Extensive Interaction Network Research Articles

Applications of Functional Polymeric Eutectogels.

Over the past two decades, deep eutectic solvents (DESs) have captured significant attention as an emergent class of solvents that have unique properties and applications in differing fields of chemistry. One area where DES systems find utility is the design of polymeric gels, often referred to as "eutectogels," which can be prepared either using a DES to replace a traditional solvent, or where monomers form part of the DES themselves. Due to the extensive network of intramolecular interactions (e.g., hydrogen bonding) and ionic species that exist in DES systems, polymeric eutectogels often possess appealing material properties-high adhesive strength, tuneable viscosity, rapid polymerization kinetics, good conductivity, as well as high strength and flexibility. In addition, non-covalent crosslinking approaches are possible due to the inherent interactions that exist in these materials. This review considers several key applications of polymeric eutectogels, including organic electronics, wearable sensor technologies, 3D printing resins, adhesives, and a range of various biomedical applications. The design, synthesis, and properties of these eutectogels are discussed, in addition to the advantages of this synthetic approach in comparison to traditional gel design. Perspectives on the future directions of this field are also highlighted.

Computational biophysical characterization of the effect of gatekeeper mutations on the binding of ponatinib to the FGFR kinase

Fibroblast Growth Factor Receptor (FGFR) is connected to numerous downstream signalling cascades regulating cellular behavior. Any dysregulation leads to a plethora of illnesses, including cancer. Therapeutics are available, but drug resistance driven by gatekeeper mutation impedes the treatment. Ponatinib is an FDA-approved drug against BCR-ABL kinase and has shown effective results against FGFR-mediated carcinogenesis. Herein, we undertake molecular dynamics simulation-based analysis on ponatinib against all the FGFR isoforms having Val to Met gatekeeper mutations. The results suggest that ponatinib is a potent and selective inhibitor for FGFR1, FGFR2, and FGFR4 gatekeeper mutations. The extensive electrostatic and van der Waals interaction network accounts for its high potency. The FGFR3_VM mutation has shown resistance towards ponatinib, which is supported by their lesser binding affinity than wild-type complexes. The disengaged molecular brake and engaged hydrophobic spine were believed to be the driving factors for weak protein-ligand interaction. Taken together, the inhibitory and structural characteristics exhibited by ponatinib may aid in thwarting resistance based on Val-to-Met gatekeeper mutations at an earlier stage of treatment and advance the design and development of other inhibitors targeted at FGFRs harboring gatekeeper mutations.

Unraveling the Binding Mode of Cyclic Adenosine-Inosine Monophosphate (cAIMP) to STING through Molecular Dynamics Simulations.

The stimulator of interferon genes (STING) plays a significant role in immune defense and protection against tumor proliferation. Many cyclic dinucleotide (CDN) analogues have been reported to regulate its activity, but the dynamic process involved when the ligands activate STING remains unclear. In this work, all-atom molecular dynamics simulations were performed to explore the binding mode between human STING (hSTING) and four cyclic adenosine-inosine monophosphate analogs (cAIMPs), as well as 2',3'-cGMP-AMP (2',3'-cGAMP). The results indicate that these cAIMPs adopt a U-shaped configuration within the binding pocket, forming extensive non-covalent interaction networks with hSTING. These interactions play a significant role in augmenting the binding, particularly in interactions with Tyr167, Arg238, Thr263, and Thr267. Additionally, the presence of hydrophobic interactions between the ligand and the receptor further contributes to the overall stability of the binding. In this work, the conformational changes in hSTING upon binding these cAIMPs were also studied and a significant tendency for hSTING to shift from open to closed state was observed after binding some of the cAIMP ligands.

Contributors to fatigue among nurses working in critical care units: A qualitative study.

