Moroidin

Overcoming Complexities in Chemical Synthesis of Moroidin

The total synthesis of this compound presents considerable challenges primarily due to its intricate bicyclic peptide structure and the presence of unusual amino acid cross-links. google.comacs.org Specifically, the formation of the C-C bond between leucine and tryptophan and the C-N bond between tryptophan and histidine requires highly selective and efficient synthetic methodologies. google.comacs.orgwikipedia.org Low isolation yields from natural sources further underscore the need for effective synthetic routes for research and potential therapeutic development. google.comacs.orgnih.gov While the total synthesis of this compound itself had not been fully described in some reports, significant progress has been made on the synthesis of its structural analogue, celogentin C, which shares the key cross-linkages. google.comwikipedia.org

Strategic Approaches for Constructing Bicyclic Peptide Scaffolds

Constructing the bicyclic peptide scaffold of this compound involves forming two macrocyclic rings through side-chain cross-linkages. google.comwikipedia.org This typically requires strategic peptide coupling reactions and selective bond formations at the desired amino acid residues. Approaches to synthesizing similar bicyclic peptides often involve building the linear peptide chain first, followed by sequential or simultaneous macrocyclization steps. The specific challenges in this compound synthesis lie in the non-standard C-C and C-N linkages involving the indole and imidazole rings. google.comacs.orgwikipedia.org

Partial Syntheses and Targeted Investigations of Specific Cross-Linkages

Due to the complexity of the full structure, research has often focused on the partial synthesis of this compound, specifically targeting the formation of the key cross-linkages. Partial syntheses including the Leu-Trp and Trp-His linkages have been achieved. wikipedia.org

Specific approaches for the Leu-Trp cross-link have included methods involving intermolecular Knoevenagel condensation followed by radical conjugate addition and nitro reduction, although this can yield diastereomeric mixtures. wikipedia.org Another strategy for the Leu-Trp crosslink involves a palladium-catalyzed C-H functionalization reaction to stereoselectively form the bond. google.com

For the Trp-His cross-link (C2 of tryptophan indole to N1 of histidine imidazole), oxidative coupling methods, such as using N-chlorosuccinimide (NCS), have been employed. wikipedia.org Careful control of reaction conditions is necessary to prevent over-oxidation or side reactions. wikipedia.org These partial syntheses have been crucial in developing the methodologies required for assembling the full bicyclic system.

Development of Biomimetic and Chemoenzymatic Synthetic Routes

Recognizing the challenges of purely organic synthesis and the low natural abundance, researchers have explored alternative approaches, including biomimetic and chemoenzymatic routes. mdpi.com Studies into the biosynthesis of this compound in plants like Dendrocnide moroides and Celosia argentea have revealed that these peptides are ribosomally synthesized and post-translationally modified (RiPPs). google.commit.eduacs.org A key enzyme in this process is a copper-dependent BURP-domain peptide cyclase, such as KjaBURP from Kerria japonica, which catalyzes the formation of the tryptophan-indole-centered macrocyclic bonds. mit.eduacs.orgumich.edu

This understanding of the biosynthetic pathway has paved the way for chemoenzymatic strategies. One notable development is the in planta biosynthesis of this compound and its analogues by expressing the relevant precursor genes, such as the predicted precursor gene from K. japonica, in heterologous plant systems like tobacco. mdpi.com This approach leverages the plant's own machinery, guided by the introduced gene, to perform the complex enzymatic cyclization and modifications, allowing for the production of this compound and diverse analogues on a milligram scale without extensive traditional organic synthesis. mdpi.com While in vitro reconstitution of the biosynthesis using the BURP domain has also been explored, challenges remain in optimizing the speed and yield of the enzymatic cyclization for large-scale production.

Synthetic Modulations for Analog Generation

Synthetic studies are not limited to the total synthesis of the parent compound but also extend to the generation of this compound analogues. This is important for exploring structure-activity relationships and developing potential therapeutics. mit.edu Both traditional organic synthesis and biosynthetic approaches offer avenues for creating analogues.

