ACS Chemical Biology最新文献

{"title":"Unraveling the Molecular Jam: How Crowding Shapes Protein Aggregation in Neurodegenerative Disorders","authors":"Shashi Prakash Patel,&nbsp;Tejas Nikam,&nbsp;Bhargavi Sreepathi,&nbsp;Vijayshree S. Karankar,&nbsp;Ankita Jaiswal,&nbsp;Salumuri Vamsi Vardhan,&nbsp;Anika Rana,&nbsp;Vanshu Toga,&nbsp;Nidhi Srivastava,&nbsp;Shubhini A Saraf and Saurabh Awasthi*,&nbsp;","doi":"10.1021/acschembio.4c0036510.1021/acschembio.4c00365","DOIUrl":"https://doi.org/10.1021/acschembio.4c00365https://doi.org/10.1021/acschembio.4c00365","url":null,"abstract":"<p >Protein misfolding and aggregation are the hallmarks of neurodegenerative diseases including Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, and prion diseases. A crowded cellular environment plays a crucial role in modulating protein aggregation processes <i>in vivo</i> and the pathological aggregation of proteins linked to different neurodegenerative disorders. Here, we review recent studies examining the effects of various crowding agents, such as polysaccharides, polyethylene glycol, and proteins like BSA and lysozyme on the behaviors of aggregation of several amyloidogenic peptides and proteins, including amylin, huntingtin, tau, α-synuclein, prion, and amyloid-β. We also summarize how the aggregation kinetics, thermodynamic stability, and morphology of amyloid fibrils are altered significantly in the presence of crowding agents. In addition, we also discuss the molecular basis underlying the modulation of amyloidogenic aggregation, focusing on changes in the protein conformation, and the nucleation mechanism. The molecular understanding of the effects of macromolecular crowding on amyloid aggregation is essential for revealing disease pathologies and identifying possible therapeutic targets. Thus, this review offers a perspective on the complex interplay between protein aggregation and the crowded cellular environment <i>in vivo</i> and explains the relevance of crowding in the context of neurodegenerative disorders.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450860","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Glucose Transporter-Targeting Chimeras Enabling Tumor-Selective Degradation of Secreted and Membrane Proteins","authors":"Chengyu Yun,&nbsp;Na Li,&nbsp;Yishu Zhang,&nbsp;Tong Fang,&nbsp;Jing Ma,&nbsp;Zhenting Zheng,&nbsp;Subing Zhou and Xiaoqing Cai*,&nbsp;","doi":"10.1021/acschembio.4c0058410.1021/acschembio.4c00584","DOIUrl":"https://doi.org/10.1021/acschembio.4c00584https://doi.org/10.1021/acschembio.4c00584","url":null,"abstract":"<p >Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Glucose Transporter-Targeting Chimeras Enabling Tumor-Selective Degradation of Secreted and Membrane Proteins.","authors":"Chengyu Yun, Na Li, Yishu Zhang, Tong Fang, Jing Ma, Zhenting Zheng, Subing Zhou, Xiaoqing Cai","doi":"10.1021/acschembio.4c00584","DOIUrl":"https://doi.org/10.1021/acschembio.4c00584","url":null,"abstract":"<p><p>Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386348","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unraveling the Molecular Jam: How Crowding Shapes Protein Aggregation in Neurodegenerative Disorders.","authors":"Shashi Prakash Patel, Tejas Nikam, Bhargavi Sreepathi, Vijayshree S Karankar, Ankita Jaiswal, Salumuri Vamsi Vardhan, Anika Rana, Vanshu Toga, Nidhi Srivastava, Shubhini A Saraf, Saurabh Awasthi","doi":"10.1021/acschembio.4c00365","DOIUrl":"https://doi.org/10.1021/acschembio.4c00365","url":null,"abstract":"<p><p>Protein misfolding and aggregation are the hallmarks of neurodegenerative diseases including Huntington's disease, Parkinson's disease, Alzheimer's disease, and prion diseases. A crowded cellular environment plays a crucial role in modulating protein aggregation processes <i>in vivo</i> and the pathological aggregation of proteins linked to different neurodegenerative disorders. Here, we review recent studies examining the effects of various crowding agents, such as polysaccharides, polyethylene glycol, and proteins like BSA and lysozyme on the behaviors of aggregation of several amyloidogenic peptides and proteins, including amylin, huntingtin, tau, α-synuclein, prion, and amyloid-β. We also summarize how the aggregation kinetics, thermodynamic stability, and morphology of amyloid fibrils are altered significantly in the presence of crowding agents. In addition, we also discuss the molecular basis underlying the modulation of amyloidogenic aggregation, focusing on changes in the protein conformation, and the nucleation mechanism. The molecular understanding of the effects of macromolecular crowding on amyloid aggregation is essential for revealing disease pathologies and identifying possible therapeutic targets. Thus, this review offers a perspective on the complex interplay between protein aggregation and the crowded cellular environment <i>in vivo</i> and explains the relevance of crowding in the context of neurodegenerative disorders.