Diverse Ligands Research Articles

Profiling the Binding Activities of Peptides and Inhibitors to the U2 Auxiliary Factor Homology Motif (UHM) Domains.

RNA splicing is a biological process to generate mature mRNA (mRNA) by removing introns and annexing exons in the nascent RNA transcript and is executed by a multiprotein complex called spliceosome. To aid RNA splicing, a class of splicing factors use an atypical RNA recognition domain (UHM) to bind with U2AF ligand motifs (ULMs) in proteins to form modules that recognize splice sites and splicing regulatory elements on mRNA. Mutations of UHM containing splicing factors have been found frequently in myeloid neoplasms. To profile the selectivity of UHMs for inhibitor development, we established binding assays to measure the binding activities between UHM domains and ULM peptides and a set of small-molecule inhibitors. Additionally, we computationally analyzed the targeting potential of the UHM domains by small-molecule inhibitors. Our study provided the binding assessment of UHM domains to diverse ligands that may guide development of selective UHM domain inhibitors in the future.

Turning high-throughput structural biology into predictive inhibitor design

A common challenge in drug design pertains to finding chemical modifications to a ligand that increases its affinity to the target protein. An underutilized advance is the increase in structural biology throughput, which has progressed from an artisanal endeavor to a monthly throughput of hundreds of different ligands against a protein in modern synchrotrons. However, the missing piece is a framework that turns high-throughput crystallography data into predictive models for ligand design. Here, we designed a simple machine learning approach that predicts protein-ligand affinity from experimental structures of diverse ligands against a single protein paired with biochemical measurements. Our key insight is using physics-based energy descriptors to represent protein-ligand complexes and a learning-to-rank approach that infers the relevant differences between binding modes. We ran a high-throughput crystallography campaign against the SARS-CoV-2 main protease (MPro), obtaining parallel measurements of over 200 protein-ligand complexes and their binding activities. This allows us to design one-step library syntheses which improved the potency of two distinct micromolar hits by over 10-fold, arriving at a noncovalent and nonpeptidomimetic inhibitor with 120 nM antiviral efficacy. Crucially, our approach successfully extends ligands to unexplored regions of the binding pocket, executing large and fruitful moves in chemical space with simple chemistry.

Mechanism of activation and biased signaling in complement receptor C5aR1

The complement system plays an important role in the innate immune response to invading pathogens. The complement fragment C5a is one of its important effector components and exerts diverse physiological functions through activation of the C5a receptor 1 (C5aR1) and associated downstream G protein and β-arrestin signaling pathways. Dysfunction of the C5a-C5aR1 axis is linked to numerous inflammatory and immune-mediated diseases, but the structural basis for activation and biased signaling of C5aR1 remains elusive. Here, we present cryo-electron microscopy structures of the activated wild-type C5aR1–Gi protein complex bound to each of the following: C5a, the hexapeptidic agonist C5apep, and the G protein-biased agonist BM213. The structures reveal the landscape of the C5a–C5aR1 interaction as well as a common motif for the recognition of diverse orthosteric ligands. Moreover, combined with mutagenesis studies and cell-based pharmacological assays, we deciphered a framework for biased signaling using different peptide analogs and provided insight into the activation mechanism of C5aR1 by solving the structure of C5aR1I116A mutant–Gi signaling activation complex induced by C089, which exerts antagonism on wild-type C5aR1. In addition, unusual conformational changes in the intracellular end of transmembrane domain 7 and helix 8 upon agonist binding suggest a differential signal transduction process. Collectively, our study provides mechanistic understanding into the ligand recognition, biased signaling modulation, activation, and Gi protein coupling of C5aR1, which may facilitate the future design of therapeutic agents.