Fatigue is a growing concern among nurses who provide care to unstable and critically ill patients in critical care wards. It has various negative consequences for both nurses and patients. To explore and describe contributors to fatigue among nurses working in critical care units. A qualitative content analysis. Participants were 21 nurses chosen using purposeful sampling. They were working in different critical care units at different hospitals in nine urban areas of Iran. Semi-structured interviews were used to collect data, and conventional content analysis was used to develop categories and subcategories. The analysis of data on the nurses' perspectives and experiences led to developing nine categories as contributors to fatigue: 'compassion fatigue', 'extensive network of interactions', 'sound and alarm fatigue', 'psychological tensions', 'managerial and organizational tensions', 'lack of motivation and incentives', 'individual characteristics of nurses', 'physical and mental pressure', and requirements of special care and situational complexity'. Understanding the factors that influence the experience of fatigue among nurses working in critical care units is essential for maintaining a reliable and high-quality health care environment within health care facilities. The experience of fatigue by critical care nurses can increase the possibility of practice errors, reduce patient safety and quality of care, and enhance staff burnout and turnover. Proactive policies should be formulated to assess nurses' fatigue levels and implement strategies for effective fatigue management. This approach aims to enhance both patient safety and job satisfaction in the workplace.

Identification and Analysis of lncRNA and circRNA Related to Wheat Grain Development.

The role of lncRNA and circRNA in wheat grain development is still unclear. The objectives of this study were to characterize the lncRNA and circRNA in the wheat grain development and to construct the interaction network among lncRNA, circRNA, and their target miRNA to propose a lncRNA-circRNA-miRNA module related to wheat grain development. Full transcriptome sequencing on two wheat varieties (Annong 0942 and Anke 2005) with significant differences in 1000-grain weight at 10 d (days after pollination), 20 d, and 30 d of grain development were conducted. We detected 650, 736, and 609 differentially expressed lncRNA genes, and 769, 1054, and 1062 differentially expressed circRNA genes in the grains of 10 days, 20 days and 30 days after pollination between Annong 0942 and Anke 2005, respectively. An analysis of the lncRNA-miRNA and circRNA-miRNA targeting networks reveals that circRNAs exhibit a more complex and extensive interaction network in the development of cereal grains and the formation of grain shape. Central to these interactions are tae-miR1177, tae-miR1128, and tae-miR1130b-3p. In contrast, lncRNA genes only form a singular network centered around tae-miR1133 and tae-miR5175-5p when comparing between varieties. Further analysis is conducted on the underlying genes of all target miRNAs, we identified TaNF-YB1 targeted by tae-miR1122a and TaTGW-7B targeted by miR1130a as two pivotal regulatory genes in the development of wheat grains. The quantitative real-time PCR (qRT-PCR) and dual-luciferase reporter assays confirmed the target regulatory relationships between miR1130a-TaTGW-7B and miR1122a-TaNF-YB1. We propose a network of circRNA and miRNA-mediated gene regulation in the development of wheat grains.

Myocardial B cells have specific gene expression and predicted interactions in dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy.

Growing evidence from animal models indicates that the myocardium hosts a population of B cells that play a role in the development of cardiomyopathy. However, there is minimal data on human myocardial B cells in the context of cardiomyopathy. We integrated single-cell and single-nuclei datasets from 45 healthy human hearts, 70 hearts with dilated cardiomyopathy (DCM), and 8 hearts with arrhythmogenic right ventricular cardiomyopathy (ARVC). Interactions between B cells and other cell types were investigated using the CellChat Package. Differential gene expression analysis comparing B cells across conditions was performed using DESeq2. Pathway analysis was performed using Ingenuity, KEGG, and GO pathways analysis. We identified 1,100 B cells, including naive B cells and plasma cells. Cells showed an extensive network of interactions within the healthy myocardium that included outgoing signaling to macrophages, T cells, endothelial cells, and pericytes, and incoming signaling from endothelial cells, pericytes, and fibroblasts. This niche relied on ECM-receptor, contact, and paracrine interactions; and changed significantly in the context of cardiomyopathy, displaying disease-specific features. Differential gene expression analysis showed that in the context of DCM both naive and plasma B cells upregulated several pathways related to immune activation, including upregulation of oxidative phosphorylation, upregulation of leukocyte extravasation, and, in naive B cells, antigen presentation. The human myocardium contains naive B cells and plasma cells, integrated into a diverse and dynamic niche that has distinctive features in healthy, DCM, and ARVC. Naive myocardial-associated B cells likely contribute to the pathogenesis of human DCM.

