Cholestane-3ß,5α,6ß-triol Induces Multiple Cell Death in A549 Cells via ER Stress and Autophagy Activation.

Cholestane-3ß,5α,6ß-triol (CT) and its analogues are abundant in natural sources and are reported to demonstrate cytotoxicity toward different kinds of tumor cells without a deep probe into their mechanism of action. CT is also one of the major metabolic oxysterols of cholesterol in mammals and is found to accumulate in various diseases. An extensive exploration of the biological roles of CT over the past few decades has established its identity as an apoptosis inducer. In this study, the effects of CT on A549 cell death were investigated through cell viability assays. RNA-sequencing analysis and western blot of CT-treated A549 cells revealed the role of CT in inducing endoplasmic reticulum (ER) stress response and enhancing autophagy flux, suggesting a putative mechanism of CT-induced cell-death activation involving reactive oxygen species (ROS)-mediated ER stress and autophagy. It is reported for the first time that the upregulation of autophagy induced by CT can serve as a cellular cytotoxicity response in accelerating CT-induced cell death in A549 cells. This research provides evidence for the effect of CT as an oxysterol in cell response to oxidative damage and allows for a deep understanding of cholesterol in its response in an oxidative stress environment that commonly occurs in the progression of various diseases.

Studying the trafficking of labeled trodusquemine and its application as nerve marker for light-sheet and expansion microscopy.

Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.

Vitamin D Increases Irisin Serum Levels and the Expression of Its Precursor in Skeletal Muscle.

Irisin is a myokine synthesized by skeletal muscle, which performs key actions on whole-body metabolism. Previous studies have hypothesized a relationship between irisin and vitamin D, but the pathway has not been thoroughly investigated. The purpose of the study was to evaluate whether vitamin D supplementation affected irisin serum levels in a cohort of 19 postmenopausal women with primary hyperparathyroidism (PHPT) treated with cholecalciferol for six months. In parallel, to understand the possible link between vitamin D and irisin, we analyzed the expression of the irisin precursor, Fndc5, in the C2C12 myoblast cell line treated with a biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Our results demonstrate that vitamin D supplementation resulted in a significant increase in irisin serum levels (p = 0.031) in PHPT patients. In vitro, we show that vitamin D treatment on myoblasts enhanced Fndc5 mRNA after 48 h (p = 0.013), while it increased mRNAs of sirtuin 1 (Sirt1) (p = 0.041) and peroxisome proliferator-activated receptor γ coactivator 1α (Pgc1α) (p = 0.017) over a shorter time course. Overall, our data suggest that vitamin-D-induced modulation of Fndc5/irisin occurs through up-regulation of Sirt1, which together with Pgc1α, is an important regulator of numerous metabolic processes in skeletal muscle.

Reduced High-Density Lipoprotein Cholesterol Is an Independent Determinant of Altered Bone Quality in Women with Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is associated with an increased fracture risk. Our study aimed to explore differences in bone alterations between T2DM women and controls and to assess clinical predictors of bone impairment in T2DM. For this observational case control study, we recruited 126 T2DM female patients and 117 non-diabetic, age- and BMI-comparable women, who underwent clinical examination, routine biochemistry and dual-energy X-ray absorptiometry (DXA) scans for bone mineral density (BMD) and trabecular bone score (TBS) assessment-derived indexes. These were correlated to metabolic parameters, such as glycemic control and lipid profile, by bivariate analyses, and significant variables were entered in multivariate adjusted models to detect independent determinants of altered bone status in diabetes. The T2DM patients were less represented in the normal bone category compared with controls (5% vs. 12%; p = 0.04); T2DM was associated with low TBS (OR: 2.47, C.I. 95%: 1.19-5.16, p = 0.016) in a regression model adjusted for age, menopausal status and BMI. In women with T2DM, TBS directly correlated with plasma high-density lipoprotein cholesterol (HDL-c) (p = 0.029) and vitamin D (p = 0.017) levels. An inverse association was observed with menopausal status (p < 0.001), metabolic syndrome (p = 0.014), BMI (p = 0.005), and waist circumference (p < 0.001). In the multivariate regression analysis, lower HDL-c represented the main predictor of altered bone quality in T2DM, regardless of age, menopausal status, BMI, waist circumference, statin treatment, physical activity, and vitamin D (p = 0.029; R2 = 0.47), which likely underlies common pathways between metabolic disease and bone health in diabetes.

