinkage and the coupled two-dimensional problem was solved using the Arbitrary Lagrangian-Eulerian method. Results revealed that the three-parameter Maxwell model accurately described the viscoelastic behaviour of the bread during VC and was superior to the linear elastic model. Additionally, the model prediction was accurate with R 2 values and maximum errors of 96.35% and 9.56% for moisture variation, and 99.65% and 13.51% for areal shrinkage, respectively. Sensitivity analysis revealed that the deformation model was more sensitive to water transport parameters than the viscoelastic model. Notably, the hydrous compressibility factor had the highest sensitivity to the deformation model. The developed model could be used for quality index evaluation to better understand and optimize the VC of porous foods, thereby improving the product's quality. • Shrinkage during vacuum cooling (VC) of steamed bread was modelled. • Conjugate transport and deformation modelling during VC was developed. • Steamed bread was accurately represented using linear viscoelastic model. • The model prediction was accurate for moisture variation and shrinkage. • Water transport parameters strongly influenced structural deformation.

ATP-Binding Cassette (ABC) Transporters in Fusarium Specific Mycoparasite Sphaerodes mycoparasitica during Biotrophic Mycoparasitism

Recent transcriptomic profiling has revealed importance membrane transporters such as ATP-binding cassette (ABC) transporters in fungal necrotrophic mycoparasites. In this study, RNA-Seq allowed rapid detection of ABC transcripts involved in biotrophic mycoparasitism of Sphaerodes mycoparasitica against the phytopathogenic and mycotoxigenic Fusarium graminearum host, the causal agent of Fusarium head blight (FHB). Transcriptomic analyses of highly expressed S. mycoparasitica genes, and their phylogenetic relationships with other eukaryotic fungi, portrayed the ABC transporters’ evolutionary paths towards biotrophic mycoparasitism. Prior to the in silico phylogenetic analyses, transmission electron microscopy (TEM) was used to confirm the formation of appressorium/haustorium infection structures in S. mycoparasitica during early (1.5 d and 3.5 d) stages of mycoparasitism. Transcripts encoding biotrophy-associated secreted proteins did uncover the enrolment of ABC transporter genes in this specific biocontrol mode of action, while tandem ABC and BUB2 (non-ABC) transcripts seemed to be proper for appressorium development. The next-generation HiSeq transcriptomic profiling of the mycoparasitic hypha samples, revealed 81 transcripts annotated to ABC transporters consisting of a variety of ABC-B (14%), ABC-C (22%), and ABC-G (23%), and to ABC-A, ABC-F, aliphatic sulfonates importer (TC 3.A.1.17.2), BtuF, ribose importer (TC 3.A.1.2.1), and unknown families. The most abundant transcripts belonged to the multidrug resistance exporter (TC 3.A.1.201) subfamily of the ABC-B family, the conjugate transporter (TC 3.A.1.208) subfamily of the ABC-C family, and the pleiotropic drug resistance (PDR) (TC 3.A.1.205) subfamily of the ABC-G family. These findings highlight the significance of ABC transporter genes that control cellular detoxification against toxic substances (e.g., chemical pesticides and mycotoxins) in sustaining a virulence of S. mycoparasitica for effective biotrophic mycoparasitism on the F. graminearum host. The findings of this study provide clues to better understand the biotrophic mycoparasitism of S. mycoparasitica interacting with the Fusarium host, which implies that the ABC transporter group of key proteins is involved in the mycoparasite’s virulence and multidrug resistance to toxic substances including cellular detoxification.

Unique TLR9 Activation by Helicobacter pylori Depends on the cag T4SS, But Not on VirD2 Relaxases or VirD4 Coupling Proteins

