Ligand-receptor Binding Research Articles

Membrane-proximal motifs encode differences in signaling strength between type I and III interferon receptors.

Interferons (IFNs) play crucial roles in antiviral defenses. Despite using the same Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) signaling cascade, type I and III IFN receptors differ in the magnitude and dynamics of their signaling in terms of STAT phosphorylation, gene transcription, and antiviral responses. These differences are not due to ligand-binding affinity and receptor abundance. Here, we investigated the ability of the intracellular domains (ICDs) of IFN receptors to differentiate between type I and III IFN signaling. We engineered synthetic, heterodimeric type I and III IFN receptors that were stably expressed at similar amounts in human cells and responded to a common ligand. We found that our synthetic type I IFN receptors stimulated STAT phosphorylation and gene expression to greater extents than did the corresponding type III IFN receptors. Furthermore, we identified short "box motifs" within ICDs that bind to JAK1 that were sufficient to encode differences between the type I and III IFN receptors. Together, our results indicate that specific regions within the ICDs of IFN receptor subunits encode different downstream signaling strengths that enable type I and III IFN receptors to produce distinct signaling outcomes.

THU596 Heteromerization Of The Angiotensin II Type 1 Receptor And The Bradykinin Type 2 Receptor

Abstract Disclosure: E.K. Johnstone: Grant Recipient; Self; Australian Research Council. K.D. Pfleger: Grant Recipient; Self; Australian Research Council. Stock Owner; Self; Dimerix Bioscience Pty Ltd. Background: Receptor heteromerization is the phenomenon whereby two different receptors form a functional complex that attains unique pharmacological properties. Consequently, receptor heteromers can be considered novel drug targets, with the potential to achieve greater therapeutic selectivity and specificity. In a landmark study, the first receptor heteromer to be associated with a disease was the heteromer that forms between the angiotensin II type 1 (AT1) receptor and the bradykinin type 2 (B2) receptor (1). The AT1-B2 heteromer reportedly led to increased angiotensin II signalling, which was implicated in the angiotensin II hypersensitivity involved in pre-eclampsia. However, the validity of this study was questioned by a collaboration of several groups who were unable to find any evidence for a physical or a function interaction between the two receptors (2). As a consequence of these conflicting studies, the existence of the AT1-B2 receptor heteromer has remained controversial. Aim: Investigation of evidence for heteromerization of the AT1 receptor and the B2 receptor. Methods: AT1 receptor and B2 receptor pharmacology and potential heteromerization was investigated in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, as well as Dimerix’s Receptor-Heteromer Investigation Technology (Receptor-HIT) (3). Receptor-HIT enables the investigation of receptor heteromers through their specific ligand-induced proximity to interacting proteins. BRET assays, including in the Receptor-HIT configuration, allow real time, live cell monitoring of proximity between biomolecules of interest. Results: Using the Receptor-HIT assay, we confirmed the existence of the AT1-B2 receptor heteromer in HEK293FT cells. We found that the heteromer was able to recruit various GPCR interacting proteins, such as G proteins and arrestin in a bradykinin-dependent manner. Additionally, the heteromer also internalised and trafficked through the cell upon treatment with bradykinin. The close proximity of the two receptors was confirmed using a variation of the Receptor-HIT assay, which monitors receptor-ligand binding rather than protein-protein interactions. Discussion: Our study supports the existence of the AT1-B2 receptor heteromer and identifies various pharmacological properties of the complex. Further studies are required to relate these molecular properties to physiological consequences of heteromerization. (1)AbdAlla et al. Nat Med2001, 7 (9), 1003 1009.(2)Hansen et al. J Biol Chem2009;284(3):1831 1839.(3)Johnstone et al. Biochem Soc T. 2021;49:1555–65. Presentation: Thursday, June 15, 2023

Interplay between G protein-coupled receptors and nanotechnology.