A Scalable Synthesis of Chiral Himbert Diene Ligands for Asymmetric Catalysis

AbstractChiral dienes are important ligands in asymmetric catalysis but they are less accessible than other commonly used ligands such as chiral bisphosphines. Here, we show that intramolecular [4+2] cycloaddition of a simply attained chiral allenecarboxanilide readily affords pseudoenantiomeric bicyclo[2.2.2]octa‐2,5‐dienes containing an alkenyl bromide, which can be readily functionalized to give diverse chiral diene ligands. The synthesis is straightforward and easily conducted on multigram scales. These ligands exhibit high performance in nine types of enantioselective Rh(I)‐catalyzed 1,4‐addition or 1,2‐addition reactions.magnified image

High-throughput binding free energy simulations: Applications in drug discovery.

Computational drug discovery methods are becoming more and more prevalent in the drug discovery pipeline. Binding free energy (BFE) simulations are a class of molecular dynamics-based free energy methods that utilize a nonphysical “alchemical” thermodynamic pathway to efficiently and rigorously compute protein-ligand complex binding free energies. With the advent of GPU computing, BFE simulations can be performed on a timescale reasonable for small molecule drug discovery applications. Combining these methods with machine learning methods such as active learning, one can rapidly explore large chemical spaces with a fraction of the cost of brute force simulations. Herein, we describe a framework for the high-throughput and highly automated screening of a large number of chemically diverse small molecule ligands for the LRRK2 WDR domain using BFE calculations with thermodynamic integration. LRRK2 is the most commonly mutated gene in familial Parkinson's Disease, and its WDR domain is an understudied drug target with no known molecular inhibitors. This work was done as a submission to the Critical Assessment of Computational Hit-Finding Experiments (CACHE) Challenge. We also discuss various methods to minimize computational cost while maintaining accuracy and make recommendations for future high-throughput screening campaigns.

A Novel and Functionally Diverse Class of Acetylcholine-Gated Ion Channels.

Fast cholinergic neurotransmission is mediated by acetylcholine-gated ion channels; in particular, excitatory nicotinic acetylcholine receptors play well established roles in virtually all nervous systems. Acetylcholine-gated inhibitory channels have also been identified in some invertebrate phyla, yet their roles in the nervous system are less well understood. We report the existence of multiple new inhibitory ion channels with diverse ligand activation properties in Caenorhabditis elegans We identify three channels, LGC-40, LGC-57, and LGC-58, whose primary ligand is choline rather than acetylcholine, as well as the first evidence of a truly polymodal channel, LGC-39, which is activated by both cholinergic and aminergic ligands. Using our new ligand-receptor pairs we uncover the surprising extent to which single neurons in the hermaphrodite nervous system express both excitatory and inhibitory channels, not only for acetylcholine but also for the other major neurotransmitters. The results presented in this study offer new insight into the potential evolutionary benefit of a vast and diverse repertoire of ligand-gated ion channels to generate complexity in an anatomically compact nervous system.SIGNIFICANCE STATEMENT Here we describe the diversity of cholinergic signaling in the nematode Caenorhabditis elegans We identify and characterize a novel family of ligand-gated ion channels and show that they are preferentially gated by choline rather than acetylcholine and expressed broadly in the nervous system. Interestingly, we also identify one channel gated by chemically diverse ligands including acetylcholine and aminergic ligands. By using our new knowledge of these ligand-gated ion channels, we built a model to predict the synaptic polarity in the C. elegans connectome. This model can be used for generating hypotheses on neural circuit function.

The increase of europium-based OLED luminance through reducing the excited state lifetime by mixed-ligand complex formation.

An approach to the luminance increase of the europium-based OLED is proposed through the formation of the mixed-ligand complex. The introduction of two diverse anionic ligands around one europium ion forming a mixed-ligand complex is confirmed by powder X-ray diffraction, 1H and 19F NMR spectroscopy, MALDI MS spectroscopy, and luminescence spectroscopy. A decrease in the symmetry of the coordination environment leads to a 50% reduction of the lifetime of the excited state. The obtained OLEDs based on mixed ligand europium complexes are significantly superior in luminance to OLEDs based on individual complexes.

Ligand entry pathways control the chemical space recognized by GPR183.