Exploration of Binding Affinities of a 3ß,6ß-Diacetoxy-5α-cholestan-5-ol with Human Serum Albumin: Insights from Synthesis, Characterization, Crystal Structure, Antioxidant and Molecular Docking.

The present study describes the synthesis, characterization, and in vitro molecular interactions of a steroid 3ß,6ß-diacetoxy-5α-cholestan-5-ol. Through conventional and solid-state methods, a cholestane derivative was successfully synthesized, and a variety of analytical techniques were employed to confirm its identity, including high-resolution mass spectrometry (HRMS), Fourier transforms infrared (FT-IR), nuclear magnetic resonance (NMR), elemental analysis, and X-ray single-crystal diffraction. Optimizing the geometry of the steroid was undertaken using density functional theory (DFT), and the results showed great concordance with the data from the experiments. Fluorescence spectral methods and ultraviolet-vis absorption titration were employed to study the in vitro molecular interaction of the steroid regarding human serum albumin (HSA). The Stern-Volmer, modified Stern-Volmer, and thermodynamic parameters' findings showed that steroids had a significant binding affinity to HSA and were further investigated by molecular docking studies to understand the participation of active amino acids in forming non-bonding interactions with steroids. Fluorescence studies have shown that compound 3 interacts with human serum albumin (HSA) through a static quenching mechanism. The binding affinity of compound 3 for HSA was found to be 3.18 × 104 mol-1, and the Gibbs free energy change (ΔG) for the binding reaction was -9.86 kcal mol-1 at 298 K. This indicates that the binding of compound 3 to HSA is thermodynamically favorable. The thermodynamic parameters as well as the binding score obtained from molecular docking at various Sudlow's sites was -8.2, -8.5, and -8.6 kcal/mol for Sites I, II, and III, respectively, supporting the system's spontaneity. Aside from its structural properties, the steroid demonstrated noteworthy antioxidant activity, as evidenced by its IC50 value of 58.5 µM, which is comparable to that of ascorbic acid. The findings presented here contribute to a better understanding of the pharmacodynamics of steroids.

Novel Synthesis of IMC-48 and Affinity Evaluation with Different i-Motif DNA Sequences.

During the last decade, the evidence for the biological relevance of i-motif DNA (i-DNA) has been accumulated. However, relatively few molecules were reported to interact with i-DNA, and a controversy concerning their binding mode, affinity, and selectivity persists in the literature. In this context, the cholestane derivative IMC-48 has been reported to modulate bcl-2 gene expression by stabilizing an i-motif structure in its promoter. In the present contribution, we report on a novel, more straightforward, synthesis of IMC-48 requiring fewer steps compared to the previous approach. Furthermore, the interaction of IMC-48 with four different i-motif DNA sequences was thoroughly investigated by bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy. Surprisingly, our results show that IMC-48 is a very weak ligand of i-DNA as no quantifiable interaction or significant stabilization of i-motif structures could be observed, stimulating a quest for an alternative mechanism of its biological activity.

The aminosterol Claramine inhibits ß-secretase 1-mediated insulin receptor cleavage.