G protein-coupled receptors (GPCRs), as the largest family of membrane receptors, actively modulate plasma membrane and endosomal signalling. Importantly, GPCRs are naturally nanosized, and spontaneously formed nanoaggregates of GPCRs (natural nano-GPCRs) may enhance GPCR-related signalling and functions. Although GPCRs are the molecular targets of the majority of marketed drugs, the poor pharmacokinetics and physicochemical properties of GPCR ligands greatly limit their clinical applicability. Nanotechnology, as versatile techniques, can encapsulate GPCR ligands to assemble synthetic nano-GPCRs to overcome their obstacles, robustly elevating drug efficacy and safety. Moreover, endosomal delivery of GPCR ligands by nanoparticles can precisely initiate sustained endosomal signal transduction, while nanotechnology has been widely utilized for isolation, diagnosis, and detection of GPCRs. In turn, due to overexpression of GPCRs on the surface of various types of cells, GPCR ligands can endow nanoparticles with active targeting capacity for specific cells via ligand-receptor binding and mediate receptor-dependent endocytosis of nanoparticles. This significantly enhances the potency of nanoparticle delivery systems. Therefore, emerging evidence has revealed the interplay between GPCRs and nanoparticles, although investigations into their relationship have been inadequate. This review aims to summarize the interaction between GPCRs and nanotechnology for understanding their mutual influences and utilizing their interplay for biomedical applications. It will provide a fundamental platform for developing powerful and safe GPCR-targeted drugs and nanoparticle systems. STATEMENT OF SIGNIFICANCE: GPCRs as molecular targets for the majority of marketed drugs are naturally nanosized, and even spontaneously form nano aggregations (nano-GPCRs). Nanotechnology has also been applied to construct synthetic nano-GPCRs or detect GPCRs, while endosomal delivery of GPCR ligands by nanoparticles can magnify endosomal signalling. Meanwhile, molecular engineering of nanoparticles with GPCRs or their ligands can modulate membrane binding and endocytosis, powerfully improving the efficacy of nanoparticle system. However, there are rare summaries on the interaction between GPCRs and nanoparticles. This review will not only provide a versatile platform for utilizing nanoparticles to modulate or detect GPCRs, but also facilitate better understanding of the designated value of GPCRs for molecular engineering of biomaterials with GPCRs in therapeutical application.

Structure-Based Virtual Screening of Helicobacter pylori SecA Inhibitors.

The human bacterial pathogen Helicobacter pylori causes a range of gastric diseases. The killing rate of Helicobacter pylori is declining year by year because of high antibiotics resistance. It is urgent to develop new target and novel anti- Helicobacter pylori drugs. As an "energy pump" for bacterial cells, SecA is essential for bacterial growth and drives bacterial protein transmembrane transport, moreover SecA is absent in mammals, all of which nominate SecA as an attractive antimicrobial target. Here, we provided a structure-based virtual screening method to screen the 3D-diversity natural-product-like screening library against SecA for novel anti- Helicobacter pylori inhibitors with novel scaffolds. In this study, homology modeling was used to construct the three-dimensional structure of Helicobacter pylori SecA. Two rounds of molecular docking were then used to find new small-molecule inhibitors of SecA, identifying six lead candidates that maintained key interactions with the binding pocket. After that, molecular dynamics simulations were used to explore more accurate ligand-receptor binding modes in states close to natural conditions. Encouragingly, all six compounds were relatively stable during the simulation. Apart from that the binding free energy calculation based on MM/PBSA demonstrated favorable results of < -13.642 kcal/mol. Finally, ADME-T analysis indicated that these compounds were also sufficiently druggable. All six compounds can be well combined with the crystal structure, which further facilitate the development of SecA inhibitors and lead compounds against Helicobacter pylori.

Inhibitory effect of adenosine on adaptive antitumor immunity and intervention strategies.

