In vitro studies, as well as analysis of patient samples, have been performed.
Investigators at the National Heart, Lung and Blood Institute have developed modified defensins that are resistant to degradation, have improved characteristics compared to unmodified defensins, and are promising candidates for pulmonary disease therapeutics.
Defensins are small cationic peptides that defend the lung against pathogenic microorganisms and play an important role in innate immunity. However, during lung inflammation, defensin concentrations can reach levels that are cytotoxic for airway epithelial cells. Therefore, the development of methods to produce modified defensins that exhibit reduced cytotoxicity, while retaining the ability to stimulate the innate immune response, would be of potential therapeutic benefit for pulmonary diseases.
The inventors have previously shown that a defensin, human neutrophil peptide 1 (HNP-1), is elevated in samples from the lungs of patients with inflammatory lung disease, and that the HNP-1 in these samples is ADP-ribosylated at one or both of two arginine residues within the protein. In vitro studies by the inventors show that ADP-ribosyl-HNP-1 has reduced cytotoxic activity compared to HNP-1, while retaining its T cell chemotactic properties and ability to promote neutrophil recruitment, and thus ADP-ribosyl-HNP-1 may play an important role as a regulator of the inflammatory response. These properties would also be useful for treatment of pulmonary inflammation and lung diseases. However, ADP-ribosylated HNP-1 and other defensins are degraded rapidly in vivo due to the susceptibility of the ADP-ribose moiety to attack by hydrolases and pyrophosphatases, which limits their therapeutic potential.
The inventors have recently discovered that the ADP-ribosylated arginine residues in HNP-1 can be converted to ornithine through a non-enzymatic process that results in a peptide with an altered pharmacological profile. The investigators have also successfully generated ornithine-substituted ADP-ribosyl HNP-1 and ornithine-HNP-1 in vitro, which are currently being characterized. Thus, ornithine-substituted ADP-ribosyl HNP-1 and ornithine-HNP-1 may be promising candidates for the development of therapeutics to treat pulmonary disease, and the strategy of replacing ADP-ribosylated residues with ornithine to enhance stability and therapeutic efficacy may also be extended to other defensins.
Through an earlier, related invention, the inventors have also demonstrated that recombinant proteins wherein tryptophan or phenylalanine residues substitute for ADP-ribosylarginine have a similar stabilizing impact on polypeptides, making them more suitable as therapeutic agents.
The inventors also hypothesize that it would be possible to develop a treatment that increases levels of an ADP-ribosylated therapeutic protein, such as HNP-1, in the lung via inhalation administration of the therapeutic protein in conjunction with nicotinamide adenine dinucleotide (NAD), which is required for ADP-ribosylation. This could represent a unique therapeutic strategy for treating pulmonary disease.
Development of defensin-based therapeutics that enhance the immune response in pulmonary disease patients, without damaging the epithelial cells lining the airway.
- Modified defensins are less cytotoxic, while retaining ability to stimulate innate immunity.
- Ornithine-substituted defensins are resistant to enzymatic degradation, making them more promising as drug candidates.