Receptor Extracellular Domain Research Articles

Landscape of FLT3 Variations Associated with Structural and Functional Impact on Acute Myeloid Leukemia: A Computational Study.

Acute myeloid leukemia (AML) is hallmarked by the clonal proliferation of myeloid blasts. Mutations that result in the constitutive activation of the fms-like tyrosine kinase 3 (FLT3) gene, coding for a class III receptor tyrosine kinase, are significantly associated with this heterogeneous hematologic malignancy. The fms-related tyrosine kinase 3 ligand binds to the extracellular domain of the FLT3 receptor, inducing homodimer formation in the plasma membrane, leading to autophosphorylation and activation of apoptosis, proliferation, and differentiation of hematopoietic cells in bone marrow. In the present study, we evaluated the association of FLT3 as a significant biomarker for AML and tried to comprehend the effects of specific variations on the FLT3 protein's structure and function. We also examined the effects of I836 variants on binding affinity to sorafenib using molecular docking. We integrated multiple bioinformatics tools, databases, and resources such as OncoDB, UniProt, COSMIC, UALCAN, PyMOL, ProSA, Missense3D, InterProScan, SIFT, PolyPhen, and PredictSNP to annotate the structural, functional, and phenotypic impact of the known variations associated with FLT3. Twenty-nine FLT3 variants were analyzed using in silico approaches such as DynaMut, CUPSAT, AutoDock, and Discovery Studio for their impact on protein stability, flexibility, function, and binding affinity. The OncoDB and UALCAN portals confirmed the association of FLT3 gene expression and its mutational status with AML. A computational structural analysis of the deleterious variants of FLT3 revealed I863F mutants as destabilizers of the protein structure, possibly leading to functional changes. Many single-nucleotide variations in FLT3 have an impact on its structure and function. Thus, the annotation of FLT3 SNVs and the prediction of their deleterious pathogenic impact will facilitate an insight into the tumorigenesis process and guide experimental studies and clinical implications.

Synthesis of R-GABA Derivatives via Pd(II) Catalyzed Enantioselective C(sp3)-H Arylation and Virtual Validation with GABAB1 Receptor for Potential leads.

GABA (γ-amino butyric acid) analogues like baclofen, tolibut, phenibut, etc., are well-known GABAB1 inhibitors and pharmaceutically important drugs. However, there is a huge demand for more chiral GABA aryl analogues with promising pharmacological actions. Here, we demonstrate the chiral ligand acetyl-protected amino quinoline (APAQ) mediated enantioselective synthesis of GABAB1 inhibitor drug scaffolds from easily accessible GABA via Pd-catalyzed C(sp3)-H activation. The synthetic methodology shows moderate to good yields, up to 74% of ee. We have successfully demonstrated the deprotection and removal of the directing group to synthesize R-tolibut in 86% yield. Further, we employed computation to probe the binding of R-GABA analogues to the extracellular domain of the human GABAB1 receptor. Our Rosetta-based molecular docking calculations show better binding for four R-enantiomers of GABA analogues than R-baclofen and R-phenibut. In addition, we employed GROMACS MD simulations and MMPB(GB)SA calculations to identify per-residue contribution to binding free energy. Our computational results suggest analogues (3R)-4-amino-3-(3,4-dimethylphenyl) butanoic acid, (3R)-4-amino-3-(3-fluorophenyl) butanoic acid, (3R)-3-(4-acetylphenyl)-4-aminobutanoic acid, (3R)-4-amino-3-(4-methoxyphenyl) butanoic acid, and (3R)-4-amino-3-phenylbutanoic acid are potential leads which could be synthesized from our methodology reported here.

Glycine Receptor β-Targeting Autoantibodies Contribute to the Pathology of Autoimmune Diseases.

Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit-binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRβ subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology. In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRβ. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRβ binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRβ aAb binding were resolved by whole-cell patch-clamp recordings. Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRβ aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRβ colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRβ aAb from both patients to its target impair glycine efficacy. Our study establishes GlyRβ as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRβ impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRβ aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization.

Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL.

Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3ECD) are the 2 core features of CADASIL, a common cerebral small vessel disease caused by highly stereotyped dominant mutations in NOTCH3. Yet the relationship between NOTCH3 receptor activity, Notch3ECD accumulation, and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using dedicated histopathological and multiscale imaging modalities, we could detect and quantify previously undetectable CADASIL-driven arterial SMC loss in the CNS of mice expressing the archetypal Arg169Cys mutation. We found that arterial pathology was more severe and Notch3ECD accumulation greater in transgenic mice overexpressing the mutation on a wild-type Notch3 background (TgNotch3R169C) than in knockin Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. Notably, expression of Notch3-regulated genes was essentially unchanged in TgNotch3R169C arteries. We further showed that wild-type Notch3ECD coaggregated with mutant Notch3ECD and that elimination of 1 copy of wild-type Notch3 in TgNotch3R169C was sufficient to attenuate Notch3ECD accumulation and arterial pathology. These findings suggest that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, is a major driver of arterial SMC loss in CADASIL, paving the way for NOTCH3-lowering therapeutic strategies.

Inhaled delivery of cetuximab-conjugated immunoliposomes loaded with afatinib: A promising strategy for enhanced non-small cell lung cancer treatment.

Afatinib (AT), an FDA-approved aniline-quinazoline derivative, is a first-line treatment for metastatic non-small cell lung cancer (NSCLC). Combining it with cetuximab (CX), a chimeric human-murine derivative immunoglobulin-G1 monoclonal antibody (mAb) targeting the extracellular domain of epidermal growth factor receptor (EGFR), has shown significant improvements in median progression-free survival. Previously, we developed cetuximab-conjugated immunoliposomes loaded with afatinib (AT-MLP) and demonstrated their efficacy against NSCLC cells (A549 and H1975). In this study, we aimed to explore the potential of pulmonary delivery to mitigate adverse effects associated with oral administration and intravenous injection. We formulated AT-MLP dry powders (AT-MLP-DPI) via freeze drying using tert-butanol and mannitol as cryoprotectants in the hydration medium. The physicochemical and aerodynamic properties of dry powders were well analyzed firstly. In vitro cellular uptake and cytotoxicity study revealed concentration- and time-dependent cellular uptake behavior and antitumor efficacy of AT-MLP-DPI, while Transwell assay demonstrated the superior inhibitory effects on NSCLC cell invasion and migration. Furthermore, in vivo pharmacokinetic study showed that pulmonary delivery of AT-MLP-DPI significantly increased bioavailability, prolonged blood circulation time, and exhibited higher lung concentrations compared to alternative administration routes and formulations. The in vivo antitumor efficacy study carried on tumor-bearing nude mice indicated that inhaled AT-MLP-DPI effectively suppressed lung tumor growth.

ADAM19 cleaves the PTH receptor and associates with brachydactyly type E.

Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64-65. ADAM-19 cleavage increased Gq and decreased Gs activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.

Cryo-EM structure of the extracellular domain of murine Thrombopoietin Receptor in complex with Thrombopoietin

Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric Class I cytokine receptor) and initiating cell proliferation or differentiation. Here we characterise the murine Tpo:TpoR signalling complex biochemically and structurally, using cryo-electron microscopy. Tpo uses opposing surfaces to recruit two copies of receptor, forming a 1:2 complex. Although it binds to the same, membrane-distal site on both receptor chains, it does so with significantly different affinities and its highly glycosylated C-terminal domain is not required. In one receptor chain, a large insertion, unique to TpoR, forms a partially structured loop that contacts cytokine. Tpo binding induces the juxtaposition of the two receptor chains adjacent to the cell membrane. The therapeutic agent romiplostim also targets the cytokine-binding site and the characterisation presented here supports the future development of improved TpoR agonists.

Unrevealing the multitargeted potency of 3-1-BCMIYPPA against lung cancer structural maintenance and suppression proteins through pharmacokinetics, QM-DFT, and multiscale MD simulation studies.

