Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model.

BACKGROUND: Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of 68Ga- and 99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics. RESULTS: The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both 68Ga- and 99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described. CONCLUSION: The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin.

Rifabutin but not rifampicin can partly out-balance P-glycoprotein induction by concurrent P-glycoprotein inhibition through high affinity binding to the inhibitory site.

Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = -11.5 vs -5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp.

2-Substituted quinazolines: Partial agonistic and antagonistic ligands of the constitutive androstane receptor (CAR).

Following the discovery of 2-(3-methoxyphenyl)-3,4-dihydroquinazoline-4-one and 2-(3-methoxyphenyl)quinazoline-4-thione as potent, but non-specific activators of the human Constitutive Androstane Receptor (CAR, NR1I3), a series of quinazolinones substituted at the C2 phenyl ring was prepared to examine their ability to selectively modulate human CAR activity. Employing cellular and in vitro TR-FRET assays with wild-type CAR or its variant 3 (CAR3) ligand binding domains (LBD), several novel partial human CAR agonists and antagonists were identified. 2-(3-Methylphenyl) quinazolinone derivatives 7d and 8d acted as partial agonists with the recombinant CAR LBD, the former in nanomolar units (EC50 = 0.055 µM and 10.6 µM, respectively). Moreover, 7d did not activate PXR, and did not show any signs of cytotoxicity. On the other hand, 2-(4-bromophenyl)quinazoline-4-thione 7l possessed significant CAR antagonistic activity, although the compound displayed no agonistic or inverse agonistic activities. A compound possessing purely antagonistic effect was thus identified for the first time. These and related compounds may serve as a remedy in xenobiotic intoxication or, conversely, in suppression of undesirable hepatic CAR activation.

Dietary phytochemicals as modulators of human pregnane X receptor.

As a promiscuous xenobiotic sensor, pregnane X receptor (PXR) plays a crucial role in drug metabolism. Since dietary phytochemicals exhibit the potential to modulate human PXR, this review aims to summarize the plant-derived PXR modulators, including agonists, partial agonists, and antagonists. The crystal structures of the apo and ligand-bound forms of PXR especially that of PXR complexed with binary mixtures are summarized, in order to provide the structural basis for PXR binding promiscuity and synergistic activation of PXR by composite ligands. Furthermore, this review summarizes the characterized agonists, partial agonists, and antagonists of human PXR from botanical source. Contrary to PXR agonists, there are only a few antagonists obtained from botanical source due to the promiscuity of PXR. It is worth noting that trans-resveratrol and a series of methylindoles have been identified as partial agonists of PXR, both in activating PXR function, but also inhibiting the effect of other PXR agonists. Since antagonizing PXR function plays a crucial role in the prevention of drug-drug interactions and improvement of therapeutic efficacy, further research is necessary to screen more plant-derived PXR antagonists in the future. In summary, this review may contribute to understanding the roles of phytochemicals in food-drug and herb-drug interactions.

Allosteric Antagonism of the Pregnane X Receptor (PXR): Current-State-of-the-Art and Prediction of Novel Allosteric Sites.

The pregnane X receptor (PXR, NR1I2) is a xenobiotic-activated transcription factor with high levels of expression in the liver. It not only plays a key role in drug metabolism and elimination, but also promotes tumor growth, drug resistance, and metabolic diseases. It has been proposed as a therapeutic target for type II diabetes, metabolic syndrome, and inflammatory bowel disease, and PXR antagonists have recently been considered as a therapy for colon cancer. There are currently no PXR antagonists that can be used in a clinical setting. Nevertheless, due to the large and complex ligand-binding pocket (LBP) of the PXR, it is challenging to discover PXR antagonists at the orthosteric site. Alternative ligand binding sites of the PXR have also been proposed and are currently being studied. Recently, the AF-2 allosteric binding site of the PXR has been identified, with several compounds modulating the site discovered. Herein, we aimed to summarize our current knowledge of allosteric modulation of the PXR as well as our attempt to unlock novel allosteric sites. We describe the novel binding function 3 (BF-3) site of PXR, which is also common for other nuclear receptors. In addition, we also mention a novel allosteric site III based on in silico prediction. The identified allosteric sites of the PXR provide new insights into the development of safe and efficient allosteric modulators of the PXR receptor. We therefore propose that novel PXR allosteric sites might be promising targets for treating chronic metabolic diseases and some cancers.

