Discovery of a new antifungal agent ASP2397 using a silkworm model of Aspergillus fumigatus infection

Abstract

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 antifungal pipeline: a reality check

“…As mammalian cells do not have this transporter, it is hypothesized that the compound will have excellent selective fungal toxicity. In both in vitro and in vivo studies, ASP2397 has very potent fungicidal activity against A. fumigatus 67 . Along with potent activity against both azole-susceptible and azole-resistant strains of Aspergillus spp., it also has in vitro antifungal activity against a few Candida spp.…”

Advantages of the Silkworm As an Animal Model for Developing Novel Antimicrobial Agents

“…Since then, silkworms have been utilized to study pathogenic bacterial toxins (Hossain et al, 2006); evaluate the target specificity of antibacterial agents (Kurokawa et al, 2009); identify novel S. aureus virulence genes (Kaito et al, 2005; Miyazaki et al, 2012); and identify novel probiotic bacteria that promote survival during P. aeruginosa infection (Nishida et al, 2016). Additionally, infection model of multiple pathogenic microorganisms Stenotrophomonas maltophilia (Hamamoto et al, 2004); Vibrio vulnificus (Yamamoto et al, 2016); Vibrio cholera (Kaito et al, 2002); Candida tropicalis (Hamamoto et al, 2004); Candida albicans (Hamamoto et al, 2004); Aspergillus fumigatus (Nakamura et al, 2017a); Cryptococcus neoformans (Matsumoto et al, 2012); and laboratory generated vancomycin-resistant S. aureus VR7 (Tabuchi et al, 2017) have been developed and the success of the antimicrobial to rescue the silkworms from the effect of pathogens have been reported (Table 2). These findings suggest that silkworm infection models can be utilized to mimic the infections caused by various bacterial and fungal pathogens.…”

“…Further, antibiotics and antifungal agents clinically used against those bacteria and fungi effectively cured infections in silkworms. By utilizing the silkworm infection model, we and collaborators successfully identified novel antimicrobial agents—lysocin E (Hamamoto et al, 2015), nosokomycins (Uchida et al, 2010), and ASP2397 (Nakamura et al, 2017a). The details of the discovery of lysocin E and ASP2397 are presented in this review.…”

Silkworm fungal infection model for identification of virulence genes in pathogenic fungus and screening of novel antifungal drugs

“…Silkworms die within a few days after injection with Aspergillus fumigatus, Candida albicans, Candida tropicalis, Candida glabrata, or Cryptococcus neoformans (7,(10)(11)(12)(13). Heat treatment (121°C, 15 min) of the fungi eliminates the killing ability of C. albicans or C. neoformans (12,14).…”

“…We recently reported the discovery of a novel therapeutic agent using an Aspergillus fumigatus infection model of silkworms (13). A. fumigatus killed silkworms 2 days after injection.…”