Protease Stability of the Non-Covalent Staple Temporin L Peptide
Disciplines
Biochemistry | Medicinal-Pharmaceutical Chemistry
Abstract (300 words maximum)
Peptide therapeutics are very attractive over small-molecule medications, as they are highly selective, well-tolerated, and have less adverse effects. However, there are problems with making oral medications for peptide, as various digestive enzymes such as pepsin and trypsin degrade the peptide and prevent them from reaching the bloodstream and resulting in a stronger dosage. Our previous peptidomimetic design and in-vitro investigation revealed that a methylated analogue of Temporin L (TL) demonstrated an estimated IC50 at 4.57 μM which is higher than the natural TL peptide (38 µM) against the main protease (3CLpro) of SARS-CoV-2. However, generally this peptide has low stability against protease and liver metabolism. The aim of this research is to assess the proteases stability of Temporin L and its methylated pi-pi stable peptide in gastric and intestinal fluid containing proteases employing high performance liquid chromatography coupled with mass spectrometry (LCMS). In-vitro gastric and intestinal stability assay was performed by mixing the best staple peptide with gastric fluid containing pepsin and intestinal fluid comprising pancreatin at 37°C. Samples was removed at various time points and acetonitrile was added, then centrifuged, supernatant was taken and mixed with water and analyzed by LCMS. The absolute abundance of the intact peptide in MS was monitored compared to the abundance of the peptide at zero min (100%). The methylated Temporin L peptide showed increased half-life over 60 min in-vitro gastric fluid containing pepsin, however, the non-methylated TL showed half-life less than 30 mins. Molecular docking of these peptides targeting pepsin and trypsin was conducted to reveal the mechanism behind the improved stability of the methylated staple peptide.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad Halim
Protease Stability of the Non-Covalent Staple Temporin L Peptide
Peptide therapeutics are very attractive over small-molecule medications, as they are highly selective, well-tolerated, and have less adverse effects. However, there are problems with making oral medications for peptide, as various digestive enzymes such as pepsin and trypsin degrade the peptide and prevent them from reaching the bloodstream and resulting in a stronger dosage. Our previous peptidomimetic design and in-vitro investigation revealed that a methylated analogue of Temporin L (TL) demonstrated an estimated IC50 at 4.57 μM which is higher than the natural TL peptide (38 µM) against the main protease (3CLpro) of SARS-CoV-2. However, generally this peptide has low stability against protease and liver metabolism. The aim of this research is to assess the proteases stability of Temporin L and its methylated pi-pi stable peptide in gastric and intestinal fluid containing proteases employing high performance liquid chromatography coupled with mass spectrometry (LCMS). In-vitro gastric and intestinal stability assay was performed by mixing the best staple peptide with gastric fluid containing pepsin and intestinal fluid comprising pancreatin at 37°C. Samples was removed at various time points and acetonitrile was added, then centrifuged, supernatant was taken and mixed with water and analyzed by LCMS. The absolute abundance of the intact peptide in MS was monitored compared to the abundance of the peptide at zero min (100%). The methylated Temporin L peptide showed increased half-life over 60 min in-vitro gastric fluid containing pepsin, however, the non-methylated TL showed half-life less than 30 mins. Molecular docking of these peptides targeting pepsin and trypsin was conducted to reveal the mechanism behind the improved stability of the methylated staple peptide.