Binding Of Molecules Research Articles

Screening and separation of natural anticancer active ingredients related to phospholipase C.

Based on the specific binding of drug molecules to cell membrane receptors, a screening and separation method for active compounds of natural products was established by combining phospholipase C (PLC) sensitized hollow fiber microscreening by a solvent seal with high-performance liquid chromatography technology. In the process, the factors affecting the screening were optimized. Under the optimal screening conditions, we screened honokiol (HK), magnolol (MG), negative control drug carbamazepine, and positive control drug amentoflavone, the repeatability of the method was tested. The PLC activity was determined before and after the screening. Experimental results showed that the sensitization factors of PLC of HK and MG were 61.0 and 48.5, respectively, and amentoflavone was 15.0, carbamazepine could not bind to PLC. Moreover, the molecular docking results were consistent with this measurement, indicating that HK and MG could be combined with PLC, and they were potential interacting components with PLC. This method used organic solvent to seal the PLC greatly ensuring the activity, so this method had the advantage of integrating separation, and purification with screening, it not only exhibited good reproducibility and high sensitivity but was also suitable for screening the active components in natural products by various targets in vitro.

Fangyukangsuan granules ameliorate hyperuricemia and modulate gut microbiota in rats.

Hyperuricaemia (HUA) is a metabolic disorder characterised by high blood uric acid (UA) levels; moreover, HUA severity is closely related to the gut microbiota. HUA is also a risk factor for renal damage, diabetes, hypertension, and dyslipidaemia; however, current treatments are associated with detrimental side effects. Alternatively, Fangyukangsuan granules are a natural product with UA-reducing properties. To examine their efficacy in HUA, the binding of small molecules in Fangyukangsuan granules to xanthine oxidase (XOD), a key factor in UA metabolism, was investigated via molecular simulation, and the effects of oral Fangyukangsuan granule administration on serum biochemical indices and intestinal microorganisms in HUA-model rats were examined. Overall, 24 small molecules in Fangyukangsuan granules could bind to XOD. Serum UA, creatinine, blood urea nitrogen, and XOD levels were decreased in rats treated with Fangyukangsuan granules compared to those in untreated HUA-model rats. Moreover, Fangyukangsuan granules restored the intestinal microbial structure in HUA-model rats. Functional analysis of the gut microbiota revealed decreased amino acid biosynthesis and increased fermentation of pyruvate into short-chain fatty acids in Fangyukangsuan granule-treated rats. Together, these findings demonstrate that Fangyukangsuan granules have anti-hyperuricaemic and regulatory effects on the gut microbiota and may be a therapeutic candidate for HUA.

Selective Bacterial Growth Inactivation by pH-Sensitive Sulfanilamide Functionalized Carbon Dots.

Carbon dots (CDs) were synthesized hydrothermally by mixing citric acid (CA) and an antifolic agent, sulfanilamide (SNM), employed for pH sensing and bacterial growth inactivation. Sulfanilamide is a prodrug; aromatic hetero cyclization of the amine moiety along with other chemical modifications produces an active pharmacological compound (chloromycetin and miconazole), mostly administered for the treatment of various microbial infections. On the other hand, the efficacy of the sulfanilamide molecule as a drug for antimicrobial activity was very low. We anticipated that the binding of the sulfanilamide molecule on the carbon dot (CD) surface may form antibacterial CDs. Citric acid was hybridized with sulfanilamide during the hydrothermal preparation of the CDs. The molecular fragments of bioactivated sulfanilamide molecule play a crucial role in bacterial growth inactivation for Gram-positive and Gram-negative bacteria. The functional groups of citric acid and sulfanilamide were conserved during the CD formation, facilitating the zwitterionic behavior of CDs associated with its photophysical activity. At low concentrations of CDs, the antibacterial activity was apparent for Gram-positive bacteria only. This Gram-positive bacteria selectivity was also rationalized by zeta potential measurement.

Multivalent DNAzyme agents for cleaving folded RNA.

Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.

