Dissociation Constant Research Articles

Pin1 WW Domain Ligand Library Synthesized with an Easy Solid-Phase Phosphorylating Reagent.

Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr-cis/trans-Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, R1CO-pSer-Pro-NHR2, was designed to identify ligands specific for the Pin1 WW domain. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite functional group was synthesized in one step from Wang resin. The SPPR was used in the preparation of the library by parallel synthesis. The final 315-member library was screened with our WW-domain-specific, enzyme-linked enzyme-binding assay (ELEBA). Four of the best hits were resynthesized, and the competitive dissociation constants were measured by ELEBA. NMR chemical-shift perturbations (CSP) of ligands with 15N-labeled Pin1 were used to measure Kd for the best four ligands directly, demonstrating that they were specific Pin1 WW domain ligands. Models of the ligands bound to the Pin1 WW domain were used to visualize the mode of binding in the WW domain.

Effects of SARS-CoV-2 Main Protease Mutations at Positions L50, E166, and L167 Rendering Resistance to Covalent and Noncovalent Inhibitors.

SARS-CoV-2 propagation under nirmatrelvir and ensitrelvir pressure selects for main protease (MPro) drug-resistant mutations E166V (DRM2), L50F/E166V (DRM3), E166A/L167F (DRM4), and L50F/E166A/L167F (DRM5). DRM2-DRM5 undergoes N-terminal autoprocessing to produce mature MPro with dimer dissociation constants (Kdimer) 2-3 times larger than that of the wildtype. Co-selection of L50F restores catalytic activity of DRM2 and DRM4 from ∼10 to 30%, relative to that of the wild-type enzyme, without altering Kdimer. Binding affinities and thermodynamic profiles that parallel the drug selection pressure, exhibiting significant decreases in affinity through entropy/enthalpy compensation, were compared with GC373. Reorganization of the active sites due to mutations observed in the inhibitor-free DRM3 and DRM4 structures as compared to MProWT may account for the reduced binding affinities, although DRM2 and DRM3 complexes with ensitrelvir are almost identical to MProWT-ensitrelvir. Chemical reactivity changes of the mutant active sites due to differences in electrostatic and protein dynamics effects likely contribute to losses in binding affinities.

Effective Dose of Dexmedetomidine with Nalbuphine Hydrochloride or Butorphanol Tartrate for Sedation in Buff Orpington Hens (Gallus gallus domesticus).

Chickens (Gallus gallus domesticus) are commonly used for research, food production, show, and companionship. Sedation is often necessary for sample collection, imaging, or treatment. Dexmedetomidine has been previously used to sedate birds, often with other sedatives. Butorphanol tartrate, a Schedule IV controlled substance, is commonly used but presents regulatory challenges. Nalbuphine hydrochloride, an opioid with similar receptor affinity to butorphanol, has potential as a noncontrolled alternative. Although information regarding nalbuphine use in birds is limited, its noncontrolled status makes it more accessible. The purpose of this study was to determine the effective dose to produce sedation in 50% (ED50) of patients and to estimate the calculated effective dose of dexmedetomidine in combination with either butorphanol (DexBut) or nalbuphine (DexNal) in domestic hens to sedate 99% of patients (ED99). Eighteen 33-week-old laying Buff Orpington hens were divided into 2 groups: one receiving DexBut (n = 9) and the second receiving DexNal (n = 9). Each hen was sedated with varying doses of intramuscular dexmedetomidine with a constant dose of either 2 mg/kg IM butorphanol or 12.5 mg/kg IM nalbuphine by an up-and-down design. Sedation was determined using a clinically applicable scoring system. The ED50 values of dexmedetomidine with 2 mg/kg IM of butorphanol, calculated by both the up-and-down method and logistic regression, were 38 and 49 µg/kg, respectively, while the ED50 values of dexmedetomidine in combination with 12.5 mg/kg IM of nalbuphine were 19 and 18 µg/kg, respectively. The estimated dexmedetomidine ED99 values with butorphanol or nalbuphine were 51 and 19 µg/kg, respectively. Multiple chickens in both groups exhibited open-mouth breathing and comb pallor but no lasting morbidity or mortality occurred. Combinations of DexBut or DexNal should be considered for sedation of domestic chickens.

