Molecular Pharmacology最新文献

Membrane-bound adenylyl cyclases (ACs) function as vital enzymes that convert external signals into intracellular responses through the second messenger cAMP. Traditionally viewed as monomers, ACs are now recognized to form oligomers, introducing new regulatory mechanisms. This minireview synthesizes emerging structural and biochemical evidence for AC oligomerization and explores its functional significance. Oligomerization plays a critical role in the localization of ACs by regulating retention in the endoplasmic reticulum, membrane targeting, and distribution within signaling microdomains. Functional data suggest that dimer/oligomer interfaces act as regulatory nodes, although isoform-specific differences in oligomer architecture and functional consequences remain poorly understood. Defining the mechanisms underlying these differences is a critical area for future investigation. Importantly, the structural variability of oligomer interfaces, relative to conserved catalytic domains, offers new therapeutic potential that may enable isoform-selective modulation of AC activity. By integrating past and current research, this review frames oligomerization as a fundamental, yet underexplored, determinant of cAMP signaling. Advancing our understanding of the assembly, regulation, and dynamics of AC oligomers may open new avenues for precise control of cAMP signaling in both physiological and pathological contexts. SIGNIFICANCE STATEMENT: Oligomerization of membrane adenylyl cyclases adds regulatory complexity to cAMP signaling by modulating catalytic activity, localization, and compartmentalization. Distinct homo- and hetero-oligomers may underlie isoform-specific functions and offer new therapeutic opportunities.

The class III antiarrhythmic drug amiodarone (AMIO) inhibits the rapidly activating delayed rectifier K⁺ current that is conducted by the human ether-a-go-go-related gene (hERG) encoded channel. Like other class III antiarrhythmic drugs, AMIO can cause long QT syndrome. In the present study, we investigated the effects of AMIO and its major metabolite, desethylamiodarone, on hERG channels expressed in human embryonic kidney (HEK)293 (hERG-HEK) as well as in cardiomyocyte-derived H9c2 cells. Our results show that after acute inhibition of hERG current (I_hERG) by AMIO (IC₅₀ of 0.2 μM) or desethylamiodarone (IC₅₀ of 0.5 μM), continuous washout of the drug for 20 to 25 minutes during whole-cell patch clamp recordings did not lead to any current recovery. Furthermore, when hERG-HEK cells were cultured with AMIO overnight, and I_hERG was recorded in a drug-free bath solution, AMIO treatment resulted in a concentration-dependent inhibition of I_hERG with an IC₅₀ of 0.3 μM. In contrast, such overnight treatments did not affect the expression of hERG channels shown by Western blot analyses. However, the mature hERG protein of AMIO-pretreated cells cultured in a drug-free medium degraded faster than that of control cells, indicating that AMIO treatment modified the property of mature hERG channels, making them permanently nonconductive and less stable. Consistently, our results showed that following AMIO-mediated inhibition, recovery of I_hERG during cell culture in drug-free conditions resulted from newly made channels, and a full recovery took up to 20 hours. Thus, AMIO-mediated hERG inhibition may persist for tens of hours after drug discontinuation, which has clinical importance. SIGNIFICANCE STATEMENT: Amiodarone (AMIO) is a frequently used antiarrhythmic drug that blocks human ether-a-go-go-related gene (hERG) potassium channels. The present study revealed that, unlike other hERG-interacting drugs, AMIO irrecoverably inhibits hERG channel currents. Upon removal of AMIO after hERG inhibition, recovery of hERG currents relies on newly made channels in a process of up to 20 hours. Thus, lingering effects on hERG channels after AMIO discontinuation are anticipated, which have important clinical implications.