In organic synthesis, modifications can be introduced by using modified amino acid building blocks or by altering the cyclization strategies to form different ring sizes or incorporate different residues.

Using the insights from biosynthesis, analogue generation can be achieved by modifying the core peptide sequences within the precursor genes expressed in host plants. mit.edu By varying the amino acid sequence that corresponds to the this compound peptide motif in the precursor protein, researchers have demonstrated the feasibility of producing diverse this compound-like peptides. mit.eduacs.org This gene-guided approach allows for the exploration of a library of this compound-based peptides, facilitating the discovery of analogues with potentially improved properties. acs.org

Anti-mitotic Activity and Microtubule Dynamics Modulation

Moroidin's anti-mitotic activity stems from its ability to modulate the dynamic behavior of microtubules. wikipedia.orgresearchgate.net Microtubules, composed of α- and β-tubulin heterodimers, are essential components of the cytoskeleton and play vital roles in maintaining cell structure, intracellular transport, and chromosome segregation during mitosis. researchgate.net Agents that disrupt microtubule dynamics interfere with the formation and proper function of the mitotic spindle, leading to impaired chromosome segregation and ultimately cell death. researchgate.net

Regulation of Cell Cycle Progression

The disruption of microtubule dynamics by this compound has direct consequences for cell cycle progression, particularly during mitosis. researchgate.net

Mechanisms of Apoptotic Induction

This compound has been shown to induce apoptosis in cancer cells through various pathways and molecular interactions. researchgate.netacs.orgnih.govchemsrc.com

Anti-migratory and Anti-invasive Effects on Cancer Cells

Beyond its apoptotic effects, this compound has also demonstrated the ability to inhibit the migration and invasion of cancer cells, particularly in glioblastoma (GBM) cells and A549 lung cancer cells. researchgate.netacs.orgnih.govresearchgate.netnih.govfigshare.comjbr-pub.org.cn Cell migration and invasion are critical processes in cancer progression and metastasis. oncotarget.com

Studies have shown that this compound significantly suppressed cell migration and invasion in A549 cells at sublethal concentrations. researchgate.netacs.orgnih.govfigshare.com In glioblastoma cells, this compound inhibited cell migration and tube formation, which is a measure of vasculogenic mimicry (VM), an alternative mechanism of blood supply in aggressive tumors. researchgate.netnih.govjbr-pub.org.cn

The anti-migratory and anti-invasive effects of this compound in glioblastoma cells have been linked to the regulation of the epithelial-mesenchymal transition (EMT) pathway and β-catenin activation. researchgate.netnih.govjbr-pub.org.cn EMT is a process by which cancer cells acquire migratory and invasive properties. researchgate.netnih.gov this compound treatment led to a concentration-dependent decrease in the expression of EMT markers such as N-cadherin and vimentin in GBM cells. researchgate.netnih.gov Furthermore, this compound significantly reduced the level of phosphorylated extracellular signal-regulated protein kinase (p-ERK) and inhibited the activation of β-catenin. researchgate.netnih.gov These findings suggest that this compound inhibits VM formation in GBM cells by regulating the ERK/β-catenin-mediated EMT pathway. researchgate.netnih.govjbr-pub.org.cn

Research findings on the anti-migratory and anti-invasive effects of this compound:

Anti-Vasculogenic Mimicry (VM) Activity

Vasculogenic mimicry (VM) is a process employed by aggressive tumor cells, particularly under conditions of hypoxia and nutrient deficiency, to form fluid-conducting channels that supplement or replace endothelial cell-lined vessels, thereby contributing to tumor blood supply and resistance to anti-angiogenic therapies. nih.govjbr-pub.org.cntandfonline.com this compound has been identified as a potential agent against VM, particularly in glioblastoma (GBM). nih.govresearchgate.netjbr-pub.org.cn

Correlating Bicyclic Ring System Architecture with Biological Activity

The bicyclic ring system is a defining feature of moroidin and the celogentins, formed by unusual non-peptide cross-connections. Research indicates that this intricate bicyclic architecture is crucial for their biological activity, specifically their interaction with tubulin. The presence of the bicyclic system, including these unique linkages, appears to be a fundamental requirement for the observed anti-mitotic effects. Studies comparing bicyclic this compound-type peptides to monocyclic analogues, such as stephanotic acid-[LV], suggest that the second ring is required for cytotoxicity against certain cancer cell lines.

