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New Spins on Old Drugs: Enhancing Activity of Antifungals
Cell Chemical Biology
Previews
New Spins on Old Drugs: Enhancing Activity of Antifungals J. Andrew Alspaugh1,* 1Department of Medicine, Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27701, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chembiol.2020.03.001
In this issue of Cell Chemical Biology, Caplan et al. (2020) describe a series of studies in the human fungal pathogen Candida albicans to identify a new target for antimicrobial drug development. Beginning with an unbiased compound screen, they identify new mechanisms to address rising resistance to currently used anti-infective agents.
Infections due to fungi generally occur in our most vulnerable patients, especially those with compromised immune function (Brown et al., 2012). These infections have become increasingly common, especially with advancing medical interventions that specifically target the immune system to treat human conditions as wide-ranging as classical auto-immune diseases and many previously untreatable cancers. Unlike their bacterial counterparts, fungal pathogens as eukaryotes are physiologically quite similar to the hosts that they infect. Therefore, one of the greatest challenges of antifungal drug discovery is to identify fungal-specific cellular features that can be specifically targeted and inhibited pharmacologically. Even more vexing is the issue of fungal cell permeability. Many compounds demonstrate potent inhibitory effects against essential fungal processes, but few of these compounds can effectively and stably enter the fungal cell. Screening of natural products for antifungal activity has historically been one of the most effective strategies for identifying compounds that can be considered good drug candidates (Roemer et al., 2011). These screening methods are designed to identify compounds that actually enter and inhibit the fungal cell. Moreover, natural product screens have identified the major classes of antifungal agents in current clinical use. These classes include two drug families, the polyenes and azoles, that disrupt different steps in the homeostasis of ergosterol, the primary sterol in fungal membranes. Limitations of this approach include the fact that the same classes of
naturally occurring antifungal compounds seem to be repeatedly identified by unbiased screening approaches (Roemer et al., 2011). The echinocandins, one of the more recently introduced classes of antifungals, inhibit the production of a major carbohydrate component of the fungal cell wall (Douglas et al., 1997). This fungalspecific feature is responsible for providing cellular structural strength and resilience among diverse fungal species. Cells with defective cell walls are compromised for growth as well as resistance to exogenous stress. In addition to its protective functions, the cell wall serves as an important antigenic surface for interaction of fungal pathogens with host immune cells. In fact, the fungal cell wall carbohydrate b-1,3-D-glucan is specifically recognized by the C-type lectin, pattern recognition receptor (PRR) dectin-1 (Brown et al., 2003). This glucanPRR recognition triggers the activation of a cascading series of signaling events resulting in immune activation to a fungal challenge. Importantly, b-1,3-D-glucan synthase is the target of echinocandins, explaining the deleterious effects of this drug class on cell wall integrity (Douglas et al., 1997). The echinocandins are exceptionally useful clinically due to their profound efficacy against many of the most common fungal pathogens of humans. These compounds typically have excellent activity for infections due to Candida albicans, a common cause of human bloodstream infections, as well as other tissue invasive infections in immunocompromised patients (reviewed in Pianalto and Alspaugh, 2016). Moreover, echinocandins have
been one of the most important last treatment options for highly drug-resistant fungi such as recently identified Candida auris (Dudiuk et al., 2019). Echinocandins also have clinical indications in infections due to Aspergillus species. However, perhaps due to the widespread use of these agents in clinical practice, resistance to echinocandins is increasingly observed, even among fungal species that were universally susceptible to these agents only a few years ago. Several investigators have defined mechanisms of increasing echinocandin tolerance and frank echinocandin resistance among diverse fungal species. These resistance mechanisms include impaired fungal cell entry as well as point mutations in the FKS1 gene encoding b-1,3-D-glucan synthase (Walker et al., 2010). Given the very limited number of antifungal agents, combatting antifungal resistance is of paramount importance. This paradigm motivated the studies outlined in the article by Caplan et al. (2020). The investigators screened a library of diverse compounds with predicted inhibitory activity against mammalian kinases, looking for those that would increase the antifungal effect of an echinocandin drug, caspofungin, against C. albicans. Therefore, this screening strategy was not designed to find new antifungal agents per se, but to identify potentiators of a currently used drug. Importantly, they identified several small molecules with a common chemical scaffold, lending great confidence to the robustness of this screening strategy. Using a combination of complementary experimental approaches, they identified the mammalian Yck2 kinase as the target of their most active compound
