As a consequence of the fast resistance spreading, a limited number of drugs are available
to treat fungal infections. Therefore, there is an urgent need to develop new antifungal
treatment strategies. The features of a disulfide bond-stabilized antifungal protein,
NFAP2 secreted by the mold Neosartorya (Aspergillus) fischeri render it to be a promising
template for future protein-based antifungal drug design, which requires knowledge
about the native disulfide linkage pattern as it is one of the prerequisites for biological
activity. However, in the lack of tryptic and chymotryptic proteolytic sites in the
ACNCPNNCK sequence, the determination of the disulfide linkage pattern of NFAP2 is
not easy with traditional mass spectrometry-based methods. According to in silico
predictions working with a preliminary nuclear magnetic resonance (NMR) solution structure,
two disulfide isomers of NFAP2 (abbacc and abbcac) were possible. Both were chemically
synthesized; and comparative reversed-phase high-performance liquid chromatography,
electronic circular dichroism and NMR spectroscopy analyses, and antifungal susceptibility
and efficacy tests indicated that the abbcac is the native pattern. This knowledge
allowed rational modification of NAFP2 to improve the antifungal efficacy and spectrum
through the modulation of the evolutionarily conserved gamma-core region, which is
responsible for the activity of several antimicrobial peptides. Disruption of the
steric structure of NFAP2 upon gamma-core modification led to the conclusions that
this motif may affect the formation of the biologically active three-dimensional structure,
and that the gamma-core modulation is not an efficient tool to improve the antifungal
efficacy or to change the antifungal spectrum of NFAP2.