The G protein-coupled receptor GPR183 is a chemotactic receptor with an important function in the immune system and association with a variety of diseases. It recognizes ligands with diverse physicochemical properties as both the endogenous oxysterol ligand 7α,25-OHC and synthetic molecules can activate the G protein pathway of the receptor. To better understand the ligand promiscuity of GPR183, we utilized both molecular dynamics simulations and cell-based validation experiments. Our work reveals that the receptor possesses two ligand entry channels: one lateral between transmembrane helices 4 and 5 facing the membrane, and one facing the extracellular environment. Using enhanced sampling, we provide a detailed structural model of 7α,25-OHC entry through the lateral membrane channel. Importantly, the first ligand recognition point at the receptor surface has been captured in diverse experimentally solved structures of different GPCRs. The proposed ligand binding pathway is supported by in vitro data employing GPR183 mutants with a sterically blocked lateral entrance, which display diminished binding and signaling. In addition, computer simulations and experimental validation confirm the existence of a polar water channel which might serve as an alternative entrance gate for less lipophilic ligands from the extracellular milieu. Our study reveals knowledge to understand GPR183 functionality and ligand recognition with implications for the development of drugs for this receptor. Beyond, our work provides insights into a general mechanism GPCRs may use to respond to chemically diverse ligands.

Modulating the Schottky barriers of metal-2D perovskite junctions through molecular engineering of spacer ligands.

The crucial role of spacer ligands in affecting the contact properties of metal-2D perovskite junctions, which can severely affect device performance, is revealed in this work. We studied the contact properties of Ag, Au, and Pt with 2D perovskites that possess ligands with different sizes and functional groups. It is found that the interface binding energy, Schottky barrier height (SBH), and tunneling property depend strongly on the ligand size and functional group type. Small-size ligands can induce effective interface coupling and result in perturbed perovskite electronic properties and a high tunneling probability. In addition, high work-function metals and more electronegative functional groups can induce more severe band shifts at the interface. The features of diverse ligands ensure a widely tunable SBH ranging from 0-1.07 eV. This study provides guidance for developing more effective 2D perovskite-based electric nanodevices by tuning the contact properties through molecular engineering of spacer ligands.

Family-wide analysis of integrin structures predicted by AlphaFold2

Recent advances in protein structure prediction using AlphaFold2, known for its high efficiency and accuracy, have opened new avenues for comprehensive analysis of all structures within a single protein family. In this study, we evaluated the capabilities of AphaFold2 in analyzing integrin structures. Integrins are heterodimeric cell surface receptors composed of a combination of 18 α and 8 β subunits, resulting in a family of 24 different members. Both α and β subunits consist of a large extracellular domain, a short transmembrane domain, and typically, a short cytoplasmic tail. Integrins play a pivotal role in a wide range of cellular functions by recognizing diverse ligands. Despite significant advances in integrin structural studies in recent decades, high-resolution structures have only been determined for a limited subsets of integrin members, thus limiting our understanding of the entire integrin family. Here, we first analyzed the single-chain structures of 18 α and 8 β integrins in the AlphaFold2 protein structure database. We then employed the newly developed AlphaFold2-multimer program to predict the α/β heterodimer structures of all 24 human integrins. The predicted structures show a high level of accuracy for the subdomains of both α and β subunits, offering high-resolution structure insights for all integrin heterodimers. Our comprehensive structural analysis of the entire integrin family unveils a potentially diverse range of conformations among the 24 members, providing a valuable structure database for studies related to integrin structure and function. We further discussed the potential applications and limitations of the AlphaFold2-derived integrin structures.

Ru3 (CO)12 -Catalyzed Modular Assembly of Hemilabile Ligands by C-H Activation of Phosphines with Isocyanates.