The tumors with a microenvironment characterized by a lack of immune cell infiltration, referred as "cold tumors", typically exhibit low responsiveness to immune therapy. Targeting the factors contributing to "cold tumors" formation and converting them into "hot tumors" would be a novel strategy for improving the efficacy of immunotherapy. Adenosine, a hydrolysis product of ATP, accumulates with a significantly higher concentration in the tumor microenvironments compared to normal tissue and exerts inhibitory effects on tumor-specific adaptive immunity. Tumor cells, dendritic cells, macrophages, and T cells express abundant adenosine receptors on their surfaces. The binding of adenosine to these receptors initiates downstream signaling pathways that suppress tumor antigen presentation and immune cell activation, consequently dampening adaptive immune responses against tumors. Adenosine downregulates the expression of major histocompatibility complex class Ⅱ and co-stimulatory factors on dendritic cells and macrophages, thereby inhibiting antigen presentation to T cells. Adenosine also inhibits ligand-receptor binding and transmembrane signaling on T cells, concomitantly suppressing the secretion of anti-tumor cytokines and impairing T cell activation. Furthermore, adenosine hinders effector T cell trafficking to tumor sites and infiltration by inhibiting chemokine secretion and KCa3.1 channels. Additionally, adenosine promotes the secretion of immunosuppressive cytokines, increases immune checkpoint protein expression, and enhances the activity of immunosuppressive cells, collectively curbing cytotoxic T cell-mediated tumor cell killing. Given the immunosuppressive role of adenosine in adaptive antitumor immunity, several inhibitors targeting adenosine generation or adenosine receptor blockade are currently in preclinical or clinical development with the aim of enhancing the effectiveness of other immunotherapies. This review provides an overview of inhibitory effects of adenosine on adaptive antitumor immunity, elucidating the molecular mechanisms involved, and summarizes the latest advances in the application of adenosine inhibition strategies in anticancer immunotherapy.

The translational value of ligand-receptor binding kinetics in drug discovery.

The translation of in vitro potency of a candidate drug, as determined by traditional pharmacology metrics (such as EC50/IC50 and KD/Ki values), to in vivo efficacy and safety is challenging. Residence time, which represents the duration of drug-target interaction, can be part of a more comprehensive understanding of the dynamic nature of drug-target interactions in vivo, thereby enabling better prediction of drug efficacy and safety. As a consequence, a prolonged residence time may help in achieving sustained pharmacological activity, while transient interactions with shorter residence times may be favourable for targets associated with side effects. Therefore, integration of residence time into the early stages of drug discovery and development has yielded a number of clinical candidates with promising in vivo efficacy and safety profiles. Insights from residence time research thus contribute to the translation of in vitro potency to in vivo efficacy and safety. Further research and advances in measuring and optimizing residence time will bring a much-needed addition to the drug discovery process and the development of safer and more effective drugs. In this review, we summarize recent research progress on residence time, highlighting its importance from a translational perspective.

In Vitro Affinity Maturation of Nanobodies against Mpox Virus A29 Protein Based on Computer-Aided Design.

Mpox virus (MPXV), the most pathogenic zoonotic orthopoxvirus, caused worldwide concern during the SARS-CoV-2 epidemic. Growing evidence suggests that the MPXV surface protein A29 could be a specific diagnostic marker for immunological detection. In this study, a fully synthetic phage display library was screened, revealing two nanobodies (A1 and H8) that specifically recognize A29. Subsequently, an in vitro affinity maturation strategy based on computer-aided design was proposed by building and docking the A29 and A1 three-dimensional structures. Ligand-receptor binding and molecular dynamics simulations were performed to predict binding modes and key residues. Three mutant antibodies were predicted using the platform, increasing the affinity by approximately 10-fold compared with the parental form. These results will facilitate the application of computers in antibody optimization and reduce the cost of antibody development; moreover, the predicted antibodies provide a reference for establishing an immunological response against MPXV.

Costimulatory capacity of CD137 mAbs on T cells depends on elaborate CRD structures but not on blocking ligand-receptor binding.

CD137 is mainly a costimulatory receptor of CD8+ T cells. Two representative CD137 antibodies, utomilumab, and urelumab, show different costimulatory capacities in clinical trials. Balancing the antitumor effect and systemic toxicity of T cells activated by CD137 signaling is a challenge that requires clinical consideration. In this study, a panel of specific anti-human CD137 monoclonal antibodies (mAbs) were prepared and their affinities, isotypes, CD137-CRD (cysteine-rich domain) binding regions, cross-reactivity to mouse and rhesus CD137, inhibition of ligand-receptor binding and costimulatory activities were analyzed. The results showed that anti-human CD137 mAbs had high cross-reactivity with rhesus CD137. MAbs fell into three clusters according to their different binding regions of the CD137 extracellular domain. They bound to CRDI+CRDII, CRDIII or CRDIV+STP. CRDIII-binding mAbs had the strongest blocking activity. Highly costimulatory CD137 mAbs showed stronger abilities to promote CD8+ T-cell proliferation. However, the costimulatory capacity of mAbs on T cells was not closely related to their ability to block CD137L-CD137 binding and may be controlled by more elaborate CRD conformational structures. This study provides additional information for the development of next-generation CD137 mAbs to meet clinical needs.