Assessing functional suitability of a lyophilized formulation containing designed ankyrin repeat proteins for radionuclide imaging of HER2/neu overexpression in malignant tumors

Aim. To study in vitro and in vivo the functional suitability of 99mTc-labeled lyophilized formulation containing designed ankyrin repeat protein (DARPin) G3-(GGGS)3Cys for radionuclide imaging of HER2/neu overexpression in malignant tumors.Materials and methods. To create a targeted protein, a modified genetic construct with the sequence encoding DARPin G3-(GGGS)3Cys was used. To generate the experimental probe, we used a lyophilized formulation containing DARPin G3-(GGGS)3Cys with auxiliary substances and 99mTc sodium pertechnetate (500 MBq) incubated at 60 °C for 30 min. Radiochemical purity of 99mTc-G3-(GGGS)3Cys was analyzed by thin-layer radiochromatography. SKOV-3, BT-474, and DU-145 cell lines were used to test binding specificity in vitro. The dissociation constant was determined via a saturation binding assay on SKOV-3 cells with a range of protein concentrations from 0.2 to 40 nM. Nu/j mice bearing HER2-positive SKOV-3 xenografts and HER2-negative Ramos xenografts were used to evaluate the targeting properties and biodistribution.Results. A radiocomplex based on 99mTc and a lyophilized formulation with DARPin G3-(GGGS)3Cys was obtained with the radiochemical purity of more than 96%. Binding of 99mTc-G3-(GGGS)3Cys to the cells was specific (KD 3.9 ± 0.5 nM) and proportional to the level of HER2/neu expression in the cells. The uptake of 99mTc-G3-(GGGS)3Cys in SKOV-3 xenografts was significantly higher than in Ramos xenografts. 99mTc-G3-(GGGS)3Cys demonstrated rapid blood and renal clearance and had low activity in the salivary glands and stomach. Liver uptake was about 5–7%ID/g. In addition, 99mTc-G3-(GGGS)3Cys exhibited very low uptakes in the lungs, muscles, small intestine, and bones.Conclusion. The 99mTc-labeled lyophilized formulation with DARPin G3-(GGGS)3Cys is functionally suitable for imaging HER2/neu overexpression in tumors, as it binds specifically to the receptor, is stable in vivo, and has favorable biodistribution in organs and tissues. The radiocomplex based on 99mTc-G3-(GGGS)3Cys was obtained by a simple method with high radiochemical purity.

Cysteine protease I29 propeptide from Calotropis procera R. Br. As a potent cathepsin L inhibitor and its suppressive activity in breast cancer metastasis.

Breast cancer metastasis is associated with a poor prognosis and a high rate of mortality. Cathepsin L (CTSL) is a lysosomal cysteine protease that promotes tumor metastasis by degrading the extracellular matrix. Gene set enrichment analysis revealed that CTSL expression was higher in tumorous than in non-tumorous tissues of breast cancer patients and that high-level CTSL expression correlated positively with the epithelial-mesenchymal transition. Therefore, we hypothesized that inhibiting CTSL activity in tumor cells would prevent metastasis. In this study, we characterized the inhibitory activity of SnuCalCpI15, the I29 domain of a CTSL-like cysteine protease from Calotropis procera R. Br., and revealed that the propeptide stereoselectively inhibited CTSL in a reversible slow-binding manner, with an inhibitory constant (Ki) value of 1.38 ± 0.71 nM, indicating its potency as an exogenous inhibitor in anti-cancer therapy. SnuCalCpI15 was localized intracellularly in MDA-MB-231 breast cancer cells and suppressed tumor cell migration and invasion. These results demonstrate the potential of SnuCalCpI15 as a novel agent to prevent breast cancer metastasis.

