RESUMO
The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.
Assuntos
Anticorpos Antineoplásicos , Neoplasias Ovarianas , Anticorpos Monoclonais , Autoanticorpos , Autoantígenos , Feminino , Humanos , Neoplasias Ovarianas/genética , Microambiente TumoralRESUMO
Patients with loss of function in the gene encoding the master regulator of central tolerance AIRE suffer from a devastating disorder called autoimmune polyendocrine syndrome type 1 (APS-1), characterized by a spectrum of autoimmune diseases and severe mucocutaneous candidiasis. Although the key mechanisms underlying the development of autoimmunity in patients with APS-1 are well established, the underlying cause of the increased susceptibility to Candida albicans infection remains less understood. Here, we show that Aire+MHCII+ type 3 innate lymphoid cells (ILC3s) could sense, internalize and present C. albicans and had a critical role in the induction of Candida-specific T helper 17 (TH17) cell clones. Extrathymic Rorc-Cre-mediated deletion of Aire resulted in impaired generation of Candida-specific TH17 cells and subsequent overgrowth of C. albicans in the mucosal tissues. Collectively, our observations identify a previously unrecognized regulatory mechanism for effective defense responses against fungal infections.
Assuntos
Doenças Autoimunes , Candidíase , Poliendocrinopatias Autoimunes , Candida albicans , Candidíase/genética , Humanos , Imunidade Inata , Poliendocrinopatias Autoimunes/genética , Células Th17RESUMO
Protection from harmful pathogens depends on activation of the immune system, which relies on tight regulation of gene expression. Recently, the RNA modification N6-methyladenosine (m6A) has been found to play an essential role in such regulation. Here, we summarize newly discovered functions of m6A in controlling various aspects of immunity, including immune recognition, activation of innate and adaptive immune responses, and cell fate decisions. We then discuss some of the current challenges in the field and describe future directions for uncovering the immunological functions of m6A and its mechanisms of action.
Assuntos
Processamento Pós-Transcricional do RNA/imunologia , RNA/genética , Imunidade Adaptativa/genética , Adenosina/análogos & derivados , Adenosina/genética , Animais , Diferenciação Celular , Humanos , Sistema Imunitário , Imunidade Inata/genética , ImunomodulaçãoRESUMO
Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer's patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Seleção Clonal Mediada por Antígeno , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Protecting the upper airways and brain from viral invasion through the olfactory mucosa is critical. Wellford et al. describe a barrier that restricts the passage of circulating antibodies and prevents them from reaching the olfactory mucosa. Instead, plasma cells are recruited into this site and prevent viral infection of the airways and the brain through local antibody production.
Assuntos
Plasmócitos , Sistema Respiratório , EncéfaloRESUMO
Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1+ monocytes into lymphoid organs during Salmonella Typhimurium (STm) infection disrupted pre-existing germinal center (GC) reactions. GC responses induced by influenza, plasmodium, or commensals deteriorated following STm infection. GC disruption was independent of the direct bacterial interactions with B cells and instead was induced through recruitment of CCR2-dependent Sca-1+ monocytes into the lymphoid organs. GC collapse was associated with impaired cellular respiration and was dependent on TNFα and IFNγ, the latter of which was essential for Sca-1+ monocyte differentiation. Monocyte recruitment and GC disruption also occurred during LPS-supplemented vaccination and Listeria monocytogenes infection. Thus, systemic activation of the innate immune response upon severe bacterial infection is induced at the expense of antibody-mediated immunity.
Assuntos
Infecções Bacterianas , Listeriose , Linfócitos B , Centro Germinativo , Humanos , MonócitosRESUMO
Nasal vaccination elicits a humoral immune response that provides protection from airborne pathogens1, yet the origins and specific immune niches of antigen-specific IgA-secreting cells in the upper airways are unclear2. Here we define nasal glandular acinar structures and the turbinates as immunological niches that recruit IgA-secreting plasma cells from the nasal-associated lymphoid tissues (NALTs)3. Using intact organ imaging, we demonstrate that nasal vaccination induces B cell expansion in the subepithelial dome of the NALT, followed by invasion into commensal-bacteria-driven chronic germinal centres in a T cell-dependent manner. Initiation of the germinal centre response in the NALT requires pre-expansion of antigen-specific T cells, which interact with cognate B cells in interfollicular regions. NALT ablation and blockade of PSGL-1, which mediates interactions with endothelial cell selectins, demonstrated that NALT-derived IgA-expressing B cells home to the turbinate region through the circulation, where they are positioned primarily around glandular acinar structures. CCL28 expression was increased in the turbinates in response to vaccination and promoted homing of IgA+ B cells to this site. Thus, in response to nasal vaccination, the glandular acini and turbinates provide immunological niches that host NALT-derived IgA-secreting cells. These cellular events could be manipulated in vaccine design or in the treatment of upper airway allergic responses.
