The elucidation of species-specific receptor pharmacology: a case study using subtype selective para- and meta-carborane estrogen receptor agonists.

Estrogen receptors are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of estrogen receptor (ER) ß is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERß without activating α is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERß-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine estrogen receptors. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single dose IV and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERß-12 for human ERß. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK293 cells expressing murine estrogen receptors revealed species-specific differences in the ER-subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. Significance Statement This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors, revealing the greater selectivity of carborane analogs for human ERß compared to the mouse homolog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.

Body conformation traits in early-lactation associated with clinical mastitis and lameness in lactating Chinese holstein cows.

BACKGROUND: Comprehending the correlation between body conformation traits of cows at the early stages of lactation and prevalent lactation diseases might facilitate the execution of selection and feeding strategies that prioritize cow health. This study aimed to evaluate the impact of body conformation traits on the incidence of clinical mastitis and lameness in Chinese Holstein cows. From a pasture herd of 1472 early lactating Chinese Holstein cows, we evaluated 20 body conformation traits. During lactation, this pasture herd was visited weekly to gather clinical mastitis and lameness data. A nine-point scale was used to determine the conformation traits of cows to clarify their linear characters, including frame capacity, rump (RU), feet and leg (FL), mammary system (MS), and dairy character. A longitudinal binary disease (0 = healthy; 1 = diseased) data structure was created by allocating disease records to adjacent official test dates. The impact of body conformation traits on the risk of developing diseases (clinical mastitis and lameness) was analyzed using the logistic regression models. RESULTS: Compared to cows with low total scores (75-79 points), those with high total scores (80-85 points) of body conformation traits had a significantly lower risk of mastitis (P < 0.001). The disease status (0 or 1: binary variable) of clinical mastitis in lactating cows was significantly impacted negatively by age (P < 0.05). The fore udder attachment (FUA), angularity, rear attachment height (RAH), and rear teat placement (RTP) were all significantly associated with clinical mastitis during lactation (P < 0.05). The rear leg-rear view (RLRV) was significantly correlated with correlated considerably (P < 0.05) with lameness during lactation. An ideal score of four points on the lameness risk dimension of the RLRV may indicate a low risk of lameness. Since the risk of mastitis decreased as this trait score increased, the RTP may be an ideal marker for mastitis risk. CONCLUSIONS: According to the study, clinical mastitis and lameness risks in cows can be estimated using their body conformation traits. Cows with more centrally located rear teats have a lower risk of mastitis. These results may help dairy farmers identify cows at high risk of disease early in lactation and aid in breeding for disease resistance in cows.

Characterization of T-cell subsets in response to foot-and-mouth disease bivalent inactivated vaccine in Chinese Holstein cows.

IMPORTANCE: Vaccination plays a crucial role in the prevention and control of FMD; however, outbreaks persist occurring worldwide. Assessing the immune response to FMD vaccines is essential for effective prevention of FMD. In this study, a seven-color flow cytometry protocol was developed to systematically evaluate the T-cell response of Chinese Holstein cows vaccinated with FMD bivalent inactivated vaccine. Our findings showed that while most T-cell subsets (%) decreased post-vaccination, a significant increase was observed in CD4+CD8+ DP T cells, which was consistent with the levels of specific foot-and-mouth disease virus (FMDV) antibodies. These findings suggested that CD4+CD8+ DP T cells could serve as a potential biomarker for the evaluation of cellular and humoral responses to FMDV vaccination. Additionally, we should be aware of the potential decline in cellular immunity among cattle during FMD vaccination, as this may increase the risk of other pathogen-related issues.

Construction of a Dual-Mode Immune Platform Based on the Photothermal Effect of AgCo@NC NPs for the Detection of α-Fetoprotein.

Compared with the accuracy of a single signal and the limitation of environmental applicability, the application value of dual-mode detection is gradually increasing. To this end, based on the photothermal effect of Ag/Co embedded N-rich mesoporous carbon nanomaterials (AgCo@NC NPs), we designed a dual-mode signal response system for the detection of α-fetoprotein (AFP). First, AgCo@NC NPs act as a photothermal immunoprobe that converts light energy into heat driven by a near-infrared (NIR) laser and obtains temperature changes corresponding to the analyte concentration on a hand-held thermal imager. In addition, this temperature recognition system can significantly improve the efficiency of Fenton-like reactions. AgCo@NC NPs act as peroxidase mimics to initiate the generation of poly N-isopropylacrylamide (PNIPAM, resistance enhancer) by cascade catalysis and the degradation of methylene blue (MB), thus enabling electrochemical testing. The dual-mode assay ranges from 0.01 to 100 and 0.001-10 ng/mL, with lower limits of detection (LOD) of 3.2 and 0.089 pg/mL, respectively, and combines visualization, portability, and high efficiency, opening new avenues for future clinical diagnostics and inhibitor studies.

