PIF-Pocket as a Target for C. albicans Pkh Selective Inhibitors

Abstract

The phosphoinositide-dependent protein kinase 1, PDK1, is a master kinase that phosphorylates the activation loop of up to 23 AGC kinases. S. cerevisiae has three PDK1 orthologues, Pkh1-3, which also phosphorylate AGC kinases (e.g., Ypk, Tpk, Pkc1, and Sch9). Pkh1 and 2 are redundant proteins involved in multiple essential cellular functions, including endocytosis and cell wall integrity. Based on similarities with the budding yeast, the Pkh of fungal infectious species was postulated as a novel target for antifungals. Here, we found that depletion of Pkh eventually induces oxidative stress and DNA double-strand breaks, leading to programmed cell death. This finding supports Pkh as an antifungal target since pharmacological inhibition of Pkh would lead to the death of yeast cells, the ultimate goal of antifungals. It was therefore of interest to further investigate the possibility to develop Pkh inhibitors with selectivity for Candida Pkh that would not inhibit the human ortholog. Here, we describe C. albicans Pkh2 biochemically, structurally and by using chemical probes in comparison to human PDK1. We found that a regulatory site on the C. albicans Pkh2 catalytic domain, the PIF-pocket, diverges from human PDK1. Indeed, we identified and characterized PS77, a new small allosteric inhibitor directed to the PIF-pocket, which has increased selectivity for C. albicans Pkh2. Together, our results describe novel features of the biology of Pkh and chemical biology approaches that support the validation of Pkh as a drug target for selective antifungals.

Depletion of yeast PDK1 orthologs triggers a stress-like transcriptional response

“…This is not unexpected, reinforcing the notion that Pkh proteins have redundant functions and that the lack of one Pkh can be probably compensated by the presence of any of the other two Pkh proteins. Now, we have constructed the SDP8 yeast strain (Table 1 ) in which PKH1 and PKH3 genes are deleted and the quantity of Pkh2 can be progressively decreased by incubation in the presence of doxycycline [ 15 ]. According to quantitative RT-PCR experiments, incubation of the cells containing the tetO 7 - PKH2 construct for 24 h in the presence of 100 μg/ml doxycycline reduced more than 200-fold the expression of PKH2 (not shown).…”

“…According to quantitative RT-PCR experiments, incubation of the cells containing the tetO 7 - PKH2 construct for 24 h in the presence of 100 μg/ml doxycycline reduced more than 200-fold the expression of PKH2 (not shown). At this point most of the cells are still viable [ 15 ], probably because enough amounts of Pkh2 protein could still be present in the cell (the estimated half-life of Pkh2 is longer than 20 h). In order to gain insight into the cellular roles of the yeast PDK1 kinases, we decided to use this cellular system to identify the transcriptomic changes triggered by progressive depletion of Pkh proteins.…”

“…Sch9 phosphorylation by Pkh regulates lifespan and oxidative stress sensitivity in a process that depends on sphingolipids [ 13 , 14 ]. Phosphorylation of Pkc1 activates the Slt2 MAPK cascade and is crucial for maintaining cell wall integrity (CWI) [ 9 , 15 ].…”

“…In the case of the essential family of Pkh protein kinases most of the information obtained has been acquired using a Pkh1 D398G temperature-sensitive allele in combination with deletion of the PKH2 gene and incubation at the restrictive temperature of 37 °C. We have recently reported an alternative genetic strategy to deplete cells of Pkh activity circumventing the incubation at stressful temperatures [ 15 ]. In the newly generated strains the expression of PKH2 , controlled by the tetO 7 promoter, is decreased by addition of doxycycline.…”

“…Our strategy allows the phenotypic analysis caused by depletion of Pkh with no need of incubation of cells at 37 °C, a temperature that triggers the activation of the CWI pathway. This approach has been successfully used to demonstrate the importance of Pkh in the activation of the CWI pathway in the absence of a heat shock [ 15 ].…”