The cleavage of the insulin receptor by ß-secretase 1 (BACE1) in the liver increases during diabetes, which contributes to reduce insulin receptor levels and impair insulin signaling. However, the precise signaling events that lead to this increased cleavage are unclear. We showed that BACE1 cleaves the insulin receptor in the early secretory pathway. Indeed, coimmunoprecipitation experiments reveal the interaction of the proforms of the two proteins. Moreover, fragments of insulin receptor are detected in the early secretory pathway and a mutated form of BACE1 that retains its prodomain cleaves an early secretory pathway-resident form of the insulin receptor. We showed that BACE1 proform levels are regulated by proteasome and/or lysosome-dependent degradation systems whose efficiencies are dependent on the O-GlcNacylation process. Our results showed that enhanced O-GlcNacylation reduces the efficiency of intracellular protein degradation systems, leading to the accumulation of the proform of BACE1 in the early secretory pathway where it cleaves the precursor of the insulin receptor. All these dysregulations are found in the livers of diabetic mice. In addition, we performed a screen of molecules according to their ability to increase levels of the insulin receptor at the surface of BACE1-overexpressing cells. This approach identified the aminosterol Claramine, which accelerated intracellular trafficking of the proform of BACE1 and increased autophagy. Both of these effects likely contribute to the reduced amount of the proform of BACE1 in the early secretory pathway, thereby reducing insulin receptor cleavage. These newly described properties of Claramine are consistent with its insulin sensitizing effect.

Squalamine and trodusquemine: two natural products for neurodegenerative diseases, from physical chemistry to the clinic.

Covering: 1993 to 2021 (mainly 2017-2021)Alzheimer's and Parkinson's diseases are neurodegenerative conditions affecting over 50 million people worldwide. Since these disorders are still largely intractable pharmacologically, discovering effective treatments is of great urgency and importance. These conditions are characteristically associated with the aberrant deposition of proteinaceous aggregates in the brain, and with the formation of metastable intermediates known as protein misfolded oligomers that play a central role in their aetiology. In this Highlight article, we review the evidence at the physicochemical, cellular, animal model and clinical levels on how the natural products squalamine and trodusquemine offer promising opportunities for chronic treatments for these progressive conditions by preventing both the formation of neurotoxic oligomers and their interaction with cell membranes.

Selective obstruction of the mTORC2 complex by a naturally occurring cholestane saponin (OSW-1) for inhibiting prostate cancer cell growth.

Ornithogalum caudatum Ait (OCA) is a natural product used in Chinese traditional medicine. The cholestane saponin OSW-1 is isolated from plant OCA and has recently been shown to have potent cytotoxic effects against different types of cancers. The therapeutic efficacy of OSW-1 on prostate cancer and its underlying mechanism are yet to be established. OSW-1 inhibited the growth of prostate cancer cells by interrupting the interaction between mTOR and Rictor/mTORC2. This mechanism showed a better therapeutic outcome than that of the conventional inhibition of mTOR and provided a basis for as sisting modern prostate cancer treatment strategies.

From Marine Metabolites to the Drugs of the Future: Squalamine, Trodusquemine, Their Steroid and Triterpene Analogues.

This review comprehensively describes the recent advances in the synthesis and pharmacological evaluation of steroid polyamines squalamine, trodusquemine, ceragenins, claramine, and their diverse analogs and derivatives, with a special focus on their complete synthesis from cholic acids, as well as an antibacterial and antiviral, neuroprotective, antiangiogenic, antitumor, antiobesity and weight-loss activity, antiatherogenic, regenerative, and anxiolytic properties. Trodusquemine is the most-studied small-molecule allosteric PTP1B inhibitor. The discovery of squalamine as the first representative of a previously unknown class of natural antibiotics of animal origin stimulated extensive research of terpenoids (especially triterpenoids) comprising polyamine fragments. During the last decade, this new class of biologically active semisynthetic natural product derivatives demonstrated the possibility to form supramolecular networks, which opens up many possibilities for the use of such structures for drug delivery systems in serum or other body fluids.

Fused Pyrroles in Cholestane and Norcholestane Side Chains: Acaricidal and Plant Growth-Promoting Effects.

Herein, we describe the synthesis and characterization of fused pyrroles in cholestane and norcholestane side chains derived from kryptogenin and diosgenin, respectively. Both conventional and microwave heating techniques were used to synthesize the steroidal pyrroles from primary amines, with the microwave method producing the highest yields. In particular, the norcholestane pyrroles were tested as acaricides against the two-spotted spider mite (Tetranychus urticae Koch) under laboratory conditions and as plant growth promoters on habanero pepper (Capsicum chinense Jacq) under greenhouse conditions.

