Pseurotin A Validation as a Metastatic Castration-Resistant Prostate Cancer Recurrence-Suppressing Lead via PCSK9-LDLR Axis Modulation.

Metastatic castration-resistant prostate cancer (mCRPC) cells can de novo biosynthesize their own cholesterol and overexpress proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 proved to contribute to mCRPC cell motility since PCSK9 knockdown (KD) in mCRPC CWR-R1ca cells led to notable reductions in cell migration and colony formation. Human tissue microarray results proved a higher immunohistoscore in patients ≥ 65 years old, and PCSK9 proved to be expressed higher at an early Gleason score of ≤7. The fermentation product pseurotin A (PS) suppressed PCSK9 expression, protein-protein interactions with LDLR, and breast and prostate cancer recurrences. PS suppressed migration and colony formation of the CWR-R1ca cells. The progression and metastasis of the CWR-R1ca-Luc cells subcutaneously (sc) xenografted into male nude mice fed a high-fat diet (HFD, 11% fat content) showed nearly 2-fold tumor volume, metastasis, serum cholesterol, low-density lipoprotein cholesterol (LDL-C), prostate-specific antigen (PSA), and PCSK9 levels versus mice fed a regular chow diet. Daily oral PS 10 mg/kg treatments prevented the locoregional and distant tumor recurrence of CWR-R1ca-Luc engrafted into nude mice after primary tumor surgical excision. PS-treated mice showed a significant reduction in serum cholesterol, LDL-C, PCSK9, and PSA levels. These results comprehensively validate PS as an mCRPC recurrence-suppressive lead by modulating the PCSK9-LDLR axis.

Towards Developing Novel Prostate Cancer Recurrence Suppressors: Acute Toxicity of Pseurotin A, an Orally Active PCSK9 Axis-Targeting Small-Molecule in Swiss Albino Mice.

The proprotein convertase subtilisin kexin type 9 (PCSK9) emerged as a molecular target of great interest for the management of cardiovascular disorders due to its ability to reduce low density lipoprotein (LDL) cholesterol by binding and targeting at LDLR for lysosomal degradation in cells. Preliminary studies revealed that pseurotin A (PsA), a spiro-heterocyclic γ-lactam alkaloid from several marine and terrestrial Aspergillus and Penicillium species, has the ability to dually suppress the PCSK9 expression and protein-protein interaction (PPI) with LDLR, resulting in an anti-hypercholesterolemic effect and modulating the oncogenic role of PCSK9 axis in breast and prostate cancers progression and recurrence. Thus, a preliminary assessment of the PsA acute toxicity represents the steppingstone to develop PsA as a novel orally active PCSK9 axis modulating cancer recurrence inhibitor. PsA studies for in vitro toxicity on RWPE-1 and CCD 841 CoN human non-tumorigenic prostate and colon cells, respectively, indicated a cellular death shown at a 10-fold level of its reported anticancer activity. Moreover, a Western blot analysis revealed a significant downregulation of the pro-survival marker Bcl-2, along with the upregulation of the proapoptotic Bax and caspases 3/7, suggesting PsA-mediated induction of cell apoptosis at very high concentrations. The Up-and-Down methodology determined the PsA LD50 value of >550 mg/kg in male and female Swiss albino mice. Animals were orally administered single doses of PsA at 10, 250, and 500 mg/kg by oral gavage versus vehicle control. Mice were observed daily for 14 days with special care over the first 24 h after dosing to monitor any abnormalities in their behavioral, neuromuscular, and autonomic responses. After 14 days, the mice were euthanized, and their body and organ weights were recorded and collected. Mice plasma samples were subjected to comprehensive hematological and biochemical analyses. Collected mouse organs were histopathologically examined. No morbidity was detected following the PsA oral dosing. The 500 mg/kg female dosing group showed a 45% decrease in the body weight after 14 days but displayed no other signs of toxicity. The 250 mg/kg female dosing group had significantly increased serum levels of liver transaminases AST and ALT versus vehicle control. Moreover, a modest upregulation of apoptotic markers was observed in liver tissues of both animal sexes at 500 mg/kg dose level. However, a histopathological examination revealed no damage to the liver, kidneys, heart, brain, or lungs. While these findings suggest a possible sex-related toxicity at higher doses, the lack of histopathological injury implies that single oral doses of PsA, up to 50-fold the therapeutic dose, do not cause acute organ toxicity in mice though further studies are warranted.

Intra-hemocoel injection of pseurotin A from Metarhizium anisopliae, induces dose-dependent reversible paralysis in the Greater Wax Moth (Galleria mellonella).

Species from the Metarhizium genus are the causal agents of the green muscardine disease of insects. These fungi have been successfully employed for the biological control of pests over decades. Besides the biocontrol applications, recent efforts for genome sequencing of species in this genus have revealed a great diversity of biosynthetic gene clusters potentially associated with secondary metabolite synthesis. Amongst such molecules are the pseurotins, compounds with several activities, as chitin synthase inhibitors, and immunoglobulin E suppressors. Here, we report, for the first time, the isolation of pseurotin A from the culture broth of M. anisopliae, as well as the characterization of the effects of this compound over the model-arthropod Galleria mellonella. Pseurotin A displayed dose-dependent reversible paralysis effects when injected into the larvae hemocoel. However, the posterior challenge of the treated insects with M. anisopliae conidia did not lead to increased mortality, suggesting that pseurotin A treatment did not increase larvae susceptibility to the green muscardine disease. Although apparent insecticidal effects were not observed for pseurotin A, the paralysis effect observed can be important in M. anisopliae infection development.

Pseurotin A as a novel suppressor of hormone dependent breast cancer progression and recurrence by inhibiting PCSK9 secretion and interaction with LDL receptor.

Hypercholesterolemia has been documented to drive hormone-dependent breast cancer (BC) progression and resistance to hormonal therapy. Proprotein convertase subtilisin/kexin type-9 (PCSK9) regulates cholesterol metabolism through binding to LDL receptor (LDLR) and targeting the receptor for lysosomal degradation. Inhibition of PCSK9 is an established strategy to treat hypercholesterolemia. Pseurotin A (PS) is a unique spiro-heterocyclic γ-lactam alkaloid isolated from the fungus Aspergillus fumigatus. Preliminary studies indicated that PS lowered PCSK9 secretion in cultured HepG2 hepatocellular carcinoma cells, with an IC50 value of 1.20 µM. Docking studies suggested the ability of PS to bind at the PCSK9 narrow interface pocket that accommodates LDLR. Surface plasmon resonance (SPR) showed PS ability to inhibit the PCSK9-LDLR interaction at a concentration range of 10-150 µM. PS showed in vitro dose-dependent reduction of PCSK9, along with increased LDLR levels in hormone-dependent BT-474 and T47D breast cancer (BC) cell lines. In vivo, daily oral 10 mg/kg PS suppressed the progression of the hormone-dependent BT-474 BC cells in orthotopic nude mouse xenograft model. Immunohistochemistry (IHC) investigation of BT-474 breast tumor tissue proved the PS ability to reduce PCSK9 expression. PS also effectively suppressed BT-474 BC cells locoregional recurrence after primary tumor surgical excision. Western blot analysis showed decreased PCSK9 expression in liver tissues of PS-treated mice compared to vehicle-treated control group. PS treatment significantly reduced PCSK9 expression and normalized LDLR levels in collected primary and recurrent breast tumors at the study end. PS-treated mice showed reduced plasma cholesterol and 17ß-estradiol levels. Inhibition of tumor recurrence was associated with significant reductions in plasma level of the human BC recurrence marker CA 15-3 in treated mice at the study end. Histopathological examination of various PS-treated mice organs indicated lack of metastatic tumor cells and any pathological changes. The results of this study provide the first evidence for the suppression of the hormone-dependent breast tumor progression and recurrence by targeting the PCSK9-LDLR axis. PS is a novel first-in-class PCSK9-targeting lead appropriate for the use to control hormone-dependent BC progression and recurrence.

Protective Effects of Prunasin A against the Differentiation of Osteoclasts and Destruction of Cartilage via the Receptor Activator of Nuclear Factor-Kappa-Β Ligand/Mitogen-Activated Protein Kinase/Osteoprotegerin Pathway in a Rat Model of Arthritis.

The present study evaluated the protective effects of pseurotin A against inflammation and the destruction of cartilage in a rat model of rheumatoid arthritis (RA). RA was induced by intradermal injections of Freund's complete adjuvant (1 mg/mL), and the treatment with pseurotin A (50 and 100 mg/kg, p.o.) was administered over 1 week. The effects of pseurotin A were assessed by estimating hind paw volume (HPV) and determining the levels of inflammatory mediators in the serum and synovial fluid of collagen-induced arthritis (CIA)-induced RA rats. Western blot and histopathological assays were performed to assess changes in synovial tissues. Additionally, in vitro analyses of receptor activator of nuclear factor-kappa-Β ligand (RANKL)-stimulated RAW264.7 cells treated with pseurotin A at different concentrations (1, 10, and 100 µg/mL) were conducted to assess the effects of pseurotin A on apoptosis ratio, real-time polymerase chain reaction data, and tartrate-resistant acid phosphatase staining. Compared to the RA group, treatment with pseurotin A significantly decreased HPV and reduced the levels of inflammatory mediators in the synovial fluid and blood. Additionally, pseurotin A ameliorated the protein expressions of osteoprotegerin, nuclear factor of activated T-cells, nuclear factor-kappa beta (NF-κB), IκBα, extracellular signal regulated kinase, and P38 as well as histopathological changes in the synovial tissue of CIA-induced RA rats. The in vitro findings revealed that pseurotin A treatment did not alter the apoptosis ratio in RANKL-stimulated RAW264.7 cells but significantly reduced the mRNA expressions of calcitonin receptor, NF-κB, and matrix metallopeptidase-9. The present findings suggest that pseurotin A ameliorated the differentiation of osteoclasts and the destruction of cartilage in RA rats via regulation of the mitogen-activated protein kinase/RANKL/NF-kB pathway.