BAK-up: the receptor kinase BAK-TO-LIFE 2 enhances immunity when BAK1 is lacking

BRI1-ASSOCIATED KINASE 1 (BAK1/SERK3) and its closest homolog BAK1-LIKE 1 (BKK1/SERK4) are leucine-rich repeat receptor kinases (LRR-RKs) belonging to the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family. They act as co-receptors of various other LRR-RKs and participate in multiple signaling events by complexing and transphosphorylating ligand-binding receptors. Initially identified as the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) co-receptor, BAK1 also functions in plant immunity by interacting with pattern recognition receptors. Mutations in BAK1 and BKK1 cause severely stunted growth and cell death, characterized as autoimmune cell death. Several factors play a role in this type of cell death, including RKs and components of effector-triggered immunity (ETI) signaling pathways, glycosylation factors, ER quality control components, nuclear trafficking components, ion channels, and Nod-like receptors (NLRs). The Shan lab has recently discovered a novel RK BAK-TO-LIFE 2 (BTL2) that interacts with BAK1 and triggers cell death in the absence of BAK1 and BKK1. This RK compensates for the loss of BAK1-mediated pattern-triggered immunity (PTI) by activating phytocytokine-mediated immune and cell death responses.

Multifunctional Nanofibrous Scaffolds Capable of Localized Delivery of Theranostic Nanoparticles for Postoperative Cancer Management.

Postoperative recovery of cancer patients can be affected by complications, such as tissue dysfunction or disability caused by tissue resection, and also cancer recurrence resulting from residual cancer cells. Despite impressive progress made for tissue engineering scaffolds that assist tissue regeneration for postoperative cancer patients, the majority of existing tissue engineering scaffolds still lack functions for monitoring and killing residual cancer cells, if there are any, upon their detection. In this study, multifunctional scaffolds that comprise biodegradable nanofibers and core-shell structured microspheres encapsulated with theranostic nanoparticles (NPs) are developed. The multifunctional scaffolds possess an extracellular matrix-like nanofibrous architecture and soft tissue-like mechanical properties, making them excellent tissue engineering patch candidates for assisting in the repair and regeneration of tissues at the cancerous sites after surgery. Furthermore, they are capable of localized delivery of theranostic NPs upon quick degradation of core-shell structured microspheres that contain theranostic NPs. Leveraging on folic acid-mediated ligand-receptor binding, surface-enhanced Raman scattering activity, and near-infrared-responsive photothermal effect of the theranostic gold NPs (AuNPs) delivered locally, the multifunctional scaffolds display excellent active targeting, diagnosis, and photothermal therapy functions for cancer cells, showing great promise for adaptive postoperative cancer management.

ADGRL1 is a glucose receptor involved in mediating energy and glucose homeostasis

