RESUMO
CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.
Assuntos
Alelos , Edição de Genes , Mutagênese , Linfócitos T , Humanos , Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Mutagênese/genética , RNA Guia de Sistemas CRISPR-Cas/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ativação Linfocitária , Citocinas/biossíntese , Citocinas/metabolismo , Mutação com Ganho de Função , Mutação com Perda de FunçãoRESUMO
Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
Assuntos
Edição de Genes , Proteínas de Ligação a RNA , Humanos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Células K562 , Poli U/genética , Poli U/metabolismo , RNA Polimerase III/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.
Assuntos
Fibrose Cística , Quimera/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/uso terapêutico , Humanos , Ligantes , Ubiquitina/metabolismoRESUMO
Targeted protein degradation (TPD) using proteolysis targeting chimeras (PROTACs) and molecular glue degraders has arisen as a powerful therapeutic modality for eliminating disease-causing proteins from cells. PROTACs and molecular glue degraders employ heterobifunctional or monovalent small molecules, respectively, to chemically induce the proximity of target proteins with E3 ubiquitin ligases to ubiquitinate and degrade specific proteins via the proteasome. Whereas TPD is an attractive therapeutic strategy for expanding the druggable proteome, only a relatively small number of E3 ligases out of the >600 E3 ligases encoded by the human genome have been exploited by small molecules for TPD applications. Here we review the existing E3 ligases that have thus far been successfully exploited for TPD and discuss chemoproteomics-enabled covalent screening strategies for discovering new E3 ligase recruiters. We also provide a chemoproteomic map of reactive cysteines within hundreds of E3 ligases that may represent potential ligandable sites that can be pharmacologically interrogated to uncover additional E3 ligase recruiters.
Assuntos
Proteínas , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Proteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , UbiquitinaçãoRESUMO
While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Cisteína , Antivirais/farmacologia , Antivirais/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Simulação de Acoplamento MolecularRESUMO
While brown adipose tissue (BAT) is well-recognized for its ability to dissipate energy in the form of heat, recent studies suggest multifaced roles of BAT in the regulation of glucose and lipid homeostasis beyond stimulating thermogenesis. One of the functions involves interorgan communication with metabolic organs, such as the liver, through BAT-derived secretory factors, a.k.a., batokine. However, the identity and the roles of such mediators remain insufficiently understood. Here, we employed proteomics and transcriptomics in human thermogenic adipocytes and identified previously unappreciated batokines, including phospholipid transfer protein (PLTP). We found that increased circulating levels of PLTP, via systemic or BAT-specific overexpression, significantly improve glucose tolerance and insulin sensitivity, increased energy expenditure, and decrease the circulating levels of cholesterol, phospholipids, and sphingolipids. Such changes were accompanied by increased bile acids in the circulation, which in turn enhances glucose uptake and thermogenesis in BAT. Our data suggest that PLTP is a batokine that contributes to the regulation of systemic glucose and lipid homeostasis as a mediator of BAT-liver interorgan communication.
Assuntos
Tecido Adiposo Marrom , Glucose , Tecido Adiposo Marrom/metabolismo , Metabolismo Energético , Glucose/metabolismo , Homeostase , Humanos , Lipídeos , Fígado , TermogêneseRESUMO
Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.
Assuntos
Autofagia/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Autofagia/fisiologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-akt , Pirazóis/farmacologiaRESUMO
Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Limoninas/farmacologia , Proteólise/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Limoninas/química , Limoninas/isolamento & purificação , Ubiquitina-Proteína LigasesRESUMO
The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation.
Assuntos
Neoplasias da Mama/química , Genoma Humano , Fases de Leitura Aberta , Peptídeos/análise , Proteoma/análise , Neoplasias da Mama/genética , Linhagem Celular , Cromatografia Líquida , Códon de Iniciação/química , Códon de Iniciação/genética , Feminino , Humanos , Células K562 , Peptídeos/química , Biossíntese de Proteínas , Proteoma/química , Espectrometria de Massas em TandemRESUMO
One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.
Assuntos
Carbono , Cisteína , Epigênese Genética , Formaldeído , Metionina Adenosiltransferase , S-Adenosilmetionina , Animais , Camundongos , Carbono/metabolismo , Epigênese Genética/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Formaldeído/metabolismo , Formaldeído/toxicidade , Exposição Ambiental , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Cisteína/metabolismo , Humanos , Células Hep G2RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 168 million cases and 3.4 million deaths to date, while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here, we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.
Assuntos
Antivirais , COVID-19 , Antivirais/farmacologia , Humanos , Pandemias , SARS-CoV-2RESUMO
Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.
