Differences In Binding Affinity Research Articles

Faricimab maintains substance integrity and sterility after compounding and storage in two different polypropylene syringe types.

Compounding and storage of intravitreal anti-vascular endothelial growth factor (anti-VEGF) agents in syringes is commonly performed in an off-label manner. However, the preservation of compound integrity and microbiological safety must be guaranteed. The aim of this study was to compare the chemical and physical stability, sterility and binding affinity to vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) of faricimab, a novel bispecific anti-VEGF/Ang-2 biologic, after compounding and storage in two different polypropylene syringe types for up to 28 days. Faricimab was compounded into silicone oil-free and silicone oil-containing polypropylene syringes under controlled aseptic clean room conditions and stored under light protection at 2-8 °C for up to 28 days. Compound integrity was analysed by size exclusion chromatography, nano differential scanning fluorimetry, UV-vis and dynamic light scattering. The analysis of the simultaneous binding of VEGF and Ang-2 was performed by grating-coupled interferometry. Additionally, samples were tested for sterility and presence of bacterial endotoxins. One-way ANOVA test was used to analyse statistical significance (p ≤ 0.05). No significant differences in VEGF and ANG-2 binding affinity were found in faricimab samples stored in either syringe type after 28 days compared to control. Chemical and physical stability testing revealed no statistically significant variation. Furthermore, sterility and the absence of bacterial endotoxins could be maintained. Our findings confirm the pharmaceutical safety of compounded faricimab after storage for up to 28 days. This may facilitate a cost-effective off-label use of faricimab in clinical practice while maintaining safety in the treatment of patients.

Identification of Marijuana Using Silver-Phosphine Ion Complexation and a Semi-Quantitative 1% Decision-Point Assay

Due to the strict federal regulations concerning ∆9-tetrahydrocannabinol (∆9-THC) content for the differentiation of hemp and marijuana outlined in the 2018 Farm Bill, the ability to quickly and reliably differentiate cannabis as marijuana or hemp is crucial within both the seized drug community and hemp industry. This study provides a novel direct mass spectrometry approach for the identification of marijuana using Ag-phosphine ion complexation and a semi-quantitative 1% decision-point assay. The main constituents of hemp and marijuana, cannabidiol (CBD) and ∆9-THC, are isomeric and cannot be differentiated using soft ionization mass spectrometry techniques alone. However, the incorporation of [Ag(PPh3)(OTf)]2 enables the formation of unique MS/MS product ions at m/z 421/423, m/z 353/355, and m/z 231 for CBD due to differences in binding affinity, allowing CBD to be differentiated from ∆9-THC. Likewise, the isomeric cannabinoid precursors ∆9-tetrahydrocannabinolic acid (∆9-THCA) and cannabidiolic acid (CBDA) can be differentiated due to the formation of unique MS/MS product ions at m/z 465/467 and m/z 379/381, which are specific to CBDA. Eight additional cannabinoids were also characterized utilizing the proposed Ag-phosphine ion complexation approach. To reduce the potential for false positives, a more conservative 1% decision-point assay was developed by fortifying 1% weight-by-volume ∆9-THC-d9 into methanolic extracts of authentic cannabis plant material, followed by assessing if the total ∆9-THC content (containing ∆9-THC and ∆9-THCA) was greater than or less than the intensity of the spiked internal standard. When the developed approach was applied to 20 methanolic extracts of authentic cannabis samples with known cannabinoid compositions, 90% of the samples were correctly identified as marijuana or not marijuana based on the 1% administrative threshold for the total ∆9-THC content. The two lone misidentifications were due to the presence of elevated ∆8-THC, which highlights the necessity to explore more selective ligands in the future. This study provides the first application of Ag-ligand ion complexation for the identification of marijuana based on a semi-quantitative 1% decision-point assay, which shows great promise as an alternative method for the rapid identification of marijuana.