Cell Chemical Biology 27, March 19, 2020 ª 2020 Elsevier Ltd. 255
Cell Chemical Biology
Previews
(GW461484A). These screening approaches included fungal haplo-insufficiency profiling, a technique built upon the premise that a reduction in the dose of a target enzyme will result in altered susceptibility to the screening compound. In their studies, Caplan et al. treated a large collection of heterozygous C. albicans mutant strains with caspofungin, observing for alterations in cell growth. In this manner, they identified many caspofungin-hypersusceptible strains with mutations in genes involved in actin-related processes, including protein trafficking and cell division. Three of these mutations were in protein kinaseencoding genes, including the casein kinase 1 family members Yck2 and Hrr25. Specific overexpression of C. albicans YCK2, and not HRR25, resulted in relative caspofungin resistance, and YCK2 repression led to enhanced drug susceptibility. The fungal genetics experiments were complemented by in vitro kinase activity assays in which GW461484A inhibited the phosphorylation of a casein peptide substrate by recombinant Yck2. Structural studies of C. albicans Yck2 in its apo-form as well as in the presence of drug suggested a precise mechanism of GW416148A activity—to competitively occupy the ATP-binding pocket of this enzyme. Fungal-specific features of this interaction were also inferred, and these observations may provide direction for future compound derivatization. One of the limitations of advancing pyrazolopyridines such as GW461148A for clinical use is their recognized vulnerability to oxidative degradation at the electron-rich C6-C7 double bond. Therefore, a number of structurally similar compounds were tested for antifungal activity and chemical stability. These studies provide a theoretical foundation to derive future compounds to retain optimal activity against the fungal kinase while promoting enhanced chemical stability. Follow-up work in this area will be critical to determine the ultimate translational relevance of these initial studies. Because of the pharmacological instability of the pyrazolopyridines, efficacy of GW461148A in a relevant animal model of fungal infection was not reported. However, this compound did enhance the
antifungal effect of caspofungin in a macrophage-fungal co-culture system, protecting the mammalian cell monolayer from fungal-mediated disruption and without resulting in obvious host cell toxicity. Consistent with prior studies (Blankenship et al., 2010; Jung et al., 2017), genetic inhibition of C. albicans Yck2 activity impaired in vitro correlates of virulence as well as the pathogenesis of a systemic infection in mice. Although these initial studies were performed primarily for C. albicans, the investigators also noted significant activity of GW461148A against other common fungal pathogens, including the drugresistant C. auris, and the echinocandintolerant neuropathogen Cryptococcus neoformans. Measurable, but more limited, activity against the mold pathogen Aspergillus fumigatus was also observed. Is there room in the clinical arena for agents that primarily enhance the activity of current agents? I propose that this question has already been answered. Aminoglycosides are used to provide antibacterial synergy with other antibiotics against many pathogens. Beta lactamase inhibitors enhance the activity of antibiotics against resistant pathogens. Combination therapy for infections as diverse as tuberculosis and cryptococcosis demonstrates superior efficacy compared to monotherapy, both to prevent the development of resistance and to more rapidly sterilize the infected tissue. In summary, these combined studies are remarkable in both their breadth and focus. Beginning with unbiased compound screening, the investigators identified a chemical activity and common scaffold with previously unrecognized potential for antifungal activity, especially to enhance the effect of available antifungal agents. These initial compounds were tested using sophisticated microbial genetics tools to infer mechanism of activity, which was subsequently confirmed biochemically. Their structural studies can be used to direct chemical modifications to enhance fungal selectivity. This is especially important given the generalizability of antifungal activity demonstrated in multiple, diverse fungal pathogens. Despite these experimental strengths, transla-
256 Cell Chemical Biology 27, March 19, 2020
tional challenges remain. Despite possessing convincing antimicrobial activity, these compounds are still not yet drugs given their current pharmacological instability. These latter observations underscore the importance of rigorous medical chemistry to provide the translational bridge from the atomic and molecular scale to the patient.
