ABSTRACT
Throughout the body, T cells monitor MHC-bound ligands expressed on the surface of essentially all cell types. MHC ligands that trigger a T cell immune response are referred to as T cell epitopes. Identifying such epitopes enables tracking, phenotyping, and stimulating T cells involved in immune responses in infectious disease, allergy, autoimmunity, transplantation, and cancer. The specific T cell epitopes recognized in an individual are determined by genetic factors such as the MHC molecules the individual expresses, in parallel to the individual's environmental exposure history. The complexity and importance of T cell epitope mapping have motivated the development of computational approaches that predict what T cell epitopes are likely to be recognized in a given individual or in a broader population. Such predictions guide experimental epitope mapping studies and enable computational analysis of the immunogenic potential of a given protein sequence region.
Subject(s)
Epitopes, T-Lymphocyte/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Biomarkers , Computational Biology/methods , Disease Susceptibility , Histocompatibility Antigens/immunology , Humans , Ligands , Machine Learning , Protein BindingABSTRACT
Given the many cell types and molecular components of the human immune system, along with vast variations across individuals, how should we go about developing causal and predictive explanations of immunity? A central strategy in human studies is to leverage natural variation to find relationships among variables, including DNA variants, epigenetic states, immune phenotypes, clinical descriptors, and others. Here, we focus on how natural variation is used to find patterns, infer principles, and develop predictive models for two areas: (a) immune cell activation-how single-cell profiling boosts our ability to discover immune cell types and states-and (b) antigen presentation and recognition-how models can be generated to predict presentation of antigens on MHC molecules and their detection by T cell receptors. These are two examples of a shift in how we find the drivers and targets of immunity, especially in the human system in the context of health and disease.
Subject(s)
Immune System , Immunity , Animals , Antigen Presentation/immunology , Biomarkers , Disease Susceptibility/immunology , Disease Susceptibility/metabolism , Epitopes/immunology , Genomics/methods , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immune System/cytology , Immune System/physiology , Ligands , Major Histocompatibility Complex/genetics , Major Histocompatibility Complex/immunology , Peptides/immunology , Protein Transport , Proteolysis , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Molecular docking has become an essential part of a structural biologist's and medicinal chemist's toolkits. Given a chemical compound and the three-dimensional structure of a molecular target-for example, a protein-docking methods fit the compound into the target, predicting the compound's bound structure and binding energy. Docking can be used to discover novel ligands for a target by screening large virtual compound libraries. Docking can also provide a useful starting point for structure-based ligand optimization or for investigating a ligand's mechanism of action. Advances in computational methods, including both physics-based and machine learning approaches, as well as in complementary experimental techniques, are making docking an even more powerful tool. We review how docking works and how it can drive drug discovery and biological research. We also describe its current limitations and ongoing efforts to overcome them.
Subject(s)
Drug Discovery , Molecular Docking Simulation , Protein Binding , Proteins , Ligands , Drug Discovery/methods , Humans , Proteins/chemistry , Proteins/metabolism , Machine Learning , Binding Sites , Drug DesignABSTRACT
The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.
Subject(s)
Allosteric Site , Cryoelectron Microscopy , Molecular Dynamics Simulation , Receptors, Nicotinic , Signal Transduction , Receptors, Nicotinic/metabolism , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Allosteric Regulation , Humans , Animals , Crystallography, X-Ray , Binding Sites , Protein Conformation , Ligands , Models, Molecular , Protein Multimerization , Nicotinic Agonists/chemistry , Nicotinic Agonists/pharmacology , Nicotinic Agonists/metabolismABSTRACT
Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.
Subject(s)
Cell Differentiation , Fibroblast Growth Factors , Receptors, Fibroblast Growth Factor , Signal Transduction , Animals , Humans , Receptors, Fibroblast Growth Factor/metabolism , Fibroblast Growth Factors/metabolism , Mice , Ligands , Calcium/metabolism , MAP Kinase Signaling SystemABSTRACT
Effective treatments for complex central nervous system (CNS) disorders require drugs with polypharmacology and multifunctionality, yet designing such drugs remains a challenge. Here, we present a flexible scaffold-based cheminformatics approach (FSCA) for the rational design of polypharmacological drugs. FSCA involves fitting a flexible scaffold to different receptors using different binding poses, as exemplified by IHCH-7179, which adopted a "bending-down" binding pose at 5-HT2AR to act as an antagonist and a "stretching-up" binding pose at 5-HT1AR to function as an agonist. IHCH-7179 demonstrated promising results in alleviating cognitive deficits and psychoactive symptoms in mice by blocking 5-HT2AR for psychoactive symptoms and activating 5-HT1AR to alleviate cognitive deficits. By analyzing aminergic receptor structures, we identified two featured motifs, the "agonist filter" and "conformation shaper," which determine ligand binding pose and predict activity at aminergic receptors. With these motifs, FSCA can be applied to the design of polypharmacological ligands at other receptors.