Unraveling the role of integrating signal peptides into natural collagen on modulating cancer cell adhesion

The signal peptides GVMGFO and GFOGER exhibit differential binding affinities towards Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and HT-1080 human fibrosarcoma cells, respectively, which in turn modulate the cell adhesion properties of natural collagen. GVMGFO demonstrates a more potent interaction with discoidin domain receptor 1(DDR1)-expressing MCF-7 cells, whereas GFOGER preferentially binds to the integrin α2β1 present on HT-1080 cells. The integration of GVMGFO into natural collagen through direct doping or crosslinking markedly enhances its association with MCF-7 cells, especially when optimal peptide concentrations and blending ratios are utilized, indicating a synergistic effect. This augmented adhesion is attributed to specific binding at the DDR1-collagen interface, facilitated by a constellation of amino acids within the collagen scaffold engaging with the DDR1 discoidin (DS) domain through polar interactions and hydrogen bonding. Conversely, the incorporation of GFOGER into natural collagen through co-assembling or crosslinking leads to a progressive increase in adherence to HT-1080 cells, as evidenced by the peptide's affinity for integrin α2β1. These findings advance the design of collagen-based biomaterials for targeted cellular interactions in the medical, pharmaceutical, and enhance our understanding of the molecular mechanisms governing peptide-collagen mediated cell adhesion processes.

Expression interplay of genes coding for calcium-binding proteins and transcription factors during the osmotic phase provides insights on salt stress response mechanisms in bread wheat

Bread wheat is an important crop for the human diet, but the increasing soil salinization is reducing the yield. The Ca2+ signaling events at the early stages of the osmotic phase of salt stress are crucial for the acclimation response of the plants through the performance of calcium-sensing proteins, which activate or repress transcription factors (TFs) that affect the expression of downstream genes. Physiological, genetic mapping, and transcriptomics studies performed with the contrasting genotypes Syn86 (synthetic, salt-susceptible) and Zentos (elite cultivar, salt-tolerant) were integrated to gain a comprehensive understanding of the salt stress response. The MACE (Massive Analysis of cDNA 3ʹ-Ends) based transcriptome analysis until 4 h after stress exposure revealed among the salt-responsive genes, the over-representation of genes coding for calcium-binding proteins. The functional and structural diversity within this category was studied and linked with the expression levels during the osmotic phase in the contrasting genotypes. The non-EF-hand category from calcium-binding proteins was found to be enriched for the susceptibility response. On the other side, the tolerant genotype was characterized by a faster and higher up-regulation of genes coding for proteins with EF-hand domain, such as RBOHD orthologs, and TF members. This study suggests that the interplay of calcium-binding proteins, WRKY, and AP2/ERF TF families in signaling pathways at the start of the osmotic phase can affect the expression of downstream genes. The identification of SNPs in promoter sequences and 3ʹ -UTR regions provides insights into the molecular mechanisms controlling the differential expression of these genes through differential transcription factor binding affinity or altered mRNA stability.

On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.

The humanization of camelid-derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity-determining regions (CDRs), Vernier and Hallmark residues, shaping the three-dimensional fold and influencing VHH structure and function. Additionally, the presence of non-canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid-derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next-generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non-canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.

Novel Bis-thiazoles with pyridine and 1,4-Dihydropyridine linkers as potential anti-Alzheimer agents

