Inhibition of Sterylglucosidases as Novel Antifungal Targets

Invasive fungal infections by fungi such as C. neoformans and A. fumigatus have devastating effects on human health and quality of life. Exposure to both C. neoformans and A. fumigatus is quite common through air-borne spores, and immunocompetent individuals mount an effective immune response that results in small granulomas within the lung that house dormant spores.^1,2 People with compromised immune systems are not able to produce an suitable immune response, which allows these fungi to grow and ultimately progress to their symptomatic diseases; cryptococcosis and aspergillosis, respectively. Aspergillosis and cryptococcosis have unique disease pathways, with C. neoformans propagating from the lungs to the central nervous system, causing inflammation and lethal meningoencephalitis, while A. fumigatus remains within the lungs and grows into large fungal tumors. Despite these differences, both C. neoformans and A. fumigatus require sterylglucosidase activity to avoid the mammalian immune response. Sterylglucosidase - a fungi-specific enzyme - hydrolyzes sterylglucosides, such as Ergosterol-3β-D-glucoside (ErgGlc) shown in Figure 1.

Figure 1. ErgGlc hydrolysis by sterylglucosidase

Sterylglucosidase expressed by both C. neoformans (Sgl1) and A. fumigatus (SglA) have been validated as anti-fungal drug targets. As shown in Figure 17, both wild type (WT) and reconstituted fungal cells (∆sgl+SGL) show steep survival curves, while the genetic ablation (∆sgl) hovers at 100% survival.^3,4 This definitively shows that sterylglucosidase activity is required for pathogenicity of both A. fumigatus and C. neoformans.

Figure 2. Sgl1 (left) and SglA (right) target validation.

Crystal structures of both Sgl1 and SglA have been resolved, and high throughput screening has yielded hit compounds.^5,3 Two of the hit compounds resulting from the Sgl1 high throughput screening were successfully crystalized with Sgl1, elucidating the catalytic site (Figure 3 and Figure 4).

Figure 3. Hit 1-Sgl1 crystal structure (PDB: 7LPP)

Figure 4. Hit 9-Sgl1 crystal structure (PDB: 7LPQ)

The SglA high throughput screening identified two unique hit compounds, as shown in Figure 5.

Figure 5. Hit compounds from high throughput screening of SglA.

Using these data, we aim to use computer-aided drug design (CADD) to develop Sgl1 and SglA inhibitors to treat cryptococcosis and aspergillosis, respectively.

Associated publications:

(1)       Kechichian, T. B.; Shea, J.; Del Poeta, M. Depletion of Alveolar Macrophages Decreases the Dissemination of a Glucosylceramide-Deficient Mutant of Cryptococcus Neoformans in Immunodeficient Mice. Infection and Immunity 2007, 75 (10), 4792–4798. https://doi.org/10.1128/IAI.00587-07.

(2)       McQuiston, T.; Luberto, C.; Del Poeta, M. Role of Host Sphingosine Kinase 1 in the Lung Response against Cryptococcosis. Infection and Immunity 2010, 78 (5), 2342–2352. https://doi.org/10.1128/IAI.01140-09.

(3)       Pereira de Sa, N.; Jayanetti, K.; Rendina, D.; Clement, T.; Soares Brauer, V.; Mota Fernandes, C.; Ojima, I.; Airola, M. V.; Del Poeta, M. Targeting Sterylglucosidase A to Treat Aspergillus Fumigatus Infections. mBio 2023, 14 (2), e0033923. https://doi.org/10.1128/mbio.00339-23.

(4)       Rella, A.; Mor, V.; Farnoud, A. M.; Singh, A.; Shamseddine, A. A.; Ivanova, E.; Carpino, N.; Montagna, M. T.; Luberto, C.; Del Poeta, M. Role of Sterylglucosidase 1 (Sgl1) on the Pathogenicity of Cryptococcus Neoformans: Potential Applications for Vaccine Development. Frontiers in Microbiology 2015, 6.

(5)       Pereira de Sa, N.; Taouil, A.; Kim, J.; Clement, T.; Hoffmann, R. M.; Burke, J. E.; Rizzo, R. C.; Ojima, I.; Del Poeta, M.; Airola, M. V. Structure and Inhibition of Cryptococcus Neoformans Sterylglucosidase to Develop Antifungal Agents. Nat Commun 2021, 12 (1), 5885. https://doi.org/10.1038/s41467-021-26163-5.