7940 Is Urocortin 2 A Mediator Of Stress-induced Suppression Of The Luteinizing Hormone Surge?

Abstract Disclosure: H.F. Bell: None. R.A. Carrasco: None. M.J. Kreisman: None. R.B. McCosh: None. K.M. Breen Church: None. Stress is thought to be a contributing factor to the reduction of reproductive function and infertility observed in many different species. Although this is a well-documented phenomenon, the neural mechanisms mediating the suppression of reproductive neuroendocrine activity in response to stress are not yet fully understood. The paraventricular nucleus (PVN) of the hypothalamus is an integral part of the neural response to stress. The PVN initiates activation of Corticotropin Releasing Hormone (CRH) neurons and, in addition to CRH, the PVN contains a population of neurons expressing Urocortin 2 (UCN2), a CRH-like family member, with high affinity for CRH receptor 2. In females, stress has been shown to inhibit both the pulse and surge modes of Luteinizing Hormone (LH) secretion. We have preliminary evidence that kisspeptin neurons, controlling either type of LH secretion, contain CRH receptor 2. In this study we tested the sufficiency of UCN2 to disrupt the estradiol-induced LH surge. Female mice were ovariectomized and given a high estradiol treatment to induce a circadian-timed LH surge. Two days later, UCN2 or Vehicle was administered into the lateral ventricle via ICV injection on the morning of the expected surge (10 AM). Blood and brains were collected in the evening (6 PM), at the time of the expected surge. Whole blood from the tail tip was measured by LH ELISA and a surge was defined as > 0.5 ng/mL. A majority of saline-treated animals expressed an LH surge (8 of 9 females). In contrast, 0% of UCN2-treated animals surged (n=7), suggesting that UCN2 impaired the positive feedback response to estradiol. Additionally, immunohistochemistry was performed to observe colocalization of c-Fos within Kisspeptin neurons in the rostral periventricular region of the third ventricle (RP3V) which are responsible for surge generation. Lower Kisspeptin/C-Fos co-expression in animals treated with UCN2 demonstrated the efficacy of UCN2 to impair estradiol-induced activation of RP3V Kisspeptin neurons which are critical for LH surge generation. These results support the hypothesis that UCN2 signaling plays a mediatory role in the stress-induced reduction of ovulatory function in the female mouse. Presentation: 6/1/2024

7018 Improved Tolerability After Transitioning from Glucagon-like Peptide-1 Receptor Agonists to Tirzepatide

Abstract Disclosure: S. Hudson: None. J. Aurora: None. F. Erenler: None. Background: Tirzepatide is a dual incretin targeting glucagon-like peptide-1 receptor agonist (GLP-1RA) and glucose-dependent insulinotropic polypeptide receptor agonist (GIP-RA) with potent reductions in HbA1c and weight, especially compared to semaglutide, a GLP-1RA (1). Gastrointestinal (GI)-related events are commonly associated with incretins. As an imbalanced dual agonist, tirzepatide has a stronger affinity for GIP compared to GLP-1 receptors (2). This stronger affinity for GIP receptors may be related to the improved tolerability of tirzepatide, as GIP is not as associated with GI effects as compared to GLP-1 (2). There is a reported dose-dependent increase in GI events and treatment discontinuation with tirzepatide compared to semaglutide in RCTs, although this has not been corroborated in real-world data (1). Additionally, there is no literature evaluating GI tolerability when switching from GLP-1 receptor agonists to tirzepatide. We present a case series to highlight improved tolerability and sustained clinical outcomes observed in patients transitioning from GLP-1RA to tirzepatide, who previously had GI intolerances. Clinical Case A retrospective review was conducted of five patients who started on GLP-1RA for either T2DM (n=3) or obesity (n=2) but stopped due to GI-related events. All patients were female with a mean age of 57 years. On average, patients with T2DM had a 23-year duration of diabetes, HbA1c 8.4%, weight 78.4 kg, and BMI 29.5 kg/m2 prior to starting tirzepatide. For patients without T2DM, the average weight was 75.9 kg and BMI 30.0 kg/m2 prior to starting tirzepatide. Before switching to tirzepatide, three patients were on maximally tolerated dulaglutide and two patients were on semaglutide. Patients were initially transitioned to tirzepatide 2.5 mg (n=2), 5 mg (n=2), and 7.5 mg (n=1). All patients tolerated tirzepatide with no GI-related events after an average of 6 months of treatment. Of the three patients with T2DM, they saw an additional average HbA1c reduction of 1.1% and an average weight loss of 3%. For patients who transitioned to tirzepatide for weight loss, they saw an additional average weight loss of 9%. Clinical Implications Tirzepatide may offer improved tolerability in patients who are intolerant of GLP-1RA drugs, while yielding further glycemic lowering and weight loss. Given tirzepatide’s stronger affinity to GIP than GLP-1 receptors, this may explain improved GI tolerability as seen in our case series. Further research is needed to evaluate tolerability improvement with tirzepatide using real-world data and larger sample sizes.

Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide-Protein Interactions.

Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (∼10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.

Effects of Ring Functionalization in Anthracene-Based Cyclophanes on the Binding Properties Toward Nucleotides and DNA.

Supramolecular recognition of nucleobases and short sequences is an emerging research field focusing on possible applications to treat many diseases. Controlling the affinity and selectivity of synthetic receptors to target desired nucleotides or short sequences is a highly challenging task. Herein, we elucidate the effect of substituents in the phenyl ring of the anthracene-benzene azacyclophane on the recognition of nucleoside triphosphates (NTPs) and double-stranded DNA. We show that introducing phenyl rings increases the affinity for NTPs 10-fold and implements groove and intercalation binding modes with double-stranded DNA. NMR studies and molecular modeling calculations support the ability of cyclophanes to encapsulate nucleobases as part of nucleotides.

Harnessing high potential benzothiazole chalcones against dengue virus NS5 protein: A multi-faceted theoretical study through molecular docking, ADME, and DFT

Chalcones bearing tetralone, indanone and benzothiazole cores were synthesized successfully using a general Claisen-Schmidt condensation protocol. The prepared compounds were purified and structurally analyzed by 1H, 13C NMR, and FT-IR techniques. A multi-faceted theoretical approach, combining Density Functional Theory (DFT), molecular docking, and ADME predictions, was employed to evaluate their therapeutic potential. DFT calculations at the B3LYP/def2-TZVP level revealed key electronic properties, with TD3 compound demonstrating the highest chemical reactivity. Molecular Electrostatic Potential (MEP) and Reduced Density Gradient (RDG) analyses provided insights into the compounds' non-covalent interactions and charge distributions. Molecular docking studies against the NS5 protein (PDB: 6KR2) showed superior binding affinities for all three compounds compared to the control ligand SAH, with TD3 exhibiting the lowest binding energy (−8.41 kcal/mol) and theoretical inhibition constant (689.31 nM). ADME predictions indicated favorable drug-like properties with concerns regarding aqueous solubility and potential P-glycoprotein interactions. Toxicity evaluations highlighted challenges, particularly in hepatotoxicity and carcinogenicity. The study identified TD3 as a promising lead compound for Dengue Virus NS5 inhibition, while also emphasizing the need for targeted modifications to address toxicity concerns. This research not only contributes to anti-dengue drug discovery efforts but also provides a robust methodological framework for the theoretical evaluation of similar small compounds in future investigations.

Interaction of Purine and its Derivatives with A1, A2-Adenosine Receptors and Vascular Endothelial Growth Factor Receptor-1 (Vegf-R1) as a Therapeutic Alternative to Treat Cancer.

There are several studies that indicate that cancer development may be conditioned by the activation of some biological systems that involve the interaction of different biomolecules, such as adenosine and vascular endothelial growth factor. These biomolecules have been targeted of some drugs for treat of cancer; however, there is little information on the interaction of purine derivatives with adenosine and vascular endothelial growth factor receptor (VEGF-R1). The aim of this research was to determine the possible interaction of purine (1: ) and their derivatives (2-31: ) with A1, A2-adenosine receptors, and VEGF-R1. Theoretical interaction of purine and their derivatives with A1, A2-adenosine receptors and VEGF-R1 was carried out using the 5uen, 5mzj and 3hng proteins as theoretical tools. Besides, adenosine, cgs-15943, rolofylline, cvt-124, wrc-0571, luf-5834, cvt-6883, AZD-4635, cabozantinib, pazopanib, regorafenib, and sorafenib drugs were used as controls. The results showed differences in the number of aminoacid residues involved in the interaction of purine and their derivatives with 5uen, 5mzj and 3hng proteins compared with the controls. Besides, the inhibition constants (Ki) values for purine and their derivatives 5: , 9: , 10: , 14: , 15: , 16: , and 20: were lower compared with the controls CONCLUSIONS: Theoretical data suggest that purine and their derivatives 5: , 9: , 10: , 14: , 15: , 16: , and 20: could produce changes in cancer cell growth through inhibition of A1, A2-adenosine receptors and VEGFR-1 inhibition. These data indicate that these purine derivatives could be a therapeutic alternative to treat some types of cancer.