A series of bis-hydrazonothiazoles with pyridine and 1,4-dihydropyridine linkers was synthesized using 2,2′-[(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)bis(ethan-1-yl-1-ylidene)]bis(hydrazine-1-carbothioamide) and 2,2′-[(2,6-dimethylpyridine-3,5-diyl)bis(ethan-1-yl-1-ylidene)]bis(hydrazine-1-carbothioamide) as starting materials, which were reacted with 1-aryl-2-bromoethanone to form the desired bis-hydrazonothiazoles. Additionally, an alternative synthetic strategy involved the simple oxidation of 1,4-dihydropyridine linkers using a mixture of aqueous sodium nitrite and acetic acid to produce pyridine linkers. The synthesized compounds were subjected to molecular docking studies against the protein 1EVE (one of the AD bio-markers) to evaluate their 3-D orientation, flexibility, and conformational changes upon binding—key factors for optimizing drug design and therapeutic efficacy. The binding affinities of the synthesized compounds ranged from −1.14 to −11.64 kcal/mol, compared to -6.78 to -10.24 kcal/mol for the reference drugs. Statistical analysis revealed no significant difference in binding affinity between Donepezil and compounds 5c, 7a, 7b, and 8a, indicating similar therapeutic potential. Differences in binding affinities among the approved drugs underscored their distinct interaction profiles with the target protein. Molecular docking and SwissADME predictions helped prioritize molecules based on drug-likeness and pharmacokinetic profiles, identifying candidates for in-vivo validation and enhancing the drug development process.

Chelator-Assisted Precipitation-Based Separation of the Rare Earth Elements Neodymium and Dysprosium from Aqueous Solutions.

The rare earth elements (REEs) are critical resources for many clean energy technologies, but are difficult to obtain in their elementally pure forms because of their nearly identical chemical properties. Here, an analogue of macropa, G-macropa, was synthesized and employed for an aqueous precipitation-based separation of Nd3+ and Dy3+. G-macropa maintains the same thermodynamic preference for the large REEs as macropa, but shows smaller thermodynamic stability constants. Molecular dynamics studies demonstrate that the binding affinity differences of these chelators for Nd3+ and Dy3+ is a consequence of the presence or absence of an inner-sphere water molecule, which alters the donor strength of the macrocyclic ethers. Leveraging the small REE affinity of G-macropa, we demonstrate that within aqueous solutions of Nd3+, Dy3+, and G-macropa, the addition of HCO3 - selectively precipitates Dy2(CO3)3, leaving the Nd3+-G-macropa complex in solution. With this method, remarkably high separation factors of 841 and 741 are achieved for 50 : 50 and 75 : 25 mixtures. Further studies involving Nd3+:Dy3+ ratios of 95 : 5 in authentic magnet waste also afford an efficient separation as well. Lastly, G-macropa is recovered via crystallization with HCl and used for subsequent extractions, demonstrating its good recyclability.

A bulge uridine in the HIF2α IRE allows IRP1 but not IRP2 to selectively regulate HIF2α expression and ensuing EPO levels

AbstractIron regulatory proteins (IRP1 and IRP2) play a pivotal role in maintaining cellular iron homeostasis by binding to iron-responsive elements (IREs) of target messenger RNAs and regulating the expression of these iron-related genes. Mice and humans who lack functional IRP1 develop erythrocytosis due to erythropoietin (EPO) overproduction, whereas those who lack IRP2 develop microcytic anemia, believed to result from iron deficiency of erythroblasts. Here, we discovered that IRP2 deficiency reduced the expression of hypoxia-inducible factor 2α (HIF2α) and its transcriptional target, EPO, thereby compromising the stress erythropoiesis response to generate red blood cells upon anemia. The distinct consequences of IRP2 and IRP1 on EPO result from the higher binding affinity of the HIF2α IRE for IRP1 than IRP2. This difference in binding affinity arises from a bulge uridine in the upper stem of HIF2α IRE that impairs the ability of IRP2 to bind the IRE. These results reveal that IRP1 and IRP2 play distinct roles in erythropoiesis and unveil an unsuspected IRE binding preference that contributes to the divergent phenotypes observed in IRP1- and IRP2-deficient mammals.