Neuronal Protein Tyrosine Phosphatase 1B Hastens Amyloid ß-Associated Alzheimer's Disease in Mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, resulting in the progressive decline of cognitive function in patients. Familial forms of AD are tied to mutations in the amyloid precursor protein, but the cellular mechanisms that cause AD remain unclear. Inflammation and amyloidosis from amyloid ß (Aß) aggregates are implicated in neuron loss and cognitive decline. Inflammation activates the protein-tyrosine phosphatase 1B (PTP1B), and this could suppress many signaling pathways that activate glycogen synthase kinase 3ß (GSK3ß) implicated in neurodegeneration. However, the significance of PTP1B in AD pathology remains unclear. Here, we show that pharmacological inhibition of PTP1B with trodusquemine or selective ablation of PTP1B in neurons prevents hippocampal neuron loss and spatial memory deficits in a transgenic AD mouse model with Aß pathology (hAPP-J20 mice of both sexes). Intriguingly, while systemic inhibition of PTP1B reduced inflammation in the hippocampus, neuronal PTP1B ablation did not. These results dissociate inflammation from neuronal loss and cognitive decline and demonstrate that neuronal PTP1B hastens neurodegeneration and cognitive decline in this model of AD. The protective effect of PTP1B inhibition or ablation coincides with the restoration of GSK3ß inhibition. Neuronal ablation of PTP1B did not affect cerebral amyloid levels or plaque numbers, but reduced Aß plaque size in the hippocampus. In summary, our preclinical study suggests that targeting PTP1B may be a new strategy to intervene in the progression of AD.SIGNIFICANCE STATEMENT Familial forms of Alzheimer's disease (AD) are tied to mutations in the amyloid precursor protein, but the cellular mechanisms that cause AD remain unclear. Here, we used a mouse model expressing human amyloid precursor protein bearing two familial mutations and asked whether activation of a phosphatase PTP1B participates in the disease process. Systemic inhibition of this phosphatase using a selective inhibitor prevented cognitive decline, neuron loss in the hippocampus, and attenuated inflammation. Importantly, neuron-targeted ablation of PTP1B also prevented cognitive decline and neuron loss but did not reduce inflammation. Therefore, neuronal loss rather than inflammation was critical for AD progression in this mouse model, and that disease progression could be ameliorated by inhibition of PTP1B.

Inhibition of lipid phosphatase SHIP1 expands myeloid-derived suppressor cells and attenuates rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of unknown cause, characterized by infiltration and accumulation of activated immune cells in the synovial joints where cartilage and bone destructions occur. Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) was shown to be involved in the regulation of MDSC differentiation. The purpose of the present study was to investigate the effect of inhibition of SHIP1 on the expansion of MDSCs in RA using a collagen-induced inflammatory arthritis (CIA) mouse model. In DBA/1 mice, treatment with a small molecule-specific SHIP1 inhibitor 3α-aminocholestane (3AC) induced a marked expansion of MDSCs in vivo. Both pretreatment with 3AC of DBA/1 mice prior to CIA induction and intervention with 3AC during CIA progression significantly reduced disease incidence and severity. Adoptive transfer of MDSCs isolated from 3AC-treated mice, but not naïve MDSCs from normal mice, into CIA mice significantly reduced disease incidence and severity, indicating that the 3AC-induced MDSCs were the cellular mediators of the observed amelioration of the disease. In conclusion, inhibition of SHIP1 expands MDSCs in vivo and attenuates development of CIA in mice. Small molecule-specific inhibition of SHIP1 may therefore offer therapeutic benefit to patients with RA and other autoimmune diseases.

Ketamine's schizophrenia-like effects are prevented by targeting PTP1B.

