Activation of class A G protein-coupled receptors (GPCRs) is commonly described through rearrangement of the transmembrane-bundle and engagement of the Gα α5 helix, whereas the conformational behavior of intracellular loop 2 (ICL2) has not been examined systematically across the structural record. Here, I classified ICL2 as folded, unfolded, or disordered in 1358 experimental class A GPCR structures using a backbone-dihedral criterion and evaluated the classification against activation state, transducer class, crystallization fusions, ICL2-proximal modulators, and resolution. The assignment was additionally assessed using Dictionary of Secondary-Structure of Proteins and a literature-anchored benchmark set. In canonical fusion-free structures, ICL2 was folded in 84% of active structures compared with 53% of inactive structures [odds ratio, 4.74; two-sided Fisher exact test; descriptive P = 7 × 10[-13]]. The association remained evident after controlling for receptor overrepresentation, within receptors represented in both states, and among active structures without an annotated transducer. Crystallization fusions were associated with increased apparent ICL2 folding in inactive structures. Folded and unfolded ICL2 also showed distinct Gα contact patterns, while sequence features displayed only modest associations with folding. A geometrically compatible contact between ICL2 position 34.53 and the conserved D3.49 residue accompanied the folded state but was not universal. These exploratory structural analyses identify ICL2 folding as an activation-associated feature of class A GPCRs without establishing a causal mechanism or a determinant of transducer selectivity. SIGNIFICANCE STATEMENT: This structure-wide analysis shows that intracellular loop 2 (ICL2) folding is strongly enriched in active compared with inactive class A GPCR structures, identifying ICL2 as an intracellular structural readout of receptor activation. The association is retained across major structural controls, while transducer analyses show that ICL2 folding is most prevalent in Gs-bound complexes but does not by itself define coupling specificity.

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that mediate the slow component of excitatory neurotransmission. Modulators of N-methyl-D-aspartate receptor function have long been evaluated as pharmacotherapies to treat neurological disorders such as Parkinson's disease, Alzheimer's disease, schizophrenia, stroke, and other neuropsychiatric and neurodegenerative disorders. We describe the mechanisms of action and explore the structural determinants underlying a phenomenon where GluN2B-specific negative allosteric modulators from the 93- and 96-series can be inverted to act as positive allosteric modulators by mutations at 2 GluN1 residues in their binding pocket located within the amino-terminal domain. Mutations at GluN1-Y109 that invert the activity could each conceivably strengthen the interaction between the residue and 93-31, resulting in a switch from enhancement to relief of tonic inhibition of the receptor by protons. The demonstration that this binding site is capable of being tuned for either inhibition or potentiation provides an opportunity to design novel positive allosteric modulators for potential therapeutic development in humans. SIGNIFICANCE STATEMENT: Mutations of 2 residues in the N-methyl-D-aspartate receptor GluN1 subunit amino-terminal domain independently interconvert GluN2B-selective negative allosteric modulators into positive allosteric modulators. This work provides a path for the discovery of the structural requirements for GluN2B-specific positive allosteric modulation.

The TREK subfamily of 2-pore domain potassium channels act as polymodal sensors of multiple physical and chemical stimuli, coupling electrical excitability to the cellular environment. A nuanced understanding of TREK channel pharmacology is necessary for the development of novel experimental tools, for potential pharmacotherapies, and for understanding potential beneficial or adverse effects of clinically used drugs. Here, we sought to determine the mechanism of action of 2 TREK channel inhibitors: the β-blocker carvedilol and the antipsychotic fluphenazine. Using patch and 2-electrode voltage-clamp electrophysiology, we show that both carvedilol and fluphenazine exhibit an activation-mode dependence, with mechanical- and temperature-activation of TREK channels reducing drug sensitivity approximately 10-fold. Mutagenesis at a key intramembrane fenestration site leucine, modulator competition experiments, and in silico docking studies are all consistent with carvedilol and fluphenazine binding at a shared, overlapping binding site. These findings establish a conserved conformation-dependent mechanism of action for structurally diverse TREK inhibitors and identify the fenestration as a promiscuous binding site for a range of clinically used drugs. SIGNIFICANCE STATEMENT: TREK potassium channels play key roles in regulating membrane excitability in a wide range of cells. Modulation of these channels has been proposed for potential antiarrhythmic and antidepressant effects. This study defined the off-target molecular mechanism of action of the clinically used drugs carvedilol and fluphenazine on TREK channels and identify the TREK channel fenestration as a promiscuous binding site for inhibitory drugs.