Significance of Unusual Leu-Trp and Trp-His Cross-Linkages in Bioactivity

This compound's structure includes two distinctive cross-links: a carbon-carbon (C-C) bond between the β-carbon of a leucine residue and the C6 of a tryptophan indole, and a carbon-nitrogen (C-N) bond connecting the C2 of the same tryptophan indole to the N1 of a C-terminal histidine imidazole. nih.govresearchgate.net These cross-linkages are integral to the formation of the bicyclic scaffold. nih.govresearchgate.net The Trp-His cross-link, in particular, forms the second ring of the bicyclic system, and its presence is necessary for the bioactivity of this compound-type peptides. The specific structure of the right-hand ring, which contains the Trp-His linkage, has been correlated with the degree of biological activity. nih.gov

Influence of Ring Size and Conformation on Target Interaction and Efficacy

The size and conformation of the bicyclic rings play a significant role in the interaction of this compound and its analogues with their biological target, tubulin. Cyclization in peptides generally leads to reduced conformational flexibility, which can enhance metabolic stability and improve binding affinity and specificity to target molecules. Bicyclic peptides, with their further constrained structures, exhibit even greater conformational rigidity compared to their linear or monocyclic counterparts. The second ring in moroidins can vary in size, typically comprising 2 to 4 amino acids. Variations in ring size and the resulting conformations are considered important factors influencing their biological activity. Synthetic efforts have explored altering the size of the right-hand ring to investigate the impact on activity.

Comparative Structure-Activity Profiles of Celogentins and other this compound-Type Analogues

The celogentins, a family of bicyclic peptides isolated from Celosia argentea, are structurally related to this compound and share its anti-mitotic activity through tubulin polymerization inhibition. Comparative studies have revealed distinct differences in potency among these compounds. Celogentin C is consistently reported as being significantly more potent in inhibiting tubulin polymerization than this compound. nih.gov While this compound demonstrates an inhibitory activity with an IC₅₀ of 3.0 µM, comparable to that of vinblastine, celogentin C exhibits a higher potency with an IC₅₀ of 0.8 µM. nih.gov This difference in potency has been linked to variations in the structure of the right-hand ring. nih.gov Specifically, celogentin C contains a proline residue in its right-hand ring, whereas this compound has a glycine residue in the corresponding position, leading to presumably different conformations of this macrocycle. Other celogentins, such as celogentins A, B, and D, have shown significantly lower activity compared to this compound and celogentin C. The presence of an additional lysine residue in celogentin B did not appear to substantially affect its inhibitory activity. Furthermore, the discovery of this compound analogues from other plant sources, such as a water chickweed analogue showing higher cytotoxicity against lung adenocarcinoma cells than this compound, highlights the potential for structural variations to influence biological activity.

Table 1: Comparative Potency of this compound, Celogentin C, and Vinblastine in Tubulin Polymerization Inhibition nih.gov

Q. What are the primary molecular mechanisms through which Moroidin exerts its anti-cancer effects in glioblastoma models?

this compound inhibits glioblastoma (GBM) progression via dual mechanisms: (1) microtubule disruption by binding to tubulin, preventing polymerization (observed in celogentin C analogs) , and (2) suppression of epithelial-mesenchymal transition (EMT) by downregulating N-cadherin, vimentin, and β-catenin signaling. RNA sequencing and GSEA analysis revealed significant downregulation of EMT-related genes (e.g., MMP9, MMP14) and inhibition of ERK/β-catenin pathways in U87/U251 cells treated with 2 µM this compound . Western blot and immunofluorescence validated dose-dependent reduction in phosphorylated ERK and nuclear β-catenin .