REFERENCES Blankenship, J.R., Fanning, S., Hamaker, J.J., and Mitchell, A.P. (2010). An extensive circuitry for cell wall regulation in Candida albicans. PLoS Pathog. 6, e1000752. Brown, G.D., Herre, J., Williams, D.L., Willment, J.A., Marshall, A.S., and Gordon, S. (2003). Dectin-1 mediates the biological effects of betaglucans. J. Exp. Med. 197, 1119–1124. Brown, G.D., Denning, D.W., Gow, N.A., Levitz, S.M., Netea, M.G., and White, T.C. (2012). Hidden killers: human fungal infections. Sci. Transl. Med. 4, 165rv13. Caplan, T., Lorente-Macı´as, A´., Stogios, P.J., Evdokimova, E., Hyde, S., Wellington, M.A., Liston, S., Iyer, K.R., Puumala, E., ShekharGuturja, T., et al. (2020). Overcoming fungal echinocandin resistance through inhibition of the nonessential stress kinase Yck2. Cell Chem. Biol. 27, this issue, 269–282. Douglas, C.M., D’Ippolito, J.A., Shei, G.J., Meinz, M., Onishi, J., Marrinan, J.A., Li, W., Abruzzo, G.K., Flattery, A., Bartizal, K., et al. (1997). Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob. Agents Chemother. 41, 2471–2479. Dudiuk, C., Berrio, I., Leonardelli, F., MoralesLopez, S., Theill, L., Macedo, D., YesidRodriguez, J., Salcedo, S., Marin, A., Gamarra, S., and Garcia-Effron, G. (2019). Antifungal activity and killing kinetics of anidulafungin, caspofungin and amphotericin B against Candida auris. J. Antimicrob. Chemother. 74, 2295–2302. Jung, S.I., Rodriguez, N., Irrizary, J., Liboro, K., Bogarin, T., Macias, M., Eivers, E., Porter, E., Filler, S.G., and Park, H. (2017). Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans. PLoS One 12, e0187721. Pianalto, K.M., and Alspaugh, J.A. (2016). New horizons in antifungal therapy. J. Fungi (Basel) 2, https://doi.org/10.3390/jof2040026. Roemer, T., Xu, D., Singh, S.B., Parish, C.A., Harris, G., Wang, H., Davies, J.E., and Bills, G.F. (2011). Confronting the challenges of natural product-based antifungal discovery. Chem. Biol. 18, 148–164. Walker, L.A., Gow, N.A., and Munro, C.A. (2010). Fungal echinocandin resistance. Fungal Genet. Biol. 47, 117–126.
Previews
New Spins on Old Drugs: Enhancing Activity of Antifungals J. Andrew Alspaugh1,* 1Department of Medicine, Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27701, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chembiol.2020.03.001
In this issue of Cell Chemical Biology, Caplan et al. (2020) describe a series of studies in the human fungal pathogen Candida albicans to identify a new target for antimicrobial drug development. Beginning with an unbiased compound screen, they identify new mechanisms to address rising resistance to currently used anti-infective agents.
Infections due to fungi generally occur in our most vulnerable patients, especially those with compromised immune function (Brown et al., 2012). These infections have become increasingly common, especially with advancing medical interventions that specifically target the immune system to treat human conditions as wide-ranging as classical auto-immune diseases and many previously untreatable cancers. Unlike their bacterial counterparts, fungal pathogens as eukaryotes are physiologically quite similar to the hosts that they infect. Therefore, one of the greatest challenges of antifungal drug discovery is to identify fungal-specific cellular features that can be specifically targeted and inhibited pharmacologically. Even more vexing is the issue of fungal cell permeability. Many compounds demonstrate potent inhibitory effects against essential fungal processes, but few of these compounds can effectively and stably enter the fungal cell. Screening of natural products for antifungal activity has historically been one of the most effective strategies for identifying compounds that can be considered good drug candidates (Roemer et al., 2011). These screening methods are designed to identify compounds that actually enter and inhibit the fungal cell. Moreover, natural product screens have identified the major classes of antifungal agents in current clinical use. These classes include two drug families, the polyenes and azoles, that disrupt different steps in the homeostasis of ergosterol, the primary sterol in fungal membranes. Limitations of this approach include the fact that the same classes of