The simple 2-state conformational selection model of G protein-coupled receptor activation suggests that, by binding to a high-affinity state, an agonist shifts receptor equilibrium in favor of active state (R∗) conformations that recruit heterotrimeric G proteins over inactive (R) conformations. Agonist binding affinity at the μ-opioid receptor is reduced in the presence of Na[+] ions, which stabilize R conformations. The binding of higher efficacy opioid agonists, such as [D-Ala[2], NMe-Phe[4], Gly-ol[5]]-enkephalin and fentanyl, is more sensitive to Na[+] ions than lower efficacy ligands. However, the binding of the highly potent agonists etorphine and dihydroetorphine is less sensitive to Na[+] ions than expected, such that the prevailing models fail to explain their pharmacology. To understand this discrepancy, experiments were performed to evaluate the binding properties and G protein activation of several high-affinity, high-potency agonists, including carfentanil, etonitazene, and several peptidomimetics in comparison to [D-Ala[2], NMe-Phe[4], Gly-ol[5]]-enkephalin, Met-enkephalin, fentanyl, and morphine in the presence of Na[+] or K[+] ions. Several very potent agonists retained high-affinity and potency in both ionic conditions, whereas the standard agonists displayed enhanced binding and signaling only in the presence of K[+] ions. Comparison of the difference in binding affinity with degree of agonism afforded a negative correlation in which several ligands with a very high degree of agonism were least sensitive to Na[+] ions. These data suggest that select highly potent μ-opioid receptor agonists have high-affinity for Na[+] bound receptor states and shift the receptors into active conformations that efficiently couple to G protein. SIGNIFICANCE STATEMENT: The theory of conformational selection suggests that agonists bind to active states (R∗) of G protein-coupled receptors, including the μ-opioid receptor, to shift the receptor equilibrium in favor of R∗ over inactive states (R). This study demonstrates that ligands with very high-affinity and potency at the μ-opioid receptor can engage low-affinity Na[+]-bound receptor states (R) and convert these to active R∗ states that efficiently couple to G protein, suggesting a conformational induction model.

The airway epithelium must constantly adapt to maintain function, as disrupted epithelial homeostasis contributes to asthma and other respiratory diseases. Toxic electrophilic aldehydes, quinones, and substituted phenols are transient receptor potential ankyrin-1 (TRPA1) channel agonists found in common air pollutants, including wood and biomass smoke, cigarette smoke, and vehicle exhaust. TRPA1 agonists damage airway epithelial cells in part by disrupting calcium homeostasis and triggering endoplasmic reticulum stress. A detailed understanding of how inhaled toxicants affect epithelial homeostasis is critical for understanding their effects and managing adverse effects. We integrated results from kinase-targeted phenotypic screening, unbiased mass spectrometry-based global proteomics and phosphoproteomics, live-cell imaging, calcium flux assays, gene expression assays, and Western blot analyses to decipher systematic changes in kinase signaling that control TRPA1 agonist-induced injury. We show that injury coincides with changes in cell structure and motility overlaid with epidermal growth factor receptor (EGFR) transactivation, and that inhibiting kinase pathways known to favor survival and wound repair, including EGFR, extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/protein kinase B, and c-Jun N-terminal kinases, exacerbated injury, while inhibiting protein kinase C and p38 mitogen-activated kinase, and others, reduced injury. We attributed the protective effects of protein kinase C and p38 mitogen-activated kinase inhibition to an attenuation of TRPA1 activity, balanced by enhanced transient receptor potential vanilloid-3 expression, resulting in a net shift toward proadaptive kinase-driven signaling downstream of EGFR. Our results support a model in which repair depends on kinase-regulated transitions through a transient injury state that could potentially be targeted prophylactically to reduce injury caused by selected inhaled toxicants. SIGNIFICANCE STATEMENT: This study shows that inhibiting protein kinase C and p38 mitogen-activated kinase reduces transient receptor potential ankyrin-1 activity and enhances transient receptor potential vanilloid-3 expression and prosurvival kinase signaling, favoring cell adaptation and repair following acute injury, offering insight into potential therapeutic approaches to limit transient receptor potential ankyrin-1 agonist-induced injury through a vital network of transient receptor potential channels.