Immunogenetic profiles of 9 human herpes virus envelope glycoproteins

Human herpes viruses (HHV) are ubiquitous and have been implicated in numerous long-term health conditions. Since the association between viral exposure and long-term health impacts is partially influenced by variation in human leukocyte antigen (HLA) genes, we evaluated in silico the binding affinities of 9 HHV envelope glycoproteins with 127 common HLA Class I and Class II molecules. The findings show substantial variability in HHV binding affinity across viruses, HLA Class, HLA genes, and HLA alleles. Specific findings were as follows: (1) the predicted binding affinities of HHVs were characterized by four distinct groupings—[HHV1, HHV2], [HHV3, HHV4, HHV5], [HHV6A], [HHV6B, HHV7, HHV8]—with relatively lower binding affinities for HHV1, HHV2, and HHV6a compared to other HHVs; (2) significantly higher binding affinity was found for HLA Class I relative to Class II; (3) analyses within each class demonstrated that alleles of the C gene (for Class I) and DRB1 gene (for Class II) had the highest binding affinities; and (4) for each virus, predicted binding affinity to specific alleles varied, with HHV6a having the lowest affinity for HHV-HLA complexes, and HHV3, HHV4, and HHV5 having the highest. Since HLA-antigen binding is the first step in initiating an immune response to foreign antigens, these relative differences in HHV binding affinities are likely to influence long-term health impacts such that the cells infected with viruses associated with higher binding affinities across common HLA alleles may be more reduced in numbers, thereby lowering the potential for long-term sequelae of their infections.

Interference with Systemic Negative Feedback Regulation as a Potential Mechanism for Nonmonotonic Dose-Responses of Endocrine-Disrupting Chemicals.

Endocrine-disrupting chemicals (EDCs) often exhibit nonmonotonic dose-response (NMDR) relationships, posing significant challenges to health risk assessment and regulations. Several molecular mechanisms operating locally in cells have been proposed, including opposing actions via different receptors, mixed-ligand heterodimer formation, and receptor downregulation. Systemic negative feedback regulation of hormone homeostasis, which is a common feature of many endocrine systems, has also been invoked as a mechanism; however, whether and how exactly such global feedback structure may underpin NMDRs is poorly understood. We hypothesize that an EDC may compete with the endogenous hormone for receptors (i) at the central site to interfere with the feedback regulation thus altering the physiological hormone level, and (ii) at the peripheral site to disrupt the hormone action; this dual-action may oppose each other, producing nonmonotonic endocrine effects. The objective here is to explore - through computational modeling - how NMDRs may arise through this potential mechanism and the relevant biological variabilities that enable susceptibility to nonmonotonic effects. We constructed a dynamical model of a generic hypothalamic-pituitary-endocrine (HPE) axis with negative feedback regulation between a pituitary hormone and a terminal effector hormone (EH). The effects of model parameters, including receptor binding affinities and efficacies, on NMDR were examined for EDC agonists and antagonists. Monte Carlo human population simulations were then conducted to systemically explore biological parameter conditions that engender NMDR. When an EDC interferes sufficiently with the central feedback action of EH, the net endocrine effect at the peripheral target site can be opposite to what is expected of an agonist or antagonist at low concentrations. J/U or Bell-shaped NMDRs arise when the EDC has differential binding affinities and/or efficacies, relative to EH, for the peripheral and central receptors. Quantitative relationships between these biological variabilities and associated distributions were discovered, which can distinguish J/U and Bell-shaped NMDRs from monotonic responses. The ubiquitous negative feedback regulation in endocrine systems can act as a universal mechanism for counterintuitive and nonmonotonic effects of EDCs. Depending on key receptor kinetic and signaling properties of EDCs and endogenous hormones, some individuals may be more susceptible to these complex endocrine effects.