Synthesis and investigation of selective human carbonic anhydrase IX, XII inhibitors using coumarins bearing a sulfonamide or biotin moiety

The role of carbonic anhydrases isoforms (CAs) IX and XII in the pathogenesis and progression of many types of solid tumors is well known. In this context, selective CA inhibitors (CAIs) towards the mentioned isoforms is a validated strategy for the development of agents to target cancer. For this purpose, novel coumarin derivatives based on the hybridization with arylsulfonamide or biotin scaffolds were synthesized and tested as inhibitors of four different human carbonic anhydrases isoforms: hCA I, II, IX and XII. Coumarin-sulfonamide derived 27, with a thiourea moiety and triazole as linker, showed the highest inhibition activity against hCA XII with an inhibition constant (KI) of 7.5 nM and afforded a very good selectivity over hCA I. Compound 32 was the most potent inhibitor against hCA IX (KI = 6.3 nM), 4-fold stronger than the drug acetazolamide AAZ (KI = 25 nM), used herein as a reference compound, and showed remarkable selectivity over hCA I and II. The coumarin-biotin derivatives 37-39 showed outstanding selectivity towards on-target enzymes (hCA IX and XII) and appear as plausible leads for designing of CAIs.

Kinetics of the inhibition of CYP3A4 and CYP2C19 activity by jabara juice and identification of the responsible inhibitory components

Some citrus fruits are known to cause clinically significant drug interactions by inhibiting intestinal cytochrome P450 (CYP) enzymes. This in vitro study aimed to investigate the kinetics of the inhibition of CYP3A4 and CYP2C19 by the juice of jabara, a Japanese citrus fruit that does not contain furanocoumarins such as 6′,7′-dihydroxybergamottin, and to identify the inhibitory compound(s).CYP3A4 and CYP2C19 activity levels were determined in vitro using recombinant CYP preparations and their respective substrates. The ethyl acetate extract (EAE) of jabara juice was separated to isolate and identify the compound(s) that inhibited CYP3A4. Then, the time-dependent kinetics of the inhibition of CYP3A4 and CYP2C19 by the EAE and its inhibitory compound(s) were analyzed.The EAE of jabara juice was found to inhibit CYP3A4 in a time-dependent manner. Two flavonoids, 3,3′,4′,5,6,7,8-heptamethoxyflavone (HpMF) and 3,3′,4′,5,6,7-hexamethoxyflavone (HxMF), were identified as the responsible compounds. HpMF and HxMF inhibited CYP3A4 activity in a concentration- and time-dependent manner, with inhibition constants (KI) of 10.0 and 7.90 µM and maximal inactivation rate constants (kinact,max) of 0.00856 and 0.0134 min−1, respectively. The EAE did not inhibit CYP2C19, even when preincubation was employed. These findings imply that jabara juice may cause food-drug interactions via time-dependent inhibition of intestinal CYP3A4.

Probe substrates assay estimates the effect of polyphyllin H on the activity of cytochrome P450 enzymes in human liver microsomes.

Cytochrome P450 enzymes (CYPs) play a crucial role in phase I metabolic reactions. The activity of CYPs would affect therapeutic efficacy and may even induce toxicity. Given the complex components of traditional Chinese medicine, it is important to understand the effect of active ingredients on CYPs activity to guide their prescription. This study aimed to evaluate the effect of polyphyllin H on the activity of CYPs major isoforms providing a reference for the clinical prescription of polyphyllin H and its source herbs. The effects of polyphyllin H were evaluated in pooled human liver microsomes using probe substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to determine their activities. The Lineweaver-Burk was used to model the inhibition, and a time-dependent inhibition experiment was performed to understand the characteristics of the inhibition. Polyphyllin H significantly suppressed the activity of CYP1A2, 2D6, and 3A4 with IC50 values of 6.44, 13.88, and 4.52 μM, respectively. The inhibition of CYP1A2 and 2D6 was best fitted with a competitive model, yielding the inhibition constant (Ki) values of 3.18 and 6.77 μM, respectively. The inhibition of CYP3A4 was fitted with the non-competitive model with the Ki value of 2.38 μM. Moreover, the inhibition of CYP3A4 was revealed to be time-dependent with the inhibition parameters inhibition constant (KI) and inactivation rate constant (Kinact) values of 2.26 μM-1 and 0.045 min-1. Polyphyllin H acted as a competitive inhibitor of CYP1A2 and 2D6 and a non-competitive and time-dependent inhibitor of CYP3A4.