Precision pharmacology has emerged from advances in cell biology, microscopy, and signal transduction, revealing that intracellular communication is highly organized within spatially restricted compartments. Hence, therapeutic approaches that manage disease-associated signaling steps within defined subcellular microdomains are attractive precision pharmacology targets. A-kinase anchoring proteins coordinate this remarkable level of spatial organization by clustering protein kinase A with phosphatases and cAMP effector proteins. These signaling islands direct second messenger responses toward selected substrates while restricting inappropriate crosstalk. Perturbation of A-kinase anchoring protein-directed signaling contributes to cardiovascular disease, endocrine dysfunction, cancer progression, chronic pain, and neurodegeneration. Agents that selectively modulate or dismantle pathological signaling complexes may offer a less invasive means to therapeutically exploit the molecular geography of diseased cells, particularly if used in combination with molecular glues, chimera-targeted protein degradation targeted protein degradation, and selective kinase inhibitors. This article is a foreword for a series of 4 minireviews commissioned by the journal from speakers at the 2025 ASPET Axelrod Symposium on spatial cAMP signaling. These contributions examine how pathological perturbations in A-kinase anchoring protein-mediated signaling networks contribute to a diverse spectrum of disease processes, including maladaptive cardiac remodeling, endocrine hyperfunction, nociceptor sensitization, and synaptic failure in AD. SIGNIFICANCE STATEMENT: Intracellular signaling is a fundamental process that governs cellular physiology and disease pathogenesis. This miniseries highlights how A-kinase anchoring proteins assemble signaling nanodomains to locally control second messenger-responsive enzymes in the cardiovascular, endocrine, and nervous systems. A-kinase anchoring protein macromolecular assemblies are emerging venues for precision pharmacology.

Sacituzumab govitecan (SG), an antibody-drug conjugate approved for the treatment of triple-negative breast cancer and hormone receptor-positive breast cancer, is currently undergoing clinical trials in multiple forms of cancer. However, there is currently no preclinical information relating to whether senescence is one component of the response to this antibody-drug conjugate in tumor cells. Our studies provide evidence for senescence in response to SG (β-galactosidase staining with quantification by flow cytometry, upregulation of p21 and downregulation of Lamin B1, and expression of interleukins IL-6, IL-8, and IL-1β) in 9 tumor cell lines derived from breast, prostate, lung, ovarian, and colorectal cancer. Senescence induction was associated with a transient growth arrest followed by proliferative recovery. Similar results were generated using SN-38, the toxic payload that is released from SG. We conclude that one central component of the tumor cell response to SG is a transient senescence-associated growth arrest that can generate a pool of surviving tumor cells with the potential to contribute to disease recurrence. SIGNIFICANCE STATEMENT: Antibody-drug conjugates (ADCs) are among the newest anticancer therapies. Current studies demonstrate, for the first time, that one component of the response to an ADC, sacituzumab govitecan, is the promotion of senescence. This raises the possibility that the inclusion of senolytics or senomorphics in combination with ADCs could improve their clinical effectiveness and interfere with disease recurrence.

Many structurally and therapeutically diverse drugs block the human ether-à-go-go-related gene (HERG) potassium channel, predisposing patients to an increased risk of arrhythmias and sudden cardiac death. Many of these drugs show state dependence of block, exhibiting a greater preference for block of the inactivated state. Four key residues, ie, T623, S624, Y652, and F656, within the central pore cavity of HERG have been implicated in drug binding. However, whether drugs bind differently to these key residues to dictate preference for binding to the inactivated over the open state is not known. We used the SyncroPatch 384PE, automated patch clamp platform to measure how drug block was impacted by point mutations at these 4 residues when introduced into wild-type (WT) HERG (preferentially occupies the inactivated state) and N588K-HERG (preferentially occupies the open state) channels at depolarized potentials. Mutations to Y652 and F656, which abolished aromatic and hydrophobic characteristics, respectively, reduced drug binding in both WT and N588K backgrounds. The S624A mutation attenuated block by cisapride, astemizole, and quinidine in the WT background but not in the N588K background. We suggest that relative movements between the polar S624 sidechain and the aromatic Y652 sidechains in WT (inactivated) compared with N588K (open) channels can explain preferential binding to the inactivated state. An improved understanding of the structural basis of where and how drugs bind to HERG channels should facilitate efforts to reduce inadvertent HERG drug block during the drug development process. SIGNIFICANCE STATEMENT: A diverse range of drugs block human ether-à-go-go-related gene (HERG) potassium channels, which can have serious side effects, including sudden death. Most problematic drugs bind to the inactivated state of HERG channels. This study shows that the polar side chain of Ser624, at the base of the selectivity filter, is critical for preferential drug block of inactivated compared with open channels. A better understanding of state-dependent drug binding should facilitate efforts to reduce inadvertent HERG drug block.