Lung cancer, a relentless and challenging disease, demands unwavering attention in drug design research. Single-target drugs have yielded limited success, unable to effectively address this malignancy's profound heterogeneity and often developed resistance. Consequently, the clarion call for lung cancer drug design echoes louder than ever, and multitargeted drug design emerges as an imperative approach in this landscape, which is done by concurrently targeting multiple proteins and pathways and offering a beacon of hope. This study is focused on the multitargeted drug designing approach by identifying drug candidates against human cyclin-dependent kinase-2, SRC-2 domains of C-ABL, epidermal growth factor and receptor extracellular domains, and insulin-like growth factor-1 receptor kinase. We performed the multitargeted molecular docking studies of Drug Bank compounds using HTVS, SP and XP algorithms and poses filter with MM\GBSA against all proteins and identified DB02504, namely [3-(1-Benzyl-3-Carbamoylmethyl-2-Methyl-1h-Indol-5-Yloxy)-Propyl-]-Phosphonic Acid (3-1-BCMIYPPA) as multitargeted lead with docking and MM\GBSA score range from -8.242 to -6.274 and -28.2 and -44.29 Kcal/mol, respectively. Further, the QikProp-based pharmacokinetic computations and QM-based DFT showed acceptance results against standard values, and interaction fingerprinting reveals that THR, MET, GLY, VAL, LEU, GLU and ASP were among the most interacting residues. The NPT ensemble-based 100ns MD simulation in a neutralised state with an SPC water model has also shown a stable performance and produced deviation and fluctuations <2Å with huge interactions, making it a promising multitargeted drug candidate-however, experimental studies are suggested.

Effects of tea catechin extracts from BB35 and purple (TRFK 306) tea clones on the gene expression of Egfr, App, Bcl2, Dnmt, Casp3, Hif1a, Gadd45b and Psmb5 genes involved in triple negative breast cancer diseases: In silico and in vitro study

Tea has been shown to contain metabolites that exhibit antioxidant, anti-inflammatory, anti-diabetic, anti-cancer and anti-cardio vascular diseases properties. This study aimed to carry out an in silico assessment of catechins on the genes previously shown to be involved in Triple Negative Breast Cancer (TNBC) disease and also to evaluate the effects of catechin extracts from purple (TRFK306) and BB35 tea clones on the expression patterns of genes in the 4T1 TNBC cell line as mentioned above. Identification and quantification of different catechin contents in these two tea clones were performed by using High-Performance Liquid Chromatography (HPLC). In silico assessment including Absorption, Distribution, Metabolism and Excretion (ADME) study for drug-likeness evaluation, Drug target prediction, Protein-protein interaction (PPI) network analysis and construction, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment and analysis for targets and Molecular docking study was conducted. Simplified Molecular Input Line Entry System (SMILES) of 4 different tea catechins showed a target on 111 genes in TNBC and have high binding affinity to the receptor-binding pocket of chain A of Epidermal Growth Factor Receptor (EGFR) Extracellular Domains. Catechin extracts promoted the downregulation of expression of 6 genes (Egfr, App, Bcl2, Dnmt, Hif1a and Psmb5) and the upregulation of 2 genes (Casp3 and Gadd45b) in the 4T1 TNBC cell line. Catechin extracts from purple tea clones showed higher activity on gene expression levels of Egfr, Bcl2 and Casp3 than catechin extracts from BB35 tea clone.

Splice-Switching Antisense Oligonucleotides Targeting Extra- and Intracellular Domains of Epidermal Growth Factor Receptor in Cancer Cells.

Cancer is one of the leading causes of death globally. Epidermal growth factor receptor is one of the proteins involved in cancer cell proliferation, differentiation, and invasion. Antisense oligonucleotides are chemical nucleic acids that bind to target messenger ribonucleic acid and modulate its expression. Herein, we demonstrate the efficacy of splice-modulating antisense oligonucleotides to target specific exons in the extracellular (exon 3) and intracellular (exon 18, 21) domains of epidermal growth factor receptor. These antisense oligonucleotides were synthesized as 25mer 2'-O methyl phosphorothioate-modified ribonucleic acids that bind to complementary specific regions in respective exons. We found that PNAT524, PNAT525, PNAT576, and PNAT578 effectively skipped exon 3, exon 18, and exon 21 in glioblastoma, liver cancer, and breast cancer cell lines. PNAT578 treatment also skipped partial exon 19, complete exon 20, and partial exon 21 in addition to complete exon 21 skipping. We also found that a cocktail of PNAT576 and PNAT578 antisense oligonucleotides performed better than their individual counterparts. The migration potential of glioblastoma cancer cells was reduced to a greater extent after treatment with these antisense oligonucleotides. We firmly believe that using these splice-modulating antisense oligonucleotides in combination with existing EGFR-targeted therapies could improve therapeutic outcomes.

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

SELEX-discovered aptamer that inhibits cellular interleukin-17/interleukin-17 receptor interaction and antagonizes interleukin-17 signaling.