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142379391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural Insights and Reaction Profile of a New Unspecific Peroxygenase from <i>Marasmius wettsteinii</i> Produced in a Tandem-Yeast Expression System.","authors":"Israel Sánchez-Moreno, Angela Fernandez-Garcia, Ivan Mateljak, Patricia Gomez de Santos, Martin Hofrichter, Harald Kellner, Julia Sanz-Aparicio, Miguel Alcalde","doi":"10.1021/acschembio.4c00504","DOIUrl":"https://doi.org/10.1021/acschembio.4c00504","url":null,"abstract":"<p><p>Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from <i>Marasmius rotula</i> (<i>Mro</i>UPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C-C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications. Here, we introduce a close ortholog of <i>Mro</i>UPO, a UPO gene from <i>Marasmius wettsteinii</i> (<i>Mwe</i>UPO-1), that has a similar reaction profile to <i>Mro</i>UPO and for which we have set up a directed evolution platform based on tandem-yeast expression. Recombinant <i>Mwe</i>UPO-1 was produced at high titers in the bioreactor (0.7 g/L) and characterized at the biochemical and atomic levels. The conjunction of soaking crystallographic experiments at a resolution up to 1.6 Å together with the analysis of reaction patterns sheds light on the substrate preferences of this promiscuous biocatalyst.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142378784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural Insights and Reaction Profile of a New Unspecific Peroxygenase from Marasmius wettsteinii Produced in a Tandem-Yeast Expression System","authors":"Israel Sánchez-Moreno,&nbsp;Angela Fernandez-Garcia,&nbsp;Ivan Mateljak,&nbsp;Patricia Gomez de Santos,&nbsp;Martin Hofrichter,&nbsp;Harald Kellner,&nbsp;Julia Sanz-Aparicio and Miguel Alcalde*,&nbsp;","doi":"10.1021/acschembio.4c0050410.1021/acschembio.4c00504","DOIUrl":"https://doi.org/10.1021/acschembio.4c00504https://doi.org/10.1021/acschembio.4c00504","url":null,"abstract":"<p >Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from <i>Marasmius rotula</i> (<i>Mro</i>UPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C–C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications. Here, we introduce a close ortholog of <i>Mro</i>UPO, a UPO gene from <i>Marasmius wettsteinii</i> (<i>Mwe</i>UPO-1), that has a similar reaction profile to <i>Mro</i>UPO and for which we have set up a directed evolution platform based on tandem-yeast expression. Recombinant <i>Mwe</i>UPO-1 was produced at high titers in the bioreactor (0.7 g/L) and characterized at the biochemical and atomic levels. The conjunction of soaking crystallographic experiments at a resolution up to 1.6 Å together with the analysis of reaction patterns sheds light on the substrate preferences of this promiscuous biocatalyst.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improved Sensitivity in a Modified Berkeley Red Sensor of Transmembrane Potential","authors":"Marisol X. Navarro,&nbsp;Nels C. Gerstner,&nbsp;Soren M. Lipman,&nbsp;Gabby E. Dolgonos and Evan W. Miller*,&nbsp;","doi":"10.1021/acschembio.4c0044210.1021/acschembio.4c00442","DOIUrl":"https://doi.org/10.1021/acschembio.4c00442https://doi.org/10.1021/acschembio.4c00442","url":null,"abstract":"<p >Voltage imaging is an important complement to traditional methods for probing cellular physiology, such as electrode-based patch clamp techniques. Unlike the related Ca²⁺ imaging, voltage imaging provides a direct visualization of bioelectricity changes. We have been exploring the use of sulfonated silicon rhodamine dyes (Berkeley Red Sensor of Transmembrane potential, BeRST) for voltage imaging. In this study, we explore the effect of converting BeRST to diEt BeRST, by replacing the dimethyl aniline of BeRST with a diethyl aniline group. The new dye, diEt BeRST, has a voltage sensitivity of 40% Δ<i>F</i>/<i>F</i> per 100 mV, a 33% increase compared to the original BeRST dye, which has a sensitivity of 30% Δ<i>F</i>/<i>F</i> per 100 mV. In neurons, the cellular brightness of diEt BeRST is about 20% as bright as that of BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to that of BeRST (800 μM). Despite this lower cellular brightness, diEt BeRST is able to record spontaneous and evoked