Heart failure is a major cause of morbidity and mortality worldwide, representing the end stage of cardiovascular diseases that induce pathological cardiac remodeling. At the cellular level, pathological remodeling includes myocyte hypertrophy and decreased survival, changes in myocyte metabolism and contractility, and interstitial myocardial fibrosis, all of which are controlled by a complex network of intracellular signaling pathways. At the outer nuclear envelope of the cardiomyocyte, A-kinase anchoring protein (AKAP) 6β (also known as mAKAPβ) organizes multimolecular signaling complexes called "signalosomes," which include over 25 different signaling enzymes and effector proteins that, in response to cAMP, calcium, and phosphoinositide second messengers, regulate gene expression. The AKAP6β scaffold also binds other scaffold proteins such as AKAP9, permitting higher-order interactions between signaling molecules at the nuclear envelope and adjacent Golgi apparatus. Together, these protein complexes present novel opportunities for compartmentalized inhibition of pathophysiological cardiac remodeling. Both basic molecular mechanisms and potential therapeutic approaches are considered, because this review highlights recent advances in the role of AKAP6 in coordinating compartmentalized signaling and the regulation of cardiomyocyte gene expression promoting heart failure. SIGNIFICANCE STATEMENT: Heart failure is a syndrome of major public health significance. Novel therapies for the treatment of cardiac disease are needed to improve patient quality of life, reduce symptoms, and manage comorbidities associated with heart disease. The A-kinase anchoring protein 6β signalosome has been shown to orchestrate multiple pathological signaling pathways in the cardiomyocyte that induce cardiac disease. Targeting this signalosome should comprise more specific therapeutics for heart failure with fewer side effects than those associated with current treatments.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease with limited therapeutic options, highlighting the need for new molecular targets and effective antifibrotic agents. In this study, we investigated the role of B-cell lymphoma 9 (BCL9) in IPF and evaluated the antifibrotic potential of ZD-4009, a novel small-molecule inhibitor targeting BCL9. BCL9 was upregulated in fibrotic lung tissues, suggesting a possible association between BCL9 and pulmonary fibrosis progression. ZD-4009 showed high-affinity binding to β-catenin (pKD = 7.36 ± 0.01, KD = 44.72 nM). In vitro, ZD-4009 inhibited fibroblast activation, suppressed extracellular matrix deposition, and reduced the expression of fibrosis-associated markers, including collagen type I alpha 1 chain (COL1A1) and alpha-smooth muscle actin (α-SMA). Mechanistically, ZD-4009 suppressed hypoxia-inducible factor 1α-associated glycolytic reprogramming and inhibited transforming growth factor-β (TGF-β)/SMAD family member (SMAD) signaling, suggesting that its antifibrotic effects are associated with coordinated regulation of metabolic and profibrotic pathways. In vivo, ZD-4009 treatment was associated with alleviated bleomycin (BLM)-induced pulmonary fibrosis, as reflected by improved survival from 55% in the BLM group to 82% in the BLM + ZD-4009 group, reduced relative collagen content from 61% to 48%, and ameliorated micro-computed tomography-assessed fibrotic changes. In the tested model, ZD-4009 showed favorable antifibrotic effects compared with nintedanib, whereas liposome-encapsulated ZD-4009 reduced treatment-related toxicity. Together, these findings nominate ZD-4009 as a promising antifibrotic candidate and highlight BCL9 as a potential therapeutic target for IPF. SIGNIFICANCE STATEMENT: ZD-4009 is a potent and selective B-cell lymphoma 9 inhibitor that suppresses fibroblast proliferation and extracellular matrix deposition in idiopathic pulmonary fibrosis. It shows superior antifibrotic efficacy compared with current therapies, positioning ZD-4009 as a promising therapeutic candidate for treating this progressive and untreatable disease.

