Leveraging Fungal Calcineurin-Inhibitor Structures, Biophysics and Dynamics to Design Selective and Non-Immunosuppressive FK506 Analogs

Abstract

Calcineurin is an attractive antifungal target due to its central role in fungal pathogenesis. The development of specific antifungals targeting calcineurin is complex, as calcineurin inhibitors, such as FK506, are immunosuppressive. Using fungal calcineurin-inhibitor crystal structures we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection. To better understand the interaction of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition at a molecular level, and guide the design of fungal-selective and nonimmunosuppressive FK506 analogs, here we report the high-resolution structures of the M. circinelloides FKBP12 bound to FK506 and the human, A. fumigatus and M. circinelloides FKBP12 proteins bound to the FK506 analog, APX879. Combining structural, genetic and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12:ligand complexes to enhance fungal-selectivity.

Interactions of FK506 and Rapamycin With FK506 Binding Protein 12 in Opportunistic Human Fungal Pathogens

“…The CnaA residue W377 is required for FK506-CnaA hydrophobic interaction and spontaneous mutation resulting in W377L was predicted to disrupt the hydrophobic pocket at the binding interface (Lee et al, 2015). Gobeil et al (2020) found that the amino acid sequence of McFKBP12 is 58% identical to HsFKBP12. The sequence variations were present in the 40, 50, and 80s extended loops surrounding the FK506 binding pocket.…”

“…The sequence variations were present in the 40, 50, and 80s extended loops surrounding the FK506 binding pocket. Interestingly, McFKBP12 sequence is 65% identical to AfFKBP12 and share structural similarity but functionally they are not equivalent in inhibiting calcineurin function (Gobeil et al, 2020). When Affkbp12 was replaced with Mcfkbp12 in A. fumigatus, FK506 was not able to inhibit calcineurin function.…”

“…As mentioned above, in the presence of FK506 the F88 residue in the 80s loop of AfFKBP12 is critical in the FK506-AfFKBP12-calcineurin interaction. Interestingly, McFKBP12 possesses a tyrosine at the 88 th residue instead of phenylalanine because of which McFKBP12-FK506 failed to inhibit calcineurin function in A. fumigatus ( Figure 3C; Gobeil et al, 2020).…”

“…These differences were exploited to generate an FK506 analog-APX879, characterized by an acetohydrazide substitution at C22ketone. The APX879 (Figure 2) interacts less favorably than FK506 with HsFKBP12 due to a higher steric clash with H88 residue (Juvvadi et al, 2019;Gobeil et al, 2020). Subsequently, APX879 was found to be 71-fold less immunosuppressive than FK506, as measured by their ability to induce IL-2 production from differentiated CD4 + T cells.…”