Chelator‐Assisted Precipitation‐Based Separation of the Rare Earth Elements Neodymium and Dysprosium from Aqueous Solutions

AbstractThe rare earth elements (REEs) are critical resources for many clean energy technologies, but are difficult to obtain in their elementally pure forms because of their nearly identical chemical properties. Here, an analogue of macropa, G‐macropa, was synthesized and employed for an aqueous precipitation‐based separation of Nd3+ and Dy3+. G‐macropa maintains the same thermodynamic preference for the large REEs as macropa, but shows smaller thermodynamic stability constants. Molecular dynamics studies demonstrate that the binding affinity differences of these chelators for Nd3+ and Dy3+ is a consequence of the presence or absence of an inner‐sphere water molecule, which alters the donor strength of the macrocyclic ethers. Leveraging the small REE affinity of G‐macropa, we demonstrate that within aqueous solutions of Nd3+, Dy3+, and G‐macropa, the addition of HCO3− selectively precipitates Dy2(CO3)3, leaving the Nd3+−G‐macropa complex in solution. With this method, remarkably high separation factors of 841 and 741 are achieved for 50 : 50 and 75 : 25 mixtures. Further studies involving Nd3+:Dy3+ ratios of 95 : 5 in authentic magnet waste also afford an efficient separation as well. Lastly, G‐macropa is recovered via crystallization with HCl and used for subsequent extractions, demonstrating its good recyclability.

Photo- and thermo-responsive poly(ionic liquid) hydrogel regulated by hierarchical host–guest interactions

Supramolecular hydrogels constructed through various host–guest interactions have attracted tremendous attention in view of their excellent dynamic and multi-stimuli responsive performances. Herein, benzyl (Bn) and azobenzene (Azo) grafted poly(ionic liquid)s (PILs) were prepared via a one-pot post-synthesis strategy. By virtue of the difference in binding affinity of Azo and Bn groups with the host compound cucurbituril [8] urea (CB[8]), the newly-developed supramolecular hydrogels exhibited distinctive photo- and thermo-responsive behaviors. Under UV light irradiation, PIL-Azo-Bn/CB[8] hydrogel got weaker, while a gel-to-sol transition was observed when treated by heating. Moreover, the above tunable behaviors were highly reversible. Variable-temperature measurements such as proton nuclear magnetic resonance and ultraviolet–visible spectroscopy illustrated that the photo- and thermo-regulated performances of PIL hydrogel were ascribed to the hierarchical host–guest interactions. As a prototype application in smart circuits, PIL hydrogel exhibited a photo- and thermo-tuned conductivity. Thus, our finding presented a novel and versatile platform to fabricate responsive functional hydrogels via host–guest interactions, which has great promising applications in smart materials and flexible sensors.

Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding.

Trans-activation response (TAR) RNA-binding protein (TRBP) has emerged as a key player in the RNA interference pathway, wherein it binds to different pre-microRNAs (miRNAs) and small interfering RNAs (siRNAs), each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12-bp dsRNA owing to the presence of critical residues and structural plasticity. Furthermore, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.

Mechanistic and structural insights into vitamin B2 metabolizing enzyme riboflavin kinase from Leishmania donovani

Leishmania donovani relies on specific vitamins and cofactors crucial for its survival and pathogenesis. Tailoring therapies to disrupt these pathways offers a promising strategy for the treatment of Visceral Leishmaniasis. Current treatment regimens are limited due to drug resistance and high costs. The dependency of Leishmania parasites on Vitamin B2 and its metabolic products is not known. In this study, we have biochemically and biophysically characterized a Vitamin B2 metabolism enzyme, riboflavin kinase from L. donovani (LdRFK) which converts riboflavin (vitamin B2) into flavin mononucleotide (FMN). Sequence comparison with human counterpart reflects 31.58 % identity only, thus opening up the possibility of exploring it as drug target. The rfk gene was cloned, expressed and the recombinant protein was purified. Kinetic parameters of LdRFK were evaluated with riboflavin and ATP as substrates which showed differential binding affinity when compared with the human RFK enzyme. Thermal and denaturant stability of the enzyme was evaluated. The rfk gene was overexpressed in the parasites and its role in growth and cell cycle was evaluated. In the absence of crystal structure, homology modelling and molecular dynamic simulation studies were performed to predict LdRFK structure. The data shows differences in substrate binding between human and parasite enzyme. This raises the possibility of exploring LdRFK for specific designing of antileishmanial molecules. Gene disruption studies can further validate its candidature as antileishmanial target.