Assuntos
Linfócitos B , Centro Germinativo , Tecido Linfoide , Conchas Nasais , Animais , Camundongos , Tecido Linfoide/imunologia , Tecido Linfoide/citologia , Linfócitos B/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Feminino , Masculino , Centro Germinativo/imunologia , Centro Germinativo/citologia , Conchas Nasais/citologia , Conchas Nasais/imunologia , Vacinação , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Plasmócitos/imunologia , Plasmócitos/citologia , Plasmócitos/metabolismo , Quimiocinas CC/metabolismo , Camundongos Endogâmicos C57BL , Administração Intranasal , Movimento CelularRESUMO
Somatic hypermutation (SHM) and class-switch recombination (CSR) increase the affinity and diversify the effector functions of antibodies during immune responses. Although SHM and CSR are fundamentally different, their independent roles in regulating B cell fate have been difficult to uncouple because a single enzyme, activation-induced cytidine deaminase (encoded by Aicda), initiates both reactions. Here, we used a combination of Aicda and antibody mutant alleles that separate the effects of CSR and SHM on polyclonal immune responses. We found that class-switching to IgG1 biased the fate choice made by B cells, favoring the plasma cell over memory cell fate without significantly affecting clonal expansion in the germinal center (GC). In contrast, SHM reduced the longevity of memory B cells by creating polyreactive specificities that were selected against over time. Our data define the independent contributions of SHM and CSR to the generation and persistence of memory in the antibody system.
Assuntos
Linfócitos B/imunologia , Citidina Desaminase/genética , Switching de Imunoglobulina/imunologia , Memória Imunológica/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Linfócitos B/citologia , Diferenciação Celular/imunologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Switching de Imunoglobulina/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hipermutação Somática de Imunoglobulina/genéticaRESUMO
Reexposure to a pathogen triggers the activation of memory T cells that have already encountered a similar microbe. These long-lived CD4 T cells either circulate through the blood and tissues or reside within organs and are referred to as tissue-resident T cells (CD4 TRM ). In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: 2250247] issue, Curham et al. found that tissue-resident memory CD4 T cells in the lung and nasal tissues can respond to noncognate immune challenges. CD4 TRM cells, which were formed in response to Bordetella pertussis, proliferated and produced IL-17A in response to a secondary challenge with heat-killed Klebsiella pneumonia or lipopolysaccharide (LPS). This bystander response depends on the presence of dendritic cells that provide inflammatory cytokines. Furthermore, post K. pneumonia, intranasal immunization with whole cell pertussis vaccine reduced bacterial burden in the nasal tissue in a CD4 T-cell-dependent manner. The study indicates that the noncognate activation of TRM may serve as an innate-like immune response that rapidly develops before establishing a new pathogen-specific adaptive immune response.
Assuntos
Linfócitos T CD4-Positivos , Células T de Memória , Humanos , Amigos , Bordetella pertussis , Vacina contra Coqueluche , Memória ImunológicaRESUMO
Enduring immunity against harmful pathogens depends on the generation of immunological memory. Serum immunoglobulins are constantly secreted by long-lived antibody-producing cells, which provide extended protection from recurrent exposures. These cells originate mainly from germinal center structures, wherein B cells introduce mutations to their immunoglobulin genes followed by affinity-based selection. Generation of high-affinity antibodies relies on physical contacts between T and B cells, a process that facilitates the delivery of fate decision signals. T-B cellular engagements are mediated through interactions between the T cell receptor and its cognate peptide presented on B cell major histocompatibility class II molecules. Here, we describe the cellular and molecular aspects of these cognate T-B interactions, and highlight exceptional cases, especially those arising at intestinal lymphoid organs, at which T cells provide help to B cells in an atypical manner, independent of T cell specificity.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Comunicação Celular/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/imunologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Imunidade Celular , Imunidade Humoral , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of "turn-on" probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.
Assuntos
Acetamidas , Inibidores de Proteínas Quinases , Camundongos , Animais , Ligantes , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact-dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34(+) progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP-protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells.
Assuntos
Células da Medula Óssea/imunologia , Quimiocina CXCL12/imunologia , Conexinas/imunologia , Junções Comunicantes/imunologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Mesenquimais/imunologia , Células Estromais/imunologia , Animais , Cálcio/imunologia , Movimento Celular/imunologia , Técnicas de Cocultura , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Proteínas ral de Ligação ao GTP/imunologiaRESUMO
Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.
