Molecular Pharmacology最新文献

{"title":"Corrigendum to \"Repurposing Treprostinil for Enhancing Hematopoietic Progenitor Cell Transplantation\".","authors":"","doi":"10.1016/j.molpha.2025.100025","DOIUrl":"10.1016/j.molpha.2025.100025","url":null,"abstract":"","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 3","pages":"100025"},"PeriodicalIF":3.2,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143476760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing remyelination in multiple sclerosis via M1 muscarinic acetylcholine receptor.","authors":"Keren Chen, Eunyoung Park, Khaled S Abd-Elrahman","doi":"10.1016/j.molpha.2025.100027","DOIUrl":"https://doi.org/10.1016/j.molpha.2025.100027","url":null,"abstract":"<p><p>Multiple sclerosis (MS) is growing in prevalence; yet, treatments that can reverse the progression of the disease are still needed. One strategy that has shown promise for reversing MS is remyelination by inhibiting the M1 receptor, a member of the muscarinic acetylcholine receptor (mAChR) family. Antagonizing the M1 mAChR is believed to be the mechanism by which clemastine, a developing drug that has been observed to enhance myelination in animal studies and phase II clinical trials, elicits its myelination-promoting effects. Recent studies have indicated that blocking M1 mAChR may promote oligodendrocyte differentiation via the extracellular signal-regulated kinase pathway, modulating Ca²⁺ concentration oscillations, and cross-talking with N-methyl-d-aspartate and Notch-1 receptors. However, clemastine has recently been found to accelerate disability in patients with MS, discouraging further progress in its clinical trials. Nevertheless, the underlying mechanisms following M1 mAChR antagonism by clemastine may still be targeted using alternative antimuscarinic drugs. This review consolidates recent advancements in our understanding of the mechanisms by which antagonizing M1 mAChR promotes remyelination and summarizes alternative antimuscarinic drugs that could be leveraged to treat MS in the future. SIGNIFICANCE STATEMENT: Current treatments for multiple sclerosis are limited to disease management, and there is a need for restorative treatments that can reverse progressive forms of the disease. This review aims to summarize the potential mechanisms by which antagonizing the M1 muscarinic acetylcholine receptor could promote remyelination and elaborate on a collection of promising antimuscarinic drugs, consolidating the knowledge needed to target these mechanisms and develop therapeutics that could reverse the progress of demyelinating diseases like multiple sclerosis.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 4","pages":"100027"},"PeriodicalIF":3.2,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143753439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Traffic control: Mechanisms of ligand-specific internalization and intracellular distribution of CCR5.","authors":"Siyi Gu, Svetlana Maurya, Alexis Lona, Leire Borrega Roman, Catherina Salanga, David J Gonzalez, Irina Kufareva, Tracy M Handel","doi":"10.1016/j.molpha.2025.100020","DOIUrl":"https://doi.org/10.1016/j.molpha.2025.100020","url":null,"abstract":"<p><p>CC chemokine receptor (CCR) 5 promotes inflammatory responses by driving cell migration and scavenging chemokine. A CCR5 inhibitor Maraviroc has been approved for blocking HIV entry; however, inhibitors for the treatment of other diseases have had limited success, likely because of the complexity of CCR5 pharmacology and biology. CCR5 is activated by natural and engineered chemokines that elicit distinct signaling and trafficking responses, including receptor sequestration inside the cell. Intracellular sequestration may be therapeutically exploitable as a strategy for receptor inhibition, but the mechanisms by which different ligands promote receptor intracellular retention versus presence on the cell membrane are poorly understood. In this study, we systematically compared the time-dependent trafficking behavior of CCR5 following stimulation with its endogenous agonist, CCL5, and 2 CCL5 variants that promote CCR5 intracellular retention. Using a broad panel of pharmacologic assays, fluorescence microscopy, and live cell ascorbic acid peroxidase proximity labeling proteomics, we identified distinct ligand-dependent CCR5 trafficking patterns with temporal and spatial resolution. All 3 chemokines internalize CCR5 via β-arrestin-dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and varying dependencies on G protein-coupled receptor kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the cellular mechanisms behind CCR5 intracellular sequestration and suggest how trafficking can be exploited for the development of functional antagonists of CCR5. SIGNIFICANCE STATEMENT: CC chemokine receptor (CCR) 5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer, and transmission of HIV. It responds to different ligands with distinct signaling and trafficking behaviors; notably, some ligands induce retention of the receptor inside the cell. This study reveals the cellular basis for receptor sequestration that can be exploited as a therapeutic strategy for inhibiting CCR5 function.