action potentials from multiple neurons simultaneously and in single trials. Far-red excitation and emission profiles enable diEt BeRST to be used alongside existing fluorescent indicators of cellular physiology, like Ca²⁺-sensitive Oregon Green BAPTA. In hippocampal neurons, simultaneous voltage and Ca²⁺ imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca²⁺ imaging methods. This study represents a first step toward describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improved Sensitivity in a Modified Berkeley Red Sensor of Transmembrane Potential.","authors":"Marisol X Navarro, Nels C Gerstner, Soren M Lipman, Gabby E Dolgonos, Evan W Miller","doi":"10.1021/acschembio.4c00442","DOIUrl":"https://doi.org/10.1021/acschembio.4c00442","url":null,"abstract":"<p><p>Voltage imaging is an important complement to traditional methods for probing cellular physiology, such as electrode-based patch clamp techniques. Unlike the related Ca²⁺ imaging, voltage imaging provides a direct visualization of bioelectricity changes. We have been exploring the use of sulfonated silicon rhodamine dyes (Berkeley Red Sensor of Transmembrane potential, BeRST) for voltage imaging. In this study, we explore the effect of converting BeRST to diEt BeRST, by replacing the dimethyl aniline of BeRST with a diethyl aniline group. The new dye, diEt BeRST, has a voltage sensitivity of 40% Δ<i>F</i>/<i>F</i> per 100 mV, a 33% increase compared to the original BeRST dye, which has a sensitivity of 30% Δ<i>F</i>/<i>F</i> per 100 mV. In neurons, the cellular brightness of diEt BeRST is about 20% as bright as that of BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to that of BeRST (800 μM). Despite this lower cellular brightness, diEt BeRST is able to record spontaneous and evoked action potentials from multiple neurons simultaneously and in single trials. Far-red excitation and emission profiles enable diEt BeRST to be used alongside existing fluorescent indicators of cellular physiology, like Ca²⁺-sensitive Oregon Green BAPTA. In hippocampal neurons, simultaneous voltage and Ca²⁺ imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca²⁺ imaging methods. This study represents a first step toward describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142363397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Leveraging Covalency to Stabilize Ternary Complex Formation For Cell-Cell \"Induced Proximity\".","authors":"Karolina Krygier, Anjalee N Wijetunge, Arthur Srayeddin, Harrison Mccann, Anthony F Rullo","doi":"10.1021/acschembio.4c00286","DOIUrl":"https://doi.org/10.1021/acschembio.4c00286","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Recent advances in the field of translational chemical biology use diverse \"proximity-inducing\" synthetic modalities to elicit new modes of \"event driven\" pharmacology. These include mechanisms of targeted protein degradation and immune clearance of pathogenic cells. Heterobifunctional \"chimeric\" compounds like Proteolysis TArgeting Chimeras (PROTACs) and Antibody Recruiting Molecules (ARMs) leverage these mechanisms, respectively. Both systems function through the formation of reversible \"ternary\" or higher-order biomolecular complexes. Critical to function are key parameters, such as bifunctional molecule affinity for endogenous proteins, target residence time, and turnover. To probe the mechanism and enhance function, covalent chemical approaches have been developed to kinetically stabilize ternary complexes. These include electrophilic PROTACs and Covalent Immune Recruiters (CIRs), the latter designed to uniquely enforce cell-cell induced proximity. Inducing cell-cell proximity is associated with key challenges arising from a combination of steric and/or mechanical based destabilizing forces on the ternary complex. These factors can attenuate the formation of ternary complexes driven by high affinity bifunctional/proximity inducing molecules. This Account describes initial efforts in our lab to address these challenges using the CIR strategy in antibody recruitment or receptor engineered T cell model systems of cell-cell induced proximity. ARMs form ternary complexes with serum antibodies and surface protein antigens on tumor cells that subsequently engage immune cells via Fc receptors. Binding and clustering of Fc receptors trigger immune cell killing of the tumor cell. We applied the CIR strategy to convert ARMs to covalent chimeras, which \"irreversibly\" recruit serum antibodies to tumor cells. These covalent chimeras leverage electrophile preorganization and kinetic effective molarity to achieve fast and selective