Preparation, In Vitro Affinity, and In Vivo Biodistribution of Receptor-Specific 68Ga-Labeled Peptides Targeting Vascular Endothelial Growth Factor Receptors.

As angiogenesis plays a key role in tumor growth and metastasis, the angiogenic process has attracted scientific interest as a target for diagnostic and therapeutic agents. Factors influencing angiogenesis include the vascular endothelial growth factor (VEGF) family and the two associated receptor types (VEGFR-1 and VEGFR-2). VEGFR-1/-2 detection and quantification in cancer lesions are essential for tumor process management. As a result of the advantageous pharmacokinetics and image contrast, peptides radiolabeled with PET emitters have become interesting tools for the visualization of VEGFR-1/-2-positive tumors. In this study, we prepared 68Ga-labeled peptides containing 15 (peptide 1) and 23 (peptide 2) amino acids as new PET tracers for tumor angiogenic process imaging. METHODS: The peptides were conjugated with NODAGA-tris(t-Bu ester) and subsequently radiolabeled with [68Ga]Ga-chloride. The prepared [68Ga]Ga-NODAGA-peptide 1 and [68Ga]Ga-NODAGA-peptide 2 were tested for radiochemical purity and saline/plasma stability. Consequently, the binding affinity toward VEGFRs was assessed in vitro on human glioblastoma and kidney carcinoma cells. The found peptide receptor affinity was compared with the calculated values in the PROtein binDIng enerGY prediction (PRODIGY) server. Finally, the biodistribution study was performed on BALB/c female mice to reveal the basic pharmacokinetic behavior of radiopeptides. RESULTS: The in vitro affinity testing of [68Ga]Ga-NODAGA-peptides 1 and 2 showed retained receptor binding as characterized by equilibrium dissociation constant (KD) values in the range of 0.5-1.2 µM and inhibitory concentration 50% (IC50) values in the range of 3.0-5.6 µM. Better binding properties of peptide 2 to VEGFR-1/-2 were found in the PRODIGY server. The biodistribution study on mice showed remarkable accumulation of both peptides in the kidneys and urinary bladder with a short half-life after intravenous application. The in vitro plasma stability of [68Ga]Ga-NODAGA-peptide 2 was superior to that of [68Ga]Ga-NODAGA-peptide 1. CONCLUSIONS: The obtained results demonstrated a high radiolabeling yield with no need for purification and preserved binding potency of 68Ga-labeled peptides 1 and 2 toward VEGFRs in cancer cells. The peptide-receptor protein interaction assessed in protein-peptide docking determined the strongest interaction of peptide 2 with domain 2 of VEGFR-2 in addition to a more acceptable plasma stability (t1/2 = 120 min) than that for peptide 1. We found both radiolabeled peptides very potent in their receptor binding, which makes them suitable imaging agents. The rapid transition of the radiopeptides into the urinary tract indicates suitable pharmacokinetic characteristics.

Gene Expression Profiling of 1α,25(OH)2 D3 Treatment in 2D/3D Human Hepatocyte Models Reveals CYP3A4 Induction but Minor Changes in Other Xenobiotic-Metabolizing Genes.

SCOPE: CYP3A4 is the most important drug-metabolizing enzyme regulated via the vitamin D receptor (VDR) in the intestine. However, less is known about VDR in the regulation of CYP3A4 and other drug-metabolizing enzymes in the liver. METHODS AND RESULTS: This study investigates whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3 ) regulates major cytochrome P450 enzymes, selected phase I and II enzymes, and transporters involved in xenobiotic and steroidal endobiotic metabolism in 2D and 3D cultures of human hepatocytes. The authors found that 1α,25(OH)2 D3 increases hepatic CYP3A4 expression and midazolam 1'-hydroxylation activity in 2D hepatocytes. The results are confirmed in 3D spheroids, where 1α,25(OH)2 D3 has comparable effect on CYP3A4 mRNA expression as 1α-hydroxyvitamin D3 , an active vitamin D metabolite. Other regulated genes such as CYP1A2, AKR1C4, SLC10A1, and SLCO4A1 display only mild changes in mRNA levels after 1α,25(OH)2 D3 treatment in 2D hepatocytes. Expression of other cytochrome P450, phase I and phase II enzyme, or transporter genes are not significantly influenced by 1α,25(OH)2 D3 . Additionally, the effect of VDR activation on CYP3A4 mRNA expression is abolished by natural dietary compound sulforaphane, a common suppressor of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). CONCLUSION: This study proposes that VDR or vitamin D supplementation is unlikely to significantly influence liver detoxification enzymes apart from CYP3A4.