Toxicity of rare earth elements (REEs) to marine organisms: Using species sensitivity distributions to establish water quality guidelines for protecting marine life

A lack of chronic rare earth element (REE) toxicity data for marine organisms has impeded the establishment of numerical REE water quality benchmarks (e.g., guidelines) to protect marine life and assess ecological risk. This study determined the chronic no (significant) effect concentrations (N(S)ECs) and median-effect concentrations (EC50s) of eight key REEs (yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), gadolinium (Gd), dysprosium (Dy) and lutetium (Lu)) for 30 coastal marine organisms (encompassing 22 phyla and five trophic levels from temperate and tropical habitats). Organisms with calcifying life stages were most vulnerable to REEs, which competitively inhibit calcium uptake. The most sensitive organism was a sea urchin, with N(S)ECs ranging from 0.64 μg/L for Y to 1.9 μg/L for La and Pr, and EC50s ranging from 4.3 μg/L for Y to 14.4 μg/L for Pr. Conversely, the least sensitive organism was a cyanobacterium, with N(S)ECs ranging from 121 μg/L for Y to 469 μg/L for Pr, and EC50s ranging from 889 μg/L for Y to 3000 μg/L for Pr. Median sensitivity varied 215-fold across all organisms. The two-fold difference in median toxicity (μmol/L EC50) among REEs (Y ∼ Gd > Lu ∼ Nd ∼ Dy ∼ Ce > La ∼ Pr) was attributed to offset differences in binding affinity (log K) to cell surface receptors and the percentage of free metal ion (REE3+) in the test waters. The toxicity (EC50) of the remaining REEs (samarium, europium, terbium, holmium, thulium and ytterbium) was predicted using a combination of physicochemical data and measured EC50s for the eight tested REEs, with good agreement between predicted and measured EC50s for selected organisms. Numerical REE water quality guidelines to protect marine life were established using species sensitivity distributions (e.g., for 95 % species protection, values ranged from 1.1 μg/L for Y to 3.0 μg/L for La, Pr or Lu).

Functional studies on tandem carbohydrate-binding modules of a multimodular enzyme possessing two catalytic domains.

Lignocellulolytic enzymes, particularly those of bacterial origin, often harbor multiple carbohydrate-binding modules (CBMs). However, the function of CBM multivalency remains poorly understood. This is especially true for enzymes that contain more than one catalytic domain (CD), as the interactions between CDs, CBMs, and CDs and CBMs can be complex. Our research demonstrates that homogeneous CBMs can have distinct functions in a multimodular enzyme. The tandem CBMs coordinate the CDs in catalytic conflict through their differences in binding affinity, ligand preference, and arrangement within the full-length enzyme. Additionally, although the synergism between mannanase and esterase is widely acknowledged, our study highlights the benefits of integrating the two enzymes into a single entity for the degradation of complex substrates. In summary, these findings enhance our understanding of the intra-synergism of a multimodular enzyme and emphasize the significance of multiple CBMs in this context.

Entropy drives the ligand recognition in G-protein-coupled receptor subtypes

Achieving ligand subtype selectivity within highly homologous subtypes of G-protein-coupled receptor (GPCR) is critical yet challenging for GPCR drug discovery, primarily due to the unclear mechanism underlying ligand subtype selectivity, which hampers the rational design of subtype-selective ligands. Herein, we disclose an unusual molecular mechanism of entropy-driven ligand recognition in cannabinoid (CB) receptor subtypes, revealed through atomic-level molecular dynamics simulations, cryoelectron microscopy structure, and mutagenesis experiments. This mechanism is attributed to the distinct conformational dynamics of the receptor's orthosteric pocket, leading to variations in ligand binding entropy and consequently, differential binding affinities, which culminate in specific ligand recognition. We experimentally validated this mechanism and leveraged it to design ligands with enhanced or ablated subtype selectivity. One such ligand demonstrated favorable pharmacokinetic properties and significant efficacy in rodent inflammatory analgesic models. More importantly, it is precisely due to the high subtype selectivity obtained based on this mechanism that this ligand does not show addictive properties in animal models. Our findings elucidate the unconventional role of entropy in CB receptor subtype selectivity and suggest a strategy for rational design of ligands to achieve entropy-driven subtype selectivity for many pharmaceutically important GPCRs.

