Bioinformatics-based stereochemical elucidation of MM 46115: an unusual antiviral spirotetronate that inhibits clathrin-mediated endocytosis

Abstract

The influenza virus is a leading cause of respiratory tract infection in humans, with the emergence of antiviral drug resistance posing an ongoing challenge to the treatment of influenza. This underscores the urgent need for effective new antiviral treatments. Spirotetronates are a family of bacterial natural products with a range of potent bioactivities. Some members of this family have antiviral activity. Here, we report the bioinformatics-based stereochemical assignment, antiviral properties, and mechanism of action of the unusual type II spirotetronate MM 46115 (renamed pellemicin). We show that pellemicin is active against both influenza A and B viruses at non-toxic concentrations, and inhibits clathrin-mediated endocytosis, which is required for virus internalisation. Given the need for new treatments for viral infections, the results of our work suggest that pellemicin and related spirotetronates could provide a basis for developing promising alternatives to existing antivirals to combat drug-resistant influenza. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/677008v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@177aacforg.highwire.dtl.DTLVardef@16f0b26org.highwire.dtl.DTLVardef@144f9dcorg.highwire.dtl.DTLVardef@16fe673_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMM 46115 (here renamed pellemicin) produced by a mycetoma pathogen is a promising antiviral C_LIO_LIThe pellemicin biosynthetic gene cluster was identified in Actinomadura pelletieri DSM 43383 C_LIO_LISequence analysis of biosynthetic enzymes enabled full stereochemical assignment C_LIO_LIPellemicin inhibits clathrin-mediated endocytosis, an early stage of the viral life cycle C_LI SignificanceThe influenza virus is a respiratory pathogen with epidemic and pandemic potential, resulting in tens of thousands of deaths each year and causing significant health, social and economic impact. The rise of resistant influenza strains against existing antiviral drugs creates a pressing need for alternative treatments. Natural products - specialised metabolites of bacteria, fungi, and plants that often have antimicrobial activity - are a promising source of therapeutics to treat human disease. Here, we identify a cryptic polyketide biosynthetic gene cluster in the genome sequence of Actinomadura pelletieri DSM 43383, an Actinomycete that causes mycetoma in humans. This is proposed to direct production of the previously reported spirotetronate MM 46115 (here renamed pellemicin), which has activity against the influenza virus. Sequence analyses of a polyketide synthase and Diels-Alderase involved in pellemicin biosynthesis, combined with comparative analysis of NMR data for other spirotetronates, enabled us to fully resolve longstanding stereochemical ambiguities in the structure of pellemicin. This underscores the emerging power of bioinformatics-based approaches, and their complementarity to traditional spectroscopic methods, for natural product structure elucidation. We also report extensive insights into the antiviral properties and mechanism of pellemicin, demonstrating that it exhibits antiviral activity at non-toxic doses for up to eight hours after administration and inhibits clathrin-mediated endocytosis, a process that many viruses exploit to enter cells. Only one other bacterial natural product, ikarugamycin, is known to inhibit clathrin-mediated endocytosis. Ikarugamycin contains a tetramate group, which bears a close structural relationship to the tetronate moiety of pellemicin, suggesting that inhibition of clathrin-mediated endocytosis may be a general mechanism of action for antiviral spirotetronates. Our findings therefore indicate that pellemicin, and related spirotetronate natural products, could form a basis for development of new drugs to combat antiviral resistance, and serve as useful tools to study the process of endocytosis.