Amberlyst-15 catalysed synthesis of novel indole derivatives under ultrasound irradiation: Their evaluation as serotonin 5-HT2C receptor agonists.

A series of indole based novel Schiff bases was designed as potential agonists of 5-HT2C receptor that was supported by docking studies in silico. These compounds were synthesized via Amberlyst-15 catalysed condensation of an appropriate pyrazole based primary amine with the corresponding indole-3-aldehyde under ultrasound irradiation at ambient temperature. A number of target Schiff bases were obtained in good yields (77-87%) under mild conditions within 1 h. Notably, the methodology afforded the corresponding pyrazolo[4,3-d]pyrimidin-7(4H)-one derivatives when the primary amine was replaced by a secondary amine. Several Schiff bases showed agonist activity when tested against human 5-HT2C using luciferase assay in HEK293T cells in vitro. The SAR (Structure-Activity-Relationship) studies suggested that the imine moiety was more favorable over its cyclic form i.e. the corresponding pyrazolopyrimidinone ring. The Schiff bases 3b (EC50 1.8 nM) and 3i (EC50 5.7 nM) were identified as the most active compounds and were comparable with Lorcaserin (EC50 8.5 nM). Also like Lorcaserin, none of these compounds were found to be PAM of 5-HT2C. With ∼24 and ∼150 fold selectivity towards 5-HT2C over 5-HT2A and 5-HT2B respectively the compound 3i that reduced locomotor activity in zebrafish (Danio rerio) larvae model emerged as a promising hit molecule for further study.

A review on pharmacological activities and synergistic effect of quercetin with small molecule agents.

BACKGROUND: Quercetin is a natural flavonoid, which widely exists in nature, such as tea, coffee, apples, and onions. Numerous studies have showed that quercetin has multiple biological activities such as anti-oxidation, anti-inflammatory, and anti-aging. Hence, quercetin has a significant therapeutic effect on cancers, obesity, diabetes, and other diseases. In the past decades, a large number of studies have shown that quercetin combined with other agents can significantly improve the overall therapeutic effect, compared to single use. PURPOSE: This work reviews the pharmacological activities of quercetin and its derivatives. In addition, this work also summarizes both in vivo and in vitro experimental evidence for the synergistic effect of quercetin against cancers and metabolic diseases. METHODS: An extensive systematic search for pharmacological activities and synergistic effect of quercetin was performed considering all the relevant literatures published until August 2021 through the databases including NCBI PubMed, Scopus, Web of Science, and Google Scholar. The relevant literatures were extracted from the databases with following keyword combinations: "pharmacological activities" OR "biological activities" OR "synergistic effect" OR "combined" OR "combination" AND "quercetin" as well as free-text words. RESULTS: Quercetin and its derivatives possess multiple pharmacological activities including anti-cancer, anti-oxidant, anti-inflammatory, anti-cardiovascular, anti-aging, and neuroprotective activities. In addition, the synergistic effect of quercetin with small molecule agents against cancers and metabolic diseases has also been confirmed. CONCLUSION: Quercetin cooperates with agents to improve the therapeutic effect by regulating signal molecules and blocking cell cycle. Synergistic therapy can reduce the dose of agents and avoid the possible toxic and side effects in the treatment process. Although quercetin treatment has some potential side effects, it is safe under the expected use conditions. Hence, quercetin has application value and potential strength as a clinical drug. Furthermore, quercetin, as the main effective therapeutic ingredient in traditional Chinese medicine, may effectively treat and prevent coronavirus disease 2019 (COVID-19).