Hemilabile ligands have been applied extensively in transition metal catalysis, but preparations of these molecules typically require multistep synthesis. Here, modular assembly of diverse phosphine-amide ligands, including related axially chiral compounds, is first reported through ruthenium-catalyzed C-H activation of phosphines with isocyanate directed by phosphorus(III) atoms. High reactivity and regioselectivity can be obtained by using a Ru3 (CO)12 catalyst with a mono-N-protected amino acid ligand. This transformation significantly expands the pool of phosphine-amide ligands, some of which have shown excellent efficiency for asymmetric catalysis. More broadly, the discovery constitutes a proof of principle for facile construction of hemilabile ligands directly from the parent monodentate phosphines by C-H activation with ideal atom, step and redox economy. Several dinuclear ruthenium complexes were characterized by single-crystal X-ray diffraction analysis revealing the key mechanistic features of this transformation.

3D-QSAR assisted identification of selective CYP1B1 inhibitors: an effective bioisosteric replacement/molecular docking/electrostatic complementarity analysis.

Cytochrome P450-1B1 is a majorly overexpressed drug-metabolizing enzyme in tumors and is responsible for inactivation and subsequent resistance to a variety of anti-cancer drugs, i.e., docetaxel, tamoxifen, and cisplatin. In the present study, a 3D quantitative structure-activity relationship (3D-QSAR) model has been constructed for the identification, design, and optimization of novel CYP1B1 inhibitors. The model has been built using a set of 148 selective CYP1B1 inhibitors. The developed model was evaluated based on certain statistical parameters including q2 and r2 which showed the acceptable predictive and descriptive capability of the generated model. The developed 3D-QSAR model assisted in understanding the key molecular fields which were firmly related to the selective CYP1B1 inhibition. A theoretic approach for the generation of new lead compounds with optimized CYP1B1 receptor affinity has been performed utilizing bioisosteric replacement analysis. These generated molecules were subjected to a developed 3D-QSAR model to predict the inhibitory activity potentials. Furthermore, these compounds were scrutinized through the activity atlas model, molecular docking, electrostatic complementarity, molecular dynamics, and waterswap analysis. The final hits might act as selective CYP1B1 inhibitors which could address the issue of resistance. This 3D-QSAR includes several chemically diverse selective CYP1B1 receptor ligands and well accounts for the individual ligand's inhibition affinities. These features of the developed 3D-QSAR model will ensure future prospective applications of the model to speed up the identification of new potent and selective CYP1B1 receptor ligands.

Enhanced protein-protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment.

Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein-protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.

RAGE ablation attenuates glioma progression and enhances tumor immune responses by suppressing galectin-3 expression.

Malignant gliomas consist of heterogeneous cellular components that have adopted multiple overlapping escape mechanisms that overcome both targeted and immune-based therapies. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that is activated by diverse proinflammatory ligands present in the tumor microenvironment. Activation of RAGE by its ligands stimulates multiple signaling pathways that are important in tumor growth and invasion. However, treatment strategies that only target the interaction of RAGE with its ligands are ineffective as cancer therapies due to the abundance and diversity of exogenous RAGE ligands in gliomas. As an alternative approach to RAGE ligand inhibition, we evaluated the genetic ablation of RAGE on the tumorigenicity of 2 syngeneic murine glioma models. RAGE expression was inhibited in the GL261 and K-Luc gliomas by shRNA and CRSPR/Cas9 techniques prior to intracranial implantation. Tumor growth, invasion, and inflammatory responses were examined by histology, survival, Nanostring, and flow cytometry. Intracellular RAGE ablation abrogated glioma growth and invasion by suppressing AKT and ERK1/2 activities and by downregulating MMP9 expression. Interestingly, RAGE inhibition in both glioma models enhanced tumor inflammatory responses by downregulating the expression of galectin-3 and potentiated immunotherapy responses to immune checkpoint blockade. We demonstrated that intracellular RAGE ablation suppresses multiple cellular pathways that are important in glioma progression, invasion, and immune escape. These findings strongly support the development of RAGE ablation as a treatment strategy for malignant gliomas.