Assuntos
Quimiocinas/metabolismo , Células Endoteliais/metabolismo , Linfócitos/imunologia , Migração Transendotelial e Transepitelial/imunologia , Vesículas Transportadoras/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Integrinas/metabolismo , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Camundongos , Receptores CCR2/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/ultraestrutura , Fator de Necrose Tumoral alfa/farmacologia , Vasculite/imunologia , Vasculite/metabolismoRESUMO
The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Stress-induced ß2-adrenergic receptor (ß2AR) activation in B cells increases IgG secretion; however, the impact of this activation on antibody affinity and the underlying mechanisms remains unclear. In the current study, we demonstrate that stress in mice following ovalbumin (OVA) or SARS-CoV-2 RBD immunization significantly increases both serum and surface-expressed IgG binding to the immunogen, while concurrently reducing surface IgG expression and B cell clonal expansion. These effects were abolished by pharmacological ß2AR blocking or when the experiments were conducted in ß2AR -/- mice. In the second part of our study, we used single B cell sorting to characterize the monoclonal antibodies (mAbs) generated following ß2AR activation in cultured RBD-stimulated B cells from convalescent SARS-CoV-2 donors. Ex vivo ß2AR activation increased the affinities of the produced anti-RBD mAbs by 100-fold compared to mAbs produced by the same donor control cultures. Consistent with the mouse experiments, ß2AR activation reduced both surface IgG levels and the frequency of expanded clones. mRNA sequencing revealed a ß2AR-dependent upregulation of the PI3K pathway and B cell receptor (BCR) signaling through AKT phosphorylation, as well as an increased B cell motility. Overall, our study demonstrates that stress-mediated ß2AR activation drives changes in B cells associated with BCR activation and higher affinity antibodies.
Assuntos
Adrenérgicos , COVID-19 , Camundongos , Animais , Fosfatidilinositol 3-Quinases , SARS-CoV-2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Imunoglobulina GRESUMO
Establishment of effective immunity against invading microbes depends on continuous generation of antibodies that facilitate pathogen clearance. Long-lived plasma cells with the capacity to produce high affinity antibodies evolve in germinal centers (GCs), where B cells undergo somatic hypermutation and are subjected to affinity-based selection. Here, we focus on the cellular interactions that take place early in the antibody immune response during GC colonization. Clones bearing B-cell receptors with different affinities and specificities compete for entry to the GC, at the boundary between the B-cell and T-cell zones in lymphoid organs. During this process, B cells compete for interactions with T follicular helper cells, which provide selection signals required for differentiation into GC cells and antibody secreting cells. These cellular engagements are long-lasting and depend on activation of adhesion molecules that support persistent interactions and promote transmission of signals between the cells. Here, we discuss how interactions between cognate T and B cells are primarily maintained by three types of molecular interactions: homophilic signaling lymphocytic activation molecule (SLAM) interactions, T-cell receptor: peptide-loaded major histocompatibility class II (pMHCII), and LFA-1:ICAMs. These essential components support a three-step process that controls clonal selection for entry into the antibody affinity maturation response in the GC, and establishment of long-lasting antibody-mediated immunity.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Modelos Imunológicos , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antígenos/imunologia , Diferenciação Celular , Humanos , Imunidade Humoral , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismoRESUMO
Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive "turn-on" fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.
Assuntos
Tirosina Quinase da Agamaglobulinemia/química , Corantes Fluorescentes/química , Ligantes , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Tirosina Quinase da Agamaglobulinemia/metabolismo , Domínio Catalítico , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Meia-Vida , Humanos , Piperidinas/química , Piperidinas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , SARS-CoV-2/enzimologiaRESUMO
Targeted covalent inhibitors are an important class of drugs and chemical probes. However, relatively few electrophiles meet the criteria for successful covalent inhibitor design. Here we describe α-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically α-substitutions inactivate acrylamides, we show that hetero α-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition-elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pKa/pKb of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition-elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and K-RasG12C. We further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK inhibitors. Altogether we show that α-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted covalent inhibitor design.
RESUMO
Proteolysis targeting chimeras (PROTACs) represent an exciting inhibitory modality with many advantages, including substoichiometric degradation of targets. Their scope, though, is still limited to date by the requirement for a sufficiently potent target binder. A solution that proved useful in tackling challenging targets is the use of electrophiles to allow irreversible binding to the target. However, such binding will negate the catalytic nature of PROTACs. Reversible covalent PROTACs potentially offer the best of both worlds. They possess the potency and selectivity associated with the formation of the covalent bond, while being able to dissociate and regenerate once the protein target is degraded. Using Bruton's tyrosine kinase (BTK) as a clinically relevant model system, we show efficient degradation by noncovalent, irreversible covalent, and reversible covalent PROTACs, with <10 nM DC50's and >85% degradation. Our data suggest that part of the degradation by our irreversible covalent PROTACs is driven by reversible binding prior to covalent bond formation, while the reversible covalent PROTACs drive degradation primarily by covalent engagement. The PROTACs showed enhanced inhibition of B cell activation compared to ibrutinib and exhibit potent degradation of BTK in patient-derived primary chronic lymphocytic leukemia cells. The most potent reversible covalent PROTAC, RC-3, exhibited enhanced selectivity toward BTK compared to noncovalent and irreversible covalent PROTACs. These compounds may pave the way for the design of covalent PROTACs for a wide variety of challenging targets.