R1205H (Vicenza) causes conformational changes in the von Willebrand factor D′D3 domains and enhances von Willebrand factor binding to clearance receptors LRP1 and SR-AI

Backgroundvon Willebrand factor (VWF)-R1205H variant (Vicenza) results in markedly enhanced VWF clearance in humans that has been shown to be largely macrophage-mediated. However, the biological mechanisms underlying this enhanced clearance remain poorly understood. ObjectivesThis study aimed to investigate the roles of (i) specific VWF domains and (ii) different macrophage receptors in regulating enhanced VWF-R1205H clearance. MethodsIn vivo clearance of full-length and truncated wild-type (WT)-VWF and VWF with R1205 substitutions was investigated in VWF−/− mice. Plate-binding assays were employed to characterize VWF binding to purified scavenger receptor class A member 1 (SR-AI), low-density lipoprotein receptor–related protein-1 (LRP1) cluster II or cluster IV receptors, and macrophage galactose-type lectin. ResultsIn full-length VWF missing the A1 domain, introduction of R1205H led to significantly enhanced clearance in VWF−/− mice compared with WT-VWF missing the A1 domain. Importantly, R1205H in a truncated VWF-D′D3 fragment also triggered increased clearance compared with WT-VWF-D′D3. Additional in vivo studies demonstrated that VWF-R1205K (which preserves the positive charge at 1205) exhibited normal clearance, whereas VWF-R1205E (which results in loss of the positive charge) caused significantly enhanced clearance, pinpointing the importance of the positive charge at VWF-R1205. In vitro plate-binding studies confirmed increased VWF-R1205H interaction with SR-AI compared with WT-VWF. Furthermore, significantly enhanced VWF-R1205H binding to LRP1 cluster IV (P < .001) and less marked enhanced binding to LRP1 cluster II (P = .034) was observed. In contrast, VWF-R1205H and WT-VWF demonstrated no difference in binding affinity to macrophage galactose-type lectin. ConclusionDisruption of the positive charge at amino acid R1205 causes conformational changes in the VWF-D′D3 domains and triggers enhanced LRP1-mediated and SR-AI–mediated clearance.

Differential strain sensitivity to low-dose anxiolytic diazepam in anxiety-related behaviour in male Wistar and Wistar-Kyoto, a rat model of endogenous depression