MiRNA-miRNA interaction network in peripheral blood of patients with myocardial infarction: a gene expression meta-analysis

In recent years, investigations have revealed that microRNAs (miRNAs) can bind together and form a miRNA-miRNA-mRNA regulatory network that alters the consequence of miRNA-mRNA interaction. If we consider the miRNA that binds to mRNA as the primary miRNA and the miRNA that binds to the primary miRNA as the secondary one, secondry miRNAs can act as master regulators upstream of primary miRNAs and their target mRNAs. One of the distinguishing characteristics of secondary miRNAs as master regulators within a diverse set of differentially expressed genes is the absence of direct target mRNA for them. Instead, these master regulators exclusively govern the regulation of miRNAs that target specific mRNAs. Through in silico analysis, we identified 18 miRNAs among 385 differentially expressed miRNAs (DEmiRNAs) with no direct target mRNAs among 58 differentially expressed mRNAs (DEmRNAs) in peripheral blood of patients with myocardial infarction (MI). Instead, these secondary miRNAs targeted 9 primary miRNAs that had 36 direct targets among 58 DEmRNAs. We found that one primary miRNA might be regulated by more than one secondary miRNAs and each secondary miRNA can target more than one primary miRNAs. Among identified miRNA-miRNA-mRNA networks miR-188-5p/miR-299-3p/natural killer cell granule protein (NKG7), miR-200a-3p/miR-199b-5p/granzyme B (GZMB), and miR-377-3p/miR-581/oviductal glycoprotein 1 (OVGP1) exhibited higher scors in terms of expression levels (>2-fold increase or decrease) and strengh of interactions (ΔG < −5). Given the extensive network of miRNA interactions, focusing on master regulators opens up avenues for identifying key regulatory nodes for more effective therapeutic strategies.

Reduced peptidoglycan synthesis capacity impairs growth of E. coli at high salt concentration.

Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli, we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCEEscherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.

Quantification of intraspecific and interspecific competition in fish species of the Aegean Sea

Abstract Competition shapes species coexistence and community assembly, playing a central role in maintaining biodiversity and ecosystem resilience. Here, we used an asymmetric competition index that quantifies the possible competitive effect of a species on any other, using southern Aegean Sea nekton biomass from the Mediterranean International Trawl Survey dataset, combined with data on six traits relevant to trophic ecology and spawning. We modeled the effect of selected haul-level covariates on the competition index, and we indicated competition variation across depth and habitats. Most species experienced stronger inter- than intra-specific competition. Both the higher-than-expected significance of interspecific competition (in comparison to intraspecific that is generally anticipated to be higher) and the existence of an extensive network of multiple competitive interactions indicated, are associated with high biodiversity, combined to the absence of dominance by one or a few species in the community (no species is disproportionally highly abundant in comparison to others); the latter may be relevant to community stability and resilience. The quantification of competition across marine ecosystems can help predict how fish communities will respond to future resource availability and environmental conditions.

De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures invitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5-/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the invivo functions of Tektin 5.

Evolutionarily conserved cysteines in plant cytosolic seryl-tRNA synthetase are important for its resistance to oxidation.

We have previously identified a unique disulfide bond in the crystal structure of Arabidopsis cytosolic seryl-tRNA synthetase involving cysteines evolutionarily conserved in all green plants. Here, we discovered that both cysteines are important for protein stability, but with opposite effects, and that their microenvironment may promote disulfide bond formation in oxidizing conditions. The crystal structure of the C244S mutant exhibited higher rigidity and an extensive network of noncovalent interactions correlating with its higher thermal stability. The activity of the wild-type showed resistance to oxidation with H2 O2 , while the activities of cysteine-to-serine mutants were impaired, indicating that the disulfide link may enable the protein to function under oxidative stress conditions which can be beneficial for an efficient plant stress response.

Navigating the integration of biotic interactions in biogeography

AbstractBiotic interactions are widely recognised as the backbone of ecological communities, but how best to study them is a subject of intense debate, especially at macro‐ecological scales. While some researchers claim that biotic interactions need to be observed directly, others use proxies and statistical approaches to infer them. Despite this ambiguity, studying and predicting the influence of biotic interactions on biogeographic patterns is a thriving area of research with crucial implications for conservation. Three distinct approaches are currently being explored. The first approach involves empirical observation and measurement of biotic interactions' effects on species demography in laboratory or field settings. While these findings contribute to theory and to understanding species' demographies, they can be challenging to generalise on a larger scale. The second approach centers on inferring biotic associations from observed co‐occurrences in space and time. The goal is to distinguish the environmental and biotic effects on species distributions. The third approach constructs extensive potential interaction networks, known as metanetworks, by leveraging existing knowledge about species ecology and interactions. This approach analyses local realisations of these networks using occurrence data and allows understanding large distributions of multi‐taxa assemblages. In this piece, we appraise these three approaches, highlighting their respective strengths and limitations. Instead of seeing them as conflicting, we advocate for their integration to enhance our understanding and expand applications in the emerging field of interaction biogeography. This integration shows promise for ecosystem understanding and management in the Anthropocene era.