Aims/hypothesisThe brain is a major consumer of glucose as an energy source and regulates systemic glucose as well as energy balance. Although glucose transporters such as GLUT2 and sodium−glucose cotransporter 2 (SGLT2) are known to regulate glucose homeostasis and metabolism, the identity of a receptor that binds glucose to activate glucose signalling pathways in the brain is unknown. In this study, we aimed to discover a glucose receptor in the mouse hypothalamus.MethodsHere we used a high molecular mass glucose–biotin polymer to enrich glucose-bound mouse hypothalamic neurons through cell-based affinity chromatography. We then subjected the enriched neurons to proteomic analyses and identified adhesion G-protein coupled receptor 1 (ADGRL1) as a top candidate for a glucose receptor. We validated glucose–ADGRL1 interactions using CHO cells stably expressing human ADGRL1 and ligand–receptor binding assays. We generated and determined the phenotype of global Adgrl1-knockout mice and hypothalamus-specific Adgrl1-deficient mice. We measured the variables related to glucose and energy homeostasis in these mice. We also generated an Adgrl1Cre mouse model to investigate the role of ADGRL1 in sensing glucose using electrophysiology.ResultsAdgrl1 is highly expressed in the ventromedial nucleus of the hypothalamus (VMH) in mice. Lack of Adgrl1 in the VMH in mice caused fasting hyperinsulinaemia, enhanced glucose-stimulated insulin secretion and insulin resistance. In addition, the Adgrl1-deficient mice had impaired feeding responses to glucose and fasting coupled with abnormal glucose sensing and decreased physical activity before development of obesity and hyperglycaemia. In female mice, ovariectomy was necessary to reveal the contribution of ADGRL1 to energy and glucose homeostasis.Conclusions/interpretationAltogether, our findings demonstrate that ADGRL1 binds glucose and is involved in energy as well as glucose homeostasis in a sex-dependent manner. Targeting ADGRL1 may introduce a new class of drugs for the treatment of type 2 diabetes and obesity.Graphical

Ab initio quantification of the oxygen-hemoglobin dissociation curve

Uptake of oxygen by hemoglobin (Hb), described by the oxygen-Hb dissociation curve, is obviously important for the existence of all vertebrates. Its sigmoidal curve shape indicates that oxygen binds more tightly if sites already are occupied, commonly referred to as the cooperative effect. The effect has been challenging to understand and quantify ever since its experimental demonstration in 1904. Here, we derive an ab initio analytical expression for the dissociation curve based on the fundamental principle of uniform oxygen chemical potential and absolute activity throughout the system at equilibrium using the grand partition function. The resulting analytical dissociation expression therefore only has four molecular oxygen-Hb binding energies as free variables, which are determined by fitting the analytical expression to measured data. The corresponding resulting negative reaction enthalpies identified in increasing magnitude are, ΔH1=−41.6, ΔH2=−48.8, ΔH3=−51.2, andΔH4=−51.8kJ/mol, in the range observed experimentally. The difference between ΔH1 and ΔH4 is ∼10kJ/mol, smaller than the maximum enthalpy difference measured experimentally, ∼16.7kJ/mol. Hence, the cooperative effect can therefore be explained, from an energy point of view, as caused by the reaction enthalpy difference between ΔH1 and the three subsequent enthalpy values. No impact of Hb’s spatial and structural properties is assumed. The finding highlights the importance of identifying the ligand-receptor molecular binding energies, and thereby the reaction enthalpies, under different conditions as a way for calculating not only the oxygen-Hb but ligand-receptor dissociation curves in general under various conditions, a priori, since the procedure for determining these curves ab initio has been established.

Benchmarking different docking protocols for predicting the binding poses of ligands complexed with cyclooxygenase enzymes and screening chemical libraries.

Non-steroidal anti-inflammatory drugs (NSAIDs) constitute an important class of pharmaceuticals acting on cyclooxygenase COX-1 and COX-2 enzymes. Due to their numerous severe side effects, it is necessary to search for new selective, safe, and effective anti-inflammatory drugs. In silico design of novel therapeutics plays an important role in nowadays drug discovery pipelines. In most cases, the design strategies require the use of molecular docking calculations. The docking procedure may require case-specific condition for a successful result. Additionally, many different docking programs are available, which highlights the importance of identifying the most proper docking method and condition for a given problem. In the current work, the performances of five popular molecular docking programs, namely, GOLD, AutoDock, FlexX, Molegro Virtual Docker (MVD) and Glide to predict the binding mode of co- crystallized inhibitors in the structures of known complexes available for cyclooxygenases were evaluated. Furthermore, the best performers, Glide, AutoDock, GOLD and FlexX, were further evaluated in docking-based virtual screening of libraries consisted of active ligands and decoy molecules for cyclooxygenase enzymes and the obtained docking scores were assessed by receiver operating characteristics (ROC) analysis. The results of docking experiments indicated that Glide program outperformed other docking programs by correctly predicting the binding poses (RMSD less than 2 Å) of all studied co-crystallized ligands of COX-1 and COX-2 enzymes (i.e., the performance was 100%). However, the performances of the other studied docking methods for correctly predicting the binding poses of the ligands were between 59% to 82%. Virtual screening results treated by ROC analysis revealed that all tested methods are useful tools for classification and enrichment of molecules targeting COX enzymes. The obtained AUCs range between 0.61-0.92 with enrichment factors of 8 - 40 folds. The obtained results support the importance of choosing appropriate docking method for predicting ligand-receptor binding modes, and provide specific information about docking calculations on COXs ligands.