The genomes of the gastric bacterial pathogen Helicobacter pylori harbor multiple type-IV secretion systems (T4SSs). Here we analyzed components of three T4SSs, the cytotoxin-associated genes (cag) T4SS, TFS3 and TFS4. The cag T4SS delivers the effector protein CagA and the LPS-metabolite ADP-heptose into gastric epithelial cells, which plays a pivotal role in chronic infection and development of gastric disease. In addition, the cag T4SS was reported to facilitate conjugative transport of chromosomal bacterial DNA into the host cell cytoplasm, where injected DNA activates intracellular toll-like receptor 9 (TLR9) and triggers anti-inflammatory signaling. Canonical DNA-delivering T4SSs in a variety of bacteria are composed of 11 VirB proteins (VirB1-11) which assemble and engage VirD2 relaxase and VirD4 coupling proteins that mediate DNA processing and guiding of the covalently bound DNA through the T4SS channel. Nevertheless, the role of the latter components in H. pylori is unclear. Here, we utilized isogenic knockout mutants of various virB (virB9 and virB10, corresponding to cagX and cagY), virD2 (rlx1 and rlx2), virD4 (cag5, traG1/2) and xerD recombinase genes in H. pylori laboratory strain P12 and studied their role in TLR9 activation by reporter assays. While inactivation of the structural cag T4SS genes cagX and cagY abolished TLR9 activation, the deletion of rlx1, rlx2, cag5, traG or xerD genes had no effect. The latter mutants activated TLR9 similar to wild-type bacteria, suggesting the presence of a unique non-canonical T4SS-dependent mechanism of TLR9 stimulation by H. pylori that is not mediated by VirD2, VirD4 and XerD proteins. These findings were confirmed by the analysis of TLR9 activation by H. pylori strains of worldwide origin that possess different sets of T4SS genes. The exact mechanism of TLR9 activation should be explored in future studies.

Transferrin receptor-mediated internalization and intracellular fate of conjugates of a DNA aptamer

Aptamers have excellent specificity and affinity in targeting cell surface receptors, showing great potential in targeted delivery of drugs, siRNA, mRNA, and various nanomaterials with therapeutic function. A better insight of the receptor-mediated internalization process of aptameric conjugates could facilitate the design of new targeted drugs. In this paper, human transferrin receptor-targeted DNA aptamer (termed HG1-9)-fluorophore conjugates were synthesized to visualize the internalization, intracellular transport, and nano-environmental pH of aptameric conjugates. Unlike transferrin that showed high recycling rate and short duration time in cells, the synthetic aptameric conjugates continuously accumulated within cells at a relatively slower rate, besides recycling back to cell surface. After long incubation (≥2 h), only very small amounts of HG1-9 conjugates (approximately 5%) entered late endosomes or lysosomes, and more than 90% of internalized HG1-9 was retained in cellular vesicles (pH 6.0–6.8), escaping from degradation. And among the internalized HG1-9 conjugates, approximately 20% was dissociated from transferrin receptor. The lower recycling ratios of HG1-9 conjugates and their dissociation from receptors promote the accurate and efficient release of their loaded drugs. These results suggest that aptamer HG1-9 could be provided as a versatile tool for specific and effective delivery of diverse therapeutic payloads.

Modified amino-dextrans as carriers of Gd-chelates for retrograde transport and visualization of peripheral nerves by magnetic resonance imaging (MRI)

Amino-dextrans (AD) conjugated with gadolinium (Gd3+) were developed as neuro-specific contrast agents (CA) for the visualization of the sciatic nerve in rats by magnetic resonance imaging (MRI). AD with 3, 10, and 70 kDa molecular weights were assessed as carrier molecules known to be transported with various speed by axonal microtubules. Detailed spectroscopic characterizations, analyses by Fast Protein Liquid Chromatography (FPLC), Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and inductively coupled plasma-mass spectrometry (ICP-MS), were carried out. For MRI, the paramagnetic Gd3+ ion was coupled as a T1 signal enhancer. The well-established linear chelator, diethylenetriaminepentaacetic acid (DTPA), was used and subsequently replaced by the more stable cyclic chelator 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In addition, a fluorescently labeled AD-DTPA-Gd was prepared to demonstrate an active transport to the spinal cord by histochemistry. After successful synthesis and characterization, molecular migration of the AD-DTPA-Gd in the sciatic nerve of healthy Sprague Dawley rats was monitored by MRI for up to seven days. Enhancement of nerve structures was evaluated by MRI and correlated with ICP-MS analyses. To investigate the distribution of CA along the neuraxis, all animals were sacrificed after the final MRI monitoring. Nerves, spinal ganglions, and corresponding spinal cord sections were harvested, to determine the localization and concentration of the paramagnetic element. This is the first report that demonstrates the active uptake and transport of AD-Gd conjugates within the sciatic nerve. This new concept may serve as a potential diagnostic tool for the direct visualization and monitoring of the continuity of injured nerves.