Assuntos
Complexos de Coordenação/química , Proteínas Nucleares/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos de Coordenação/metabolismo , Cisteína/química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios Proteicos , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Ubiquitinação , Zinco/químicaRESUMO
The initiation and execution of cell death can be regulated by various lipids. How the levels of environmental (exogenous) lipids impact cell death sensitivity is not well understood. We find that exogenous monounsaturated fatty acids (MUFAs) potently inhibit the non-apoptotic, iron-dependent, oxidative cell death process of ferroptosis. This protective effect is associated with the suppression of lipid reactive oxygen species (ROS) accumulation at the plasma membrane and decreased levels of phospholipids containing oxidizable polyunsaturated fatty acids. Treatment with exogenous MUFAs reduces the sensitivity of plasma membrane lipids to oxidation over several hours. This effect requires MUFA activation by acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) and is independent of lipid droplet formation. Exogenous MUFAs also protect cells from apoptotic lipotoxicity caused by the accumulation of saturated fatty acids, but in an ACSL3-independent manner. Our work demonstrates that ACSL3-dependent MUFA activation promotes a ferroptosis-resistant cell state.
Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Ferroptose/efeitos dos fármacos , Lipídeos/química , Animais , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Coenzima A Ligases/metabolismo , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Camundongos , Oxirredução , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Despite the completion of human genome sequencing efforts nearly 15 years ago that brought with it the promise of genome-based discoveries that would cure human diseases, most protein targets that control human diseases have remained largely untranslated, in-part because they represent difficult protein targets to drug. In addition, many of these protein targets lack screening assays or accessible binding pockets, making the development of small-molecule modulators very challenging. Here, we discuss modern methods for activity-based protein profiling-based chemoproteomic strategies to map 'ligandable' hotspots in proteomes using activity and reactivity-based chemical probes to allow for pharmacological interrogation of these previously difficult targets. We will showcase several recent examples of how these technologies have been used to develop highly selective small-molecule inhibitors against disease-related protein targets.
Assuntos
Biomarcadores/metabolismo , Desenho de Fármacos , Proteínas/metabolismo , Proteoma/metabolismo , Animais , Biomarcadores/análise , Humanos , Análise Serial de Proteínas , Proteínas/análise , Proteoma/análiseRESUMO
Most of the proteome is considered undruggable, oftentimes hindering translational efforts for drug discovery. Identifying previously unknown druggable hotspots in proteins would enable strategies for pharmacologically interrogating these sites with small molecules. Activity-based protein profiling (ABPP) has arisen as a powerful chemoproteomic strategy that uses reactivity-based chemical probes to map reactive, functional, and ligandable hotspots in complex proteomes, which has enabled inhibitor discovery against various therapeutic protein targets. Here, we report an alkyne-functionalized N-hydroxysuccinimide-ester (NHS-ester) as a versatile reactivity-based probe for mapping the reactivity of a wide range of nucleophilic ligandable hotspots, including lysines, serines, threonines, and tyrosines, encompassing active sites, allosteric sites, post-translational modification sites, protein interaction sites, and previously uncharacterized potential binding sites. Surprisingly, we also show that fragment-based NHS-ester ligands can be made to confer selectivity for specific lysine hotspots on specific targets including Dpyd, Aldh2, and Gstt1. We thus put forth NHS-esters as promising reactivity-based probes and chemical scaffolds for covalent ligand discovery.
Assuntos
Sítios de Ligação , Ésteres/química , Ligantes , Sondas Moleculares/química , Proteoma/metabolismo , Aldeído-Desidrogenase Mitocondrial/metabolismo , Alcinos/química , Glutationa Transferase/metabolismo , Humanos , Lisina/metabolismo , SuccinimidasRESUMO
Many natural products that show therapeutic activities are often difficult to synthesize or isolate and have unknown targets, hindering their development as drugs. Identifying druggable hotspots targeted by covalently acting anti-cancer natural products can enable pharmacological interrogation of these sites with more synthetically tractable compounds. Here, we used chemoproteomic platforms to discover that the anti-cancer natural product withaferin A targets C377 on the regulatory subunit PPP2R1A of the tumor-suppressor protein phosphatase 2A (PP2A) complex leading to activation of PP2A activity, inactivation of AKT, and impaired breast cancer cell proliferation. We developed a more synthetically tractable cysteine-reactive covalent ligand, JNS 1-40, that selectively targets C377 of PPP2R1A to impair breast cancer signaling, proliferation, and in vivo tumor growth. Our study highlights the utility of using chemoproteomics to map druggable hotspots targeted by complex natural products and subsequently interrogating these sites with more synthetically tractable covalent ligands for cancer therapy.