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.

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.

Evaluation of Cytotoxicity and α-Glucosidase Inhibitory Activity of Amide and Polyamino-Derivatives of Lupane Triterpenoids.

A series of two new and twenty earlier synthesized branched extra-amino-triterpenoids obtained by the direct coupling of betulinic/betulonic acids with polymethylenpolyamines, or by the cyanoethylation of lupane type alcohols, oximes, amines, and amides with the following reduction were evaluated for cytotoxicity toward the NCI-60 cancer cell line panel, α-glucosidase inhibitory, and antimicrobial activities. Lupane carboxamides, conjugates with diaminopropane, triethylenetetramine, and branched C3-cyanoethylated polyamine methyl betulonate showed high cytotoxic activity against most of the tested cancer cell lines with GI50 that ranged from 1.09 to 54.40 µM. Betulonic acid C28-conjugate with triethylenetetramine and C3,C28-bis-aminopropoxy-betulin were found to be potent micromolar inhibitors of yeast α-glucosidase and to simultaneously inhibit the endosomal reticulum α-glucosidase, rendering them as potentially capable to suppress tumor invasiveness and neovascularization, in addition to the direct cytotoxicity. Plausible mechanisms of cytotoxic action and underlying disrupted molecular pathways were elucidated with CellMinner pattern analysis and Gene Ontology enrichment analysis, according to which the lead compounds exert multi-target antiproliferative activity associated with oxidative stress induction and chromatin structure alteration. The betulonic acid diethylentriamine conjugate showed partial activity against methicillin-resistant S. aureus and the fungi C. neoformans. These results show that triterpenic polyamines, being analogs of steroidal squalamine and trodusquemine, are important substances for the search of new drugs with anticancer, antidiabetic, and antimicrobial activities.

Quaternary Alkylammonium Conjugates of Steroids: Synthesis, Molecular Structure, and Biological Studies.

The methods of synthesis as well as physical, spectroscopic (¹H-NMR, 13C-NMR, and FT-IR, ESI-MS), and biological properties of quaternary and dimeric quaternary alkylammonium conjugates of steroids are presented. The results were contrasted with theoretical calculations (PM5 methods) and potential pharmacological properties (PASS). Alkylammonium sterols exhibit a broad spectrum of antimicrobial activity comparable to squalamine.

Natural products targeting amyloid-ß oligomer neurotoxicity in Alzheimer's disease.

Alzheimer's disease (AD) constitutes a major global health issue, characterized by progressive neurodegeneration and cognitive impairment, for which no curative treatment is currently available. Current therapeutic approaches are focused on symptom management, highlighting the critical need for disease-modifying therapy. The hallmark pathology of AD involves the aggregation and accumulation of amyloid-ß (Aß) peptides in the brain. Consequently, drug discovery efforts in recent decades have centered on the Aß aggregation cascade, which includes the transition of monomeric Aß peptides into toxic oligomers and, ultimately, mature fibrils. Historically, anti-Aß strategies focused on the clearance of amyloid fibrils using monoclonal antibodies. However, substantial evidence has highlighted the critical role of Aß oligomers (AßOs) in AD pathogenesis. Soluble AßOs are now recognized as more toxic than fibrils, directly contributing to synaptic impairment, neuronal damage, and the onset of AD. Targeting AßOs has emerged as a promising therapeutic approach to mitigate cognitive decline in AD. Natural products (NPs) have demonstrated promise against AßO neurotoxicity through various mechanisms, including preventing AßO formation, enhancing clearance mechanisms, or converting AßOs into non-toxic species. Understanding the mechanisms by which anti-AßO NPs operate is useful for developing disease-modifying treatments for AD. In this review, we explore the role of NPs in mitigating AßO neurotoxicity for AD drug discovery, summarizing key evidence from biophysical methods, cellular assays, and animal models. By discussing how NPs modulate AßO neurotoxicity across various experimental systems, we aim to provide valuable insights into novel therapeutic strategies targeting AßOs in AD.

AFM reveals the interaction and nanoscale effects imposed by squalamine on Staphylococcus epidermidis.

The Gram-positive bacterium Staphylococcus epidermidis is responsible for important nosocomial infections. With the continuous emergence of antibiotic-resistant strains, the search for new treatments has been amplified in the last decades. A potential candidate against multidrug-resistant bacteria is squalamine, a natural aminosterol discovered in dogfish sharks. Despite its broad-spectrum efficiency, little is known about squalamine mode of action. Here, we used atomic force microscopy (AFM) imaging to decipher the effect of squalamine on S. epidermidis morphology, revealing the peptidoglycan structure at the bacterial surface after the drug action. Single-molecule force spectroscopy with squalamine-decorated tips shows that squalamine binds to the cell surface via the spermidine motif, most likely through electrostatic interactions between the amine groups of the molecule and the negatively-charged bacterial cell wall. We demonstrated that - although spermidine is sufficient for the initial attachment of squalamine to S. epidermidis - the integrity of the molecule needs to be conserved for its antimicrobial action. A deeper analysis of the AFM force-distance signatures suggests the implication of the accumulation-associated protein (Aap), one of the main adhesins of S. epidermidis, in the initial binding of squalamine to the bacterial cell wall. This work highlights that AFM -combined with microbiological assays at the bacterial suspension scale- is a valuable approach to better understand the molecular mechanisms behind the efficiency of squalamine antibacterial activity.

Squalamine and Its Aminosterol Derivatives: Overview of Biological Effects and Mechanisms of Action of Compounds with Multiple Therapeutic Applications.

Squalamine is a natural aminosterol that has been discovered in the tissues of the dogfish shark (Squalus acanthias). Studies have previously demonstrated that this promoter compound and its derivatives exhibit potent bactericidal activity against Gram-negative, Gram-positive bacteria, and multidrug-resistant bacteria. The antibacterial activity of squalamine was found to correlate with that of other antibiotics, such as colistin and polymyxins. Still, in the field of microbiology, evidence has shown that squalamine and its derivatives have antifungal activity, antiprotozoa effect against a limited list of protozoa, and could exhibit antiviral activity against both RNA- and DNA-enveloped viruses. Furthermore, squalamine and its derivatives have been identified as being antiangiogenic compounds in the case of several types of cancers and induce a potential positive effect in the case of other diseases such as experimental retinopathy and Parkinson's disease. Given the diverse effects of the squalamine and its derivatives, in this review we provide the different advances in our understanding of the various effects of these promising molecules and try to draw up a non-exhaustive list of the different mechanisms of actions of squalamine and its derivatives on the human organism and on different pathogens.

Squalamines in Blockade of Tumor-Associated Angiogenesis and Cancer Progression.

Mechanisms of action of squalamine in human vascular endothelial cells indicate that this compound attaches to cell membranes, potentially interacting with calmodulin, Na+/H+ exchanger isoform NHE3 and other signaling pathways involved in the angiogenic process. Thus, squalamine elicits blockade of VEGF-induced endothelial tube-like formation in vitro. Further, squalamine reduces growth of several preclinical models of human cancers in vivo and acts to stop metastatic tumor spread, actions due largely to blockade of angiogenesis induced by the tumor and tumor microenvironment. Squalamine in Phase I/II trials, alone or combined with standard care, shows promising antitumor activity with limited side-effects in patients with advanced solid cancers. Increased attention on squalamine regulation of signaling pathways with or without combination treatments in solid malignancies deserves further study.

Quantitative Measurement of the Affinity of Toxic and Nontoxic Misfolded Protein Oligomers for Lipid Bilayers and of its Modulation by Lipid Composition and Trodusquemine.

Many neurodegenerative diseases are associated with the self-assembly of peptides and proteins into fibrillar aggregates. Soluble misfolded oligomers formed during the aggregation process, or released by mature fibrils, play a relevant role in neurodegenerative processes through their interactions with neuronal membranes. However, the determinants of the cytotoxicity of these oligomers are still unclear. Here we used liposomes and toxic and nontoxic oligomers formed by the same protein to measure quantitatively the affinity of the two oligomeric species for lipid membranes. To this aim, we quantified the perturbation to the lipid membranes caused by the two oligomers by using the fluorescence quenching of two probes embedded in the polar and apolar regions of the lipid membranes and a well-defined protein-oligomer binding assay using fluorescently labeled oligomers to determine the Stern-Volmer and dissociation constants, respectively. With both approaches, we found that the toxic oligomers have a membrane affinity 20-25 times higher than that of nontoxic oligomers. Circular dichroism, intrinsic fluorescence, and FRET indicated that neither oligomer type changes its structure upon membrane interaction. Using liposomes enriched with trodusquemine, a potential small molecule drug known to penetrate lipid membranes and make them refractory to toxic oligomers, we found that the membrane affinity of the oligomers was remarkably lower. At protective concentrations of the small molecule, the binding of the oligomers to the lipid membranes was fully prevented. Furthermore, the affinity of the toxic oligomers for the lipid membranes was found to increase and slightly decrease with GM1 ganglioside and cholesterol content, respectively, indicating that physicochemical properties of lipid membranes modulate their affinity for misfolded oligomeric species.

Squalamine and Aminosterol Mimics Inhibit the Peptidoglycan Glycosyltransferase Activity of PBP1b.

Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional penicillin-binding proteins (PBPs) of class A, such as Escherichia coli PBP1b, belong to the GTase 51 family. These enzymes play an essential role in PG synthesis, and their specific inhibition by moenomycin was shown to lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds present an unexpected mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay, suggesting that they act as competitive inhibitors.

Squalamine Restores the Function of the Enteric Nervous System in Mouse Models of Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder thought to be caused by accumulation of α-synuclein (α-syn) within the brain, autonomic nerves, and the enteric nervous system (ENS). Involvement of the ENS in PD often precedes the onset of the classic motor signs of PD by many years at a time when severe constipation represents a major morbidity. Studies conducted in vitro and in vivo, have shown that squalamine, a zwitterionic amphipathic aminosterol, originally isolated from the liver of the dogfish shark, effectively displaces membrane-bound α-syn. OBJECTIVE: Here we explore the electrophysiological effect of squalamine on the gastrointestinal (GI) tract of mouse models of PD engineered to express the highly aggregating A53T human α-syn mutant. METHODS: GI motility and in vivo response to oral squalamine in PD model mice and controls were assessed using an in vitro tissue motility protocol and via fecal pellet output. Vagal afferent response to squalamine was measured using extracellular mesenteric nerve recordings from the jejunum. Whole cell patch clamp was performed to measure response to squalamine in the myenteric plexus. RESULTS: Squalamine effectively restores disordered colonic motility in vivo and within minutes of local application to the bowel. We show that topical squalamine exposure to intrinsic primary afferent neurons (IPANs) of the ENS rapidly restores excitability. CONCLUSION: These observations may help to explain how squalamine may promote gut propulsive activity through local effects on IPANs in the ENS, and further support its possible utility in the treatment of constipation in patients with PD.

Study on synthesis of key squalamine intermediate 7α, 24R-dihydroxy-5α-cholestan-3-one.

A facile novel strategy has been developed to obtain a key intermediate of squalamine, 7α, 24R -dihydroxy-5α-cholestan-3-one, starting from methyl Δ5-3ß-hydroxycholanate. The pure product was successfully synthesized and separated from the C-24 position epimers in good purity, d.e.% and yield.

Colonic Motility and Jejunal Vagal Afferent Firing Rates Are Decreased in Aged Adult Male Mice and Can Be Restored by an Aminosterol.

There is a general decline in gastrointestinal function in old age including decreased intestinal motility, sensory signaling, and afferent sensitivity. There is also increased prevalence of significant constipation in aged populations. We hypothesized this may be linked to reduced colonic motility and alterations in vagal-gut-brain sensory signaling. Using in vitro preparations from young (3 months) and old (18-24 months) male CD1 mice we report functional age-related differences in colonic motility and jejunal mesenteric afferent firing. Furthermore, we tested the effect of the aminosterol squalamine on colonic motility and jejunal vagal firing rate. Old mice had significantly reduced velocity of colonic migrating motor complexes (MMC) by 27% compared to young mice (p = 0.0161). Intraluminal squalamine increased colonic MMC velocity by 31% in old mice (p = 0.0150), which also had significantly reduced mesenteric afferent single-unit firing rates from the jejunum by 51% (p < 0.0001). The jejunal vagal afferent firing rate was reduced in aged mice by 62% (p = 0.0004). While the time to peak response to squalamine was longer in old mice compared to young mice (18.82 ± 1.37 min vs. 12.95 ± 0.99 min; p = 0.0182), it significantly increased vagal afferent firing rate by 36 and 56% in young and old mice, respectively (p = 0.0006, p = 0.0013). Our results show for the first time that the jejunal vagal afferent firing rate is reduced in aged-mice. They also suggest that there is translational potential for the therapeutic use of squalamine in the treatment of age-related constipation and dysmotility.

Anti-persister activity of squalamine against Acinetobacter baumannii.

Squalamine is a natural polycationic aminosterol extracted from the shark Squalus acanthias. Squalamine displays remarkable efficacy against antimicrobial-resistant Gram-negative and Gram-positive bacteria. Its membranolytic activity and low cytotoxicity make squalamine one of the most promising agents to fight nosocomial pathogens such as Acinetobacter baumannii. In the context of chronic diseases and therapeutic failures associated with this pathogen, the presence of dormant cells, i.e. persisters and viable but non-culturable cells (VBNCs), highly tolerant to antimicrobial compounds is problematic. The aim of this study was to investigate the antibacterial activity of squalamine against this bacterial population of A. baumannii. Bacterial dormancy was induced by cold shock and nutrient starvation in the presence of high doses of either colistin, ciprofloxacin or squalamine. Persisters and VBNCs induced by these treatments were then challenged with 100 mg/L squalamine. The efficacy of each treatment was determined by evaluating culturability on agar medium, membrane integrity (LIVE/DEAD®BacLightTM staining) and respiratory activity (BacLightTM RedoxSensorTM CTC staining) of bacteria. A. baumannii ATCC 17978 generated persisters as well as VBNCs in the presence of high doses of ciprofloxacin but not colistin or squalamine. Squalamine at 100 mg/L (below its haemolytic concentration) was able to kill dormant cells. Squalamine did not induce persister cell or VBNC formation in A. baumannii ATCC 17978. Interestingly, squalamine was significantly active against this type of dormant population generated by ciprofloxacin, making it a very promising anti-persister agent.