Probing excited state 1Hα chemical shifts in intrinsically disordered proteins with a triple resonance-based CEST experiment: Application to a disorder-to-order switch.

Over 40% of eukaryotic proteomes and 15% of bacterial proteomes are predicted to be intrinsically disordered based on their amino acid sequence. Intrinsically disordered proteins (IDPs) exist as heterogeneous ensembles of interconverting conformations and pose a challenge to the structure-function paradigm by apparently functioning without possessing stable structural elements. IDPs play a prominent role in biological processes involving extensive intermolecular interaction networks and their inherently dynamic nature facilitates their promiscuous interaction with multiple structurally diverse partner molecules. NMR spectroscopy has made pivotal contributions to our understanding of IDPs because of its unique ability to characterize heterogeneity at atomic resolution. NMR methods such as Chemical Exchange Saturation Transfer (CEST) and relaxation dispersion have enabled the detection of 'invisible' excited states in biomolecules which are transiently and sparsely populated, yet central for function. Here, we develop a 1Hα CEST pulse sequence which overcomes the resonance overlap problem in the 1Hα-13Cα plane of IDPs by taking advantage of the superior resolution in the 1H-15N correlation spectrum. In this sequence, magnetization is transferred after 1H CEST using a triple resonance coherence transfer pathway from 1Hα (i) to 1HN(i+1) during which the 15N(t1) and 1HN(t2) are frequency labelled. This approach is integrated with spin state-selective CEST for eliminating spurious dips in CEST profiles resulting from dipolar cross-relaxation. We apply this sequence to determine the excited state 1Hα chemical shifts of the intrinsically disordered DNA binding domain (CytRN) of the bacterial cytidine repressor (CytR), which transiently acquires a functional globally folded conformation. The structure of the excited state, calculated using 1Hα chemical shifts in conjunction with other excited state NMR restraints, is a three-helix bundle incorporating a helix-turn-helix motif that is vital for binding DNA.

Intrinsic Disorder in the Human Tear Proteome.

We aimed to characterize the proteome of human tears and assess for the presence of intrinsically disordered proteins (IDPs). IDPs, despite lacking a rigid three-dimensional structure, maintain biological functionality and could shed light on the molecular interactions within tears. We analyzed a dataset of 1475 proteins identified in the tear film of three healthy subjects. We employed several computational tools, including the Compositional Profiler, Rapid Intrinsic Disorder Analysis Online, Search Tool for the Retrieval of Interacting Genes, and Database of Disordered Protein Predictors to evaluate the intrinsic disorder, protein interactions, and functional characterization of the disordered regions within this proteome. Our analysis showed a notable inclination toward intrinsic disorder. Two out of 10 order-promoting residues and five out of 10 disorder-promoting residues were found enriched. Using the Predictor of Natural Disordered Regions (PONDR) VSL2 output, 95% of these proteins were classified as highly or moderately disordered. We revealed an extensive protein-protein interaction network with significant interaction enrichment. The most disordered proteins exhibited higher disorder binding sites and diverse posttranslational modifications compared to the most ordered ones. To the best of our knowledge, our study is the first comprehensive analysis of intrinsic disorder in the human tear film proteome, and it revealed an abundance of IDPs and their role in protein function and interaction networks. These findings suggest that variations in the intrinsic disorder of a tear film could be impacted by systemic and ocular conditions, offering promising avenues for disease biomarker identification and drug target development. Further research is needed to understand the implications of these findings in human health and disease.

Persuading the Non-canonical Intercalated-Motif DNA to Reveal Its Structure.