Subanesthetic doses of ketamine induce schizophrenia-like behaviors in mice including hyperlocomotion and deficits in working memory and sensorimotor gating. Here, we examined the effect of in vivo ketamine administration on neuronal properties and endocannabinoid (eCB)-dependent modulation of synaptic transmission onto layer 2/3 pyramidal neurons in brain slices of the prefrontal cortex, a region tied to the schizophrenia-like behavioral phenotypes of ketamine. Since deficits in working memory and sensorimotor gating are tied to activation of the tyrosine phosphatase PTP1B in glutamatergic neurons, we asked whether PTP1B contributes to these effects of ketamine. Ketamine increased membrane resistance and excitability of pyramidal neurons. Systemic pharmacological inhibition of PTP1B by Trodusquemine restored these neuronal properties and prevented each of the three main ketamine-induced behavior deficits. Ketamine also reduced mobilization of eCB by pyramidal neurons, while unexpectedly reducing their inhibitory inputs, and these effects of ketamine were blocked or occluded by PTP1B ablation in glutamatergic neurons. While ablation of PTP1B in glutamatergic neurons prevented ketamine-induced deficits in memory and sensorimotor gating, it failed to prevent hyperlocomotion (a psychosis-like phenotype). Taken together, these results suggest that PTP1B in glutamatergic neurons mediates ketamine-induced deficits in eCB mobilization, memory and sensorimotor gating whereas PTP1B in other cell types contributes to hyperlocomotion. Our study suggests that the PTP1B inhibitor Trodusquemine may represent a new class of fast-acting antipsychotic drugs to treat schizophrenia-like symptoms.

Tyrosine phosphatase PTP1B impairs presynaptic NMDA receptor-mediated plasticity in a mouse model of Alzheimer's disease.

Mutations in the beta-amyloid protein (APP) cause familial Alzheimer's disease. In hAPP-J20 mice expressing mutant APP, pharmacological inhibition or genetic ablation of the tyrosine phosphatase PTP1B prevents CA3 hippocampus neuron loss and cognitive decline. However, how targeting PTP1B affects the cellular mechanisms underlying these cognitive deficits remains unknown. Changes in synaptic strength at the hippocampus can affect information processing for learning and memory. While prior studies have focused on post-synaptic mechanisms to account for synaptic deficits in Alzheimer's disease models, presynaptic mechanisms may also be affected. Here, using whole cell patch-clamp recording, coefficient of variation (CV) analysis suggested a profound presynaptic deficit in long-term potentiation (LTP) of CA3:CA1 synapses in hAPP-J20 mice. While the membrane-impermeable ionotropic NMDA receptor (NMDAR) blocker norketamine in the post-synaptic recording electrode had no effect on LTP, additional bath application of the ionotropic NMDAR blockers MK801 could replicate the deficit in LTP in wild type mice. In contrast to LTP, the paired-pulse ratio and short-term facilitation (STF) were aberrantly increased in hAPP-J20 mice. These synaptic deficits in hAPP-J20 mice were associated with reduced phosphorylation of NMDAR GluN2B and the synaptic vesicle recycling protein NSF (N-ethylmaleimide sensitive factor). Phosphorylation of both proteins, together with synaptic plasticity and cognitive function, were restored by PTP1B ablation or inhibition by the PTP1B-selective inhibitor Trodusquemine. Taken together, our results indicate that PTP1B impairs presynaptic NMDAR-mediated synaptic plasticity required for spatial learning in a mouse model of Alzheimer's disease. Since Trodusquemine has undergone phase 1/2 clinical trials to treat obesity, it could be repurposed to treat Alzheimer's disease.

A new homo-aro-cholestane glycoside from the rhizome of Paris polyphylla var. chinensis.

A new homo-aro-cholestane glycoside parispolyside H, along with nine known compounds, were isolated from 75% ethanolic extract of the rhizome of Paris polyphylla var. chinensis. Their chemical structures were elucidated on the basic of analysis of detailed spectroscopic and physicochemical properties. In addition, the isolated compounds (1, 6-9) were evaluated for their cytotoxic activity against HepG2 human liver cancer cell lines. Among them, four known compounds (6-9) showed cytotoxicity with IC50 values ranging from 0.41 to 3.6 µM.