Understanding the Roles of Excipients in Moisture Management in Solid Dosage Forms.

Excipients are ubiquitous in pharmaceutical products, and often, they can also play a critical role in maintaining product quality. For a product containing a moisture-sensitive drug, moisture can be deleterious to the product stability during storage. Therefore, using excipients that interact with moisture in situ can potentially alleviate product stability issues. In this study, the interactive behavior of starch with moisture was augmented by coprocessing maize starch with sodium chloride (NaCl) or magnesium nitrate hexahydrate [Mg(NO3)2·6H2O] at different concentrations (5 and 10%, w/w). The effect of the formulation on drug stability was assessed through the degradation of acetylsalicylic acid, which was used as the model drug. The results showed that coprocessing of the starch with either NaCl or Mg(NO3)2·6H2O impacted the number of water molecule binding sites on the starch and how the sorbed moisture was distributed. The coprocessed excipients also resulted in lower drug degradation and lesser changes in tablet tensile strength during post-compaction storage. However, corresponding tablet formulations containing physical mixtures of starch and salts did not yield promising outcomes. This study demonstrated the advantageous concomitant use of common excipients by coprocessing to synergistically mitigate the adverse effects of moisture and promote product stability when formulating a moisture-sensitive drug. In addition, the findings could help to improve the understanding of moisture-excipient interactions and allow for the judicious choice of excipients when designing formulations containing moisture-sensitive drugs.

Genome assembly of Melilotus officinalis provides a new reference genome for functional genomics

BackgroundSweet yellow clover (Melilotus officinalis) is a diploid plant (2n = 16) that is native to Europe. It is an excellent legume forage. It can both fix nitrogen and serve as a medicine. A genome assembly of Melilotus officinalis that was collected from Best corporation in Beijing is available based on Nanopore sequencing. The genome of Melilotus officinalis was sequenced, assembled, and annotated.ResultsThe latest PacBio third generation HiFi assembly and sequencing strategies were used to produce a Melilotus officinalis genome assembly size of 1,066 Mbp, contig N50 = 5 Mbp, scaffold N50 = 130 Mbp, and complete benchmarking universal single-copy orthologs (BUSCOs) = 96.4%. This annotation produced 47,873 high-confidence gene models, which will substantially aid in our research on molecular breeding. A collinear analysis showed that Melilotus officinalis and Medicago truncatula shared conserved synteny. The expansion and contraction of gene families showed that Melilotus officinalis expanded by 565 gene families and shrank by 56 gene families. The contacted gene families were associated with response to stimulus, nucleotide binding, and small molecule binding. Thus, it is related to a family of genes associated with peptidase activity, which could lead to better stress tolerance in plants.ConclusionsIn this study, the latest PacBio technology was used to assemble and sequence the genome of the Melilotus officinalis and annotate its protein-coding genes. These results will expand the genomic resources available for Melilotus officinalis and should assist in subsequent research on sweet yellow clover plants.

125I]IPC-Lecanemab: Synthesis and Evaluation of Aβ-Plaque-Binding Antibody and Comparison with Small-Molecule [18F]Flotaza and [125I]IBETA in Postmortem Human Alzheimer's Disease.

