Molecular Pharmacology最新文献

Organic anion transporting polypeptide (OATP) 1B1 is crucial for hepatic uptake of many drugs and endogenous substrates. The clinically relevant OATP1B1 c.521 T>C (V174A) polymorphism exhibits reduced transport activity in vitro and in vivo in humans. Previously, we reported increased total phosphorylation of V174A-OATP1B1 compared to wild-type (WT)-OATP1B1, although the differentially phosphorylated sites remain to be identified. Lysine-acetylation, a key posttranslational modification (PTM), has not been investigated in OATP1B1. This study aimed to identify differential PTMs of WT-OATP1B1 and V174A-OATP1B1 by quantitatively comparing the relative abundance of modified peptides using liquid chromatography-tandem mass spectrometry-based proteomics and to assess the impact of these PTMs on OATP1B1 transport function using [³H]-estradiol-17-β-D-glucuronide as substrate in transporter-expressing human embryonic kidney 293 cells. We discovered that OATP1B1 is lysine-acetylated at 13 residues. Compared to WT-OATP1B1, V174A-OATP1B1 has increased concurrent phosphorylation at S659 and S663 and concurrent phosphorylation (at S659 and S663) and lysine-acetylation (at K650) (P < .05). Variants mimicking concurrent phosphorylation (S659E-S663E-OATP1B1) and concurrent phosphorylation and acetylation (K650Q-659E-S663E-OATP1B1) both demonstrated reduced substrate transport by 0.86 ± 0.055-fold and 0.65 ± 0.047-fold of WT-OATP1B1 (both P < .05), respectively. Single-site mimics of phosphorylation or lysine-acetylation at K650, S659, and S663 did not affect OATP1B1 transport function, indicating cooperative effects on OATP1B1 by concurrent PTMs. All variants and WT-OATP1B1 were primarily localized to the plasma membrane and colocalized with plasma membrane protein Na/K-ATPase as determined by immunofluorescent staining and confocal microscopy. The current study elucidates a novel mechanism in which concurrent serine-phosphorylation and lysine-acetylation impair OATP1B1-mediated transport, suggesting potential interplay between these PTMs in regulating OATP1B1. SIGNIFICANCE STATEMENT: Understanding organic anion transporting polypeptide (OATP1B1) regulation is key to predicting altered drug disposition. The Val174Ala-OATP1B1 polymorphism exhibits reduced transport activity and is the most effective predictor of statin-induced myopathy. Val174Ala-OATP1B1 was found to be associated with increased serine-phosphorylation at Ser659 and Ser663 and lysine-acetylation at Lys650; concurrent PTMs at these sites reduce OATP1B1 function. These findings revealed a novel mechanism involved in transporter regulation, suggesting potential interplay between these PTMs in governing hepatic drug transport and response.

G protein-coupled receptors (GPCRs) comprise a family of heptahelical membrane proteins that mediate intracellular and intercellular transmembrane signaling. Defects in GPCR signaling pathways are implicated in the pathophysiology of many diseases, including cardiovascular disease, endocrinopathies, immune disorders, and cancer. Although GPCRs are attractive drug targets, only a small number of Food and Drug Administration-approved anticancer therapeutics target GPCRs. Targeted protein degradation (TPD) technology allows for the direct modulation of the cellular expression level of a protein of interest. TPD methods such as proteolysis-targeting chimeras (PROTACs) use the ubiquitin-proteasome system to degrade a protein of interest selectively. Although the PROTAC system has not been widely applied to GPCRs and other membrane proteins, there is evidence that PROTACs or other TPD methods could be applied to the GPCRome. Current GPCR PROTACs show the feasibility of using PROTACs to degrade GPCRs; however, the degradation mechanism for some of these GPCR PROTACs is uncertain. Additional studies aimed at elucidating the degradation mechanism of GPCRs with PROTACs are necessary. Discovery of new allosteric intracellular small molecule binders of GPCRs will be required for the development of intracellularly oriented PROTACs. Promising early results in targeted degradation of GPCRs suggest that TPD drug discovery platforms will be useful in developing PROTACs targeting pathological GPCRs. SIGNIFICANCE STATEMENT: Aberrant signaling of G protein-coupled receptors (GPCRs) can contribute to the pathophysiology of cancer. Although GPCRs are generally highly attractive drug targets, many individual GPCRs are currently undrugged using traditional drug discovery approaches. Targeted protein degradation technologies, such as proteolysis-targeting chimeras, provide a new approach to drug discovery for targeting previously undruggable GPCRs relevant to the molecular pathophysiology of cancer.