Cerebrovascular inflammation is prevalent in a majority of patients with Alzheimer disease. Elevated levels of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), circulating in the plasma have been shown to cause the inflammation of blood-brain barrier (BBB) endothelium lining the cerebral microvasculature in Alzheimer disease. The BBB inflammation triggered by exposure of TNF-α in the peripheral circulation can aggravate the accumulation of Aβ peptides in Alzheimer disease brain. In the current study, we have shown that infusion of wild-type mice with TNF-α led to an increase in permeability and influx of Aβ42 into the mice brain using dynamic single-photon emission computed tomography/computed tomography imaging. To corroborate these findings, we demonstrated that TNF-α increases Aβ42 accumulation in vitro in human cerebral microvascular endothelial cells /D3 and primary porcine brain endothelial cells. In addition, our results in human cerebral microvascular endothelial cells/D3 polarized monolayers show that TNF-α alters the expression of cofilin, actin, and dynamin, which are critical components for Aβ endocytosis by BBB endothelial cells. These results suggest a mechanistic pathway by which TNF-α may promote Aβ accumulation at the BBB and the underlying interactions between inflammation and Aβ exposure that drives BBB dysfunction. Hence, a therapeutic intervention aimed at addressing elevated TNF-α levels in Alzheimer disease may potentially reduce Aβ-related cerebrovascular dysfunction in Alzheimer disease brain. SIGNIFICANCE STATEMENT: Elevated plasma tumor necrosis factor-α drives Aβ pathology in Alzheimer disease, promoting cerebrovascular inflammation, but its role in endothelial Aβ uptake in the brain is unclear. This study shows tumor necrosis factor-α increases Aβ42 accumulation in the blood-brain barrier endothelium by altering the expression of blood-brain barrier endocytic proteins cofilin, actin, and dynamin.

The universal posttranslational modification ubiquitination was originally discovered in the 1980s as a tag appended to cellular proteins to mark them for degradation by the 26S proteasomal complex. Subsequent discoveries have established ubiquitination as a process that directs spatial and temporal activities of proteins that are integral to all biochemical pathways in cells, in addition to its canonical role in facilitating life-death decisions of proteins. Protein ubiquitination is counterbalanced by ∼100 enzymes called deubiquitinases (DUBs) expressed in human cells. Although ubiquitination affects a plethora of cellular functions, the role of DUBs was initially linked to neuronal development, and later to cancer, where deregulation of protein degradation coincided with both increased expression of DUBs and worsening of disease pathology. As such, inhibition of DUBs has been regarded as a therapeutic approach for various cancers, and major investments of time and resources have been dedicated for developing DUB inhibitors for cancer therapy. The important roles of DUBs in the cardiovascular system have only recently been appreciated. DUBs play a protective anti-inflammatory role in the vascular smooth muscle, endothelium, as well as macrophages, and also serve to mitigate pathological remodeling of the myocardium in mice. Activation or positive allosteric modulation of DUBs could provide therapeutic benefit in the 2 major cardiovascular diseases, atherosclerosis and heart failure. In this review, we highlight the roles of select DUBs that have been characterized for their critical functions in the heart and vasculature. SIGNIFICANCE STATEMENT: Cardiovascular diseases represent a major global health burden and remain the leading cause of morbidity and mortality, accounting for approximately 18 million deaths annually worldwide. Among these, atherosclerosis and heart failure are 2 interrelated conditions with multifactorial, chronic, and complex etiologies, necessitating the development of more effective therapeutic strategies to reduce mortality and improve quality of life. Emerging evidence suggests that several deubiquitinases exert protective effects by attenuating atherosclerosis and cardiac dysfunction in murine models, highlighting the potential of deubiquitinase modulators to serve as novel therapeutic agents for cardiovascular diseases.

The opioid crisis showcases a need for novel therapeutic avenues to treat drug abuse. GPR83, a recently deorphanized G protein-coupled receptor shown to blunt morphine reward learning and regulate pain, is activated by the neuropeptide PEN and peptides derived from cholecystokinin. To identify small molecule ligands, we generated a homology model based on the crystal structure of related receptors and used virtual screening on a library of 7 million compounds. The top 50 hits were screened in a cell-based assay that identified 2 selective GPR83 agonists, CPD1 and CPD27, and 1 antagonist, CPD25. The model was validated by site-directed mutagenesis of GPR83 residues predicted to interact with these ligands; these mutations disrupted ligand binding to GPR83. The molecular pharmacological properties of these compounds were characterized, and their GPR83 specificity validated using knockdown cells generated using GPR83 short hair pin RNA. Peripheral antagonist (CPD25) administration to wild-type and GPR83 knockout mice blocked morphine conditioned place preference only in the wild-type mice supporting that CPD25-mediated blockade is through GPR83 antagonism. Interestingly, morphine antinociception was blunted by the GPR83 agonist (CPD1) and enhanced by CPD25 with a medium to large effect size estimate, demonstrating a role for GPR83 in regulating morphine analgesia. Taken together, we identified and validated small molecule modulators of GPR83 that could be used to probe its role in neuropsychiatric disorders. Our in vivo studies highlight GPR83 as a target that could be used to limit the addictive effects of opioids in the treatment of pain. SIGNIFICANCE STATEMENT: There is a need to identify targets that limit opioid abuse potential while maintaining the pain-relieving effects. This study identifies small molecule GPR83 ligands that block opioid reward learning while enhancing their pain-relieving effect.