N-Acetyl-L-Cysteine (NAC) Blunts Axitinib-Related Adverse Effects in Preclinical Models of Glioblastoma.

Axitinib is a tyrosine kinase inhibitor characterized by a strong affinity for Vascular Endothelial Growth Factor Receptors (VEGFRs). It was approved in 2012 by Food and Drug Administration and European Medicines Agency as a second line treatment for advanced renal cell carcinoma and is currently under evaluation in clinical trial for the treatment of other cancers. Glioblastoma IDH-wild type (GBM) is a highly malignant brain tumor characterized by diffusely infiltrative growth pattern and by a prominent neo-angiogenesis. In GBM, axitinib has demonstrated a limited effectiveness as a monotherapy, while it was recently shown to significantly improve its efficacy in combination treatments. In preclinical models, axitinib has been reported to trigger cellular senescence both in tumor as well as in normal cells, through a mechanism involving intracellular reactive oxygen species (ROS) accumulation and activation of Ataxia Telangiectasia Mutated kinase (ATM). Limiting axitinib-dependent ROS increase by antioxidants prevents senescence specifically in normal cells, without affecting tumor cells. We used brain tumor xenografts obtained by engrafting Glioma Stem Cells (GSCs) into the brain of immunocompromised mice, to investigate the hypothesis that the antioxidant molecule N-Acetyl-L-Cysteine (NAC) might be used to reduce senescence-associated adverse effects of axitinib treatment without altering its anti-tumor activity. We demonstrate that the use of the antioxidant molecule N-Acetyl-Cysteine (NAC) in combination with axitinib stabilizes tumor microvessels in GBM tumor orthotopic xenografts, eventually resulting in vessel normalization, and protects liver vasculature from axitinib-dependent toxicity. Overall, we found that NAC co-treatment allows vessel normalization in brain tumor vessels and exerts a protective effect on liver vasculature, therefore minimizing axitinib-dependent toxicity.

Purification and structural analysis of β-glucosidase from Lactobacillus acidophilus GIM1.208 and its interaction with rosamultin in Rosa roxburghii Tratt

Lactobacillus acidophilus GIM1.208 (LA) β-glucosidase (BGL) converts glycosides from Rosa roxburghii Tratt (RRT) to release more active aglycones. This study purified LA BGL and investigated it interaction with rosamultin in RRT. The isolated and purified BGL in the study had higher purity and activity, and the proportion of ordered structure in its secondary structure was greater than 60%, indicating better structural stability. The hydrolysis of purified BGL hydrolyzed rosamultin to tormentic acid at a rate of 25.2% (p < 0.01), and the hypoglycemic activity of tormentic acid was higher than that of rosamultin. Rosamultin causes BGL to undergo massive aggregation and enhances the hydrogen bonding network structure. Thermodynamic analyses indicate that their binding is a non-spontaneous exothermic entropy-decreasing reaction, with the main forces being hydrogen bonding, van der Waals forces, or protonation forces. Additionally, their thermodynamic binding constant Kd = 2.976 × 10−5 mol/L and dissociation constant KD = 2.67 × 10−5 mol/L indicate that the two have high affinity and strong interaction. In conclusion, BGL has the ability to hydrolyze rosamultin to release tormentic acid and they have a strong interaction. The results of this study can provide a reference for the study of glycoside changes and their interactions in RRT fermented by LA.

Differentiation of puerarin chelate from salt by phase solubility test

Different from salt, metal chelate is a novel state of drug constructed by more separate coordinate bonds to form a chelating circle. Due to their composition similarity, it is hard to distinguish them except identifying ionic bond (i.e., salt) or coordinate bond (i.e., chelate) in the single crystal structure. In this study, sodium chelate (CDCC No: 1865670) and lithium salt (CDCC No: 2161617) of puerarin (PUE) was prepared. In addition to difference in single crystal structure, it was found that they showed totally different phase solubility behaviors: lithium salt demonstrated a typical inverse proportion curve as other common salts, while sodium chelate exhibited disordered scatters. However, when incorporating the unit PUE-Na complex in solution state and complexation constant K11 in chemical equation, the scatters in phase solubility diagram of chelate could be well fitted and the value of K11 was dramatically higher with orders of magnitude than the dissociation constant Kc; while processing phase solubility curve of lithium salt by incorporating complex item, it could not well match the curve at all. PUE sodium chelate is more likely to be a weak electrolyte with partial dissociation, while PUE lithium salt acted as a strong electrolyte with complete dissociation. The phase solubility test would be served as a surrogate tool for differentiation of chelates from salts when single crystal was not available.