Molecular Glues: The Adhesive Connecting Targeted Protein Degradation to the Clinic.

Targeted protein degradation is a rapidly exploding drug discovery strategy that uses small molecules to recruit disease-causing proteins for rapid destruction mainly via the ubiquitin-proteasome pathway. It shows great potential for treating diseases such as cancer and infectious, inflammatory, and neurodegenerative diseases, especially for those with "undruggable" pathogenic protein targets. With the recent rise of the "molecular glue" type of protein degraders, which tighten and simplify the connection of an E3 ligase with a disease-causing protein for ubiquitination and subsequent degradation, new therapies for unmet medical needs are being designed and developed. Here we use data from the CAS Content Collection and the publication landscape of recent research on targeted protein degraders to provide insights into these molecules, with a special focus on molecular glues. We also outline the advantages of the molecular glues and summarize the advances in drug discovery practices for molecular glue degraders. We further provide a thorough review of drug candidates in targeted protein degradation through E3 ligase recruitment. Finally, we highlight the progression of molecular glues in drug discovery pipelines and their targeted diseases. Overall, our paper provides a comprehensive reference to support the future development of molecular glues in medicine.

Mantuoluosides A-H, new steroids isolated from the leaves of Datura stramonium L.

Eight new steroids, designated mantuoluosides A-H (1-8), were obtained from the enrichment of steroids of the Datura stramonium L. using HPD-BJQH macroporous resin. Their respective structures were elucidated based on spectroscopic methods and comparison data with literature. The anti-inflammatory activities of these compounds were evaluated on (LPS)-induced RAW264.7 cells with inhibition ratio. It was found that most isolates showed potential anti-inflammatory activity with varying degrees of inhibition rate in a concentration-dependent manner at non-cytotoxic concentrations.

Exogenous Aß1-42 monomers improve synaptic and cognitive function in Alzheimer's disease model mice.

Growing evidence has suggested the poor correlation between brain amyloid plaque and Alzheimer's disease (AD). Presenilin1 (PS1) and presenilin2 (PS2) conditional double knockout (cDKO) mice exhibited the reduced 42-amino acid amyloid-ß peptide (Aß1-42) level and AD-like symptoms, indicating a different pathological mechanism from the amyloid cascade hypothesis for AD. Here we found that exogenous synthetic Aß1-42 monomers could improve the impaired memory not only in cDKO mice without Aß1-42 deposition but also in the APP/PS1/Tau triple transgenic 3 × Tg-AD mice with Aß1-42 deposition, which were mediated by α7-nAChR. Our findings demonstrate for the first time that reduced soluble Aß1-42 level is the main cause of cognitive dysfunction in cDKO mice, and support the opinions that low soluble Aß1-42 level due to Aß1-42 deposition may also cause cognitive deficits in 3 × Tg-AD mice. Therefore, "loss-of-function" of Aß1-42 should be avoided when designing therapies aimed at reducing Aß1-42 burden in AD.

αCaMKII in the lateral amygdala mediates PTSD-Like behaviors and NMDAR-Dependent LTD.

Post-traumatic stress disorder (PTSD) is a psychiatric disorder that afflicts many individuals. However, its molecular and cellular mechanisms remain largely unexplored. Here, we found PTSD susceptible mice exhibited significant up-regulation of alpha-Ca2+/calmodulin-dependent kinase II (αCaMKII) in the lateral amygdala (LA). Consistently, increasing αCaMKII in the LA not only caused PTSD-like behaviors such as impaired fear extinction and anxiety-like behaviors, but also attenuated N-methyl-D-aspartate receptor (NMDAR)-dependent long-term depression (LTD) at thalamo-lateral amygdala (T-LA) synapses, and reduced GluA1-Ser845/Ser831 dephosphorylation and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization. Suppressing the elevated αCaMKII to normal levels completely rescued both PTSD-like behaviors and the impairments in LTD, GluA1-Ser845/Ser831 dephosphorylation, and AMPAR internalization. Intriguingly, deficits in GluA1-Ser845/Ser831 dephosphorylation and AMPAR internalization were detected not only after impaired fear extinction, but also after attenuated LTD. Our results suggest that αCaMKII in the LA may be a potential molecular determinant of PTSD. We further demonstrate for the first time that GluA1-Ser845/Ser831 dephosphorylation and AMPAR internalization are molecular links between fear extinction and LTD.