IMMU-08. RAGE ABLATION ATTENUATES GLIOMA PROGRESSION AND ENHANCES TUMOR IMMUNE RESPONSES BY SUPPRESSING GALECTIN-3 EXPRESSION

Abstract INTRODUCTION Malignant gliomas consist of heterogenous cellular components that have adopted multiple overlapping escape mechanisms that overcome both targeted and immune-based therapies. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that is activated by diverse proinflammatory ligands present in the tumor microenvironment (TME). Activation of RAGE by its ligands stimulates multiple signaling pathways that are important in tumor growth, invasion, and immune escape. OBJECTIVE We aimed to demonstrate that blockade of RAGE expression may tackle resistance pathways by inhibiting GBM progression, invasion, and immune evasion. METHODS We evaluated genetic ablation of RAGE on the tumorigenicity of two syngeneic murine glioma models. RAGE expression was inhibited in the GL261 and K-Luc gliomas by shRNA and CRSPR/Cas9 techniques prior to intracranial implantation. Tumor growth, invasion, and inflammatory responses were examined by immunohistochemistry, qPCR, ELISA, western blotting, NanoString, flow cytometry, and in vivo survival analysis. RESULTS Intracellular RAGE ablation abrogated glioma growth and invasion by suppressing AKT and ERK1/2 activities and by downregulating MMP9 expression. Interestingly, RAGE inhibition also enhanced tumor inflammatory responses by downregulating the expression of galectin-3, rendered the immunotherapy resistant K-Luc gliomas responsive to immune checkpoint blockade therapy, and significantly increased survival. CONCLUSION This study is the first to link RAGE expression with galactin-3 as an immune modulator in glioblastoma and revealed that the effect of glioma RAGE expression on TME was mediated through downregulation of Gal-3 and subsequent polarization of Tumor-associated macrophages. The therapeutic significance of RAGE downregulation was demonstrated by sensitizing a highly invasive and immune-resistant glioma model to immune checkpoint blockade and strongly supports the development of RAGE ablation as a complementary treatment strategy for malignant gliomas.

Human Vδ2 T Cells and Their Versatility for Immunotherapeutic Approaches.

Gamma/delta (γδ) T cells are innate-like immune effectors that are a critical component linking innate and adaptive immune responses. They are recognized for their contribution to tumor surveillance and fight against infectious diseases. γδ T cells are excellent candidates for cellular immunotherapy due to their unique properties to recognize and destroy tumors or infected cells. They do not depend on the recognition of a single antigen but rather a broad-spectrum of diverse ligands through expression of various cytotoxic receptors. In this manuscript, we review major characteristics of the most abundant circulating γδ subpopulation, Vδ2 T cells, their immunotherapeutic potential, recent advances in expansion protocols, their preclinical and clinical applications for several infectious diseases and malignancies, and how additional modulation could enhance their therapeutic potential.

Ligand-switchable nanoparticles resembling viral surface for sequential drug delivery and improved oral insulin therapy

Mutual interference between surface ligands on multifunctional nanoparticles remains a significant obstacle to achieving optimal drug-delivery efficacy. Here, we develop ligand-switchable nanoparticles which resemble viral unique surfaces, enabling them to fully display diverse functions. The nanoparticles are modified with a pH-responsive stretchable cell-penetrating peptide (Pep) and a liver-targeting moiety (Gal) (Pep/Gal-PNPs). Once orally administered, the acidic environments trigger the extension of Pep from surface in a virus-like manner, enabling Pep/Gal-PNPs to traverse intestinal barriers efficiently. Subsequently, Gal is exposed by Pep folding at physiological pH, thereby allowing the specific targeting of Pep/Gal-PNPs to the liver. As a proof-of-concept, insulin-loaded Pep/Gal-PNPs are fabricated which exhibit effective intestinal absorption and excellent hepatic deposition of insulin. Crucially, Pep/Gal-PNPs increase hepatic glycogen production by 7.2-fold, contributing to the maintenance of glucose homeostasis for effective diabetes management. Overall, this study provides a promising approach to achieving full potential of diverse ligands on multifunctional nanoparticles.