Targeting neurons in the gastrointestinal tract to treat Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is associated with α-synuclein (αS) aggregation within the enteric nervous system (ENS) and constipation. Squalamine displaces proteins that are electrostatically bound to intracellular membranes and through this mechanism suppresses aggregation of αS monomers into neurotoxic oligomers. OBJECTIVE: We sought to evaluate the safety of ENT-01 oral tablets (a synthetic squalamine salt), its pharmacokinetics, and its effect on bowel function in PD patients with constipation. METHODS: In Stage 1, 10 patients received escalating single doses from 25 to 200 mg/day or maximum tolerated dose (MTD). In Stage 2, 34 patients received daily doses escalating from 75 to a maximum of 250 mg/day, a dose that induced change in bowel function or MTD, followed by a fixed dose for 7 days, and a 2-week washout. Primary efficacy endpoint was defined as an increase of 1 complete spontaneous bowel movement (CSBM)/week, or 3 CSBM/week over the baseline period, as defined by FDA guidelines for prokinetic agents. Safety was also assessed. RESULTS: Over 80% of patients achieved the primary efficacy endpoint, with the mean number of CSBM/week increasing from 1.2 at baseline to 3.6 during fixed dosing (p = 1.2 × 10-7). Common adverse events included nausea in 21/44 (47%) and diarrhea in 18/44 (40%) patients. Systemic absorption was <0.3%. CONCLUSIONS: Orally administered ENT-01 was safe and significantly improved bowel function in PD, suggesting that the ENS is not irreversibly damaged in PD. Minimal systemic absorption suggests that improvements result from local stimulation of the ENS. A double-blind, placebo-controlled study is now ongoing.

Squalamine blocks tumor-associated angiogenesis and growth of human breast cancer cells with or without HER-2/neu overexpression.

Angiogenesis is critical for breast cancer progression. Overexpression of HER-2/neu receptors occur in 25-30% of breast cancers, and treatment with trastuzumab inhibits HER-2-overexpressing tumor growth. Notably, HER-2-mediated signaling enhances vascular endothelial growth factor (VEGF) secretion to increase tumor-associated angiogenesis. Squalamine (aminosterol compound) suppresses VEGF-induced activation of kinases in vascular endothelial cells and inhibits tumor-associated angiogenesis. We assessed antitumor effects of squalamine either alone or with trastuzumab in nude mice bearing breast tumor xenografts without (MCF-7) or with HER2-overexpression (MCF-7/HER-2). Squalamine alone inhibited progression of MCF-7 tumors lacking HER2 overexpression, and squalamine combined with trastuzumab elicited marked inhibition of MCF-7/HER2 growth exceeding that of trastuzumab alone. MCF-7/HER-2 cells secrete higher levels of VEGF than MCF-7 cells, but squalamine elicited no growth inhibition of either MCF-7/HER-2 or MCF-7 cells in vitro. However, squalamine did stop growth of human umbilical vein endothelial cells (HUVECs) and reduced VEGF-induced endothelial tube-like formations in vitro. These effects correlated with blockade of focal adhesion kinase phosphorylation and stress fiber assembly in HUVECs. Thus, squalamine effectively inhibits growth of breast cancers with or without HER-2-overexpression, an effect due in part to blockade of tumor-associated angiogenesis.

Hot Topics in Pharmacotherapy for Neovascular Age-Related Macular Degeneration.

BACKGROUND: The preferred approach for the treatment of neovascular age-related macular degeneration (AMD) is frequent intravitreal injections of the anti-vascular endothelial growth factor (VEGF) agents. However, considering the limitations of current anti-VEGF approaches, including the need for frequent injections, inadequate response in some patients, and a relatively short duration of effect, several new therapeutic modalities are under evaluation. METHODS: A comprehensive review of the literature was performed on the new treatment modalities for neovascular AMD, and the relevant studies were discussed. RESULTS: The treatment modalities for neovascular AMD include new anti-VEGF drugs, new drug delivery systems and new targets in the pathogenic cascade of choroidal neovascularization. These new modalities are in different phases of clinical development. CONCLUSION: The results of the completed studies reporting the new therapeutic modalities for neovascular AMD thus far are promising.