Role of the cholesterol hydroxyl group in the chemical exchange saturation transfer signal at -1.6 ppm.

Chemical exchange saturation transfer (CEST) can provide metabolite-weighted images in the clinical setting; therefore, understanding the origin of each CEST signal is essential to revealing the changes in diseases at the molecular level, which would provide further insight for diagnoses and treatments. The CEST signal at -1.6 ppm is attributed to the choline methyl group of phosphatidylcholines. The methyl groups have no exchangeable protons, so the corresponding CEST signals must result from the relayed nuclear Overhauser effect (rNOE); however, the detailed mechanism remains unclear. Cholesterol is a major component of biological membranes, and its content is closely related to the dynamics and phases of these lipids. However, cholesterol has a hydroxyl group, which could participate in proton exchange to complete the rNOE process. In this study, we used liposomes containing cholesterol and its analogs (5α-cholestane and progesterone), which presumably have similar capabilities of influencing lipid bilayers, and found that the steroid hydroxyl group is the key to inducing the rNOE at -1.6 ppm. Our results suggest that the origin of the rNOE at -1.6 ppm likely requires an intermolecular NOE between the proton of the choline methyl group and that of the cholesterol hydroxyl group, and a chemical exchange between the cholesterol hydroxyl group and bulk water. However, the phenomenon in which the rNOE at -1.6 ppm appears when the cholesterol concentration is high seems to contradict the in vivo results, suggesting a more complicated mechanism associated with the rNOE at -1.6 ppm in biological membranes.

Galtonosides A-E: Antiproliferative and Antiplasmodial Cholestane Glycosides from Galtonia regalis.

An extract of Galtonia regalis from the Natural Products Discovery Institute showed moderate antiplasmodial activity, with an IC50 value less than 1.25 µg/mL. The two known cholestane glycosides 1 and 2 and the five new cholestane glycosides galtonosides A-E (3-7) were isolated after bioassay-directed fractionation. The structures of the new compounds were determined by interpretation of their NMR and mass spectra. Among these compounds, galtonoside B (4) displayed the most potent antiplasmodial activity, with an IC50 value of 0.214 µM against the drug-resistant Dd2 strain of Plasmodium falciparum.

Seven new 1-oxygenated cholestane glycosides from Ornithogalum saundersiae.

As the continuous scientific research, seven new 1-oxygenated cholestane glycosides named osaundersiosides 1 A - 1 G were isolated from an EtOH extract of the bulbs of Ornithogalum saundersiae. Their structures were deduced by means of spectroscopic data, chemical evidence and the results of hydrolytic cleavage. The cytotoxicity and anti-inflammatory effects of osaundersiosides 1 A - 1 G were evaluated, but none of them displayed significant activities. [Formula: see text].

A Total of Eight Novel Steroidal Glycosides Based on Spirostan, Furostan, Pseudofurostan, and Cholestane from the Leaves of Cestrum newellii.

Previously, various steroidal glycosides were reported from plants of Cestrum species. However, phytochemical investigation has not been conducted on Cestrum newellii. A systematic phytochemical investigation of the leaves of C. newellii resulted in the isolation of eight novel steroidal glycosides (1-8), which were classified into three spirostanol glycosides (1-3), two furostanol glycosides (4 and 5), two pseudofurostanol glycosides (6 and 7), and one cholestane glycoside (8). In addition, three known cholestane glycosides (9-11) were isolated and identified. The structures of the new compounds were determined based on spectroscopic data and chemical transformations. Compounds 1 and 2 are spirostanol glycosides having hydroxy groups at C-2, C-3, C-12, and C-24 of the aglycone moiety. Although C. newellii is known to be a poisonous plant, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay exhibited that none of the isolated compounds were cytotoxic to HL-60 human promyelocytic leukemia cells.