Subject(s)
Cheminformatics , Drug Design , Polypharmacology , Animals , Mice , Humans , Cheminformatics/methods , Ligands , Receptor, Serotonin, 5-HT2A/metabolism , Receptor, Serotonin, 5-HT2A/chemistry , Receptor, Serotonin, 5-HT1A/metabolism , Receptor, Serotonin, 5-HT1A/chemistry , Male , Binding SitesABSTRACT
Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin ß-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.
Subject(s)
Integrins , Talin , Animals , Humans , Mice , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/chemistry , Cell Adhesion , CHO Cells , Cricetulus , Integrins/metabolism , Integrins/chemistry , Ligands , Protein Binding , Protein Conformation , Signal Transduction , Single Molecule Imaging , Talin/metabolism , Talin/chemistryABSTRACT
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.
Subject(s)
Cysteine , Neoplasms , Animals , Humans , Mice , Cell Line, Tumor , Cysteine/metabolism , Cysteine/chemistry , Ligands , Melanoma/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , NF-kappa B/chemistry , NF-kappa B/metabolism , Oxidation-Reduction , Signal Transduction , SOXE Transcription Factors/chemistry , SOXE Transcription Factors/metabolismABSTRACT
The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked â¼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.
Subject(s)
Aminophenols , Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Molecular Docking Simulation , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Cystic Fibrosis/drug therapy , Cystic Fibrosis/metabolism , Aminophenols/pharmacology , Aminophenols/chemistry , Aminophenols/therapeutic use , Drug Discovery , Cryoelectron Microscopy , Quinolones/pharmacology , Quinolones/chemistry , Quinolones/therapeutic use , Allosteric Site/drug effects , Animals , LigandsABSTRACT
Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception. On target cells, a signaling circuit composed of a cholesterol transporter and sensor regulates transmission of HH signals across the plasma membrane to the cytoplasm. The repeated use of cholesterol in signaling supports the view that the HH pathway likely evolved by coopting ancient systems to regulate the abundance or organization of sterol-like lipids in membranes.
Subject(s)
Cholesterol , Hedgehog Proteins , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Ligands , Cholesterol/metabolism , Signal Transduction , Sterols/metabolismABSTRACT
The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogenetically conserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of the kidney and brain. Here, we report high-resolution cryoelectron microscopy structures of LRP2 isolated from mouse kidney, at extracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligand binding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformation of which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleterious missense variants in humans appears to impair homodimer assembly. These observations lay the foundation for further understanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRP receptor subfamily.
Subject(s)
Endocytosis , Low Density Lipoprotein Receptor-Related Protein-2 , Animals , Humans , Mice , Cryoelectron Microscopy , Kidney/metabolism , Ligands , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Low Density Lipoprotein Receptor-Related Protein-2/metabolismABSTRACT
Receptors of the innate immune system detect conserved determinants of microbial and viral origin. Activation of these receptors initiates signaling events that culminate in an effective immune response. Recently, the view that innate immune signaling events rely on and operate within a complex cellular infrastructure has become an important framework for understanding the regulation of innate immunity. Compartmentalization within this infrastructure provides the cell with the ability to assign spatial information to microbial detection and regulate immune responses. Several cell biological processes play a role in the regulation of innate signaling responses; at the same time, innate signaling can engage cellular processes as a form of defense or to promote immunological memory. In this review, we highlight these aspects of cell biology in pattern-recognition receptor signaling by focusing on signals that originate from the cell surface, from endosomal compartments, and from within the cytosol.
Subject(s)
Immunity, Innate/physiology , Receptors, Pattern Recognition/metabolism , Animals , Biosynthetic Pathways , Cell Membrane/metabolism , Endosomes/metabolism , Humans , Ligands , Signal TransductionABSTRACT
The TAM receptor tyrosine kinases (RTKs)-TYRO3, AXL, and MERTK-together with their cognate agonists GAS6 and PROS1 play an essential role in the resolution of inflammation. Deficiencies in TAM signaling have been associated with chronic inflammatory and autoimmune diseases. Three processes regulated by TAM signaling may contribute, either independently or collectively, to immune homeostasis: the negative regulation of the innate immune response, the phagocytosis of apoptotic cells, and the restoration of vascular integrity. Recent studies have also revealed the function of TAMs in infectious diseases and cancer. Here, we review the important milestones in the discovery of these RTKs and their ligands and the studies that underscore the functional importance of this signaling pathway in physiological immune settings and disease.
