Abstract
Lopinavir and ritonavir (LPV/r) are the primary anti-human immunodeficiency virus (HIV) drugs recommended by the World Health Organization for treating children aged 3 years and above who ...are infected with the HIV. These drugs are typically available in liquid formulations to aid in dosing for children who cannot swallow tablets. However, the strong bitter taste associated with these medications can be a significant obstacle to adherence, particularly in young children, and can jeopardize the effectiveness of the treatment. Studies have shown that poor palatability can affect the survival rate of HIV-infected children. Therefore, developing more child-friendly protease inhibitor formulations, particularly those with improved taste, is critical for children with HIV. The molecular mechanism by which lopinavir and ritonavir activate bitter taste receptors, TAS2Rs, is not yet clear. In this study, we utilized a calcium mobilization assay to characterize the activation of bitter taste receptors by lopinavir and ritonavir. We discovered that lopinavir activates TAS2R1 and TAS2R13, while ritonavir activates TAS2R1, TAS2R8, TAS2R13, and TAS2R14. The development of bitter taste blockers that target these receptors with a safe profile would be highly desirable in eliminating the unpleasant bitter taste of these anti-HIV drugs.
Mitochondrial reactive oxygen species (mtROS) are signaling molecules, which drive inflammatory cytokine production and T cell activation. In addition, cardiovascular diseases, cancers, and ...autoimmune diseases all share a common feature of increased mtROS level. Both mtROS and ATP are produced as a result of electron transport chain activity, but it remains enigmatic whether mtROS could be generated independently from ATP synthesis. A recent study shed light on this important question and found that, during endothelial cell (EC) activation, mtROS could be upregulated in a proton leak-coupled, but ATP synthesis-uncoupled manner. As a result, EC could upregulate mtROS production for physiological EC activation without compromising mitochondrial membrane potential and ATP generation, and consequently without causing mitochondrial damage and EC death. Thus, a novel pathophysiological role of proton leak in driving mtROS production was uncovered for low grade EC activation, patrolling immunosurveillance cell trans-endothelial migration and other signaling events without compromising cellular survival. This new working model explains how mtROS could be increasingly generated independently from ATP synthesis and endothelial damage or death. Mapping the connections among mitochondrial metabolism, physiological EC activation, patrolling cell migration, and pathological inflammation is significant towards the development of novel therapies for inflammatory diseases and cancers.
G protein–sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have ...been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-2-(3,4-dimethoxyphenyl)-2-oxoethyl-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel–phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity.
Monocyte adhesion assay, a fluorescence-based method, enables the detection and quantification of monocyte adhesion to endothelial cell (EC) monolayers in vitro and measures EC activation. We ...describe in this chapter a monocyte adhesion assay based on two published papers from our laboratory that can be effectively used in studying the mechanisms of both pro- and anti-inflammatory cytokines in EC activation. Endothelial cell monolayers are cultured and treated with desired drug, cytokines, or other stimuli and incubated with fluorescently labeled monocytes.