1,1-Bis(3'-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) compounds are dual receptor ligands that bind both orphan nuclear receptor 4A1 (NR4A1) and NR4A2. Knockdown of NR4A1 or NR4A2 by RNA interference in glioblastoma (GBM) cells decreased growth and induced apoptosis and comparable effects were observed for DIM-3,5 analogs, which exhibit inverse agonist activity and inhibit NR4A1- and NR4A2-mediated pro-oncogenic activity. Knockdown of NR4A1 or NR4A2 or treatment with DIM-3,5 analogs also decreased expression of TWIST1 mRNA and protein in GBM cells by 40%-90%.The proximal region of the TWIST1 gene promoter contains functional GC-rich binding sites that bind Sp1 and Sp4, and knockdown of these transcription factors also decreased TWIST1 expression in GBM cells. Further analysis by chromatin immunoprecipitation, protein-protein coimmunoprecipitation, and binding assays demonstrated that NR4A1/NR4A2 coregulate TWIST1 gene expression as ligand-dependent cofactors of Sp1 and Sp4, which interact with cis proximal GC-rich sites in the TWIST1 gene promoter. In vivo studies show that DIM-3,5 dual NR4A1/2 inverse agonists also reduced intratumoral TWIST1 expression while significantly prolonging survival of mice in a syngeneic mouse model of GBM, demonstrating that these ligands are promising new agents for targeting TWIST1 and treating GBM. SIGNIFICANCE STATEMENT: The TWIST1 gene is a pro-oncogenic factor that regulates epithelial-to-mesenchymal transition in glioblastoma cells. This paper shows that the orphan nuclear receptor 4A1 (NR4A1) and NR4A2 regulate TWIST1 expression, which can be targeted by bis-indole-derived dual NR4A1/2 inverse agonists.

The Hsp90 and Hsp70 chaperones act as a protein quality control system for several hundred client proteins, including many implicated in neurodegenerative disorders. Hsp90 and Hsp70 are widely thought to be important drug targets. Although many structurally distinct compounds have been developed to target Hsp90, relatively few are known to target Hsp70 and even fewer have been tested in protein quality control systems. To address this, we describe a high-throughput thermal shift-based screen to find compounds that bind and stabilize Hsp70 and then employ assays with misfolded forms of a well-established client protein, neuronal NO synthase (nNOS), to identify compounds that enhance ubiquitination of client proteins. The ubiquitination assay employed a quantitative ELISA method to measure Hsp70:CHIP-dependent ubiquitination of heme-deficient nNOS, which is a model of a misfolded client, in reaction mixtures containing purified E1, E2, Hsp70, CHIP, and ubiquitin. We screened 44,447 molecules from the Maybridge and ChemDiv libraries and found one compound, protein folding disease compound 15 (PFD-15), that enhanced in vitro nNOS ubiquitination with an EC₅₀ of approximately 8 μM. PFD-15 was tested in human embryonic kidney 293 cells stably transfected with a C331A nNOS, a mutation that makes nNOS a preferred client protein for ubiquitination. In this model, PFD-15 decreased steady-state levels of C331A nNOS, but not the wild-type nNOS, in a time- and concentration-dependent manner by a process attenuated by lactacystin, an inhibitor to the proteasome. PFD-15 appears to enhance binding of Hsp70 and CHIP to client proteins without interference of protein quality control mechanisms, enabling the selective clearance of misfolded proteins. SIGNIFICANCE STATEMENT: There are few treatment options for neurodegenerative diseases, which are widely thought to be caused by formation of toxic misfolded proteins. One novel approach is to enhance the Hsp90/Hsp70 protein quality control machinery to remove these misfolded proteins. Targeting Hsp70 may have advantages over targeting Hsp90, but fewer compounds targeting Hsp70 have been developed relative to those for Hsp90. The current study provides a novel approach to enhance the number of compounds targeting the Hsp70's role in protein quality control.