Therapeutic antibodies for reducing Aβ plaque load in Alzheimer's disease (AD) is currently making rapid progress. The diagnostic imaging of Aβ plaque load in AD has been underway and is now used in clinical studies. Here, we report our preliminary findings on imaging a therapeutic antibody, Lecanemab, in a postmortem AD brain anterior cingulate. [125I]5-iodo-3-pyridinecarboxamido-Lecanemab ([125I]IPC-Lecanemab) was prepared by coupling N-succinimidyl-5-([125I]iodo)-3-pyridinecarboxylate with Lecanemab in modest yields. The distinct binding of [125I]IPC-Lecanemab to Aβ-rich regions in postmortem human AD brains was higher in grey matter (GM) containing Aβ plaques compared to white matter (WM) (GM/WM was 1.6). Anti-Aβ immunostaining was correlated with [125I]IPC-Lecanemab regional binding in the postmortem AD human brains. [125I]IPC-Lecanemab binding was consistent with the binding of Aβ small molecules, [18F]flotaza and [125I]IBETA, in the same subjects. [18F]Flotaza and [125I]IBETA, however, exhibited significantly higher GM/WM ratios (>20) compared to [125I]IPC-Lecanemab. Our results suggest that radiolabeled [125I]IPC-Lecanemab retains the ability to bind to Aβ in human AD and may therefore be useful as a PET imaging radiotracer when labeled as [124I]IPC-Lecanemab. The ability to directly visualize in vivo a promising therapeutic antibody for AD may be useful in treatment planning and dosing and could be complimentary to small-molecule diagnostic imaging to assess outcomes of therapeutic interventions.

How much does TRPV1 deviate from an ideal MWC-type protein?

Many ion channels are known to behave as an allosteric protein, coupling environmental stimuli captured by specialized sensing domains to the opening of a central pore. The classic Monod-Wyman-Changeux (MWC) model, originally proposed to describe binding of gas molecules to hemoglobin, has been widely used as a framework for analyzing ion channel gating. Here, we address the issue of how accurately the MWC model predicts activation of the capsaicin receptor TRPV1 by vanilloids. Taking advantage of a concatemeric design that makes it possible to lock TRPV1 in states with zero to four bound vanilloid molecules, we showed quantitatively that the overall gating behavior is satisfactorily predicted by the MWC model. There is, however, a small yet detectable subunit position effect: ligand binding to two kitty-corner subunits is 0.3–0.4 kcal/mol more effective in inducing opening than binding to two neighbor subunits. This difference—less than 10% of the overall energetic contribution from ligand binding—might be due to the restriction on subunit arrangement imposed by the planar membrane; if this is the case, then the position effect is not expected in hemoglobin, in which each subunit is related equivalently to all the other subunits.

Abstract LB436: Using FCCS to monitor target occupancy in cells and patient biopsies

Abstract Lack of target engagement is one of the major reasons for failures in clinical studies. We developed an assay platform to detect and quatify target occupancy values in patient and animal biopsies to guide the development and dosing during the drug development process. FCCS (fluorescence cross correlation spectroscopy) is a powerful tool to characterize drug target interaction in physiological environments. Over the last decade this technology was used to understand the binding and kinetics of small molecules and antibodies to challenging target proteins in great details and accuracy. Over the last decade Intana Bioscience expanded the application of FCCS from affinity measurements to comprehensive interaction studies including kinetics, multi component complex formation, assessing stoichiometry and quantification of aggregation, PK studies and target engagement. This presentation will highlight examples of results obtained with FCCS in projects ranging from early stages in the drug discovery up to the support of clinical trials to quantify target occupancies in patient samples. Citation Format: Frank Becker. Using FCCS to monitor target occupancy in cells and patient biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB436.

Modelling non-local cell-cell adhesion: a multiscale approach

Cell-cell adhesion plays a vital role in the development and maintenance of multicellular organisms. One of its functions is regulation of cell migration, such as occurs, e.g. during embryogenesis or in cancer. In this work, we develop a versatile multiscale approach to modelling a moving self-adhesive cell population that combines a careful microscopic description of a deterministic adhesion-driven motion component with an efficient mesoscopic representation of a stochastic velocity-jump process. This approach gives rise to mesoscopic models in the form of kinetic transport equations featuring multiple non-localities. Subsequent parabolic and hyperbolic scalings produce general classes of equations with non-local adhesion and myopic diffusion, a special case being the classical macroscopic model proposed in Armstrong et al. (J Theoret Biol 243(1): 98–113, 2006). Our simulations show how the combination of the two motion effects can unfold. Cell-cell adhesion relies on the subcellular cell adhesion molecule binding. Our approach lends itself conveniently to capturing this microscopic effect. On the macroscale, this results in an additional non-linear integral equation of a novel type that is coupled to the cell density equation.

