RESUMO
RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. MSI1 is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb, APC, and P21WAF-1, key regulators of Notch/Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1-mRNA interactions, which could block the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA. One cluster of hit compounds is the derivatives of secondary metabolites from Aspergillus nidulans. One of the top hits, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance spectroscopy, which demonstrated that Aza-9 binds directly to MSI1, and the binding is at the RNA binding pocket. We also show that Aza-9 binds to Musashi-2 (MSI2) as well. To test whether Aza-9 has anti-cancer potential, we used liposomes to facilitate Aza-9 cellular uptake. Aza-9-liposome inhibits proliferation, induces apoptosis and autophagy, and down-regulates Notch and Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1/2 function, while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.
RESUMO
Prenylated indole alkaloids such as the calmodulin-inhibitory malbrancheamides and anthelmintic paraherquamides possess great structural diversity and pharmaceutical utility. Here, we report complete elucidation of the malbrancheamide biosynthetic pathway accomplished through complementary approaches. These include a biomimetic total synthesis to access the natural alkaloid and biosynthetic intermediates in racemic form and in vitro enzymatic reconstitution to provide access to the natural antipode (+)-malbrancheamide. Reductive cleavage of an L-Pro-L-Trp dipeptide from the MalG non-ribosomal peptide synthetase (NRPS) followed by reverse prenylation and a cascade of post-NRPS reactions culminates in an intramolecular [4+2] hetero-Diels-Alder (IMDA) cyclization to furnish the bicyclo[2.2.2]diazaoctane scaffold. Enzymatic assembly of optically pure (+)-premalbrancheamide involves an unexpected zwitterionic intermediate where MalC catalyses enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels-Alderase. The crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrate how MalC and PhqE (its homologue from the paraherquamide pathway) catalyse diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites.
Assuntos
Ascomicetos/química , Alcaloides Indólicos/metabolismo , Oxirredutases/metabolismo , Biocatálise , Reação de Cicloadição , Alcaloides Indólicos/química , Modelos Moleculares , Estrutura MolecularRESUMO
The RNA-binding protein Hu antigen R (HuR) binds to AU-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs. The HuR-ARE interactions stabilize many oncogenic mRNAs that play important roles in tumorigenesis. Thus, small molecules that interfere with the HuR-ARE interaction could potentially inhibit cancer cell growth and progression. Using a fluorescence polarization (FP) competition assay, we identified the compound azaphilone-9 (AZA-9) derived from the fungal natural product asperbenzaldehyde, binds to HuR and inhibits HuR-ARE interaction (IC50 ~1.2 µM). Results from surface plasmon resonance (SPR) verified the direct binding of AZA-9 to HuR. NMR methods mapped the RNA-binding interface of HuR and identified the involvement of critical RNA-binding residues in binding of AZA-9. Computational docking was then used to propose a likely binding site for AZA-9 in the RNA-binding cleft of HuR. Our results show that AZA-9 blocks key RNA-binding residues of HuR and disrupts HuR-RNA interactions in vitro. This knowledge is needed in developing more potent AZA-9 derivatives that could lead to new cancer therapy.
Assuntos
Benzopiranos/farmacologia , Produtos Biológicos/farmacologia , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Fungos/química , Pigmentos Biológicos/farmacologia , RNA/metabolismo , Regiões 3' não Traduzidas/efeitos dos fármacos , Elementos Ricos em Adenilato e Uridilato/efeitos dos fármacos , Antígenos de Superfície/metabolismo , Sítios de Ligação/efeitos dos fármacos , Polarização de Fluorescência/métodos , Humanos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The aggregation of the microtubule-associated protein tau is a seminal event in many neurodegenerative diseases, including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activities. We have previously screened Aspergillus nidulans secondary metabolites for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol. One aggregation inhibitor identified was asperbenzaldehyde, an intermediate in azaphilone biosynthesis. We therefore tested 11 azaphilone derivatives to determine their tau assembly inhibition properties in vitro. All compounds tested inhibited tau filament assembly to some extent, and four of the 11 compounds had the advantageous property of disassembling preformed tau aggregates in a dose-dependent fashion. The addition of these compounds to the tau aggregates reduced both the total length and number of tau polymers. The most potent compounds were tested in in vitro reactions to determine whether they interfere with tau's normal function of stabilizing microtubules (MTs). We found that they did not completely inhibit MT assembly in the presence of tau. These derivatives are very promising lead compounds for tau aggregation inhibitors and, more excitingly, for compounds that can disassemble pre-existing tau filaments. They also represent a new class of anti-tau aggregation compounds with a novel structural scaffold.
Assuntos
Benzopiranos/farmacologia , Pigmentos Biológicos/farmacologia , Proteínas tau/efeitos dos fármacos , Proteínas tau/metabolismo , Aspergillus nidulans , Benzopiranos/síntese química , Técnicas In Vitro , Microtúbulos/efeitos dos fármacos , Pigmentos Biológicos/síntese químicaRESUMO
Sclerotiorin, an azaphilone polyketide, is a bioactive natural product known to inhibit 15-lipoxygenase and many other biological targets. To readily access sclerotiorin and analogs, we developed a 2-3 step semisynthetic route to produce a variety of azaphilones starting from an advanced, putative azaphilone intermediate (5) overproduced by an engineered strain of Aspergillus nidulans. The inhibitory activities of the semisynthetic azaphilones against 15-lipoxygenase were evaluated with several compounds displaying low micromolar potency.
Assuntos
Aspergillus nidulans/metabolismo , Inibidores de Lipoxigenase/metabolismo , Pigmentos Biológicos/biossíntese , Araquidonato 15-Lipoxigenase/metabolismo , Aspergillus nidulans/química , Benzopiranos/química , Benzopiranos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Concentração Inibidora 50 , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacologia , Engenharia Metabólica , Estrutura Molecular , Pigmentos Biológicos/química , Pigmentos Biológicos/farmacologia , Relação Estrutura-AtividadeRESUMO
This review studies the impact of whole genome sequencing on Aspergillus secondary metabolite research. There has been a proliferation of many new, intriguing discoveries since sequencing data became widely available. What is more, the genomes disclosed the surprising finding that there are many more secondary metabolite biosynthetic pathways than laboratory research had suggested. Activating these pathways has been met with some success, but many more dormant genes remain to be awakened.
Assuntos
Aspergillus/genética , Genoma Fúngico , Aspergillus/metabolismo , Humanos , Estrutura MolecularRESUMO
An StcA-AfoE hybrid polyketide synthase (PKS), generated by swapping the AfoE (asperfuranone biosynthesis) SAT domain with the StcA (sterigmatocystin biosynthesis) SAT domian, produced a major new metabolite with the same chain length as the native AfoE product. Structure elucidation allowed us to propose a likely pathway, and feeding studies supported the hypothesis that the chain length of PKS metabolites may be under precise control of KS and PT domains.