Fibrosis is implicated in nearly all forms of cardiomyopathy and significantly influences disease severity and outcomes. The primary cell responsible for fibrosis is the cardiac fibroblast, which remains understudied relative to cardiomyocytes in the context of cardiomyopathy. The development of induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs) allows for the modeling of patient-specific disease characteristics and provides a scalable source of fibroblasts. iPSC-CFs are invaluable for understanding molecular pathways that affect disease progression and outcomes. This review explores various aspects of cardiomyopathy, with a focus on dilated cardiomyopathy, that can be modeled using iPSC-CFs and their application in drug discovery, given the current lack of approved therapies for cardiac fibrosis. We examine how iPSC-CFs can be utilized to study heart development, fibroblast heterogeneity, and activation, with the ultimate goal of developing better therapies for patients with cardiomyopathies. SIGNIFICANCE STATEMENT: We explore how induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs) are used to study the fibrotic component of dilated cardiomyopathy. Most research has focused on cardiomyocytes, but iPSC-CFs serve as a valuable tool to elucidate molecular pathways leading to fibrosis and paracrine interactions with cardiomyocytes. Gaining insights into these events could aid in the development of new therapies and enable the use of patient-derived iPSC-CFs for precision medicine, ultimately improving patient outcomes.

Endocytosis of the μ-type opioid receptor (MOR) is a fundamentally important cellular regulatory process that is characteristically driven less effectively by partial relative to full agonist ligands. Such agonist-selective endocytic discrimination depends on how strongly drugs promote MOR binding to β-arrestins, and this, in turn, depends on how strongly they stimulate phosphorylation of the MOR cytoplasmic tail by G protein-coupled receptor kinases (GRKs) from the GRK2/3 subfamily. While these relatively "downstream" steps in the agonist selective endocytic pathway are now well defined, it remains unclear how agonist-bound receptors are distinguished "upstream" by GRKs. Focusing on GRK2 as a prototype, we show that this single GRK subtype can distinguish the endocytic activities of different MOR agonists in cells lacking other GRKs and that agonist selectivity is introduced at the most upstream step of GRK2 binding to MOR. This interaction requires prior membrane recruitment of GRK2 by its conserved Pleckstrin homology domain and is enhanced by phosphorylation of the MOR tail, but neither reaction can explain the high degree of agonist selectivity in the observed interaction of GRK2 with MOR. We identify the N-terminal domain (NTD) of GRK2, which is identical in GRK3, as a discrete element required for the full agonist selectivity of MOR-GRK2 interaction and show that the NTD is also required for GRK2 to promote MOR endocytosis after it is bound. We propose a simple cellular mechanism of upstream agonist discrimination that is organized as a series of biochemical checkpoints and uses the NTD as an agonist-selective sensor. SIGNIFICANCE STATEMENT: This study investigates how G protein-coupled receptor kinases (GRKs) distinguish the effects of opioid agonist drugs on regulated endocytosis of the μ-type opioid receptor (MOR). It shows that a single GRK subtype is sufficient to determine the agonist selectivity of MOR internalization, agonists are distinguished by how strongly they promote GRK2 recruitment by MOR, and the GRK2/3 N terminus is a key determinant of agonist discrimination.