NMR characterization and ligand binding site of the stem-loop 2 motif from the Delta variant of SARS-CoV-2.

The stem-loop 2 motif (s2m) in SARS-CoV-2 (SCoV-2) is located in the 3'-UTR. Although s2m has been reported to display characteristics of a mobile genomic element that might lead to an evolutionary advantage, its function has remained unknown. The secondary structure of the original SCoV-2 RNA sequence (Wuhan-Hu-1) was determined by NMR in late 2020, delineating the base-pairing pattern and revealing substantial differences in secondary structure compared to SARS-CoV-1 (SCoV-1). The existence of a single G29742-A29756 mismatch in the upper stem of s2m leads to its destabilization and impedes a complete NMR analysis. With Delta, a variant of concern has evolved with one mutation compared to the original sequence that replaces G29742 by U29742. We show here that this mutation results in a more defined structure at ambient temperature accompanied by a rise in melting temperature. Consequently, we were able to identify >90% of the relevant NMR resonances using a combination of selective RNA labeling and filtered 2D NOESY as well as 4D NMR experiments. We present a comprehensive NMR analysis of the secondary structure, (sub)nanosecond dynamics, and ribose conformation of s2m Delta based on heteronuclear 13C NOE and T 1 measurements and ribose carbon chemical shift-derived canonical coordinates. We further show that the G29742U mutation in Delta has no influence on the druggability of s2m compared to the Wuhan-Hu-1 sequence. With the assignment at hand, we identify the flexible regions of s2m as the primary site for small molecule binding.

Comparison of Commonly Used and New Methods to Determine Small Molecule Non-Specific Binding to Human Liver Microsomes

Accurate measurement of non-specific binding of a drug candidate to human liver microsomes (HLM) can be critical for the accurate determination of key enzyme kinetic parameters such as Michaelis-Menton (Km), reversible inhibition (Ki), or inactivation (KI) constants. Several methods have been developed to determine non-specific binding of small molecules to HLM, such as rapid equilibrium dialysis (RED), ultrafiltration (UF), HLM bound to magnetizable beads (HLM-beads), ultracentrifugation (UC), the linear extrapolation stability assay (LESA), and the Transil™ system. Despite various differences in methodology between these methods, it is generally presumed that similar free fraction values (fu,mic) should be generated. To evaluate this hypothesis, a test set of 9 compounds were selected, representing low (high fu,mic value) and significant (low fu,mic value) HLM binding, respectively, across HLM concentrations tested in this manuscript. The fu,mic values were determined using a single compound concentration (1.0 µM) and three HLM concentrations (0.025, 0.50, and 1.0 mg/mL). When the HLM non-specific binding event is not extensive resulting in high fu,mic values, all methods generated similar fu,mic values. However, fu,mic values varied markedly across assay formats when high binding to HLM occurred, where fu,mic values differed by up to 33-fold depending on the method used. Potential causes for such discrepancies across the various methods employed, practical implications related to conduct the different assays, and implications to clinical drug-drug interaction (DDI) predictions are discussed.

Citric Acid Controls the Activity of YopH Bacterial Tyrosine Phosphatase.

Citric acid (CA) is a tricarboxylic acid with antioxidant and antimicrobial properties. Based on previous studies, the small compound with its three carboxylic groups can be considered a protein tyrosine phosphatase inhibitor. YopH, a protein tyrosine phosphatase, is an essential virulence factor in Yersinia bacteria. We performed enzymatic activity assays of YopH phosphatase after treatment with citric acid in comparison with the inhibitory compound trimesic acid, which has a similar structure. We also measured the cytotoxicity of these compounds in Jurkat T E6.1 and macrophage J774.2 cell lines. We performed molecular docking analysis of the binding of citric acid molecules to YopH phosphatase. Citric acid and trimesic acid reversibly reduced the activity of YopH enzyme and decreased the viability of Jurkat and macrophage cell lines. Importantly, these two compounds showed greater inhibitory properties against bacterial YopH activity than against human CD45 phosphatase activity. Molecular docking simulations confirmed that citric acid could bind to YopH phosphatase. Citric acid, a known antioxidant, can be considered an inhibitor of bacterial phosphatases.