Structure-Activity Relationship of para-Carborane Selective Estrogen Receptor ß Agonists.

Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERß, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17ß-estradiol geometry in the design of ERß selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERß selective structure-activity relationship. We report ERß agonists with low nanomolar potency, greater than 200-fold selectivity for ERß over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERß selective agonists measure favorably against clinically developed ERß agonists and support further evaluation of carborane-based selective estrogen receptor modulators.

Oxyberberine Prevented Lipopolysaccharide-Induced Acute Lung Injury through Inhibition of Mitophagy.

Acute lung injury (ALI) is a serious respiratory syndrome characterized with uncontrolled inflammatory response. Oxyberberine has strong potential for clinical usage since it showed strong anti-inflammatory, antifungal, and antiarrhythmic effects in various diseases. In the present study, we evaluated whether oxyberberine can inhibit lipopolysaccharide- (LPS-) induced ALI in vivo and further evaluated the possible involvement of mitophagy in vitro by using A549 cells, a human lung epithelial cell line. Our in vivo study shows that oxyberberine significantly inhibited LPS-induced lung pathological injury and lung edema, as indicated by the changes in lung wet/dry ratio and total protein levels in the BALF in mice. Moreover, oxyberberine inhibited inflammation, as indicated by the changes of neutrophil accumulation and production of proinflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6 in both the lung and bronchoalveolar lavage fluid (BALF) in ALI mice. Our in vitro study shows that LPS significantly decreased the protein level of mitochondrial proteins, including cytochrome c oxidase subunit IV (COX IV), p62, and mitofusin-2 (Mfn2) in A549 cells. In addition, LPS induced significant Parkin1 translocation from cytoplasm to mitochondria. These changes were significantly inhibited by oxyberberine. Notably, the inhibitory effect of oxyberberine was almost totally lost in the presence of lysosome fusion inhibitor bafilomycin A1 (Baf), a mitophagy inhibitor. In conclusion, the present study demonstrated that oxyberberine alleviated LPS-induced inflammation in ALI via inhibition of Parkin-mediated mitophagy.

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 µM, and Caco-2 data suggested low permeability with active efflux at 2 µM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUClast (26.1 µM*h), and Cmax (0.899 µM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.

[Xuebijing injection attenuates hydrogen sulfide-induced endothelial barrier dysfunction by upregulating claudin-5 expression].

OBJECTIVE: To study the new mechanism of Xuebijing injection improving the function of pulmonary vascular barrier from the perspective of claudin-5 protein. METHODS: Acute lung injury (ALI) model was induced by hydrogen sulfide (H2S) exposure. (1) In vivo study: Sprague-Dawley (SD) rats were divided into control group, H2S exposure group (exposure to 300×10-6 H2S for 3 hours), Xuebijing control group (Xuebijing injection 4 mL/kg , twice a day, for 3 days), and Xuebijing intervention group (H2S exposure after pretreatment of Xuebijing injection) according to random number method, with 6 rats in each group. At different time points (0, 6, 12 and 24 hours) after the model was made successfully, the total protein content in plasma and bronchoalveolar lavage fluid (BALF) of rats were detected respectively, and the pulmonary permeability index (PPI) was calculated (PPI = protein content in BALF/protein content in plasma), lung dry/wet weight ratio (W/D) was detected, and claudin-5 mRNA expression in lung tissue was measured by real time-polymerase chain reaction. (2) In vitro test: human pulmonary microvascular endothelial cells (HPMECs) were divided into blank control group, NaHS treatment group (co-incubated with 500 µmol/L NaHS for 12 hours), Xuebijing control group (2 g/L Xuebijing injection for 24 hours), and Xuebijing intervention group (2 g/L Xuebijing injection pre-treated for 24 hours, then co-incubated with 500 µmol/L NaHS for 12 hours). The HPMECs claudin-5 protein expression and monolayer permeability changes were measured at different co-incubation time (1, 3, 6, 12 and 24 hours) by Western Blot and fluoresceinsodium. RESULTS: (1) In vivo study: compared with the control group, the lung W/D ratio increased significantly at 6 hours and peaked at 12 hours after H2S exposure in rats (4.67±0.11 vs. 4.26±0.06, P < 0.01). The expression of claudin-5 mRNA in lung tissue was significantly decreased, which was 89% of control group 6 hours after exposure (P < 0.01). The total protein content in BALF and PPI at 12 hours after exposure were significantly higher than those in the control group [total protein content (mg/L): 262.31±14.24 vs. 33.30±3.09, PPI: (11.72±0.57)×10-3 vs. (1.21±0.08)×10-3, both P < 0.01], while the results in Xuebijing intervention group were significantly decreased [total protein content (mg/L): 153.25±7.32 vs. 262.31±14.24, PPI: (5.79±0.23)×10-3 vs. (11.72±0.57)×10-3, both P < 0.01]. (2) In vitro test: compared with the blank control group, after incubating HPMECs with NaHS, the permeability of monolayer endothelial cells gradually increased, reaching the highest level in 12 hours, about twice of that in the blank control group, while claudin-5 protein expression decreased to the lowest level at 12 hours (claudin-5/ß-actin: 0.42±0.03 vs. 1.03±0.05, P < 0.01). After intervention with Xuebijing, the permeability of endothelial cells was significantly improved (fluorescence intensity of fluorescein sodium: 1.46±0.10 vs. 1.89±0.11, P < 0.01), and the decrease of claudin-5 protein was reduced (claudin-5/ß-actin: 0.68±0.04 vs. 0.38±0.03, P < 0.01). CONCLUSIONS: Xuebijing injection may improve pulmonary vascular barrier function in ALI by upregulating claudin-5 expression.