Exploring EGFR inhibitors with the aid of virtual screening, docking, and dynamics simulation studies

In humans, Epidermal Growth Factor Receptor (EGFR) is linked to small-cell lung cancer, breast cancer, and glioblastoma. Receptor kinase inhibitors against EGFR have become a standard treatment option for non-small cell lung cancer (NSCLC), breast cancer patients, and even for those with EGFR mutations or resistance. About 2734 FDA-approved medication compounds were subjected to virtual screening for EGFR kinase inhibitory activity. The top 30 molecules were chosen based on the binding affinity scores and subjected to extra-precision docking and binding free energy analysis. The ADMET profile of the top three hit molecules was verified to confirm their druggability nature. Top three hits- compound 1047 (ZINC000001550477), 1302 (ZINC00003781952), and 2332 (ZINC000019632618) were identified on account of their MMGBSA binding affinity values. The top three hit compounds were subjected to molecular dynamics (MD) simulation for 100 ns. The dynamic nature of the ligand-protein complex was analyzed which corroborated the results of molecular docking and MMGBSA analysis studies. All the top three hits were further subjected to steered MD studies for testing the strength of these ligand-receptor binding in the presence of an external force. Compound 2332 (ZINC000019632618) was identified as the best hit molecule that can be used as a lead to develop newer derivatives of EGFR kinase inhibitors. Communicated by Ramaswamy H. Sarma

Ligand-based adoptive T cell targeting CA125 in ovarian cancer

BackgroundOvarian cancer (OC) is a highly aggressive gynecological malignancy prevalent worldwide. Most OC cases are typically diagnosed at advanced stages, which has led to a 5-year overall survival rate of less than 35% following conventional treatment. Furthermore, immune checkpoint inhibitor therapy has shown limited efficacy in the treatment of patients with OC, and CAR-T therapy has also demonstrated modest results owing to inadequate T cell infiltration. Therefore, novel strategies must be developed to enhance T cell persistence and trafficking within the OC tumor microenvironment.MethodsIn this study, we developed a novel adoptive T-cell therapy for ovarian cancer based on a chimeric antigen receptor structure. We used a ligand-receptor binding motif to enhance the therapeutic effect of targeting CA125. Since mesothelin can naturally bind to CA125 with high affinity, we concatenated the core-binding fragment of mesothelin with the 4-1BB and CD3ζ signal fragments to assemble a novel CA125-targeting chimeric receptor (CR). The CAR structure targeting CA125 derived from the 4H11 antibody was also constructed. CR- and CAR-encoding RNA were electroporated into T cells to evaluate their antitumor activity both in vitro and in vivo.ResultsWhile CR-T or CAR-T cells exhibited moderate activity against two ovarian cancer cell lines, T cells co-expressing CR and CAR exhibited a superior killing effect compared to T cells expressing either CR or CAR alone. Furthermore, upon interaction with ovarian tumors, the ability of CR and CAR T cells to release activation markers and functional cytokines increased significantly. Similarly, CR and CAR co-expressing T cells persistently controlled the growth of transplanted ovarian cancer tumors in NSG mice and significantly prolonged the overall survival of tumor-challenged mice. Transcriptome sequencing revealed that the survival and cytotoxicity of T cells co-expressing CR and CAR were significantly altered compared with those of T cells expressing either CR or CAR.ConclusionOur findings demonstrate that CA125 targeting CR and CAR can synergistically kill ovarian cancer cells, indicating that CA125 targeting by the two binding motifs simultaneously in tumors may improve the therapeutic outcomes of ovarian cancer treatment.