Subject(s)
Homeostasis , Immunity/physiology , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Animals , Disease Susceptibility , Humans , Ligands , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/geneticsABSTRACT
The observation that a subset of cancer patients show evidence for spontaneous CD8+ T cell priming against tumor-associated antigens has generated renewed interest in the innate immune pathways that might serve as a bridge to an adaptive immune response to tumors. Manipulation of this endogenous T cell response with therapeutic intent-for example, using blocking antibodies inhibiting PD-1/PD-L1 (programmed death-1/programmed death ligand 1) interactions-is showing impressive clinical results. As such, understanding the innate immune mechanisms that enable this T cell response has important clinical relevance. Defined innate immune interactions in the cancer context include recognition by innate cell populations (NK cells, NKT cells, and γδ T cells) and also by dendritic cells and macrophages in response to damage-associated molecular patterns (DAMPs). Recent evidence has indicated that the major DAMP driving host antitumor immune responses is tumor-derived DNA, sensed by the stimulator of interferon gene (STING) pathway and driving type I IFN production. A deeper knowledge of the clinically relevant innate immune pathways involved in the recognition of tumors is leading toward new therapeutic strategies for cancer treatment.
Subject(s)
Immunity, Innate , Neoplasms/immunology , Neoplasms/metabolism , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Complement System Proteins/immunology , Complement System Proteins/metabolism , Cytotoxicity, Immunologic , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Immune System/cytology , Immunotherapy , Ligands , Macrophage Activation , Macrophages/immunology , Macrophages/metabolism , Microbiota , Neoplasms/microbiology , Neoplasms/therapy , Signal TransductionABSTRACT
Integrin receptors are established drug targets, but many of the drugs that have been developed act as partial agonists, inducing the receptor into a high-affinity, ligand-binding state. Lin et al. discovered a general mechanism to circumvent this problem-stabilizing a key water molecule that prevents receptor activation. Their findings are likely to impact future therapeutic development.
Subject(s)
Integrins , Water , Integrins/chemistry , LigandsABSTRACT
Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.
Subject(s)
Aedes , Olfactory Receptor Neurons , Aedes/genetics , Animals , Humans , Ligands , OdorantsABSTRACT
Cerebrospinal fluid (CSF) contains a tightly regulated immune system. However, knowledge is lacking about how CSF immunity is altered with aging or neurodegenerative disease. Here, we performed single-cell RNA sequencing on CSF from 45 cognitively normal subjects ranging from 54 to 82 years old. We uncovered an upregulation of lipid transport genes in monocytes with age. We then compared this cohort with 14 cognitively impaired subjects. In cognitively impaired subjects, downregulation of lipid transport genes in monocytes occurred concomitantly with altered cytokine signaling to CD8 T cells. Clonal CD8 T effector memory cells upregulated C-X-C motif chemokine receptor 6 (CXCR6) in cognitively impaired subjects. The CXCR6 ligand, C-X-C motif chemokine ligand 16 (CXCL16), was elevated in the CSF of cognitively impaired subjects, suggesting CXCL16-CXCR6 signaling as a mechanism for antigen-specific T cell entry into the brain. Cumulatively, these results reveal cerebrospinal fluid immune dysregulation during healthy brain aging and cognitive impairment.
Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Neurodegenerative Diseases , Humans , Middle Aged , Aged , Aged, 80 and over , Ligands , Brain , Aging , Lipids , BiomarkersABSTRACT
Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.
Subject(s)
Receptors, Antigen, T-Cell , Virus Internalization , Humans , Biology , Epitopes , Ligands , Peptides , Receptors, Antigen, T-Cell/metabolism , Single-Cell Analysis , GenomicsABSTRACT
Different opioid ligands can result in biased µ-opioid signaling, differentially activating signal cascades which produce analgesia, tolerance, or adverse effects. In this issue of Cell, Xu et al. used cryo-EM and computational simulations to understand how different µ-opioid receptor selective-ligands interact with key residues to produce downstream signaling.
Subject(s)
Analgesics, Opioid , Pain , Humans , Analgesics, Opioid/adverse effects , Ligands , Signal Transduction , Receptors, Opioid, mu/metabolismABSTRACT
Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.