20
2.4 Blocking Siderophore as an Alternative Approach
Toward Antifungal Therapy
The physiological conditions of host cell are not favorable for the growth of pathogen under scarcity of free iron concentration due to the presence of iron-binding
proteins, e.g., transferrin and ferritin. Further, the condition is more aggravated by
the release of iron chelators and iron sequestering agents which compete with fungal siderophores for the acquisition of iron. However, the immune function of host
is misbalanced in condition of severe fungal infections where the recruitment of
iron deficiency itself in the pathogen can be a way to bring fungicidal effect. Such
condition can be generated by blocking the synthesis of siderophore where the following discussed approaches were proven to be highly beneficial in mitigating the
fungal pathogenesis and its virulence.
2.4.1 Enzymatic Inhibitors Blocking the Production
of Siderophore
The highly impactful approach to inhibit iron-dependent fungal infection is to target
iron requirement itself which will act as a fungicidal condition, preventing the
growth of fungus. One of the best ways to achieve this condition is to block the biosynthesis of siderophore. The generalized protocol adopted for identifying compounds which can inhibit siderophore production was to grow the pathogen
(A. fumigatus, biomonitor organism) in iron-limiting and iron-rich medium as
described earlier in Sect. 2.3.1 (Pinto and Moore 2009). Catalytic site inhibitors of
important enzymes which are involved in biosynthetic pathway of siderophore production are method of choice followed by many research groups who are active in
this area. The initial step of N5 hydroxylation of L-ornithine in siderophore biosynthetic pathway is a crucial step and catalyzed by NMO encoded by SidA gene which
can be a target of choice in A. fumigatus due to absence of homologue of SidA in
humans. The crystal structure of this protein with and without ligand is present in
Protein Data Bank (PDB) which can be utilized for designing site-directed inhibitors
of NMO. By using fluorescence polarization (FP) binding assay, screening of library
of putative inhibitors of NMO has been carried out which results in identification of
sanguinarine sulfate as a competitive inhibitor in A. fumigates (Olucha and Lamb
2011). The developed assay exhibits good tolerance to temperature, incubation time,
and concentration of dimethylsulfoxide (Qi et al. 2012; Robinson et al. 2015).
Subrado and coworkers utilized the homologous approach and screened 2320 compounds using FP-based high-throughput screening assay system for identifying
inhibitors of NMO. The study suggested that celastrol (a natural quinone analogue)
was found to inhibit NMO in a noncompetitive fashion by reported MIC of 2 μM
(Martín del Campo et al. 2016). In biological evaluation, the compound shows no
growth of pathogen (A. fumigatus) on fungal growth medium. Absence of growth
clearly reflects that the celastrol binding leads to SidA inhibition and, thus, blocks
S. Bhatia and S. Singh
2.4 Blocking Siderophore as an Alternative Approach
Toward Antifungal Therapy
The physiological conditions of host cell are not favorable for the growth of pathogen under scarcity of free iron concentration due to the presence of iron-binding
proteins, e.g., transferrin and ferritin. Further, the condition is more aggravated by
the release of iron chelators and iron sequestering agents which compete with fungal siderophores for the acquisition of iron. However, the immune function of host
is misbalanced in condition of severe fungal infections where the recruitment of
iron deficiency itself in the pathogen can be a way to bring fungicidal effect. Such
condition can be generated by blocking the synthesis of siderophore where the following discussed approaches were proven to be highly beneficial in mitigating the
fungal pathogenesis and its virulence.
2.4.1 Enzymatic Inhibitors Blocking the Production
of Siderophore
The highly impactful approach to inhibit iron-dependent fungal infection is to target
iron requirement itself which will act as a fungicidal condition, preventing the
growth of fungus. One of the best ways to achieve this condition is to block the biosynthesis of siderophore. The generalized protocol adopted for identifying compounds which can inhibit siderophore production was to grow the pathogen
(A. fumigatus, biomonitor organism) in iron-limiting and iron-rich medium as
described earlier in Sect. 2.3.1 (Pinto and Moore 2009). Catalytic site inhibitors of
important enzymes which are involved in biosynthetic pathway of siderophore production are method of choice followed by many research groups who are active in
this area. The initial step of N5 hydroxylation of L-ornithine in siderophore biosynthetic pathway is a crucial step and catalyzed by NMO encoded by SidA gene which
can be a target of choice in A. fumigatus due to absence of homologue of SidA in
humans. The crystal structure of this protein with and without ligand is present in
Protein Data Bank (PDB) which can be utilized for designing site-directed inhibitors
of NMO. By using fluorescence polarization (FP) binding assay, screening of library
of putative inhibitors of NMO has been carried out which results in identification of
sanguinarine sulfate as a competitive inhibitor in A. fumigates (Olucha and Lamb
2011). The developed assay exhibits good tolerance to temperature, incubation time,
and concentration of dimethylsulfoxide (Qi et al. 2012; Robinson et al. 2015).
Subrado and coworkers utilized the homologous approach and screened 2320 compounds using FP-based high-throughput screening assay system for identifying
inhibitors of NMO. The study suggested that celastrol (a natural quinone analogue)
was found to inhibit NMO in a noncompetitive fashion by reported MIC of 2 μM
(Martín del Campo et al. 2016). In biological evaluation, the compound shows no
growth of pathogen (A. fumigatus) on fungal growth medium. Absence of growth
clearly reflects that the celastrol binding leads to SidA inhibition and, thus, blocks
S. Bhatia and S. Singh