This research is based on a Systematic Evolution of Ligands by EXponential enrichment, also referred to as in vitro selection against the extracellular domain of human interleukin-17 receptor A (IL-17RA). Pull-down assay via quantitative polymerase chain reaction and chemiluminescence detection showed that the cloned RNA with the enriched sequence bound to human IL-17RA and inhibited the interaction between IL-17RA and human interleukin-17A (IL-17A). We also revealed that the newly discovered IL-17RA-binding RNA aptamer bound to cellular IL-17RA, inhibited the cellular IL-17RA/IL-17A interaction, and antagonized cellular IL-17A signaling.

TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors

Notch signaling is an evolutionarily conserved mechanism required for numerous types of cell fate decisions in metazoans. It mediates short-range communication between cells with receptors and ligands, both of which are expressed on the cell surfaces. In response to the ligand-receptor interaction, the ligand and the extracellular domain of the Notch receptor (NECD) in the complex are internalized into ligand-expressing cells by endocytosis, a prerequisite process for the conformational change of the membrane proximal region of Notch to induce critical proteolytic cleavages for its activation. Here we report that overexpression of transmembrane 2 (TM2) domain containing 3 (TM2D3), a mammalian homologue of Drosophila melanogaster Almondex (Amx), activates Notch1. This activation requires the ligand-binding domain in Notch1 and the C-terminal region containing TM2 domain in TM2D3. TM2D3 physically associates with Notch1 at the region distinct from the ligand-binding domain and enhances expression of Notch1 on the cell surface. Furthermore, cell surface expression of Notch1 and Notch2 is reduced in Tm2d3-deficient cells. Finally, amx-deficient Drosophila early embryos exhibit impaired endocytosis of NECD and Delta ligand, for which surface presentation of Notch is required. These results indicate that TM2D3 is an element involved in Notch signaling through the surface presentation.

Pneumococcus downregulates the molecular weight of the extracellular domain of the epidermal growth factor receptor of alveolar epithelial cells.

Pneumococcus is themajor cause of bacterial and invasive pneumococcal infections. Disrupting the alveolarepithelial barrier is an important step in the pathogenesis of invasivepneumococcal infections. The epidermal growth factor receptor (EGFR) maintainsthe integrity of the alveolar epithelial barrier. In this study, we showed that secretory pneumococcal molecules decrease the molecular weight of EGFR without peptide degradation and inhibit alveolar epithelial cell proliferation via EGFR.

A large in-frame deletion of the calcium-sensing receptor extracellular domain causes familial hypocalciuric hypercalcaemia type 1 (FHH1) and is partially responsive to cinacalcet

A large in-frame deletion of the calcium-sensing receptor extracellular domain causes familial hypocalciuric hypercalcaemia type 1 (FHH1) and is partially responsive to cinacalcet

Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis

The interleukin 6 (IL6) signaling pathway plays pleiotropic roles in regulating the inflammatory milieu that contributes to arthritis development. Here, we show that activation of IL6 trans-signaling induces phenotypic transitions in tissue-resident cells toward an inflammatory state. The establishment of arthritis increases the serum number of extracellular vesicles (EVs), while these EVs express more IL6 signal transducer (IL6ST, also known as gp130) on their surface. Transferring these EVs can block IL6 trans-signaling invitro by acting as decoys that trap hyper IL6 and prevent inflammatory amplification in recipient arthritic mice. By genetically fusing EV-sorting domains with extracellular domains of receptors, we engineered EVs that harbor a higher quantity of signaling-incompetent decoy receptors. These exogenous decoy EVs exhibit significant potential in eliciting efficient anti-inflammatory effects invivo. Our findings suggest an inherent resistance of decoy EVs against inflammation, highlighting the therapeutic potential of efficient decoy EVs in treating inflammatory diseases.