2,5-dimethoxy-4-iodoamphetamine (DOI) is a phenethylamine psychedelic with high affinity for 5-HT₂ receptors. In 2022 and 2023, the US Drug Enforcement Administration proposed to place DOI, along with a similar compound, 2,5-dimethoxy-4-chloroamphetamine, in Schedule I of the Controlled Substances Act based on their psychoactivity and alleged abuse potential. Here, we describe the history of DOI, its utility in preclinical neuroscience research, and how it has significantly advanced the study of 5-HT_2A and 5-HT_2C receptors. Finally, we suggest alternative compounds for studying 5-HT₂ receptors, should obtaining DOI for research become restricted. SIGNIFICANCE STATEMENT: 2,5-Dimethoxy-4-iodoamphetamine, the key pharmacological tool for studying 5-HT_2A receptor function and localization, has been used in more 1200 publications across 5 decades. This review covers its utility, research barriers if the Drug Enforcement Administration schedules it, and alternatives for continued investigation of serotonin receptors.

G protein-coupled receptor kinases (GRKs) are a class of serine/threonine kinases that shut down active signaling mediated by agonist-bound G protein-coupled receptors. Of all the diseases that arise from dysfunctional G protein-coupled receptor-GRK interactions, this review will focus on the roles of the 2 most highly expressed GRKs in heart failure (HF)-GRK2 and GRK5. Both are upregulated in human and mouse HF heart samples. Because both GRK2 and GRK5 are expressed in all cardiac cell types-cardiac fibroblasts, endothelial cells, vascular smooth muscle cells, and cardiomyocytes-it is essential to examine their role in these individual cell types for identifying specific cardiomyopathies and targeting them accordingly. Seminal work from our laboratory over the last 3 decades has uncovered multiple crucial aspects of GRK2- and GRK5-mediated interactions that lead to HF. Based on that, several GRK2 and GRK5 inhibitors have been identified/generated that have high potency and have been tested in multiple animal models of HF. One of the GRK2 inhibitors, paroxetine, has also been evaluated in 2 clinical trials. Similarly, potent GRK5 inhibitors have also been recently generated, and it remains to be seen how they affect cardiac structure and function in vivo. Lastly, assessing cell-specific fine differences in GRK2 and GRK5 inhibition will pave the way for identifying ideal patient cohorts for clinical trials in which selective GRK2 and GRK5 inhibitors can be evaluated as a new class of drugs for HF. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases 2 and 5 play a central role in heart failure (HF) onset and progression. They have critical significance in all cardiac cells, which contribute to the pathophysiology of HF, namely, cardiomyocytes, cardiac fibroblasts, endothelial cells, and vascular smooth muscle cells. Dysfunction in their canonical G protein-coupled receptor-related function or noncanonical function must be sufficiently investigated and addressed to evaluate their inhibitors as a new class of drugs for HF.