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 4","pages":"100020"},"PeriodicalIF":3.2,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Voltage sensors.","authors":"Lily Jan","doi":"10.1016/j.molpha.2024.100011","DOIUrl":"https://doi.org/10.1016/j.molpha.2024.100011","url":null,"abstract":"<p><p>Widely distributed in all kingdoms of life, voltage sensors in the membrane serve important functions via their movements driven by changes in voltage across the membrane (membrane potential). A voltage sensor domain contains 4 transmembrane segments (S1-S4). The S1-S3 helices form a hydrophobic constriction site (HCS, also known as the gating charge transfer center) that spans roughly one-third of the membrane thickness. Flanked by aqueous vestibules connected to the extracellular solution above the HCS or cytoplasmic solution below the HCS, the HCS forms a gating pore for the S4 segment bearing multiple basic residues. Membrane potential changes cause S4 to move through the HCS in a 3₁₀ helical conformation. This S4 translocation generates a gating current as the positively charged S4 basic residues traverse the membrane electric field, transferring these gating charges from one aqueous vestibule to the other. For voltage-gated ion channels with their voltage sensor domains connected to pore domains, the HCS in the voltage sensor domain allows S4 but not ions to go through, while the channel pore formed by the pore domains mediates ion permeation. Voltage sensor mutations could result in ω currents that are conducted through the gating pore of mutant voltage-gated ion channels. These ω currents may cause pathological consequences in patients with periodic paralysis. Besides voltage-gated ion channels, the sperm-specific Na⁺/H⁺ exchanger and voltage-sensing phosphatases contain voltage sensors for membrane potential regulation. Notably, voltage-gated proton channels that are important for pH homeostasis are formed solely by the voltage sensor domain, which mediates proton permeation. SIGNIFICANCE STATEMENT: Voltage sensors mediate voltage regulation of ion channels, transporters, and phosphatases. The voltage sensor domain composed of 4 transmembrane segments (S1-S4) focuses the membrane electric field to the hydrophobic constriction site. To mediate voltage regulation, S4 basic residues within a 3₁₀ helix move across the hydrophobic constriction site without concurrent ion flow through this gating pore. As a counterexample, voltage-gated proton channels are formed by the voltage sensor to mediate proton permeation. These ingeniously engineered voltage sensors are conserved throughout evolution.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 2","pages":"100011"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537393","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A brief history of nerve action potentials after 1600.","authors":"Bertil Hille","doi":"10.1016/j.molpha.2024.100012","DOIUrl":"https://doi.org/10.1016/j.molpha.2024.100012","url":null,"abstract":"<p><p>Action potentials of individual nerve axons are the electrical signals that propagate nervous information quickly around the brain and the body. This essay discusses milestones, from the definition of electricity in 1600 to the recent elucidation of the molecular structures of ion channels and membrane proteins that underlie action potential initiation and propagation. There were several key steps. The theory of electricity and electromagnetism had to be developed enough to allow discovery and measurement of animal electricity by biophysically minded physiologists. The theory of ions and electrochemistry had to be developed enough to allow prediction and verification of an ionic basis for animal electricity. Methods to amplify electrical signals with vacuum tubes and transistors were required for quantitative measurement and display of the action potentials and currents. Physiologists had to move from extracellular recording using nerve trunks to intracellular recording using single nerve fibers. Electronic feedback and mathematical modeling were needed to recognize the conductance changes of nerve membranes during activity. Pharmacology with neurotoxins allowed recognition of underlying voltage-gated ion channels. Protein purification, cloning, and sequencing identified the molecular basis of ion channels, and atomic structures showed in graphic detail how they work. SIGNIFICANCE STATEMENT: This is a brief scientific history of the action potential, the quintessential electrical message of our nerves. As with other histories in biology, this one reiterates that major scientific advances depend on advances in physics and physical chemistry, development of the right preparations and instruments, and the experimental genius and conceptual insights of clever scientists and their students.