We performed a database mining on 102 transcriptomic datasets for the expressions of 29 msup.6A-RNA methylation (epitranscriptomic) regulators (msup.6A-RMRs) in 41 diseases and cancers and made ...significant findings: (1) a few msup.6A-RMRs were upregulated; and most msup.6A-RMRs were downregulated in sepsis, acute respiratory distress syndrome, shock, and trauma; (2) half of 29 msup.6A-RMRs were downregulated in atherosclerosis; (3) inflammatory bowel disease and rheumatoid arthritis modulated msup.6A-RMRs more than lupus and psoriasis; (4) some organ failures shared eight upregulated msup.6A-RMRs; end-stage renal failure (ESRF) downregulated 85% of msup.6A-RMRs; (5) Middle-East respiratory syndrome coronavirus infections modulated msup.6A-RMRs the most among viral infections; (6) proinflammatory oxPAPC modulated msup.6A-RMRs more than DAMP stimulation including LPS and oxLDL; (7) upregulated msup.6A-RMRs were more than downregulated msup.6A-RMRs in cancer types; five types of cancers upregulated ≥10 msup.6A-RMRs; (8) proinflammatory M1 macrophages upregulated seven msup.6A-RMRs; (9) 86% of msup.6A-RMRs were differentially expressed in the six clusters of CD4sup.+Foxp3sup.+ immunosuppressive Treg, and 8 out of 12 Treg signatures regulated msup.6A-RMRs; (10) immune checkpoint receptors TIM3, TIGIT, PD-L2, and CTLA4 modulated msup.6A-RMRs, and inhibition of CD40 upregulated msup.6A-RMRs; (11) cytokines and interferons modulated msup.6A-RMRs; (12) NF-κ B and JAK/STAT pathways upregulated more than downregulated msup.6A-RMRs whereas TP53, PTEN, and APC did the opposite; (13) methionine-homocysteine-methyl cycle enzyme Mthfd1 downregulated more than upregulated msup.6A-RMRs; (14) msup.6A writer RBM15 and one msup.6A eraser FTO, H3K4 methyltransferase MLL1, and DNA methyltransferase, DNMT1, regulated msup.6A-RMRs; and (15) 40 out of 165 ROS regulators were modulated by msup.6A eraser FTO and two msup.6A writers METTL3 and WTAP. Our findings shed new light on the functions of upregulated msup.6A-RMRs in 41 diseases and cancers, nine cellular and molecular mechanisms, novel therapeutic targets for inflammatory disorders, metabolic cardiovascular diseases, autoimmune diseases, organ failures, and cancers.
We took an experimental database mining analysis to determine the expression of 28 co-signaling receptors in 32 human tissues in physiological/pathological conditions. We made the following ...significant findings:
co-signaling receptors are differentially expressed in tissues;
heart, trachea, kidney, mammary gland and muscle express co-signaling receptors that mediate CD4
T cell functions such as priming, differentiation, effector, and memory;
urinary tumor, germ cell tumor, leukemia and chondrosarcoma express high levels of co-signaling receptors for T cell activation;
expression of inflammasome components are correlated with the expression of co-signaling receptors;
CD40, SLAM, CD80 are differentially expressed in leukocytes from patients with trauma, bacterial infections, polarized macrophages and in activated endothelial cells;
forward and reverse signaling of 50% co-inhibition receptors are upregulated in endothelial cells during inflammation; and
STAT1 deficiency in T cells upregulates MHC class II and co-stimulation receptors. Our results have provided novel insights into co-signaling receptors as physiological regulators and potentiate identification of new therapeutic targets for the treatment of sterile inflammatory disorders.
Depending on its expression level, RUNX1 can act as a tumor promoter or suppressor in hematological malignancies. The clinical impact of RUNX1 expression in cytogenetically normal acute myeloid ...leukemia (CN-AML) remained unknown, however. We evaluated the prognostic significance of RUNX1 expression using several public microarray datasets. In the testing group (n = 157), high RUNX1 expression (RUNX1high) was associated with poorer overall survival (OS; P = 0.0025) and event-free survival (EFS; P = 0.0025) than low RUNX1 expression (RUNX1low). In addition, the prognostic significance of RUNX1 was confirmed using European Leukemia Net (ELN) genetic categories and multivariable analysis, which was further validated using a second independent CN-AML cohort (n = 162, OS; P = 0.03953). To better understand the mechanisms of RUNX1, we investigated genome-wide gene/microRNAs expression signatures and cell signaling pathways associated with RUNX1 expression status. Several known oncogenes/oncogenic microRNAs and cell signaling pathways were all up-regulated, while some anti-oncogenes and molecules of immune activation were down-regulated in RUNX1high CN-AML patients. These findings suggest RUNX1high is a prognostic biomarker of unfavorable outcome in CN-AML, which is supported by the distinctive gene/microRNA signatures and cell signaling pathways.
Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy ...and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects.
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