373
of AMP LLKKK18 has been explored. These nanogels were found to be less cytotoxic as well as more resistant to degradation by host proteases, thereby increasing
the potential for its use as an antimycobacterial agent (Silva et al. 2016).
Another analog of LL37 peptide called D5 was designed by Jiang et al. (2011).
In D5, three alanine residues of LL37 were substituted with more hydrophobic leucine residues, and a valine at position 16 is substituted with lysine.
Five peptide analogs, D1–D5, were synthesized de novo by Jiang et al. (2011).
The peptides were synthesized using t-butyloxy-carbonyl (t-Boc) chemistry and
4-methylbenzhydrylamine resin by solid-phase synthesis methodology. The peptides were cleaved from the resins and purified by reversed-phase high-performance
liquid chromatography (RP-HPLC) on a Zorbax 300 SB-C8 column. A gradient of
0.2% aqueous trifluoroacetic acid (TFA), pH 2, and 0.2% TFA in acetonitrile was
used as the mobile phase. The purity of the peptides was verified by analytical
RP-HPLC, and the peptides were characterized by mass spectrometry (LC/MS) and
amino acid analysis. Characterization of helical structure was done using circular
dichroism (CD) spectroscopy, and peptide concentrations were determined by
amino acid analysis.
Antimycobacterial activity of peptides was determined by adding known concentrations of peptides to fresh mycobacterial suspension. After initial incubation
for a week, these suspensions were added to 7H11 solid plates, and colony-forming
units were enumerated after 3 weeks of growth. Peptide D5 (Iwatsuki et al. 2007)
had the highest antimycobacterial activity compared to LL37 and all its derivatives.
The aforementioned substitutions also resulted in decreased hydrophobicity,
amphipathicity, helicity, self-association ability and hemolytic activity of the peptides. The mechanism of action of D5 includes membrane disruption and biofilm
disassembly (McGrath et al. 2013; Carmona et al. 2013; Kolodkin-Gal et al. 2010).
15.4 Envelope Inhibitors
15.4.1 RNase 3
RNase 3 was first identified as an eosinophil secretion product and named eosinophil cationic protein (ECP) (Ahlstedt 1995). Activated eosinophils secrete ECP during inflammation, and its levels in biological fluids are used as a marker for the
diagnosis of allergy and eosinophilia disorders. RNase 3 is a small cationic protein
belonging to RNase A superfamily. It shows potent antibacterial and antiparasitic
activities (Venge et al. 1999; Bystrom et al. 2011). It was shown that eosinophils can
mediate their antibacterial effect through the release of cationic granule proteins
(Linch et al. 2009). Antibacterial action of ECP was shown by binding to anionic
sites, i.e., LPS on bacterial membranes (Boix et al. 2012). In addition to its antibacterial activity, ECP also has immunomodulatory activity; it regulates fibroblasts,
induces mucous secretions and exerts cytotoxic and neurotoxic activities in mammalian cells (de Oliveira et al. 2012). It was shown that in combination with a
15 Antimycobacterial Peptides
of AMP LLKKK18 has been explored. These nanogels were found to be less cytotoxic as well as more resistant to degradation by host proteases, thereby increasing
the potential for its use as an antimycobacterial agent (Silva et al. 2016).
Another analog of LL37 peptide called D5 was designed by Jiang et al. (2011).
In D5, three alanine residues of LL37 were substituted with more hydrophobic leucine residues, and a valine at position 16 is substituted with lysine.
Five peptide analogs, D1–D5, were synthesized de novo by Jiang et al. (2011).
The peptides were synthesized using t-butyloxy-carbonyl (t-Boc) chemistry and
4-methylbenzhydrylamine resin by solid-phase synthesis methodology. The peptides were cleaved from the resins and purified by reversed-phase high-performance
liquid chromatography (RP-HPLC) on a Zorbax 300 SB-C8 column. A gradient of
0.2% aqueous trifluoroacetic acid (TFA), pH 2, and 0.2% TFA in acetonitrile was
used as the mobile phase. The purity of the peptides was verified by analytical
RP-HPLC, and the peptides were characterized by mass spectrometry (LC/MS) and
amino acid analysis. Characterization of helical structure was done using circular
dichroism (CD) spectroscopy, and peptide concentrations were determined by
amino acid analysis.
Antimycobacterial activity of peptides was determined by adding known concentrations of peptides to fresh mycobacterial suspension. After initial incubation
for a week, these suspensions were added to 7H11 solid plates, and colony-forming
units were enumerated after 3 weeks of growth. Peptide D5 (Iwatsuki et al. 2007)
had the highest antimycobacterial activity compared to LL37 and all its derivatives.
The aforementioned substitutions also resulted in decreased hydrophobicity,
amphipathicity, helicity, self-association ability and hemolytic activity of the peptides. The mechanism of action of D5 includes membrane disruption and biofilm
disassembly (McGrath et al. 2013; Carmona et al. 2013; Kolodkin-Gal et al. 2010).
15.4 Envelope Inhibitors
15.4.1 RNase 3
RNase 3 was first identified as an eosinophil secretion product and named eosinophil cationic protein (ECP) (Ahlstedt 1995). Activated eosinophils secrete ECP during inflammation, and its levels in biological fluids are used as a marker for the
diagnosis of allergy and eosinophilia disorders. RNase 3 is a small cationic protein
belonging to RNase A superfamily. It shows potent antibacterial and antiparasitic
activities (Venge et al. 1999; Bystrom et al. 2011). It was shown that eosinophils can
mediate their antibacterial effect through the release of cationic granule proteins
(Linch et al. 2009). Antibacterial action of ECP was shown by binding to anionic
sites, i.e., LPS on bacterial membranes (Boix et al. 2012). In addition to its antibacterial activity, ECP also has immunomodulatory activity; it regulates fibroblasts,
induces mucous secretions and exerts cytotoxic and neurotoxic activities in mammalian cells (de Oliveira et al. 2012). It was shown that in combination with a
15 Antimycobacterial Peptides