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 2","pages":"100012"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537358","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Insights of direct and indirect regulation of PXR through phosphorylation in fatty liver disease.","authors":"Veronia Basaly, Anisha Bhattacharya, Grace L Guo","doi":"10.1016/j.molpha.2024.100014","DOIUrl":"https://doi.org/10.1016/j.molpha.2024.100014","url":null,"abstract":"<p><p>The pregnane X receptor (PXR), a ligand-activated nuclear receptor, regulates the transcription of several genes that encode many enzymes and transporters related to drug metabolism. PXR also performs an important role as a physiological sensor in the modulation of endobiotic metabolism for hormones, bile acids, cholesterol, fatty acids, and glucose. Dysregulation of these PXR-mediated pathways is implicated in the progression of metabolic dysfunction-associated steatohepatitis (MASH), contributing to the complex interplay of factors involved in chronic liver disease development and exacerbation affecting millions worldwide. This review highlights the current knowledge of PXR expression and its role in endobiotic metabolism related to MASH development, which is associated with diverse causes and dire outcomes. This review focuses on elucidating the molecular pathways associated with PXR activation directly or indirectly and PXR interaction with other regulatory factors. Although there is still much to comprehend about the intricate details of these pathways, the conclusion is drawn that PXR exerts a crucial role in the pathological and physiological pathways of hepatic cellular processes, which holds promise as a potential pharmacological target for exploring novel therapeutic approaches for MASH treatment and/or prevention. SIGNIFICANCE STATEMENT: The pregnane X receptor (PXR) plays a fundamental role in regulating gene expression involved in xenobiotic and endobiotic metabolism. Dysregulation of PXR-mediated pathways is related to the development of metabolic dysfunction-associated steatohepatitis. The ligand-independent pathways regulating PXR hepatic functions through phosphorylation shed light on possible indirect molecular mechanisms and pathways that regulate PXR activity and function. Understanding these pathways may provide insight into new pharmaceutical interventions for metabolic dysfunction-associated steatohepatitis development.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 2","pages":"100014"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"G protein-coupled receptor-targeted proteolysis-targeting chimeras in cancer therapeutics.","authors":"Victoria R Saca, Thomas Huber, Thomas P Sakmar","doi":"10.1016/j.molpha.2024.100013","DOIUrl":"https://doi.org/10.1016/j.molpha.2024.100013","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 2","pages":"100013"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537362","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Regulation of organic anion transporting polypeptide 1B1 transport function by concurrent phosphorylation and lysine-acetylation: A novel posttranslational regulation mechanism.","authors":"Vishakha Tambe, Erik J Soderblom, Ruhul Kayesh, Vikram Aditya, Chao Xu, Wei Yue","doi":"10.1016/j.molpha.2024.100007","DOIUrl":"10.1016/j.molpha.2024.100007","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 2","pages":"100007"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934288/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537378","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual nuclear receptor 4A1 (NR4A1/NR4A2) ligands inhibit glioblastoma growth and target TWIST1.","authors":"Srijana Upadhyay, Miok Lee, Lei Zhang, Arafat Rahman Oany, Svetlana A Mikheeva, Andrei M Mikheev, Robert C Rostomily, Stephen Safe","doi":"10.1016/j.molpha.2024.100009","DOIUrl":"10.1016/j.molpha.2024.100009","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 2","pages":"100009"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11881746/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput screening identifies a novel small-molecule modulator of Hsp70 that selectively enhances ubiquitination and degradation of misfolded neuronal NO synthase.","authors":"Anthony M Garcia, Amanda K Davis, Cristian Martinez-Ramos, Yoshihiro Morishima, Miranda Lau, Emily Xu, Arya Sunil, Haoming Zhang, Andrew Alt, Andrew P Lieberman, Yoichi Osawa","doi":"10.1016/j.molpha.2024.100008","DOIUrl":"10.1016/j.molpha.2024.100008","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":"107 2","pages":"100008"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}