Machine learning and statistical physics modeling of tetracycline adsorption using activated carbon derived from Cynometra ramiflora fruit biomass

The current investigation reports the usage of adaptive neuro-fuzzy inference system (ANFIS) and artificial neural network (ANN), the two recognized machine learning techniques in modelling tetracycline (TC) adsorption onto Cynometra ramiflora fruit biomass derived activated carbon (AC). Many characterization methods utilized, confirmed the porous structure of synthesized AC. ANN and ANFIS models utilized pH, dose, initial TC concentration, mixing speed, time duration, and temperature as input parameters, whereas TC removal percentage was designated as the output parameter. The optimized configuration for the ANN model was determined as 6-8-1, while the ANFIS model employed trimf input and linear output membership functions. The obtained results showed a strong correlation, indicated by high R2 values (ANNR2: 0.9939 & ANFISR2: 0.9906) and low RMSE values (ANNRMSE: 0.0393 & ANFISRMSE: 0.0503). Apart from traditional isotherms, the dataset was fitted to statistical physics models wherein, the double-layer with a single energy satisfactorily explained the physisorption mechanism of TC adsorption. The sorption energy was 21.06 kJ/mol, and the number of TC moieties bound per site (n) was found to be 0.42, conclusive of parallel binding of TC molecules to the adsorbent surface. The adsorption capacity at saturation (Qsat) was estimated to be 466.86 mg/g – appreciably more than previously reported values. These findings collectively demonstrate that the AC derived from C. ramiflora fruit holds great potential for efficient removal of TC from a given system, and machine learning approaches can effectively model the adsorption processes.

Long period grating based molecularly imprinted fiber optic sensor for the label-free detection of bisphenol A

The molecularly imprinted fiber optic (MIFO) sensors are recognized for its reliability, sensitivity and selective analyte identification. In this study, we report a Long-Period Grating (LPG) based MIFO sensor developed for the label-free detection of Bisphenol A (BPA) in water samples. The sensor is fabricated by polymerizing hydrogel precursor containing BPA on the LPG, followed by etching BPA molecules. The rebinding of the BPA molecule to the molecularly imprinted long period grating Bisphenol A (MILPG-BPA) sensor causes the hydrogel to swell, leading to a peak shift in the LP07 resonant mode of the sensor. This shift is linear across BPA concentrations ranging from femto molar to nano molar range with a low limit of detection of 0.146 fM. The non-imprinted long period grating (NILPG) sensor showed no selective binding of the BPA molecule, underscoring the significance of the microenvironment established through molecular imprinting for effective sensing. The MILPG-BPA sensor exhibits high selectivity in detecting the BPA molecules among the potential interferents. Furthermore, it has a rapid response time and reusability, thus holding promise for a wide range of applications.