Vitamin B12-peptide nucleic acids use the BtuB receptor to pass through the Escherichia coli outer membrane

Short modified oligonucleotides that bind in a sequence-specific way to messenger RNA essential for bacterial growth could be useful to fight bacterial infections. One such promising oligonucleotide is peptide nucleic acid (PNA), a synthetic DNA analog with a peptide-like backbone. However, the limitation precluding the use of oligonucleotides, including PNA, is that bacteria do not import them from the environment. We have shown that vitamin B12, which most bacteria need to take up for growth, delivers PNAs to Escherichia coli cells when covalently linked with PNAs. Vitamin B12 enters E. coli via a TonB-dependent transport system and is recognized by the outer-membrane vitamin B12-specific BtuB receptor. We engineered the E. coli ΔbtuB mutant and found that transport of the vitamin B12-PNA conjugate requires BtuB. Thus, the conjugate follows the same route through the outer membrane as taken by free vitamin B12. From enhanced sampling all-atom molecular dynamics simulations, we determined the mechanism of conjugate permeation through BtuB. BtuB is a β-barrel occluded by its luminal domain. The potential of mean force shows that conjugate passage is unidirectional and its movement into the BtuB β-barrel is energetically favorable upon luminal domain unfolding. Inside BtuB, PNA extends making its permeation mechanically feasible. BtuB extracellular loops are actively involved in transport through an induced-fit mechanism. We prove that the vitamin B12 transport system can be hijacked to enable PNA delivery to E. coli cells.

The subgroup of 2′-hydroxy-flavonoids: Molecular diversity, mechanism of action, and anticancer properties

Flavonoids are abundant in nature, structurally very diversified and largely investigated. However, the subgroup of 2′-hydroxyflavonoids is much less known and not frequently studied. The present review identifies the major naturally-occurring and synthetic 2′-hydroxyflavonoid derivatives and discusses their structural characteristics and biological properties, with a focus on anticancer activities. The pharmacological properties of 2′-hydroxyflavone (2′-HF) and 2′-hydroxyflavanone (2′-HFa) are detailed. Upon binding to the Ral-interacting protein Rlip implicated in the transport of glutathione conjugates, 2′-HFa inhibits tumor cell proliferation and restrict tumor growth, in particular in breast cancer models. Among the synthetic derivatives, the characteristics of the anticancer product 2D08 (2′,3′,4′-trihydroxy flavone) are detailed to shed light on the molecular mechanism of action of this compound, as a regulator of protein SUMOylation. Inhibition of protein SUMOylation by 2D08 blocks cancer cell migration and invasion, and the compound greatly enhances the anticancer effects of conventional cytotoxic drugs like etoposide. The structural role of the 2′-hydroxyl group on the phenyl C-ring of the flavonoid is discussed, notably the capacity to engage intramolecular H-bonding interactions with the O1 atom on the B-ring of the chromone unit (or the oxygen of a 3-OH group when it is presents). The 2′-hydroxyl group of flavonoid appears as a regulator of the conformational freedom between the bicyclic A-B unit and the appended phenyl C-ring, favoring the planarity of the molecule. It is an essential group accounting for the biological properties of 2′-HF, 2′-HFa and structurally related compounds. This review shed light on 2′-hydroxyflavonoids to encourage their use and chemical development.

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers.

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.

Multidrug Resistance-Associated Protein 3 Is Responsible for the Efflux Transport of Curcumin Glucuronide from Hepatocytes to the Blood.

Curcumin, a major polyphenol present in turmeric, is predominantly converted to curcumin-O-glucuronide (COG) in enterocytes and hepatocytes via glucuronidation. COG is a principal metabolite of curcumin in plasma and feces. It appears that the efflux transport of the glucuronide conjugates of many compounds is mediated largely by multidrug resistance-associated protein (MRP) 3, the gene product of the ATP-binding cassette, subfamily C, member 3. However, it is currently unknown whether this was the case with COG. In this study, Mrp3 knockout (KO) and wild-type (WT) mice were used to evaluate the pharmacokinetics profiles of COG, the liver-to-plasma ratio of COG, and the COG-to-curcumin ratio in plasma, respectively. The ATP-dependent uptake of COG into recombinant human MRP3 inside-out membrane vesicles was measured for further identification, with estradiol-17β-d-glucuronide used in parallel as the positive control. Results showed that plasma COG concentrations were extremely low in KO mice compared with WT mice, that the liver-to-plasma ratios of COG were 8-fold greater in KO mice than in WT mice, and that the ATP-dependent uptake of COG at 1 or 10 μM was 5.0- and 3.1-fold greater in the presence of ATP than in the presence of AMP, respectively. No significant differences in the Abcc2 and Abcg2 mRNA expression levels were seen between Mrp3 KO and WT mice. We conclude that Mrp3 is identified to be the main efflux transporter responsible for the transport of COG from hepatocytes into the blood. SIGNIFICANCE STATEMENT: This study was designed to determine whether multidrug resistance-associated protein (Mrp) 3 could be responsible for the efflux transport of curcumin-O-glucuronide (COG), a major metabolite of curcumin present in plasma and feces, from hepatocytes into the blood using Mrp3 knockout mice. In this study, COG was identified as a typical Mrp3 substrate. Results suggest that herb-drug interactions would occur in patients concomitantly taking curcumin and either an MRP3 substrate/inhibitor or a drug that is predominantly glucuronidated by UDP-glucuronosyltransferases.