The emerging picture of G protein-coupled receptor function suggests that the global signaling response is an integrated sum of a multitude of individual receptor responses, each regulated by their local protein environment. The β2 adrenergic receptor (B2AR) has long served as an example receptor in the development of this model. However, the mechanism and the identity of the protein-protein interactions that govern the availability of receptors competent for signaling remain incompletely characterized. To address this question, we characterized the interactome of agonist-stimulated B2AR in human embryonic kidney 293 cells using FLAG coimmunoprecipitation coupled to stable isotope labeling by amino acids in cell culture and mass spectrometry. Our B2AR cross-linked interactome identified 190 high-confidence proteins, including almost all known interacting proteins and 6 out of 7 isoforms of the 14-3-3 family of scaffolding proteins. Inhibiting 14-3-3 proteins with the peptide difopein enhanced isoproterenol-stimulated adrenergic signaling via cAMP approximately 3-fold and increased both miniGs and arrestin recruitment to B2AR more than 2-fold each, without noticeably changing EC₅₀ with respect to cAMP signaling or effector recruitment upon stimulation. Our results show that 14-3-3 proteins negatively regulate downstream signaling by inhibiting access of B2AR to effector proteins. We propose that 14-3-3 proteins maintain a dynamic pool of B2AR that has reduced signaling efficacy in response to acute agonist stimulation, limiting the number of signaling-competent receptors at the plasma membrane. SIGNIFICANCE STATEMENT: This study presents a new interactome of the agonist-stimulated β2 adrenergic receptor, a paradigmatic G protein-coupled receptor that is both a model system for members of this class and an important signaling protein in respiratory, cardiovascular, and metabolic regulation. We identify 14-3-3 proteins as responsible for restricting β2 adrenergic receptor access to signaling effectors and maintaining a receptor population that is insensitive to acute stimulation by agonists.

The patch-clamp technique has been the gold standard for analysis of excitable cells. Since its development in the 1980s, it has contributed immensely to our understanding of neurons, muscle cells, and cardiomyocytes and the ion channels and receptors that reside within them. This technique, predicated on Ohm's law, enables precise measurement of macroscopic excitability patterns and assessment of ionic and gating conductance, even to the single channel level. Over the years, patch-clamp electrophysiology has undergone extensive modifications, with the introduction of new applications that have enhanced its power and reach. The most recent evolution of this technique occurred with the introduction of robotic high-throughput automated platforms that enable high-quality simultaneous recordings, in both voltage- and current-clamp modes, from tens to hundreds of cells, including cells freshly isolated from their native tissues. Combined with new dynamic-clamp applications, these new methods provide increasingly powerful tools for studying the contributions of ion channels and receptors to electrogenesis. In this brief review, we provide an overview of these enhanced patch-clamp techniques, followed by some of the applications presently being pursued, and a perspective into the potential future of the patch-clamp method. SIGNIFICANCE STATEMENT: The patch-clamp technique, introduced in the 1980s, has revolutionized the understanding of electrogenesis. Predicated on Ohm's law, this approach facilitates exploration of ionic conductances, gating mechanisms of ion channels and receptors, and their roles in neuronal, muscular, and cardiac excitability. Robotic platforms for high-throughput patch-clamp and dynamic-clamp have recently expanded their reach. Here, we outline new advances in patch-clamp including high-throughput analysis of freshly isolated neurons and discuss the increasingly powerful trajectory of new patch-clamp techniques.