PSMA2 promotes glioma proliferation and migration via EMT

BackgroundGliomas advance rapidly and are associated with a poor prognosis. Epithelial-mesenchymal transition (EMT) accelerates the progression of gliomas, exerting a pivotal role in glioma development. Proteasome subunit alpha type-2 (PSMA2) exhibits high expression levels in gliomas. however, its specific involvement in glioma progression and its correlation with EMT remain elusive. This study aims to elucidate the role of PSMA2 in glioma progression and its potential association with EMT. MethodsOnline tools were employed to analyze the expression patterns and survival curves of PSMA2 in gliomas. The relationship between PSMA2 and various characteristics of glioma patients was investigated using data from the TCGA and CGGA databases. In vitro, cell proliferation and migration were assessed through CCK-8, colony formation, and transwell assays. Furthermore, a tumor xenograft model in nude mice was established to evaluate in vivo tumorigenesis. Protein binding to PSMA2 was scrutinized using co-immunoprecipitation MS (co-IP MS). The potential biological functions and molecular pathways associated with PSMA2 were explored through GO analysis and KEGG analysis, and the correlation between PSMA2 and EMT was validated through correlation analysis and Western blot experiments. ResultsBioinformatics analysis revealed a significant upregulation of PSMA2 across various cancers, with particularly heightened expression in gliomas. Moreover, elevated PSMA2 levels were correlated with advanced tumor stages and diminished survival rates among glioma patients. Inhibition of PSMA2 demonstrated a pronounced suppressive effect on glioma cell proliferation, both in vitro and in vivo. Knockdown of PSMA2 also impeded the migratory capacity of glioma cells. GO and KEGG enrichment analyses indicated that PSMA2-binding proteins (identified through Co-IP-MS) were associated with cell adhesion molecule binding and cadherin binding. Western blot results further confirmed the role of PSMA2 in promoting epithelial-mesenchymal transition (EMT) in glioma cells. ConclusionOur study provides evidence supporting the role of PSMA2 as a regulatory factor in EMT and suggests its potential as a prognostic biomarker for glioma progression.

DrugMGR: a deep bioactive molecule binding method to identify compounds targeting proteins.

Understanding the intermolecular interactions of ligand-target pairs is key to guiding the optimization of drug research on cancers, which can greatly mitigate overburden workloads for wet labs. Several improved computational methods have been introduced and exhibit promising performance for these identification tasks, but some pitfalls restrict their practical applications: (i) first, existing methods do not sufficiently consider how multigranular molecule representations influence interaction patterns between proteins and compounds; and (ii) second, existing methods seldom explicitly model the binding sites when an interaction occurs to enable better prediction and interpretation, which may lead to unexpected obstacles to biological researchers. To address these issues, we here present DrugMGR, a deep multigranular drug representation model capable of predicting binding affinities and regions for each ligand-target pair. We conduct consistent experiments on three benchmark datasets using existing methods and introduce a new specific dataset to better validate the prediction of binding sites. For practical application, target-specific compound identification tasks are also carried out to validate the capability of real-world compound screen. Moreover, the visualization of some practical interaction scenarios provides interpretable insights from the results of the predictions. The proposed DrugMGR achieves excellent overall performance in these datasets, exhibiting its advantages and merits against state-of-the-art methods. Thus, the downstream task of DrugMGR can be fine-tuned for identifying the potential compounds that target proteins for clinical treatment. https://github.com/lixiaokun2020/DrugMGR.

Classification and Molecular Functions of Heparan Sulfate Proteoglycans and Their Molecular Mechanisms with the Receptor

Heparan sulfate proteoglycans are highly glycosylated proteins in which heparan sulfate, a glycosaminoglycan sugar chain, is an acidic sugar chain consisting of a repeating disaccharide structure of glucuronic acid and N-acetylglucosamine is locally sulfated. Syndecan, one of the transmembrane HSPGs, functions as a receptor that transmits signals from the extracellular microenvironment to the inside of the cell. In the vascular system, heparan sulfate proteoglycans, a major component of the glycocalyx, enable the binding of various plasma-derived molecules due to their diversity, epimerization of glycosaminoglycans chains, long chains, and sulfation. Heparan sulfate proteoglycans present in the extracellular matrix serve as a reservoir for bioactive molecules such as chemokines, cytokines, and growth factors. Aberrant expression of heparan sulfate proteoglycans, heparanase, and sulfatase is observed in many pathological conditions. Therefore, it can be applied to therapeutic strategies for a wide range of fields including Alzheimer’s disease, heart failure, cancer, organ transplants, diabetes, chronic inflammation, aging, and autoimmune diseases.