Prediction of the Peptide-TIM3 Binding Site in Inhibiting TIM3-Galectin 9 Binding Pathways.

T-cell immunoglobulin and mucin domain-containing protein-3 (TIM3) is an important receptor protein that modulates the immune system. The binding of TIM3 with Galectin 9 (GAL9) triggers immune system suppression, but the TIM3-GAL9 binding can be inhibited by binding of the peptide P26 to TIM3. A fast and accurate prediction of the P26-TIM3 binding site is crucial and a prerequisite for the investigation of P26-TIM3 interactions and TIM3-GAL9 binding pathways. Here, we present a machine learning approach, which considers protein conformational changes, to quickly identify the ligand-binding site on TIM3. Our results show that the P26 binding site is located near the C″-D loop of TIM3. Further simulations show that the binding pose is stabilized by a variety of electrostatic and hydrophobic interactions. Binding of P26 can alter the conformations of nearby glycan side chains on TIM3, providing possible mechanisms of how P26 inhibits TIM3-GAL9 binding pathways. The insights from this work will facilitate the identification of other peptides or antibodies that may also inhibit the TIM3-GAL9 pathways and eventually lead to improved attempts in the modulation of the TIM3-GAL9 immunosuppression pathways. The strategies and machine learning method can be generalized to study ligand-receptor binding when the conformational changes during the binding are important.

Synthesis of N-aminophalimides derived from α-amino acids: Theoretical study to find them as HDAC8 inhibitors by docking simulations and in vitro assays.

Phthalimides are valuable for synthesis and biological properties. New acetamides 3(a-c) and 4(a-c) were synthesized and characterized as precursors for novel N-aminophalimides 5(a-c) and 6(a-c). Structures of 4a, 5(a-b), and 6(a-b) were confirmed by single crystal X-ray. Docking studies identified compounds with favorable Gibbs free energy values for binding to histone deacetylase 8 (HDAC8), an enzyme targeted for anticancer drug development. These compounds bound to both the orthosteric and allosteric pockets of HDAC8, similar to Trichostatin A (TSA), an HDAC8 inhibitor. 6(a-c) contain hydroxyacetamide moiety as a zinc-binding group, a phthalimide moiety as a capping group, and aminoacetamide moiety as a linker group, which are important for ligand-receptor binding. ΔG values indicated that compounds 5b, 6b, and 6c had higher affinity for HDAC8 in the allosteric pocket compared to TSA. In vitro evaluation of inhibitory activities on HDAC8 revealed that compounds 3(a-c) and 5(a-c) showed similar inhibitory effects (IC50 ) ranging from 0.445 to 0.751 μM. Compounds 6(a-c) showed better affinity, with 6a (IC50 = 28 nM) and 6b (IC50 = 0.18 μM) showing potent inhibitory effects slightly lower than TSA (IC50 = 26 nM). These findings suggest that the studied compounds hold promise as potential candidates for further biological investigations.

Analgesic Effect of the Lysine-Containing Short Peptide Is Due to Modulation of the NaV1.8 Channel Activation Gating System.

The present work continues our recent series of articles that aim to elucidate the ligand-receptor binding mechanism of short cationic peptides to the NaV1.8 channel in the nociceptive neuron. The applied methodological approach has involved several methods: the patch-clamp experimental evaluation of the effective charge of the NaV1.8 channel activation gating system, the organotypic tissue culture method, the formalin test, and theoretical conformational analysis. The lysine-containing short peptide Ac-KEKK-NH2 has been shown to effectively modulate the NaV1.8 channel activation gating system. As demonstrated by the organotypic tissue culture method, the studied short peptide does not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects. Conformational analysis of the Ac-KEKK-NH2 molecule has revealed that the distances between the positively charged amino groups of the lysine side chains are equal to 11-12 Å. According to the previously suggested mechanism of ligand-receptor binding of short peptides to the NaV1.8 channel molecule, Ac-KEKK-NH2 should exhibit an analgesic effect, which has been confirmed by the formalin test. The data obtained unequivocally indicate that the studied lysine-containing short peptide is a promising candidate for the role of a novel analgesic medicinal substance.