naturally occurring antifungal compounds seem to be repeatedly identified by unbiased screening approaches (Roemer et al., 2011). The echinocandins, one of the more recently introduced classes of antifungals, inhibit the production of a major carbohydrate component of the fungal cell wall (Douglas et al., 1997). This fungalspecific feature is responsible for providing cellular structural strength and resilience among diverse fungal species. Cells with defective cell walls are compromised for growth as well as resistance to exogenous stress. In addition to its protective functions, the cell wall serves as an important antigenic surface for interaction of fungal pathogens with host immune cells. In fact, the fungal cell wall carbohydrate b-1,3-D-glucan is specifically recognized by the C-type lectin, pattern recognition receptor (PRR) dectin-1 (Brown et al., 2003). This glucanPRR recognition triggers the activation of a cascading series of signaling events resulting in immune activation to a fungal challenge. Importantly, b-1,3-D-glucan synthase is the target of echinocandins, explaining the deleterious effects of this drug class on cell wall integrity (Douglas et al., 1997). The echinocandins are exceptionally useful clinically due to their profound efficacy against many of the most common fungal pathogens of humans. These compounds typically have excellent activity for infections due to Candida albicans, a common cause of human bloodstream infections, as well as other tissue invasive infections in immunocompromised patients (reviewed in Pianalto and Alspaugh, 2016). Moreover, echinocandins have
been one of the most important last treatment options for highly drug-resistant fungi such as recently identified Candida auris (Dudiuk et al., 2019). Echinocandins also have clinical indications in infections due to Aspergillus species. However, perhaps due to the widespread use of these agents in clinical practice, resistance to echinocandins is increasingly observed, even among fungal species that were universally susceptible to these agents only a few years ago. Several investigators have defined mechanisms of increasing echinocandin tolerance and frank echinocandin resistance among diverse fungal species. These resistance mechanisms include impaired fungal cell entry as well as point mutations in the FKS1 gene encoding b-1,3-D-glucan synthase (Walker et al., 2010). Given the very limited number of antifungal agents, combatting antifungal resistance is of paramount importance. This paradigm motivated the studies outlined in the article by Caplan et al. (2020). The investigators screened a library of diverse compounds with predicted inhibitory activity against mammalian kinases, looking for those that would increase the antifungal effect of an echinocandin drug, caspofungin, against C. albicans. Therefore, this screening strategy was not designed to find new antifungal agents per se, but to identify potentiators of a currently used drug. Importantly, they identified several small molecules with a common chemical scaffold, lending great confidence to the robustness of this screening strategy. Using a combination of complementary experimental approaches, they identified the mammalian Yck2 kinase as the target of their most active compound
Cell Chemical Biology 27, March 19, 2020 ª 2020 Elsevier Ltd. 255
Cell Chemical Biology
Previews
(GW461484A). These screening approaches included fungal haplo-insufficiency profiling, a technique built upon the premise that a reduction in the dose of a target enzyme will result in altered susceptibility to the screening compound. In their studies, Caplan et al. treated a large collection of heterozygous C. albicans mutant strains with caspofungin, observing for alterations in cell growth. In this manner, they identified many caspofungin-hypersusceptible strains with mutations in genes involved in actin-related processes, including protein trafficking and cell division. Three of these mutations were in protein kinaseencoding genes, including the casein kinase 1 family members Yck2 and Hrr25. Specific overexpression of C. albicans YCK2, and not HRR25, resulted in relative caspofungin resistance, and YCK2 repression led to enhanced drug susceptibility. The fungal genetics experiments were complemented by in vitro kinase activity assays in which GW461484A inhibited the phosphorylation of a casein peptide substrate by recombinant Yck2. Structural studies of C. albicans Yck2 in its apo-form as well as in the presence of drug suggested a precise mechanism of GW416148A activity—to competitively occupy the ATP-binding pocket of this enzyme. Fungal-specific features of this interaction were also inferred, and these observations may provide direction for future compound derivatization. One of the limitations of advancing pyrazolopyridines such as GW461148A for clinical use is their recognized vulnerability to oxidative degradation at the electron-rich C6-C7 double bond. Therefore, a number of structurally similar compounds were tested for antifungal activity and chemical stability. These studies provide a theoretical foundation to derive future compounds to retain optimal activity against the fungal kinase while promoting enhanced chemical stability. Follow-up work in this area will be critical to determine the ultimate translational relevance of these initial studies. Because of the pharmacological instability of the pyrazolopyridines, efficacy of GW461148A in a relevant animal model of fungal infection was not reported. However, this compound did enhance the
