Natural products are the major source of currently available drugs. However, screening natural product presents several challenges, including the time-consuming and labor-intensive steps required for the isolation of a drug from crude extracts as well as the differences between the activities of compounds in vitro and in vivo. To address these challenges, we used silkworm larvae infected with Aspergillus fumigatus to screen a natural products library for potent drugs to treat invasive aspergillosis. A rationally designed library was constructed using numerous, geographically diverse fungal species and then screened to collect extracts of microorganisms that had detectable anti-Aspergillus activity. We evaluated this library using cultures of A. fumigatus and a silkworm model system of A. fumigatus infection. With this model, we identified the novel antifungal compound ASP2397 that not only cured infected silkworm larvae but also increased the rates of survival of mice infected with A. fumigatus. These findings strongly support the utility of the silkworm screening system for the simple and rapid isolation of antibiotics from natural products libraries.
The novel antifungal agent ASP2397 (Vical's compound ID VL-2397) is produced by the fungal strain MF-347833 that was isolated from Malaysian leaf litter and is identified here as an Acremonium species based on its morphology, physiological properties and 28S ribosomal DNA sequence. Because of its potential importance for producing novel antifungal agents, we determined the taxonomic and biologic properties of MF-347833. We show here that ASP2397 is a cyclic hexapeptide that chelates aluminum ion and is therefore similar to ferrichrome, a hydroxamate siderophore. However, ASP2397 differs structurally from licensed antifungal agents such as amphotericin B, triazoles and echinocandins. To understand the relationship between chemical structure and biological function, we isolated certain ASP2397 derivatives from the culture broth, and we further chemically converted the metal-free form to other derivatives.
Current therapies against invasive pulmonary aspergillosis (IPA) have a limited cure rate. Given that a delay in treatment initiation may be fatal, a new drug with rapid-onset and potent fungicidal activity is needed. The novel cyclic hexapeptide ASP2397 (currently known as VL-2397) exhibited antifungal activity against Aspergillus fumigatus (including azole-sensitive and azole-resistant isolates), A. terreus, and A. flavus at an MIC range of 1 to 4 μg/ml in human serum. Time-kill curve experiments showed that ASP2397 reduced germinated conidia of A. fumigatus by more than 1 log10 CFU within 6 h. In addition, ASP2397 inhibited hyphal elongation from germinated conidia of A. fumigatus, A. terreus, and A. flavus more rapidly than voriconazole. Under conditions of delayed treatment initiation in an IPA mouse model, ASP2397 had efficacy superior to that of posaconazole, with 100% survival and over 1 log10 CFU/g reduction in lung fungal burden. Histopathological investigation of lungs also showed that ASP2397 markedly suppressed disease progression. To clarify its mechanism of action, we generated a UV-induced mutant of A. fumigatus with low susceptibility to ASP2397. The mutant had a point mutation in the siderophore transporter gene sit1, which is absent in mammalian cells. These findings suggest that ASP2397 may improve clinical treatment options for IPA.
We found that the IgA1 produced in tonsillar tissue differed from serum IgA1. Furthermore, an overproduction of asialo IgA1 resulted from the disordered balance between IgA- and IgG-producing cells in the tonsils from the IgA nephropathy patient. Although it is unclear how such asialo IgA1 molecules are transferred from tonsil tissue to serum, a tonsillar source may produce a few micrograms of aberrant IgA1 that then appears in serum.
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