Improvement of intellectual outcomes in 20 children with refractory epilepsy after individualized surgery.

BACKGROUND: Refractory epilepsy is a common and troublesome neurosurgical disease. This study is designed to compare seizure control and degrees in intellectual outcome in children with refractory epilepsy after surgical treatment. METHODS: 20 children with refractory epilepsy were treated with tailored epilepsy surgery or vagus nerve stimulation (VNS). We used the Engel Epilepsy Surgery Outcome Scale to evaluate seizure control and the Wechsler Intelligence Scale for Children, fourth edition (WISC-IV), to test the children's intellectual outcomes 7-day preoperative and 3-, 6-, and 12-month postoperative. RESULTS: In total, 14 cases were seizure free (Engel I) and 2 cases to have suffered few attacks since surgery (Engel II). In two cases, the frequency of seizures decreased by >90% (Engel III). In the remaining two cases, the effects of surgery on seizure control were not obvious (Engel IV). All children completed the WISC-IV test. On average, postoperative intelligence quotient (IQ) increased by 6.35 points 12-month postsurgery compared with the results of the preoperative tests (P < 0.01). Second, intellectual outcomes after surgery in the Engel I and II groups increased by >3.88 points compared with in the Engel III and IV groups (P < 0.05). Finally, there were no fatal complications over the long-term follow-up except for intracranial infection of two cases; postoperative subcutaneous hematoma occurred in one case and hoarseness in one case. CONCLUSION: Individualized epilepsy surgery is safe and effective for children with refractory epilepsy. It can control or reduce the frequency of postoperative attacks as well as improve postoperative intellectual outcomes to different degrees.

Preclinical Pharmacokinetics Study of R- and S-Enantiomers of the Histone Deacetylase Inhibitor, AR-42 (NSC 731438), in Rodents.

AR-42, a new orally bioavailable, potent, hydroxamate-tethered phenylbutyrate class I/IIB histone deacetylase inhibitor currently is under evaluation in phase 1 and 2 clinical trials and has demonstrated activity in both hematologic and solid tumor malignancies. This report focuses on the preclinical characterization of the pharmacokinetics of AR-42 in mice and rats. A high-performance liquid chromatography-tandem mass spectrometry assay has been developed and applied to the pharmacokinetic study of the more active stereoisomer, S-AR-42, when administered via intravenous and oral routes in rodents, including plasma, bone marrow, and spleen pharmacokinetics (PK) in CD2F1 mice and plasma PK in F344 rats. Oral bioavailability was estimated to be 26 and 100% in mice and rats, respectively. R-AR-42 was also evaluated intravenously in rats and was shown to display different pharmacokinetics with a much shorter terminal half-life compared to that of S-AR-42. Renal clearance was a minor elimination pathway for parental S-AR-42. Oral administration of S-AR-42 to tumor-bearing mice demonstrated high uptake and exposure of the parent drug in the lymphoid tissues, spleen, and bone marrow. This is the first report of the pharmacokinetics of this novel agent, which is now in early phase clinical trials.

Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function.

We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone - chaperone and chaperone - client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 -dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 -induced activation of ER stress signaling and maintained mTOR activity; AR-12 -mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.

AMPK reverses the mesenchymal phenotype of cancer cells by targeting the Akt-MDM2-Foxo3a signaling axis.

In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKß-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis.