RSK1 vs. RSK2 Inhibitory Activity of the Marine β-Carboline Alkaloid Manzamine A: A Biochemical, Cervical Cancer Protein Expression, and Computational Study

Manzamines are complex polycyclic marine-derived β-carboline alkaloids with reported anticancer, immunostimulatory, anti-inflammatory, antibacterial, antiviral, antimalarial, neuritogenic, hyperlipidemia, and atherosclerosis suppression bioactivities, putatively associated with inhibition of glycogen synthase kinase-3, cyclin-dependent kinase 5, SIX1, and vacuolar ATPases. We hypothesized that additional, yet undiscovered molecular targets might be associated with Manzamine A’s (MZA) reported pharmacological properties. We report here, for the first time, that MZA selectively inhibited a 90 kDa ribosomal protein kinase S6 (RSK1) when screened against a panel of 30 protein kinases, while in vitro RSK kinase assays demonstrated a 10-fold selectivity in the potency of MZA against RSK1 versus RSK2. The effect of MZA on inhibiting cellular RSK1 and RSK2 protein expression was validated in SiHa and CaSki human cervical carcinoma cell lines. MZA’s differential binding and selectivity toward the two isoforms was also supported by computational docking experiments. Specifically, the RSK1-MZA (N- and C-termini) complexes appear to have stronger interactions and preferable energetics contrary to the RSK2–MZA ones. In addition, our computational strategy suggests that MZA binds to the N-terminal kinase domain of RSK1 rather than the C-terminal domain. RSK is a vertebrate family of cytosolic serine-threonine kinases that act downstream of the ras-ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which phosphorylates substrates shown to regulate several cellular processes, including growth, survival, and proliferation. Consequently, our findings have led us to hypothesize that MZA and the currently known manzamine-type alkaloids isolated from several sponge genera may have novel pharmacological properties with unique molecular targets, and MZA provides a new tool for chemical-biology studies involving RSK1.

Model-Free Approach for the Configurational Analysis of Marine Natural Products

The NMR-based configurational analysis of complex marine natural products is still not a routine task. Different NMR parameters are used for the assignment of the relative configuration: NOE/ROE, homo- and heteronuclear J couplings as well as anisotropic parameters. The combined distance geometry (DG) and distance bounds driven dynamics (DDD) method allows a model-free approach for the determination of the relative configuration that is invariant to the choice of an initial starting structure and does not rely on comparisons with (DFT) calculated structures. Here, we will discuss the configurational analysis of five complex marine natural products or synthetic derivatives thereof: the cis-palau’amine derivatives 1a and 1b, tetrabromostyloguanidine (1c), plakilactone H (2), and manzamine A (3). The certainty of configurational assignments is evaluated in view of the accuracy of the NOE/ROE data available. These case studies will show the prospective breadth of application of the DG/DDD method.

Effect of the Marine β-carboline Alkaloid Manzamine A on RSK1 vs RSK2 Inhibition: a Biochemical and Computational Study

Manzamines are complex polycyclic marine-derived β-carboline alkaloids with reported anticancer, immunostimulatory, anti-inflammatory, antibacterial, antiviral, antimalarial, neuritogenic, hyperlipidemia and atherosclerosis suppression bioactivities, putatively associated with inhibition of glycogen synthase kinase-3, cyclin-dependent kinase 5, and vacuolar ATPases. We hypothesized that additional and yet undiscovered molecular targets might be associated with Manzamine A (MZA) reported pharmacological properties. We report herein for the first time to our knowledge that MZA inhibited a 90kDa ribosomal protein kinase S6 (RSK1) when screened against a panel of 30 protein kinases. Furthermore in vitro RSK kinase assays demonstrated a 10-fold selectivity in potency of MZA against RSK1 versus RSK2. MZA’s differential binding and selectivity toward the two isoforms is also supported by computational docking experiments. Specifically, the RSK1-MZA (N- and C-termini) complexes appear to have stronger interactions and preferable energetics contrary to the RSK2-MZA ones. In addition, our computational strategy suggests that MZA binds to the N-terminal kinase domain of RSK1 rather than the C-terminal domain. RSK is a vertebrate family of cytosolic serine-threonine kinases that act downstream of the ras-ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which phosphorylates substrates shown to regulate several cellular processes including growth, survival and proliferation. Consequently, our findings have lead us to hypothesize that MZA and the 80 currently known manzamine-type alkaloids isolated from several sponge genera, may have novel pharmacological properties.

EXTH-72. MANZAMINE A-DERIVED COMPOUNDS AS POTENTIAL ANTI-BRAIN TUMOR AGENTS AND IT MECHANISM STUDY

Glioma is a severe disease with poor prognosis. Chemotherapy plays an important role but very few drugs are effective. There are two major issues: (1) the specialized blood-brain barrier blocks most large molecules from penetrating into target area; (2) tumor cells frequently develop drug resistant. Therefore, developing new chemotherapeutic agents for malignant brain tumor treatment may significantly improve the current difficulties. Compounds derived from nature products have been considered as potential sources for new discovery of anti-cancer drug. Manzamine A-derived compounds, showed significant anticancer activities against colon adenocarcinoma DLD cells, lung large cell carcinoma NCI-H661 cells, and hepatoma HepG2/A2 cells. In this study, we used in vitro model for screening these compounds and discovered both cytotoxic and anti-proliferative effects against brain tumor cells, such as A172, U87MG, and GL261 cell lines. We then used animal models to test the toxicities and treatment effect of compound A3 in vivo, and delivered the agent by convection-enhanced delivery to improve drug concentration in brain. Survival benefit for brain tumor-bearing animal was found for optimal dosage of compound A3. Details of anti-tumor mechanism need further evaluation.