Aldehyde oxidase (AOX1) is a cytosolic molybdo-flavoenzyme that metabolizes azaheterocyclic drugs. Erlotinib and gefitinib are azaheterocyclic drugs. We deployed structural analogs to investigate the molecular interaction between these drugs and AOX1. Erlotinib, O-desmethylerlotinib, and O-didesmethylerlotinib, but not gefitinib, O-desmethylgefitinib, or O-desmorpholinopropylgefitinib, decreased carbazeran 4-oxidation by liver cytosol (human, rat, and mouse) and human recombinant AOX1. Erlotinib, O-desmethylerlotinib, and O-didesmethylerlotinib exhibited time- and concentration-dependent inactivation with unbound inactivation potency (K_I,u) of 1.52, 4.41, and 1.67 μM, respectively. The inactivation was not reversed after dialysis, not protected by nucleophilic trapping agents or scavengers of reactive oxygen species, not affected by an oxidizing or reducing agent, but was attenuated by an alternative AOX1 substrate (O⁶-benzylguanine) and competitive AOX1 inhibitor (gefitinib). The terminal alkyne group of erlotinib was essential for AOX1 inactivation, as suggested by the findings for 3-vinylerlotinib (less potent inactivator) and tetrahydroerlotinib (no inactivation). Molecular docking results predicted covalent binding of erlotinib, O-desmethylerlotinib, and O-didesmethylerlotinib to the molybdenum cofactor. Adding a 4'-methyl group to erlotinib increased the inactivation potency but decreased inactivation efficiency, whereas blocking the C₂-position of erlotinib with a hydroxy group or a methyl group decreased inactivation potency and efficiency, suggesting that the C₂-position of erlotinib plays a role in AOX1 inactivation. In mice, erlotinib increased carbazeran (Aox substrate) and decreased 4-oxo-carbazeran (metabolite) levels in blood, liver, and kidneys. Overall, our study provides molecular insights into the mechanism-based inactivation of AOX1 by erlotinib, O-desmethylerlotinib, and O-didesmethylerlotinib and the irreversible AOX1 inactivation by erlotinib on the pharmacokinetics of AOX1-metabolized drugs. SIGNIFICANCE STATEMENT: This study shows that erlotinib and select metabolites are mechanism-based inactivators of AOX1, provides insights into the mechanism of the inactivation by deploying structural analogs and molecular docking, and demonstrates the in vivo impact on AOX1-mediated drug metabolism.

Transient receptor potential vanilloid 3 (TRPV3) is a thermosensitive Ca²⁺-permeable ion channel that plays essential roles in epithelial barrier function. Although its expression and function have been well characterized in the skin and, to a lesser extent, in the gastrointestinal tract, its role in the urinary bladder has remained unexplored. In this study, TRPV3 was identified in human bladder cancer cell lines, and its functional activation was demonstrated, using a novel small-molecule agonist activator of TRPV3 channel 1 (AV3-1), discovered through medium-throughput screening. AV3-1 activated mouse and human TRPV3 channels with higher potency than known TRPV3 activators in Ca²⁺ assays and electrophysiological recordings. TRPV3 activation in the KU-19-19 bladder cancer cells stimulated ATP release, which was abolished by pharmacological TRPV3 blockade, confirming target specificity. Cholesterol supplementation further enhanced TRPV3 activity in KU-19-19 cells, a finding of potential relevance given the known dysregulation of cholesterol metabolism in bladder cancer. These results provide the first evidence of functional TRPV3 expression in bladder cancer cells and suggest that TRPV3 may contribute to Ca²⁺- and cholesterol-dependent signaling pathways. Collectively, these findings support further investigation of TRPV3 as a potential pharmacological target and exploratory biomarker in urothelial carcinoma. SIGNIFICANCE STATEMENT: TRPV3 is an ion channel mainly found in the skin. This study has identified the small molecule AV3-1 as a novel TRPV3 activator. Using AV3-1, this study demonstrates TRPV3 expression in bladder cancer cells. TRPV3 activation in these cells triggers ATP release, a signal potentially promoting cancer progression.

Recent advancements in the study of orphan G protein-coupled receptors (oGPCRs) have revealed a large number with high levels of constitutive G protein signaling. Structural studies have suggested a new paradigm in which many constitutively active oGPCRs are auto-activated by their own intrinsic protein motifs, which act as auto-agonists. This includes extracellular loop 2 acting as auto-agonist to promote active-state conformations and G protein signaling. In some cases, the oGPCRs lack inhibitory microswitches that may drive a high level of constitutive activity. In this brief review, we discuss oGPCR constitutive activity, highlighting the auto-activating orphan receptors and overview structural underpinnings of constitutive activity. A discussion into the pharmacological and cell signaling implications of oGPCR constitutive activity is provided. We also propose a new concept in which orphan GPCR constitutive activity sets the baseline tone for cellular signaling and allows for dynamic regulation of cAMP signaling. Taken together, recent mechanistic studies with many oGPCRs indicate high constitutive activity is a common phenomenon that modulates cellular signaling and that can be tuned with pharmacology. SIGNIFICANCE STATEMENT: Recent literature describes a subset of orphan Class A G protein-coupled receptors with high constitutive signaling that auto-activate by their own intrinsic protein motifs. Herein, a new concept is proposed in which oGPCR constitutive activity allows dynamic regulation of cAMP signaling. Recently solved structures and functional studies of constitutively active oGPCRs provide fresh insights into oGPCR signaling with relevance for both health and disease.