Mechanistic Impact of Different Ligand Scaffolds on FXR Modulation Suggests Avenues to Selective Modulators.

The bile-acid sensing nuclear farnesoid X receptor (FXR) is an attractive target for the treatment of hepatic and metabolic diseases, but application of this chemotherapeutic concept remains limited due to adverse effects of FXR activation observed in clinical trials. To elucidate the mechanistic basis of FXR activation at the molecular level, we have systematically studied FXR co-regulator interactions and dimerization in response to seven chemically diverse FXR ligands. Different molecular effects on FXR activation mediated by different scaffolds were evident and aligned with characteristic structural changes within the ligand binding domain of FXR. A partial FXR agonist acted mainly through co-repressor displacement from FXR and caused an FXR-regulated gene expression pattern markedly differing from FXR agonist effects. These results suggest selective modulation of FXR dimerization and co-regulator interactions for different ligands, offering a potential avenue for the design of gene- or tissue-selective FXR modulators.

G protein coupled pattern recognition receptors expressed in neutrophils: Recognition, activation/modulation, signaling and receptor regulated functions.

Neutrophils, the most abundant white blood cell in human blood, express receptors that recognize damage/microbial associated pattern molecules of importance for cell recruitment to sites of inflammation. Many of these receptors belong to the family of G protein coupled receptors (GPCRs). These receptor-proteins span the plasma membrane in expressing cells seven times and the down-stream signaling rely in most cases on an activation of heterotrimeric G proteins. The GPCRs expressed in neutrophils recognize a number of structurally diverse ligands (activating agonists, allosteric modulators, and inhibiting antagonists) and share significant sequence homologies. Studies of receptor structure and function have during the last 40 years generated important information on GPCR biology in general; this knowledge aids in the overall understanding of general pharmacological principles, governing regulation of neutrophil function and inflammatory processes, including novel leukocyte receptor activities related to ligand recognition, biased/functional selective signaling, allosteric modulation, desensitization, and reactivation mechanisms as well as communication (receptor transactivation/cross-talk) between GPCRs. This review summarizes the recent discoveries and pharmacological hallmarks with focus on some of the neutrophil expressed pattern recognition GPCRs. In addition, unmet challenges, including recognition by the receptors of diverse ligands and how biased signaling mediate different biological effects are described/discussed.

Replication Protein A Utilizes Differential Engagement of Its DNA-Binding Domains to Bind Biologically Relevant ssDNAs in Diverse Binding Modes.

Replication protein A (RPA) is a ubiquitous ssDNA-binding protein that functions in many DNA processing pathways to maintain genome integrity. Recent studies suggest that RPA forms a highly dynamic complex with ssDNA that can engage with DNA in many modes that are orchestrated by the differential engagement of the four DNA-binding domains (DBDs) in RPA. To understand how these modes influence RPA interaction with biologically relevant ligands, we performed a comprehensive and systematic evaluation of RPA's binding to a diverse set of ssDNA ligands that varied in sequence, length, and structure. These equilibrium binding data show that WT RPA binds structured ssDNA ligands differently from its engagement with minimal ssDNAs. Next, we investigated each DBD's contributions to RPA's binding modes through mutation of conserved, functionally important aromatic residues. Mutations in DBD-A and -B have a much larger effect on binding when ssDNA is embedded into DNA secondary structures compared to their association with unstructured minimal ssDNA. As our data support a complex interplay of binding modes, it is critical to define the trimer core DBDs' role in binding these biologically relevant ligands. We found that DBD-C is important for engaging DNA with diverse binding modes, including, unexpectedly, at short ssDNAs. Thus, RPA uses its constituent DBDs to bind biologically diverse ligands in unanticipated ways. These findings lead to a better understanding of how RPA carries out its functions at diverse locations of the genome and suggest a mechanism through which dynamic recognition can impact differential downstream outcomes.