Comparison of the Effects of OPRM1 A118G Polymorphism Using Different Opioids: A Prospective Study

Contextμ-opioid receptor gene (OPRM1) A118G polymorphism (rs1799971) causes loss of N-glycosylation sites at the extracellular domain of μ-opioid receptors. G-allele carriers show a limited response to morphine; however, studies investigating the impact of A118G polymorphism on the efficacy of opioids other than morphine are limited. ObjectiveTo compare the impact of A118G polymorphism on the efficacy of various opioids. MethodsThis prospective cohort study enrolled 222 in-patients administered one of the following opioid therapies for cancer pain as part of an opioid introduction or rotation strategy: tapentadol extended-release tablets, methadone tablets, hydromorphone controlled-release tablets, oxycodone controlled-release tablets, or transdermal fentanyl patches. The impact of A118G polymorphism on the difference in the Brief Pain Inventory-Short Form score on days three, seven, and 14 from baseline was compared among the groups. ResultsOverall, 81, 74, and 67 patients had the AA, AG, and GG genotypes, respectively, with an OPRM1 A118G G-allele variant frequency of 0.47. The reduction in the Brief Pain Inventory-Short Form score after opioid therapy initiation did not differ significantly among the patients with the three A118G genotypes treated with tapentadol (p = 0.84) or methadone (p = 0.97), whereas it was significantly smaller in G-allele carriers than that in AA homozygous patients treated with hydromorphone (p < 0.001), oxycodone (p = 0.031), or fentanyl (p < 0.001). ConclusionTapentadol and methadone may be more suitable than hydromorphone, oxycodone, and fentanyl for G-allele carriers due to their dual mechanism of action and low susceptibility to OPRM1 A118G polymorphism.

A novel GPI-anchored dominant-negative TGF-β receptor II renders T cells unresponsive to TGF-β signaling

Transforming growth factor β (TGF-β) is a pleiotropic cytokine expressed by a wide range of cell types and is known for hampering the effectiveness of cancer immune cell therapeutic approaches. We have designed a novel construct containing the extracellular domain of the TGF-β receptor II linked to a glycosylphosphatidylinositol (GPI) anchor (GPI-ecto-TβRII) lacking the transmembrane and cytoplasmic signaling domain of TGF-β receptor II (TβRII). Tcells transduced with lentivirus expressing the GPI-ecto-TβRII construct show 5 to 15 times higher membrane expression compared with a previously established dominant-negative receptor carrying a truncated signaling domain. GPI-ecto-TβRII expression renders Tcells unresponsive to TGF-β-induced signaling seen by a lack of SMAD phosphorylation upon exogeneous TGF-β treatment. Transduced Tcells continue to express high levels of IFNγ and granulocyte-macrophage colony-stimulating factor (GM-CSF), among other cytokines, in the presence of TGF-β while cytokine expression in untransduced Tcells is being markedly suppressed. Furthermore, Tcells expressing GPI-ecto-TβRII constructs have been shown to efficiently capture and inactivate TGF-β from their environment. These results indicate the potential benefits of GPI-ecto-TβRII expressing cytotoxic Tcells (CTLs) in future cell therapies.

Reconciling intrinsic properties of activating TNF receptors by native ligands versus synthetic agonists.

The extracellular domain of tumor necrosis factor receptors (TNFR) generally require assembly into a homotrimeric quaternary structure as a prerequisite for initiation of signaling via the cytoplasmic domains. TNF receptor homotrimers are natively activated by similarly homo-trimerized TNF ligands, but can also be activated by synthetic agonists including engineered antibodies and Fc-ligand fusion proteins. A large body of literature from pre-clinical models supports the hypothesis that synthetic agonists targeting a diverse range of TNF receptors (including 4-1BB, CD40, OX40, GITR, DR5, TNFRSF25, HVEM, LTβR, CD27, and CD30) could amplify immune responses to provide clinical benefit in patients with infectious diseases or cancer. Unfortunately, however, the pre-clinical attributes of synthetic TNF receptor agonists have not translated well in human clinical studies, and have instead raised fundamental questions regarding the intrinsic biology of TNF receptors. Clinical observations of bell-shaped dose response curves have led some to hypothesize that TNF receptor overstimulation is possible and can lead to anergy and/or activation induced cell death of target cells. Safety issues including liver toxicity and cytokine release syndrome have also been observed in humans, raising questions as to whether those toxicities are driven by overstimulation of the targeted TNF receptor, a non-TNF receptor related attribute of the synthetic agonist, or both. Together, these clinical findings have limited the development of many TNF receptor agonists, and may have prevented generation of clinical data which reflects the full potential of TNF receptor agonism. A number of recent studies have provided structural insights into how different TNF receptor agonists bind and cluster TNF receptors, and these insights aid in deconvoluting the intrinsic biology of TNF receptors with the mechanistic underpinnings of synthetic TNF receptor agonist therapeutics.

Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer.

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available.