Chemokine receptors CCR3, CCR4, and CCR5 are G protein-coupled receptors implicated in diseases like cancer, Alzheimer's, asthma, human immunodeficiency virus (HIV), and macular degeneration. Recently, CCR3 and CCR4 have emerged as potential stroke targets. Although only the CCR5 antagonist maraviroc is US Food and Drug Administration-approved (for HIV), we curated data on CCR3, CCR4, and CCR5 antagonists from ChEMBL to develop and validate machine learning models. The top 5-fold cross-validation statistics for these models were high for both classification and regression models for CCR3 (receiver operating characteristic [ROC], 0.94; R² = 0.8), CCR4 (ROC, 0.98; R² = 0.57), and CCR5 (ROC, 0.96; R² = 0.78). The models for CCR3/4 were used to screen a small library of US Food and Drug Administration-approved drugs and 17 were initially tested in vitro against both CCR3/4 receptors. A promising compound lapatinib, a dual tyrosine kinase inhibitor, was identified as an antagonist for CCR3 (IC₅₀, 0.7 μM) and CCR4 (IC₅₀, 1.8 μM). Additional testing also identified it as an CCR5 antagonist (IC₅₀, 0.9 μM), and it showed moderate in vitro HIV I inhibition. We demonstrated how machine learning can be used to identify molecules for repurposing as antagonists for G protein-coupled receptors such as CCR3, CCR4, and CCR5. Lapatinib may represent a new orally available chemical probe for these 3 receptors, and it provides a starting point for further chemical optimization for multiple diseases impacting human health. SIGNIFICANCE STATEMENT: We describe the building of machine learning models for the chemokine receptors CCR3, CCR4, and CCR5 trained on data from the ChEMBL database. Using these models, we identified lapatinib as a potent inhibitor of CCR3, CCR4, and CCR5. Our study illustrates the potential of machine learning in identifying molecules for repurposing as antagonists for G protein-coupled receptors, including CCR3, CCR4, and CCR5, which have various therapeutic applications.

Reintroduction of tumor-suppressive microRNA-7-5p (miR-7) that is depleted in non-small cell lung cancer (NSCLC) represents a new therapeutic approach, whereas previous studies mainly used miR-7 mimics chemoengineered in vitro. Here we aim to establish the pharmacological actions and therapeutic potential of novel bioengineered RNA bearing a payload miR-7 (BioRNA/miR-7) molecule produced in vivo. First, through confocal imaging and immunoblot studies, we revealed that BioRNA/miR-7 altered NSCLC cell mitochondrial morphology accompanied by the downregulation of known target genes, epidermal growth factor receptor (EGFR), mitochondrial solute carrier family 25A37 (SLC25A37), and import inner membrane translocase subunit (TIM50). Second, through luciferase reporter and immunoblot studies, we validated mitochondrial acylglycerol kinase (AGK) as a new direct target for miR-7. Third, through real-time live-cell analyses, we revealed BioRNA/miR-7 to modulate mitochondrial respiration and glycolytic capacity. Fourth, live-cell and endpoint viability studies demonstrated that the combination of BioRNA/miR-7 with pemetrexed (PEM) elicited a strong synergistic effect to inhibit NSCLC cell growth, associated with an increased intracellular PEM accumulation, as quantified by a liquid chromatography tandem mass spectrometry method. Finally, through in vivo therapy study using NSCLC patient-derived xenograft mouse model, we demonstrated the efficacy and tolerability of BioRNA/miR-7 monotherapy and combination therapy with PEM to control tumor progression. Our collective works establish a role for miR-7 in NSCLC metabolism and PEM disposition and support our novel, in vivo produced BioRNA/miR-7-5p for molecular pharmacological research. Our findings further illustrate the potential of BioRNA/miR-7 plus PEM combination as a potential treatment to combat NSCLC tumor progression. SIGNIFICANCE STATEMENT: MiR-7 is a tumor-suppressive microRNA depleted in non-small cell lung cancer (NSCLC), and in vitro chemoengineered miR-7 mimics were shown to inhibit tumor growth in NSCLC cell-derived xenograft mice. Here, a novel in vivo bioengineered miR-7 molecule, namely BioRNA/miR-7, was used to effectively control target gene expression and NSCLC cell metabolism. Furthermore, BioRNA/miR-7 was demonstrated to remarkably improve pemetrexed antitumor activity in NSCLC patient-derived tumor mice, supporting the role of miR-7 in NSCLC metabolism and potential for BioRNA/miR-7 to improve NSCLC therapy.