The mechanosensitive Piezo1 channel is crucial for the regulation of calcium (Ca[2+]) signaling within myeloid tissue macrophages. However, the current lack of comprehensive pharmacological characterization for existing Piezo1 agonists complicates the assessment of Piezo1 agonism as a viable therapeutic strategy. We report inhibition of the voltage-gated Kv1.3 potassium (K[+]) channel as a critical off-target effect of the most widely used Piezo1 agonists, Yoda1 and Yoda2. Kv1.3 is essential for the activation and innate immune functions of tissue macrophages. Patch-clamp electrophysiological experiments demonstrate that Yoda1 (IC50 = 9.0 μM) and Yoda2 (IC50 = 5.9 μM) state-independently inhibit Kv1.3 by accelerating channel inactivation. Functionally, blockade of Kv1.3 by Yoda2 in Piezo1-deficient bone marrow-derived macrophages depolarizes membrane potential and reduces store-operated calcium entry and interleukin-1β release. Using RosettaLigand docking and molecular dynamics simulations, we identified a network of key residues near the Kv1.3 outer pore as the potential binding site. Our results suggest that Kv1.3 blockade likely contributes substantially to the reported anti-inflammatory effects of Yoda1 and Yoda2. This provides a plausible mechanism that aligns with the established principle that reducing Ca[2+] signaling is anti-inflammatory, while helping to reconcile existing literature discrepancies regarding Piezo1 activation. SIGNIFICANCE STATEMENT: Targeting Ca[2+] channels such as Piezo1 is vital for controlling chronic inflammation, but selectivity is challenging. This study reveals that the best-known Piezo1 agonists exhibit a critical off-target effect by blocking the Kv1.3 channel, which is essential for macrophage and microglial immune function. This concurrent inhibition reduces inflammatory Ca[2+] signaling and interleukin-1β secretion. This work underscores the necessity for rigorous selectivity screening of Piezo1 drugs and establishes Kv1.3 inactivation modification as a viable strategy for designing novel anti-inflammatory therapeutics.

G protein-coupled receptor 3 (GPR3) is a constitutively active G protein-coupled receptor known to regulate β-amyloid deposition in the brain and metabolism in brown adipose tissue. Although multiple compounds have been proposed to activate GPR3 signaling, it is still considered an orphan receptor. Recently, structural evidence revealed a hydrophobic tunnel in GPR3, suggesting that a lipid may be an endogenous agonist for GPR3. We sought to characterize the reported agonists for GPR3 to determine which might be authentic agonists and found that only the synthetic ligand, diphenyleneiodonium chloride, and the endogenous lipids, oleoylethanolamide (OEA) and oleamide, stimulated cAMP generation in a GPR3-dependent manner. Although treatment of cells with diphenyleneiodonium chloride stimulated rapid cAMP generation, we observed gradual increases in GPR3 activation over multiple hours after OEA treatment. Additionally, we found that chronic stimulation with OEA caused an increase in GPR3 cell surface expression. Coupled with the slow activation kinetics of OEA-stimulated GPR3 signaling, these findings suggest that OEA does not behave strictly as a classical agonist for GPR3. Indeed, we found that the OEA-mediated increase in GPR3 on the cell surface can be blocked by inhibiting receptor forward trafficking through the endoplasmic reticulum and Golgi, which positions OEA as a pharmacological chaperone that increases GPR3 signaling by promoting receptor trafficking to the cell surface. These findings suggest a novel mechanism for the regulation of the constitutively active receptor GPR3, whereby the amount of receptor at the plasma membrane, and therefore, the receptor's signaling activity, can be regulated by lipid binding in the endoplasmic reticulum/Golgi. SIGNIFICANCE STATEMENT: This study proposes a novel mechanism for the regulation of the activity of the G protein-coupled receptor, G protein-coupled receptor 3. This study found that the endogenous lipid oleoylethanolamide functions as a pharmacological chaperone by promoting the accumulation of G protein-coupled receptor 3 at the plasma membrane through increased forward trafficking, thereby inducing increased receptor signaling activity.

Phosphorylation participates in the signaling, localization, and trafficking of G protein-coupled receptors. In this study, the amino acids known to be phosphorylated in α1A-adrenergic receptors were substituted with nonphosphorylatable residues in intracellular loop 3 (IL3) mutant, the carboxyl terminus (CTerm) mutant, and both structural regions (IL3-CTerm mutant). Their functions were compared with those of the wild-type (WT) receptor. Agonist- and protein kinase C-induced receptor phosphorylation was markedly decreased in the IL3-CTerm mutant, confirming the major roles of both domains in this process. Surprisingly, the substitutions did not affect the agonist-induced increase in intracellular calcium. Similarly, neither the WT nor the mutant receptors were desensitized by protein kinase C activation; however, agonist stimulation markedly desensitized the receptors, and this effect was diminished in the IL3 mutant, suggesting a role for this domain in this process. Extracellular signal-regulated kinase 1/2 phosphorylation was strong but transient in cells expressing the WT receptor, strong and sustained in those expressing the CTerm mutant, and markedly reduced in those expressing the IL3 and IL3-CTerm mutants. These findings suggest that IL3 phosphorylation sites modulate extracellular signal-regulated kinase activity. A weak and slow agonist-induced receptor-β-arrestin interaction was detected in cells expressing the WT receptor but was barely detectable in any of the mutants. Consistent with these findings, agonist- and phorbol ester-induced receptor internalization was observed in cells expressing the WT receptor but was markedly decreased in cells expressing the distinct mutants. SIGNIFICANCE STATEMENT: Phosphorylation sites in both intracellular loop 3 (IL3) and carboxyl terminus domains affect receptor function in distinct but complementary ways. The IL3 mutant showed decreased agonist-induced desensitization of the calcium response and agonist-activated extracellular signal-regulated kinase phosphorylation. α1A-Adrenergic receptor internalization and interaction with β-arrestin were decreased in all mutants containing phosphorylation site substitutions. Phosphorylation sites in the IL3 and carboxyl terminus domains appear to be functionally relevant for this receptor. The approach employed could be used to expand knowledge on other G protein-coupled receptors.