Q. Which experimental assays are commonly employed to evaluate this compound's inhibitory effects on glioblastoma cell migration and vasculogenic mimicry (VM)?

Key assays include:

• Wound scratch assay : Cells cultured to 80% confluency are scratched and treated with this compound (0.5–2 µM). Migration distance is quantified via ImageJ after 24 hours .
• Transwell migration : Cells in this compound-treated upper chambers are fixed, stained, and counted after 24 hours .
• Matrigel tube formation : VM inhibition is assessed by counting tubular networks in GBM cells seeded on Matrigel. This compound (2 µM) reduces network complexity by 55% (U87) and 34% (U251) .
• qRT-PCR/Western blot : Validates downregulation of EMT markers (e.g., MMP9, vimentin) using primers/probes listed in Table 1 of .

Advanced Research Questions

Q. How can researchers reconcile contradictory findings regarding this compound's role in pain induction versus its anti-cancer properties?

While this compound was initially linked to neuropathic pain in Dendrocnide moroides, recent studies suggest its isolated form induces less pain than crude plant extracts . This discrepancy may arise from synergistic interactions with other plant toxins (e.g., histamines) or differences in in vivo vs. in vitro models. To resolve this, comparative metabolomics of plant extracts and purified this compound, paired with in vivo nociception assays (e.g., mouse paw-licking tests), are recommended .

Q. What methodologies are recommended for optimizing heterologous production of this compound analogs in non-native plant systems?

The Weng group’s gene-guided approach includes:

• BURP domain mining : Screening 793 plant transcriptomes for this compound-like motifs to identify functional cyclization enzymes .
• Copper-dependent catalysis : Utilizing copper ions to enable cyclization of tryptophan-indole macrocycles in engineered tobacco .
• Diversification : Modifying core peptide sequences in Nicotiana benthamiana to produce analogs like celogentin C, which showed selective cytotoxicity in H1437 lung adenocarcinoma cells (IC50: 1.2 µM) .

Q. How can researchers integrate multi-omics data to elucidate this compound's dual role in microtubule targeting and EMT inhibition?

A proposed workflow:

Transcriptomics : RNA-seq of this compound-treated GBM cells to identify EMT pathway suppression .

Network analysis : STRING database integration to map protein-protein interactions between EMT markers (e.g., MMP9, β-catenin) and microtubule regulators .

Functional validation : CRISPR knockdown of β-catenin in GBM cells to test if this compound’s anti-migratory effects persist .

Methodological Considerations for Data Contradiction

Q. What statistical approaches are critical for analyzing dose-dependent effects of this compound in migration assays?

• Two-way ANOVA with Dunnett’s post hoc test : Used to compare migration distances across multiple doses (0.5–2 µM) and cell lines (U87 vs. U251) .
• Dose-response modeling : IC50 calculations for VM inhibition using nonlinear regression (e.g., GraphPad Prism) .

Q. How should researchers address variability in this compound's bioactivity across different glioblastoma cell lines?

• Line-specific profiling : Compare baseline expression of β-catenin and EMT markers (e.g., U87 has higher vimentin than U251) .
• Normalization to housekeeping genes : Use GAPDH or β-actin for qRT-PCR/Western blot to control for loading differences .

Emerging Research Directions

Q. Can this compound analogs be engineered to enhance blood-brain barrier (BBB) penetration for glioblastoma therapy?

• Structural modifications : Introduce lipid-soluble groups (e.g., methyl esters) to celogentin C’s macrocyclic backbone .
• In vitro BBB models : Test permeability using co-cultures of brain endothelial cells and astrocytes, measuring transendothelial electrical resistance (TEER) .

Q. What in vivo models are suitable for validating this compound's anti-VM efficacy?

• Orthotopic GBM xenografts : Intracranial implantation of luciferase-tagged U87 cells in nude mice, with this compound administered via osmotic pump (2 mg/kg/day) .
• Dynamic contrast-enhanced MRI : Monitor VM reduction by tracking gadolinium leakage in tumors .