22
NRPS enzymes have various domains each with specific functions like adenylation, thiolation, condensation, etc. These domains are either arranged in linear or
nonlinear (as present in fungal NRPS) fashion. Researchers are also exploring the
inhibitors of adenylating enzymes by employing rational drug design strategy to
inhibit stand-alone adenylating domain. So far, this approach is utilized for inhibiting the bacterial species like Mycobacterium tuberculosis, Pseudomonas aeruginosa, and E. Coli (Finking et al. 2003; Ferreras et al. 2005). On similar lines, the
inhibitors of salicylate adenylation enzymes (salicyl-AMS) were also examined in
the hope that there mechanism of blocking the substitution of acylphosphate group
in adenylating enzymes will prove to be a novel way to curb siderophore biosynthesis. However, this strategy is having limitation in terms of toxicity as this enzyme is
having close homology with aminoacyl-tRNA synthetase of host which is an important regulating enzyme in ribosomal protein synthesis. This problem can be overcome by targeting selectively NRPS-adenylating domains of pathogen by employing
ligands carrying macrocyclic structural framework, and thus, these ligands inhibit
the enzymes in different conformational fashion as compared to aminoacyl-tRNA
synthetases, and thus such selective inhibition approaches open new routes to
develop novel antibiotics (Cisar et al. 2007).
In case of fungi, the several approaches adopted to inhibit adenylate domain
specifically are still in the emerging phase (Stack et al. 2007). Lee et al. have
reported the crystal structure of the third domain of siderophore-synthesizing NRPS
enzyme of Neotyphodium lolii fungal pathogen (Lee et al. 2010a, b). The structural
insights revealed the large binding pocket of adenylating enzymes for binding bulky
peptidal substrate. The active site was found to have 17 amino acid residues with the
divergence in signature sequences which were found in comparison to prokaryotes.
In such scenario, the rational use of homology modeling is quite effective for comparing the structure and sequence of unknown domains with sequence of known
specificity. Thus, the major focus was to develop precise methods for identifying
substrate specificity toward domains of unknown specificity in NRPS enzymes. In
spite of available literature on structural features of eukaryotic NRPS and its mechanism of regulation, more such studies are needed to understand thoroughly the
nature of this complex process, in order to design domain-specific inhibitors against
fungal NRPS (Lee et al. 2010a, b).
2.4.2 Statins
Drugs belonging to statin class are often prescribed for lowering blood cholesterol levels. HMG-CoA reductase is the rate-controlling enzyme for mevalonate pathway which
leads to biosynthesis of cholesterol. Thus, statins block the synthesis of mevalonate by
inhibiting HMG-CoA reductase through competitive inhibition as shown in Fig. 2.4.
During biosynthesis of siderophores, mevalonate motif acts as an important structural
template in structural framework of extracellular siderophores. Hence, mevalonate
S. Bhatia and S. Singh
NRPS enzymes have various domains each with specific functions like adenylation, thiolation, condensation, etc. These domains are either arranged in linear or
nonlinear (as present in fungal NRPS) fashion. Researchers are also exploring the
inhibitors of adenylating enzymes by employing rational drug design strategy to
inhibit stand-alone adenylating domain. So far, this approach is utilized for inhibiting the bacterial species like Mycobacterium tuberculosis, Pseudomonas aeruginosa, and E. Coli (Finking et al. 2003; Ferreras et al. 2005). On similar lines, the
inhibitors of salicylate adenylation enzymes (salicyl-AMS) were also examined in
the hope that there mechanism of blocking the substitution of acylphosphate group
in adenylating enzymes will prove to be a novel way to curb siderophore biosynthesis. However, this strategy is having limitation in terms of toxicity as this enzyme is
having close homology with aminoacyl-tRNA synthetase of host which is an important regulating enzyme in ribosomal protein synthesis. This problem can be overcome by targeting selectively NRPS-adenylating domains of pathogen by employing
ligands carrying macrocyclic structural framework, and thus, these ligands inhibit
the enzymes in different conformational fashion as compared to aminoacyl-tRNA
synthetases, and thus such selective inhibition approaches open new routes to
develop novel antibiotics (Cisar et al. 2007).
In case of fungi, the several approaches adopted to inhibit adenylate domain
specifically are still in the emerging phase (Stack et al. 2007). Lee et al. have
reported the crystal structure of the third domain of siderophore-synthesizing NRPS
enzyme of Neotyphodium lolii fungal pathogen (Lee et al. 2010a, b). The structural
insights revealed the large binding pocket of adenylating enzymes for binding bulky
peptidal substrate. The active site was found to have 17 amino acid residues with the
divergence in signature sequences which were found in comparison to prokaryotes.
In such scenario, the rational use of homology modeling is quite effective for comparing the structure and sequence of unknown domains with sequence of known
specificity. Thus, the major focus was to develop precise methods for identifying
substrate specificity toward domains of unknown specificity in NRPS enzymes. In
spite of available literature on structural features of eukaryotic NRPS and its mechanism of regulation, more such studies are needed to understand thoroughly the
nature of this complex process, in order to design domain-specific inhibitors against
fungal NRPS (Lee et al. 2010a, b).
2.4.2 Statins
Drugs belonging to statin class are often prescribed for lowering blood cholesterol levels. HMG-CoA reductase is the rate-controlling enzyme for mevalonate pathway which
leads to biosynthesis of cholesterol. Thus, statins block the synthesis of mevalonate by
inhibiting HMG-CoA reductase through competitive inhibition as shown in Fig. 2.4.
During biosynthesis of siderophores, mevalonate motif acts as an important structural
template in structural framework of extracellular siderophores. Hence, mevalonate
S. Bhatia and S. Singh