LAMC2 promotes EGFR cell membrane localization and acts as a novel biomarker for tyrosine kinase inhibitors (TKIs) sensitivity in lung cancer

The epidermal growth factor receptor (EGFR) is one of the first and most prominent driver genes known to promote malignant lung cancer. Investigating regulatory mechanisms beyond ligand-receptor binding, phosphorylation, and receptor kinase activation as means of EGFR signaling activation is important for improving EGFR-targeted therapy. Here, we report that Laminin-5γ-2 (LAMC2) retained high oncogenic capacity in lung cancer, silencing LAMC2 inhibited EGFR-induced cell proliferation and tumor growth in vivo. Deletion mutation experiments showed that both the EGF-Lam and LamB regions of LAMC2 are necessary for EGFR receptor binding, and that LAMC2 and EGFR were found to co-localize at the endoplasmic reticulum (ER) membrane. In addition, LAMC2 overexpression enhanced EGFR membrane deposition and promoted EGFR transport from the ER. Moreover, LAMC2 was necessary for preventing EGFR protein degradation via ubiquitination. Lastly, our study showed that high LAMC2 expression is positively associated with response to gefitinib (EGFR tyrosine kinase inhibitor) treatment. Overall, our study revealed a new regulatory mechanism of LAMC2 in promoting EGFR protein expression and stability by facilitating ER transport and preventing protein degradation via ubiquitination. Moreover, LAMC2 may serve as a stratifying biomarker for patients suitable for EGFR-TKI treatment.

Bioinformatic Analysis Identifies Potential Extracellular Matrix Related Genes in the Pathogenesis of Early Onset Preeclampsia.

Early-onset preeclampsia (EOPE) is a complex pregnancy complication that poses significant risks to the health of both mothers and fetuses, and research on its pathogenesis and pathophysiology remains insuffcient. This study aims to explore the role of candidate genes and their potential interaction mechanisms in EOPE through bioinformatics analysis techniques. Two gene expression datasets, GSE44711 and GSE74341, were obtained from the Gene Expression Omnibus (GEO) to identify differentially expressed genes (DEGs) between EOPE and gestational age-matched preterm control samples. Functional enrichment analysis was performed utilizing the kyoto encyclopedia of genes and genomes (KEGG), gene ontology (GO), and gene set enrichment analysis (GSEA). A protein-protein interaction (PPI) network was constructed using the STRING database, and hub DEGs were identified through Cytoscape software and comparative toxicogenomics database (CTD) analysis. Furthermore, a diagnostic logistic model was established using these hub genes, which were confirmed through reverse transcription polymerase chain reaction (RT-PCR). Finally, immune cell infiltration was analyzed using CIBERSORT. In total, 807 DEGs were identified in the GSE44711 dataset (451 upregulated genes and 356 downregulated genes), and 787 DEGs were identified in the GSE74341 dataset (446 upregulated genes and 341 downregulated genes). These DEGs were significantly enriched in various molecular functions such as extracellular matrix structural constituent, receptor-ligand activity binding, cytokine activity, and platelet-derived growth factor. KEGG and GSEA annotation revealed significant enrichment in pathways related to ECM-receptor interaction, PI3K-AKT signaling, and focal adhesion. Ten hub genes were identified through the CytoHubba plugin in Cytoscape. Among these hub genes, three key DEGs (COL1A1, SPP1, and THY1) were selected using CTD analysis and various topological methods in Cytoscape. The diagnostic logistic model based on these three genes exhibited high efficiency in predicting EOPE (AUC = 0.922). RT-PCR analysis confirmed the downregulation of these genes in EOPE, and immune cell infiltration analysis suggested the significant role of M1 and M2 macrophages in EOPE. In conclusion, this study highlights the association of three key genes (COL1A1, SPP1, and THY1) with EOPE and their contribution to high diagnostic efficiency in the logistic model. Additionally, it provides new insights for future research on EOPE and emphasizes the diagnostic value of these identified genes. More research is needed to explore their functional and diagnostic significance in EOPE.