cAMP signaling is a central regulator of dorsal root ganglion neuron excitability, with spatial and functional specificity conferred by A-kinase anchoring proteins (AKAPs). Here, we examine how cAMP signaling and AKAP-associated signalosomes control nociceptor sensitization and contribute to persistent pain states. We emphasize differential roles of adenylyl cyclase isoforms in dorsal root ganglion neurons and their regulation within AKAP79/150 complexes that coordinate protein kinases, ion channels, and phosphatases to modulate excitability and synaptic plasticity. In pathological conditions such as spinal cord injury, these networks promote sustained hyperexcitability and diminished opioid potency. In particular, AKAP79/150-associated L-type voltage-gated calcium channels are central to circuits involving local calcium influx, extracellular signal-regulated kinase activation, and cAMP signaling that reduce sensitivity to inhibitory Gαi and thus opioids in pathological pain states. These insights identify AKAP-centered signaling hubs as critical integrators of nociceptor function and mechanistically link altered cAMP compartmentation to reduced analgesic efficacy. Targeting these complexes, including modulation of calcium channel activity, represents a promising strategy to restore opioid sensitivity and improve treatment of chronic pain. SIGNIFICANCE STATEMENT: Chronic neuropathic pain remains difficult to treat and is often refractory to standard therapies. This review highlights a critical need to define the molecular mechanisms that sustain nociceptor hyperexcitability. A-kinase anchoring protein-organized signaling complexes in sensory ganglia spatially coordinate adenylyl cyclase, calcium channels, and downstream effectors to drive persistent excitability and reduced opioid sensitivity. Thus, these signalosomes represent promising targets for more effective pain therapeutics.

The pregnane X receptor (PXR) is a key chemosensory protein that helps the organism adapt to its chemical environment. Indeed, humans are continuously exposed to a wide range of external chemicals, known as xenobiotics, which include environmental pollutants, food components, cosmetics, and pharmaceuticals. PXR has the unique property to sense a large variety of xenobiotics and regulate the expression of detoxifying enzymes and transporters, facilitating the clearance of these chemicals. However, prolonged activation of this pathway can lead to negative effects, such as drug-drug interactions, chemoresistance, endocrine disruption, a heightened risk of metabolic diseases, or enhanced cell growth and tumor aggressiveness. Understanding how PXR interacts with xenobiotics is crucial for predicting, assessing, and preventing the potential impacts of these chemicals on human health. Here, we present the structural and functional analysis of the interaction of PXR with 2 approved drugs (nimodipine and liranaftate), a natural flavor commonly used in cosmetics and the food industry (sclareol), and an environmentally relevant halogenated derivative of the emblematic endocrine disruptor bisphenol A (2,2'-dichlorobisphenol A). Cell-based assays show that these 4 compounds display different PXR binding potencies, stimulate the expression of key target genes related to drug metabolism and cell proliferation, and promote colon cancer cell proliferation to varying degrees. Crystallographic analysis reveals their distinct mechanisms of binding to PXR. The integrated functional and structural characterization pipeline we have established yields critical insights for both environmental risk assessment and the rational development of industrial and pharmaceutical compounds with minimal harmful PXR activity. SIGNIFICANCE STATEMENT: The pregnane X receptor is an off-target of many pharmaceuticals and industrial chemicals whose activation is associated with clinically relevant drug-drug interactions. This study describes a pipeline for the identification and characterization of pregnane X receptor ligands, designed to support environmental risk assessment and the informed design of safer substitutes.