covalent engagement of the target ternary complex protein, e.g., serum antibody. Importantly, covalent engagement can proceed via diverse binding site amino acids beyond cysteine. Covalent chimeras demonstrated striking functional enhancements compared to noncovalent ARM analogs in functional immune assays. We revealed this enhancement was in fact due to the increased kinetic stability &lt;i&gt;and not&lt;/i&gt; concentration, of ternary complexes. This finding was recapitulated using analogous CIR modalities that integrate peptidic or carbohydrate binding ligands with Sulfur(VI) Fluoride Exchange (SuFEx) electrophiles to induce cell-cell proximity. Mechanistic studies in a distinct model system that uses T cells engineered with receptors that recognize covalent chimeras or ARMs, revealed covalent receptor engagement uniquely enforces downstream activation signaling. Finally, this Account discusses potential challenges and future directions for adapting and optimizing covalent chimeric/bifunctional molecules for diverse applications in","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142337252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Leveraging Covalency to Stabilize Ternary Complex Formation For Cell–Cell “Induced Proximity”","authors":"Karolina Krygier,&nbsp;Anjalee N. Wijetunge,&nbsp;Arthur Srayeddin,&nbsp;Harrison Mccann and Anthony F. Rullo*,&nbsp;","doi":"10.1021/acschembio.4c0028610.1021/acschembio.4c00286","DOIUrl":"https://doi.org/10.1021/acschembio.4c00286https://doi.org/10.1021/acschembio.4c00286","url":null,"abstract":"&lt;p &gt;Recent advances in the field of translational chemical biology use diverse “proximity-inducing” synthetic modalities to elicit new modes of “event driven” pharmacology. These include mechanisms of targeted protein degradation and immune clearance of pathogenic cells. Heterobifunctional “chimeric” compounds like Proteolysis TArgeting Chimeras (PROTACs) and Antibody Recruiting Molecules (ARMs) leverage these mechanisms, respectively. Both systems function through the formation of reversible “ternary” or higher-order biomolecular complexes. Critical to function are key parameters, such as bifunctional molecule affinity for endogenous proteins, target residence time, and turnover. To probe the mechanism and enhance function, covalent chemical approaches have been developed to kinetically stabilize ternary complexes. These include electrophilic PROTACs and Covalent Immune Recruiters (CIRs), the latter designed to uniquely enforce cell–cell induced proximity. Inducing cell–cell proximity is associated with key challenges arising from a combination of steric and/or mechanical based destabilizing forces on the ternary complex. These factors can attenuate the formation of ternary complexes driven by high affinity bifunctional/proximity inducing molecules. This Account describes initial efforts in our lab to address these challenges using the CIR strategy in antibody recruitment or receptor engineered T cell model systems of cell–cell induced proximity. ARMs form ternary complexes with serum antibodies and surface protein antigens on tumor cells that subsequently engage immune cells via Fc receptors. Binding and clustering of Fc receptors trigger immune cell killing of the tumor cell. We applied the CIR strategy to convert ARMs to covalent chimeras, which “irreversibly” recruit serum antibodies to tumor cells. These covalent chimeras leverage electrophile preorganization and kinetic effective molarity to achieve fast and selective covalent engagement of the target ternary complex protein, e.g., serum antibody. Importantly, covalent engagement can proceed via diverse binding site amino acids beyond cysteine. Covalent chimeras demonstrated striking functional enhancements compared to noncovalent ARM analogs in functional immune assays. We revealed this enhancement was in fact due to the increased kinetic stability &lt;i&gt;and not&lt;/i&gt; concentration, of ternary complexes. This finding was recapitulated using analogous CIR modalities that integrate peptidic or carbohydrate binding ligands with Sulfur(VI) Fluoride Exchange (SuFEx) electrophiles to induce cell–cell proximity. Mechanistic studies in a distinct model system that uses T cells engineered with receptors that recognize covalent chimeras or ARMs, revealed covalent receptor engagement uniquely enforces downstream activation signaling. Finally, this Account discusses potential challenges and future directions for adapting and optimizing covalent chimeric/bifunctional molecules for diverse applications in c","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}