Assessing the validity and limits of linear density models for predicting dissociation–association equilibria in supercritical water

A linear relationship between the logarithms of solute dissociation constants, log(K), and the density of water, log (ρH2O), has empirically been demonstrated for decades and raised hope for an universal formalism to describe solute thermodynamic properties over wide ranges of temperature and density at supercritical conditions. Yet, neither a theoretical foundation nor an assessment of the ranges of validity have been presented. Here, we use classical molecular dynamics (MD) simulations as a complementary tool to assess the validity of this linear relationship for the example of NaCl and reveal its limits at water densities below ca. 0.3gcm−3. The derivative ∂log(K)/∂log(ρH2O) is calculated based on the volume of reaction and water compressibility derived from the simulations performed in the isothermal–isobaric ensemble at 673K. Our results corroborate the linear dependence of log(K) vs. log(ρH2O) in the experimentally studied density range and suggest that the linear dependence also extends to higher densities. However, towards lower densities, log(K) decreases and takes on values that are lower than would be expected by simply extrapolating the linear behavior. This decrease is consistent with earlier theoretical predictions for the behavior of log(K) at vapor-like densities but questions the relevance of some indirect experimental evidence obtained at low temperatures. Although the function described by log(K) vs log(ρH2O) is non-linear in the low density range, it can be considered well-behaved even at near critical conditions.

Aptamer-functionalized gold nanoparticles for fast and selective electrochemical sensing of lead in tobacco

This study presents a novel electrochemical aptasensor for the ultrasensitive detection of lead(II) ions in tobacco products. The sensor utilizes a G-quadruplex forming DNA aptamer (5′-GGGTGGGTGGGTGGGT-3′) immobilized on gold nanoparticles (AuNPs) with an average diameter of 73.2 nm. The aptamer-Pb2+ interaction, characterized by circular dichroism and isothermal titration calorimetry, revealed a dissociation constant of 0.28 ± 0.03 μM. Optimized sensor fabrication conditions included 1 μM aptamer concentration and 16-h incubation time. The aptasensor exhibited two linear ranges: 0.2072–103.6 μg/L and 103.6–1072 μg/L, with a detection limit of 37 ng/L. The sensor maintained selectivity in the presence of interfering ions at 100-fold higher concentrations. The sensor demonstrated excellent stability, retaining 92.1 % of its initial response after 30 days, and high reproducibility with an inter-batch RSD of 5.3 %. This aptasensor offers a promising platform for rapid and sensitive Pb2+ detection in complex matrices.

Synthesis and structure-affinity relationships of spirocyclic σ1 receptor ligands with tetrahydropyran scaffold

The σ1 receptor plays a key role in the regulation of various processes in the human body; it is involved in the development of neurodegenerative and neuropsychiatric diseases and is overexpressed in several human tumors rendering it an important target for potential drug candidates. In this project, spirocyclic σ1 receptor ligands with different substituents in 4- and 9-position were synthesized and investigated for their σ1 receptor affinity and selectivity over related targets. The σ1 affinity of the ligands was correlated with their lipophilicity (logD7.4 value) giving insight into their lipophilic ligand efficiency (LLE). The (pyridin-3-yl)methyl derivative 5i showed a promising balance of high σ1 affinity (Ki(σ1) = 3.9 nM) and selectivity (> 250-fold) as well as high LLE of 5.8. 5i has a high plasma protein binding (89%) and promising metabolic stability in the presence of mouse liver microsomes and NADPH (83% intact after 90 min). Increasing the size of the piperidine ring of the spirocyclic ligands 5 to an azepane ring led to considerably increased σ1 affinity (Ki(5a) = 1.2 nM, Ki(23a) = 0.42 nM) and selectivity over σ2 receptors (5a: 45-fold, 23a: 150-fold).