Pseurotin A Inhibits Osteoclastogenesis and Prevents Ovariectomized-Induced Bone Loss by Suppressing Reactive Oxygen Species.

Rationale: Growing evidence indicates that intracellular reactive oxygen species (ROS) accumulation is a critical factor in the development of osteoporosis by triggering osteoclast formation and function. Pseurotin A (Pse) is a secondary metabolite isolated from Aspergillus fumigatus with antioxidant properties, recently shown to exhibit a wide range of potential therapeutic applications. However, its effects on osteoporosis remain unknown. This study aimed to explore whether Pse, by suppressing ROS level, is able to inhibit osteoclastogenesis and prevent the bone loss induced by estrogen-deficiency in ovariectomized (OVX) mice. Methods: The effects of Pse on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and bone resorptive function were examined by tartrate resistant acid phosphatase (TRAcP) staining and hydroxyapatite resorption assay. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to detect intracellular ROS production in vitro. Western blot assay was used to identify proteins associated with ROS generation and scavenging as well as ROS-mediated signaling cascades including mitogen-activated protein kinases (MAPKs), NF-κB pathways, and nuclear factor of activated T cells 1 (NFATc1) signaling. The expression of osteoclast-specific genes was assessed by qPCR. The in vivo potential of Pse was determined using an OVX mouse model administered with Pse or vehicle for 6 weeks. In vivo ROS production was assessed by intravenous injection of dihydroethidium (DHE) into OVX mice 24h prior to killing. After sacrifice, the bone samples were analyzed using micro-CT and histomorphometry to determine bone volume, osteoclast activity, and ROS level ex vivo. Results: Pse was demonstrated to inhibit osteoclastogenesis and bone resorptive function in vitro, as well as the downregulation of osteoclast-specific genes including Acp5 (encoding TRAcP), Ctsk (encoding cathepsin K), and Mmp9 (encoding matrix metalloproteinase 9). Mechanistically, Pse suppressed intracellular ROS level by inhibiting RANKL-induced ROS production and enhancing ROS scavenging enzymes, subsequently suppressing MAPK pathway (ERK, P38, and JNK) and NF-κB pathways, leading to the inhibition of NFATc1 signaling. Micro-CT and histological data indicated that OVX procedure resulted in a significant bone loss, with dramatically increased the number of osteoclasts on the bone surface as well as increased ROS level in the bone marrow microenvironment; whereas Pse supplementation was capable of effectively preventing these OVX-induced changes. Conclusion: Pse was demonstrated for the first time as a novel alternative therapy for osteoclast-related bone diseases such as osteoporosis through suppressing ROS level.

Antiglioma pseurotin A from marine Bacillus sp. FS8D regulating tumour metabolic enzymes.

Pseurotin A was isolated from a culture of marine Bacillus sp. FS8D and showed to be active against the proliferation of four different glioma cells with IC50 values of 0.51-29.3 µM. It has been found that pseurotin A downregulated the expression of tumour glycolytic enzymes pyruvate kinase M2 (PKM2) and lactate dehydrogenase 5 (LDH5) and upregulated the expression of pyruvate dehydrogenase beta (PDHB), adenosine triphosphate synthase beta (ATPB) and cytochrome C (Cyto-C), the important regulators for tricarboxylic acid cycle and oxidative phosphorylation. The data suggested that targeting multiple metabolic enzymes might be one of the antiglioma mechanisms of pseurotin A.

Zebrafish-Based Discovery of Antiseizure Compounds from the Red Sea: Pseurotin A2 and Azaspirofuran A.

In search for novel antiseizure drugs (ASDs), the European FP7-funded PharmaSea project used zebrafish embryos and larvae as a drug discovery platform to screen marine natural products to identify promising antiseizure hits in vivo for further development. Within the framework of this project, seven known heterospirocyclic γ-lactams, namely, pseurotin A, pseurotin A2, pseurotin F1, 11- O-methylpseurotin A, pseurotin D, azaspirofuran A, and azaspirofuran B, were isolated from the bioactive marine fungus Aspergillus fumigatus, and their antiseizure activity was evaluated in the larval zebrafish pentylenetetrazole (PTZ) seizure model. Pseurotin A2 and azaspirofuran A were identified as antiseizure hits, while their close chemical analogues were inactive. Besides, electrophysiological analysis from the zebrafish midbrain demonstrated that pseurotin A2 and azaspirofuran A also ameliorate PTZ-induced epileptiform discharges. Next, to determine whether these findings translate to mammalians, both compounds were analyzed in the mouse 6 Hz (44 mA) psychomotor seizure model. They lowered the seizure duration dose-dependently, thereby confirming their antiseizure properties and suggesting activity against drug-resistant seizures. Finally, in a thorough ADMET assessment, pseurotin A2 and azaspirofuran A were found to be drug-like. Based on the prominent antiseizure activity in both species and the drug-likeness, we propose pseurotin A2 and azaspirofuran A as lead compounds that are worth further investigation for the treatment of epileptic seizures. This study not only provides the first evidence of antiseizure activity of pseurotins and azaspirofurans, but also demonstrates the value of the zebrafish model in (marine) natural product drug discovery in general, and for ASD discovery in particular.