Human efflux transport of testosterone, epitestosterone and other androgen glucuronides

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low Km values, between 0.4 and 4 μM, while the Km values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 μM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by Km values over 100 μM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.

Endocytic Pathways and Intracellular Transport of Aptamer-Drug Conjugates in Live Cells Monitored by Single-Particle Tracking.

Aptamer-drug conjugates (ApDCs) are emerging as targeted therapeutic drugs that can effectively broaden the chemotherapeutic window with higher efficacy and less toxicity. They show promising targeted tumor-killing effects both in vitro and in vivo. However, the mechanisms underlying the cellular internalization and transport of ApDCs remain unclear, and no systematic study on this topic has been reported. Therefore, we herein investigated the endocytic internalization and subsequent transport of ApDCs in mammalian cells through single-particle tracking. We found that ApDC enters the cells mainly by caveolin-mediated endocytosis and that it exhibits cytoskeleton-dependent transport, along microfilaments and microtubules, to acidic endosomes near the cell nucleus in cytoplasm. We also found that the cellular uptake pathways of ApDCs are identical to those of the aptamer itself, confirming that aptamers play a prominent role in the internalization of ApDCs. This study extends our understanding of the internalization and transport process of ApDCs such that the results could serve as the theoretical foundation for designing new ApDCs and, in turn, promoting cancer-targeted therapy.

Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies.

Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels.

Transport of Methylmercury through the Epithelial Type Amino Acid Transporter System B0

Background: System B0 is a sodium dependent transporter that transports wide variety of neutral amino acids in the intestinal and renal proximal tubular epithelial cells. Methylmercury (MeHg) readily and non-enzymatically reacts with cysteine to form conjugate structurally similar to the amino acid methionine.&#x0D; Objective: In this study, we investigated the molecular mechanism of absorptive transport of MeHg in intestine using Xenopus oocytes expressing hB0AT1 and the uptake of metylmercry-Cys (MeHg-Cys) by heterodimeric amino acids transporter.&#x0D; Methodology: We confirmed the uptake of [14C] L-Leucine a potent substrate for the hB0AT1 amino acids transporter. The uptake of [14C] L-leucine by hB0AT1 was inhibited by MeHg-Cys conjugate, leucine, cysteine, methinine and phenylalanine in concentration–dependent manner. The IC50 of MeHg-Cys conjugate was significantly lower than that of leucine, cysteine, methinine and phenylalanine, indicating that hB0AT1 is a high affinity MeHg transporter. To assess MeHg-Cys conjugate transport, we measured [14C] MeHg uptake in Xenopus oocytes expressing hB0AT1 in presence or absence of sodium. The [14C] MeHg was transport only in the presence of cysteine and the transport was significantly sodium dependent and inhibited by a system B0 inhibitor 2-aminobicyclo-[2,21]- haptane-2-carboxylic acid (BCH).&#x0D; Result: The current findings indicate that hB0AT1 and heterodimeric amino acids absorb MeHg in the form of cysteine conjugate from the intestinal lumen across the brush-border membrane in to the cells and is supposed to be plays a critical role in the pathogenesis of Minamata disease and present results descried a major molecular mechanism by which MeHg is transported across cell membranes and indicate that metal complexes may form a novel class of substrates for amino acid carriers.&#x0D; Conclusion: In this experiment the results also suggest that uptake of Methionine and MeHg-Cys by heterodimeric amino acid transporter is significantly correlated where the uptake of Methionine and MeHg-Cys between heterodimeric amino acid transporter and hB0AT1 is not correlated.&#x0D; Journal of National Institute of Neurosciences Bangladesh, 2019;5(2): 127-136

FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer

Fluorescein isothiocyanate-labeled insulin (FITC-insulin) has been widely used for bioanalytical applications. Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature, there is still a need to develop a cost effective, reliable and quick labeling method for insulin. The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer. FITC labeling of insulin gives mono-, di- or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin. Mono-conjugate with unlabeled insulin, mixture of di- and tri-conjugate, and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h. Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer. Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient. However, tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.