antifungal effect of caspofungin in a macrophage-fungal co-culture system, protecting the mammalian cell monolayer from fungal-mediated disruption and without resulting in obvious host cell toxicity. Consistent with prior studies (Blankenship et al., 2010; Jung et al., 2017), genetic inhibition of C. albicans Yck2 activity impaired in vitro correlates of virulence as well as the pathogenesis of a systemic infection in mice. Although these initial studies were performed primarily for C. albicans, the investigators also noted significant activity of GW461148A against other common fungal pathogens, including the drugresistant C. auris, and the echinocandintolerant neuropathogen Cryptococcus neoformans. Measurable, but more limited, activity against the mold pathogen Aspergillus fumigatus was also observed. Is there room in the clinical arena for agents that primarily enhance the activity of current agents? I propose that this question has already been answered. Aminoglycosides are used to provide antibacterial synergy with other antibiotics against many pathogens. Beta lactamase inhibitors enhance the activity of antibiotics against resistant pathogens. Combination therapy for infections as diverse as tuberculosis and cryptococcosis demonstrates superior efficacy compared to monotherapy, both to prevent the development of resistance and to more rapidly sterilize the infected tissue. In summary, these combined studies are remarkable in both their breadth and focus. Beginning with unbiased compound screening, the investigators identified a chemical activity and common scaffold with previously unrecognized potential for antifungal activity, especially to enhance the effect of available antifungal agents. These initial compounds were tested using sophisticated microbial genetics tools to infer mechanism of activity, which was subsequently confirmed biochemically. Their structural studies can be used to direct chemical modifications to enhance fungal selectivity. This is especially important given the generalizability of antifungal activity demonstrated in multiple, diverse fungal pathogens. Despite these experimental strengths, transla-
256 Cell Chemical Biology 27, March 19, 2020
tional challenges remain. Despite possessing convincing antimicrobial activity, these compounds are still not yet drugs given their current pharmacological instability. These latter observations underscore the importance of rigorous medical chemistry to provide the translational bridge from the atomic and molecular scale to the patient.
REFERENCES Blankenship, J.R., Fanning, S., Hamaker, J.J., and Mitchell, A.P. (2010). An extensive circuitry for cell wall regulation in Candida albicans. PLoS Pathog. 6, e1000752. Brown, G.D., Herre, J., Williams, D.L., Willment, J.A., Marshall, A.S., and Gordon, S. (2003). Dectin-1 mediates the biological effects of betaglucans. J. Exp. Med. 197, 1119–1124. Brown, G.D., Denning, D.W., Gow, N.A., Levitz, S.M., Netea, M.G., and White, T.C. (2012). Hidden killers: human fungal infections. Sci. Transl. Med. 4, 165rv13. Caplan, T., Lorente-Macı´as, A´., Stogios, P.J., Evdokimova, E., Hyde, S., Wellington, M.A., Liston, S., Iyer, K.R., Puumala, E., ShekharGuturja, T., et al. (2020). Overcoming fungal echinocandin resistance through inhibition of the nonessential stress kinase Yck2. Cell Chem. Biol. 27, this issue, 269–282. Douglas, C.M., D’Ippolito, J.A., Shei, G.J., Meinz, M., Onishi, J., Marrinan, J.A., Li, W., Abruzzo, G.K., Flattery, A., Bartizal, K., et al. (1997). Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob. Agents Chemother. 41, 2471–2479. Dudiuk, C., Berrio, I., Leonardelli, F., MoralesLopez, S., Theill, L., Macedo, D., YesidRodriguez, J., Salcedo, S., Marin, A., Gamarra, S., and Garcia-Effron, G. (2019). Antifungal activity and killing kinetics of anidulafungin, caspofungin and amphotericin B against Candida auris. J. Antimicrob. Chemother. 74, 2295–2302. Jung, S.I., Rodriguez, N., Irrizary, J., Liboro, K., Bogarin, T., Macias, M., Eivers, E., Porter, E., Filler, S.G., and Park, H. (2017). Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans. PLoS One 12, e0187721. Pianalto, K.M., and Alspaugh, J.A. (2016). New horizons in antifungal therapy. J. Fungi (Basel) 2, https://doi.org/10.3390/jof2040026. Roemer, T., Xu, D., Singh, S.B., Parish, C.A., Harris, G., Wang, H., Davies, J.E., and Bills, G.F. (2011). Confronting the challenges of natural product-based antifungal discovery. Chem. Biol. 18, 148–164. Walker, L.A., Gow, N.A., and Munro, C.A. (2010). Fungal echinocandin resistance. Fungal Genet. Biol. 47, 117–126.