Roles of biochars’ properties in their water-holding capacity and bound water evaporation: quantitative importance and controlling mechanism

Important properties of biochar as an effective soil amendment are its high water-holding capacity (WHC) and inhibition of water evaporation. However, the mechanism and the importance of biochar properties in controlling its own WHC and bound water evaporation remain little known. In this study, wheat straw and pine sawdust biochars were pyrolyzed in N2-flow, CO2-flow, and air-limitation environments at 300–750 ℃, and a series of the produced biochars’ properties were characterized to explore the dominant controlling factors of their WHC and bound water evaporation. The results have shown that with the increasing contents of hydrogen, nitrogen, and oxygen as well as such ratios as H/C, and (O + N)/C, WHC of the biochars was also increasing while the evaporation of biochar-bound water was decreasing. With an increase in the other studied factors, such as carbon content, pH, and specific surface area (SSA), WHC of the biochars was decreasing, and the evaporation of biochar-bound water was increasing. That was connected with the fact that biochar-nitrogen was mainly in pyridinic and pyrrolic forms, while oxygen was in the form of C = O and C–O bonds. These forms of nitrogen and oxygen could be the receptors of hydrogen bonds to link to H2O molecules. Aliphatic hydrogen with a weak positive charge could be a donor of hydrogen bonds to link to H2O molecules. However, high carbon content, as well as high SSA, indicated more exposed aromatic carbon (hydrophobic sites) that could suppress the binding of H2O molecules. Additionally, high pH indicated that H2O molecules were dominated by OH–, which generated strong electrostatic repulsion with the negatively charged nitrogen- and oxygen-containing groups of biochar. It was also shown that the nitrogen-containing groups played a more important role (importance – 0.31) in WHC of the biochar than other parameters, including carbon, oxygen, hydrogen, ash contents, pH, SSA (importance from 0.02 to 0.09). Nitrogen, oxygen, and carbon contents had the most important influence on the evaporation of biochar-bound water in all studied factors. Furthermore, wheat straw biochar produced at low pyrolysis temperatures in N2 atmosphere (with high nitrogen and oxygen contents) had the highest WHC and the lowest evaporation of biochar-bound water. Consequently, it can be suggested that biochar rich in nitrogen can be an effective water retention agent and can improve agricultural soil moisture.Graphical

Abstract 2474: Antitumoral efficacy of ASKG812, a Smartkine® based bifunctional PD-1-eIL-2 molecule, through enhancing T effector cis agonism

Abstract Cytokines are potent molecules, yet their broad application as therapeutics has been significantly hampered due to short PK, severe systemic toxicity, and narrow therapeutic window. To improve the developability of cytokines, AskGene has invented a proprietary cytokine platform (Smartkine®) to achieve its overarching objective of modulating immune reactions at a disease site in a selective and controlled manner. Several antibody-cytokine fusion molecules using the SmartKine® platform have been moving into preclinical and clinical development. ASKG812 is a unique fusion molecule composed of anti-PD-1 antibody and engineered IL-2 (eIL-2). The eIL-2 moiety also comprises a specific mask such that the eIL-2 molecule binding biases towards PD-1+ T effector cells in a cis agonism manner for improved efficacy and therapeutic window. In ex vivo human PBMC assays, we demonstrated that ASKG812 significantly reduced activity in PD-1− T and NK cells, and enhanced activity in PD-1+ T cells judged by levels of pSTAT5, Ki67 and Granzyme B. In a cytotoxicity assay, ASKG812 can efficiently induce PBMC-mediated killing of MDA-MB-231 tumor cells in a dose-dependent manner. Moreover, in both anti-PD-1 therapy responsive and non-responsive syngeneic mouse tumor models, ASKG812 showed robust efficacy; without noticeable toxicity compared to a reference anti-PD-1-IL-2v fusion molecule. In conclusion, these findings provide the basis for the development of this new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the clinical treatment of cancer. Citation Format: Chunxiao Yu, Kurt Shanebeck, Shiguang Yu, Jeanine Ruiz, Shichang Li, Stone D.-H Shi, Hong Ma, Yuefeng Lu. Antitumoral efficacy of ASKG812, a Smartkine® based bifunctional PD-1-eIL-2 molecule, through enhancing T effector cis agonism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2474.