Objectives: Treatment resistant depression (TRD) prevails among individuals with major depressive disorder (MDD) with comorbid anxiety. The Wistar Kyoto (WKY) rat strain, which demonstrates exaggerated vulnerability to anxiety, has recently been suggested as a model for TRD with similar pathophysiology to MDD, non-responsiveness to antidepressants but responsive to deep brain stimulation and ketamine. At the clinical level, TRD is associated with reduced occipital cortical levels of Gamma-AminoButyric Acid (GABA), with a reduction in spontaneous GABAergic synaptic activity reported in WKY. Diazepam (DZP), a GABA agonist, is a widely used anxiolytic, so the present study was carried out to evaluate its efficacy through a low dose, oral administration in male WKY rats, with the progenitor strain Wistar, serving as vehicular control. Materials and Methods: Adult Wistar and WKY rats were treated with 1 mg/kg body weight DZP administered per os (p. o.) for 10 days. From the 6th day, rats were exposed to a comprehensive battery of behavioural paradigms, including novelty-based open field (OPF), anxiogenic elevated plus maze (EPM), light-dark box (LDB) and the stress coping behaviour assessing forced swim test (FST). Results: DZP reversed the EPM-induced anxiety in Wistars by increasing open-arm duration (P &lt; 0.05), entries (P &lt; 0.05) and exploratory behaviour (P &lt; 0.01) while concomitantly decreasing closed-arm duration (P &lt; 0.05) and entries (P &lt; 0.05), with no effect in WKY. DZP also reduced latency to the dark zone (P &lt; 0.05) in LDB and increased swimming behaviour in FST (P &lt; 0.05) in Wistars, with no effect in WKY. Baseline strain differences were observed with reduced exploratory behaviour in OPF (P &lt; 0.01), open arm entries (P &lt; 0.01) and head dips (P &lt; 0.01) in EPM and swimming (P &lt; 0.05) in FST in WKY as compared to Wistars. Strain differences persisted also in the DZP-treated groups where, as compared to matched Wistars, WKY demonstrated reduced open arm duration (P &lt; 0.05), entries (P &lt; 0.001) and head dips (P &lt; 0.001) and increased closed arm duration (P &lt; 0.001) and entries (P &lt; 0.001) in the EPM. WKY also showed reduced time spent (P &lt; 0.05) and entries (P &lt; 0.01) into the light zone and increased time spent in the dark zone (P &lt; 0.05) of LDB. Further, WKY showed increased immobility (P &lt; 0.05) during habituation and reduced swimming behaviour (P &lt; 0.001) during the test. Conclusion: Strain-specific differences and increased baseline anxiety levels in WKY, as compared to Wistars, induced differential effects of DZP with drug-induced effects observed in Wistars but not in WKY, furthering the treatment resistant aspect of this model. DZP efficacy, therefore, varies in different rat strains and manifests in differential strain-specific responses emanating from exaggerated vulnerability to stress. Results also indicated differential sensitivity of tested paradigms to the anxiolytic activity of DZP and stressed the use of a battery of tests that enable a teasing out of anxiety and depression. However, further studies are needed that would unravel GABAergic differences at the receptor level, such as differential receptor binding affinities, underlying gene polymorphisms and the implications thereof for this TRD model.

M. tuberculosis PrrA binds the dosR promoter and regulates mycobacterial adaptation to hypoxia

The PrrAB two-component system (TCS) is essential for Mycobacterium tuberculosis viability. Previously, it was demonstrated that PrrA binds DNA in the absence of PrrB-mediated transphosphorylation and that non-cognate serine/threonine-kinases phosphorylate PrrA threonine-6 (T6). Therefore, we investigated the differential binding affinity and regulatory properties of the M. tuberculosis-derived wild-type PrrA, PrrA phosphomimetic (D58E, T6E), and PrrA phosphoablative (D58A, T6A) proteins with the prrAMtb, dosRMtb, and cydAMtb genes. While we hypothesized greater DNA binding affinity and more pronounced regulation by PrrA phosphomimetic variants, recombinant, wild-type PrrAMtb bound DNA with greatest affinity. Collectively, wild-type PrrAMtb recombinant protein displayed the highest binding affinity to the dosRMtb promoter (KD 3.46 ± 2.09 nM), followed by the prrAMtb promoter (KD 9.00 ± 2.66 nM). To establish PrrAMtb regulatory activity, we constructed M. smegmatis ΔprrABMsmeg::prrAMtb strains with each of the PrrAMtb variants and extrachromosomal prrAMtb, dosRMtb, and cydAMtb promoter-mCherry reporter fusions. Our findings showed that PrrAMtb is autoregulatory and induces dosRMtb expression only during in vitro, hypoxic growth. Combined expression of prrABMtb in M. smegmatis ΔprrAB significantly induced cydAMtb promoter-mCherry expression. Our studies advanced the understanding of PrrA function and PrrAB phosphorylation-mediated regulatory mechanisms and control of mycobacterial dosR and cydA hypoxic and low-oxygen responsive genes.