Manzamine A Exerts Anticancer Activity against Human Colorectal Cancer Cells

Marine sponges are known to produce numerous bioactive secondary metabolites as defense strategies to avoid predation. Manzamine A is a sponge-derived β-carboline-fused pentacyclic alkaloid with various bioactivities, including recently reported anticancer activity on pancreatic cancer. However, its cytotoxicity and mode of action against other tumors remain unclear. In this study, we exhibit that manzamine A reduced cell proliferation in several colorectal cancer (CRC) cell lines. To further investigate the manzamine A triggered molecular regulation, we analyzed the gene expression with microarray and revealed that pathways including cell cycle, DNA repair, mRNA metabolism, and apoptosis were dysregulated. We verified that manzamine A induced cell cycle arrest at G0/G1 phase via inhibition of cyclin-dependent kinases by p53/p21/p27 and triggered a caspase-dependent apoptotic cell death through mitochondrial membrane potential depletion. Additionally, we performed bioinformatics analysis and demonstrated that manzamine A abolished epithelial–mesenchymal transition process. Several mesenchymal transcriptional factors, such as Snail, Slug, and Twist were suppressed and epithelial marker E-cadherin was induced simultaneously in HCT116 cells by manzamine A, leading to the epithelial-like phenotype and suppression of migration. These findings suggest that manzamine A may serve as a starting point for the development of an anticancer drug for the treatment of metastatic CRC.

Discovery and preliminary structure-activity relationship of the marine natural product manzamines as herpes simplex virus type-1 inhibitors

Herpes simplex virus type-1 (HSV-1) is a member of alpha-herpesviridae family and is known to cause contagious human infections. The marine habitat is a rich source of structurally unique bioactive secondary metabolites. A small library of marine natural product classes 1–10 has been screened to discover a new hit entity active against HSV-1. Manzamine A showed potent activity against HSV-1 via targeting the viral gene ICP0. Manzamine A is a β-carboline alkaloid isolated from the Indo-Pacific sponge Acanthostrongylophora species. Currently, acyclovir is the drug of choice for HSV-1 infections. Compared with 50 µM acyclovir, manzamine A at 1 µM concentration produced potent repressive effects on viral replication and release of infectious viruses in SIRC cells in recent studies. The potent anti-HSV-1 activity of manzamine A prompted a preliminary structure-activity relationship study by testing targeted manzamines. These included 8-hydroxymanzamine A (11), to test the effect of the C-8 hydroxy substitution at the β-carboline moiety; manzamine E (12), to assess the importance of substitution at the azacyclooctane ring; and ircinal A (13), to determine whether the β-carboline ring is required for the activity. Manzamine A was chemically transformed to its salt forms, manzamine A monohydrochloride (14) and manzamine A monotartrate (15), to test whether improving water solubility and hydrophilicity will positively affect the activity. Compounds were tested for activity against HSV-1 using fluorescent microscopy and plaque assay. The results showed the reduced anti-HSV-1 activity of 11, suggesting that C-8 hydroxy substitution might adversely affect the activity. Similarly, manzamines 12 and 13 showed no activity against HSV-1, indicating the preference of the unsubstituted azacylcooctane and β-carboline rings to the activity. Anti-HSV-1 activity was significantly improved for the manzamine A salts 14 and 15, suggesting that improving the overall water solubility as salt forms can significantly enhance the activity. Manzamines have significant potential for future development as anti-HSV-1 entity.

The Dixon Synthesis of Manzamine A

The pentacyclic alkaloid manzamine A 4, isolated from a sponge collected in the Okinawa Sea, displays a range of antibacterial, anticancer, and antimalarial activity. The preparation of 4 reported (J. Am. Chem. Soc. 2012, 134, 17482) by Darren J. Dixon of the University of Oxford showcases the versatility of the nitro group in organic synthesis. The nitro alkene 2 was prepared from the commercial bromide 5. Displacement with acetate followed by Swern oxidation led to the aldehyde 6, which was condensed with nitromethane to give 2. Lactam 1 was an intermediate in Professor Dixon’s synthesis (Org. Highlights May 3, 2010) of (–)-nakadomarin A. Lactam 1 was prepared from the tosylate 7, which was derived from pyroglutamic acid. The addition of 1 to the nitroalkene 2 delivered 3 as the dominant diastereomer of the four that were possible. Mannich condensation with formaldehyde and the amine 12 gave 13. The nitro group of 13 was removed by free radical reduction. Exposure of the reduced product to trimethylsilyl iodide gave, via ionization of the ketal, the primary iodide, which was carried onto the nitro compound 14. Dibal selectively reduced the δ-lactam. Partial reduction of the γ-lactam then gave an intermediate that engaged in Mannich condensation with the nitro-activated methylene to give 15. Although there are many protocols for the conversion of a nitro compound to a ketone, most of those were not compatible with the functional groups of 15. Fortunately, Ti(III) was effective. Ce-mediated addition of the Grignard reagent 16 to the ketone followed by deprotection and protection then delivered the silyl ether 17. Remarkably, the ketone 17 could be deprotonated and carried on to the enol triflate 18 without eliminating the TMSO group. Coupling with the stannane 19 then completed the synthesis of manzamine A 4. One-carbon homologation of 18 led to ircinol A, ircinal A, and methyl ircinate (not illustrated).