This invited Team Profile was created by Kevin Li and Liliya Yatsunyk, Swarthmore College PA (USA) and by John Schneekloth, Jr, (Jay) National Cancer Institute, Frederick MD (USA). They recently published an article on the first crystal structure of an intercalated motif (i-motif or iM) from the HRAS oncogene involved in many cancers. The iHRAS structure was solved to 1.8 Å resolution. It contains a tail-to-tail dimer of two iMs each with six C-C+ base pairs. The structure is unique in that only two base pairs out of 20 are canonical. The extensive network of capping and connecting interactions is unprecedented. The unique structural elements (loops/connecting region) may be targeted by ligands or proteins as cancer therapies. iHRAS represents the first crystallized iM-forming structure from a human promoter. "Crystal Structure of an iM from the HRAS Oncogene Promoter", K. S. Li, D. Jordan, L. Y. Lin, S. E. McCarthy, J. S. Schneekloth Jr., and L. A. Yatsunyk, Angew. Chem. Int. Ed. Engl. 2023, 62, e202301666.

Persuading the Non‐canonical Intercalated‐Motif DNA to Reveal Its Structure

AbstractThis invited Team Profile was created by Kevin Li and Liliya Yatsunyk, Swarthmore College PA (USA) and by John Schneekloth, Jr, (Jay) National Cancer Institute, Frederick MD (USA). They recently published an article on the first crystal structure of an intercalated motif (i‐motif or iM) from the HRAS oncogene involved in many cancers. The iHRAS structure was solved to 1.8 Å resolution. It contains a tail‐to‐tail dimer of two iMs each with six C−C+ base pairs. The structure is unique in that only two base pairs out of 20 are canonical. The extensive network of capping and connecting interactions is unprecedented. The unique structural elements (loops/connecting region) may be targeted by ligands or proteins as cancer therapies. iHRAS represents the first crystallized iM‐forming structure from a human promoter. “Crystal Structure of an iM from the HRAS Oncogene Promoter”, K. S. Li, D. Jordan, L. Y. Lin, S. E. McCarthy, J. S. Schneekloth Jr., and L. A. Yatsunyk, Angew. Chem. Int. Ed. Engl. 2023, 62, e202301666.

BRCA1/BARD1 intrinsically disordered regions facilitate chromatin recruitment and ubiquitylation.

BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits. Herein, we reveal novel interactions that support robust H2A ubiquitylation activity mediated through a high-affinity, intrinsically disordered DNA-binding region of BARD1. These interactions support BRCA1/BARD1 recruitment to chromatin and sites of DNA damage in cells and contribute to their survival. We also reveal distinct BRCA1/BARD1 complexes that depend on the presence of H2A K15-Ub, including a complex where a single BARD1 subunit spans adjacent nucleosome units. Our findings identify an extensive network of multivalent BARD1-nucleosome interactions that serve as a platform for BRCA1/BARD1-associated functions on chromatin.

Structural basis of purine nucleotide inhibition of human uncoupling protein 1.

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.

ApoM binds endotoxin contributing to neutralization and clearance by High Density Lipoprotein

BackgroundHDL possesses anti-inflammatory properties, however, the exact mechanism is not fully understood. Endotoxin is a potent inducers of TLR4 signaling, leading to inflammatory mediators’ release. It has been estimated that TLR4 recognizes about 5% of circulating lipopolysaccharide whereas 95% is cleared by plasma lipoproteins, mainly HDL. ApoM is required for HDL biogenesis and 95% of plasma ApoM is found associated with HDL, both are significantly reduced during sepsis. AimThe aim of this study is to investigate whether ApoM binds endotoxin and contributes to anti-inflammatory activity of HDL. MethodsIsothermal Titration Calorimetry (ITC) was used to determine the binding of ultrapure E. coli LPS to the recombinant ApoM protein. Purified human HDL and recombinant ApoM was used to investigate LPS neutralization using human and murine macrophages and computational simulation was performed. ResultApoM shows high affinity for E. coli LPS, forming 1:1 complexes with Kd values below 1 μΜ, as revealed by ITC. The binding process is strongly exothermic and enthalpy-driven (ΔrH = −36.5 kJ/mol), implying the formation of an extensive network of interactions between ApoM and LPS in the bound state. Computational simulation also predicted high-affinity binding between ApoM and E. coli LPS and the best scoring models showed E. coli LPS docking near the calyx of ApoM without blocking the pocket. The biological significance of this interaction was further demonstrated in macrophages where purified HDL neutralized an E. coli LPS effect and significantly reduced TNFα release from human THP-1 cells. ConclusionApoM binds LPS to facilitate endotoxin neutralization and clearance by HDL.

Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor

AbstractBruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell–driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell–driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.