Selected Ion Monitoring Based Liquid Chromatography Coupled Mass Spectrometry as a New Tool to Study Enzyme Kinetics
Disciplines
Analytical Chemistry | Medicinal-Pharmaceutical Chemistry
Abstract (300 words maximum)
The characterization of protease inhibitors is essential to the process of assessing the efficacy of novel drug candidates and is an emerging field of interest for clinical researchers. Studies to provide data on the inhibitory effects and enzymatic activity of peptide inhibitors typically rely upon Förster Resonance Energy Transfer (FRET) assay as the industry standard; however, this assay are known to suffer from low signal to noise ratio (SNR) and complications that arise from the use of fluorescent labels, driving up costs and affecting the accuracy of key data to quantify therapeutic potential. Assays analyzed using selected-ion monitoring (SIM) based LC-MS is a promising low-cost, accessible alternative due to the improved specificity and SNR in comparison to the FRET-based assays. Research to further analyze the candidacy of antimicrobial peptides effective in their inhibition of the main protease (Mpro) of SARS-CoV-2 has been necessary given the global impact of COVID-19. In this study, a new SIM based LC-MS assay was developed and tested for characterizing the mode of inhibition and kinetics of two Temporin-L analogues, TLP-1 and TLP-3. The Michaelis-Menten kinetics results showed that the velocity of the product formation significantly decreased at the concentration of 6.25, 10 and 12 μM for the TLP3 with a Ki value of 2.31 µM. Lineweaver-Burk plots were used to obtain Vmax and Km. The analysis of the Lineweaver-Burk Plot revealed a steadily decreasing Vmax value as the concentrations of TLP3 increased. However, Km values remained unchanged. This result showed that TLP3 peptide can act as a noncompetitive inhibitor which can bind to free enzyme 3CLpro as well as the 3CLpro-substrate complex. In either case, the binding of peptide prevents the formation of product. Moreover, the noncompetitive inhibition of the peptide toward 3CLpro enzyme cannot be overcome by increasing substrate concentration.
Use of AI Disclaimer
no
Academic department under which the project should be listed
CSM – Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad Halim
Selected Ion Monitoring Based Liquid Chromatography Coupled Mass Spectrometry as a New Tool to Study Enzyme Kinetics
The characterization of protease inhibitors is essential to the process of assessing the efficacy of novel drug candidates and is an emerging field of interest for clinical researchers. Studies to provide data on the inhibitory effects and enzymatic activity of peptide inhibitors typically rely upon Förster Resonance Energy Transfer (FRET) assay as the industry standard; however, this assay are known to suffer from low signal to noise ratio (SNR) and complications that arise from the use of fluorescent labels, driving up costs and affecting the accuracy of key data to quantify therapeutic potential. Assays analyzed using selected-ion monitoring (SIM) based LC-MS is a promising low-cost, accessible alternative due to the improved specificity and SNR in comparison to the FRET-based assays. Research to further analyze the candidacy of antimicrobial peptides effective in their inhibition of the main protease (Mpro) of SARS-CoV-2 has been necessary given the global impact of COVID-19. In this study, a new SIM based LC-MS assay was developed and tested for characterizing the mode of inhibition and kinetics of two Temporin-L analogues, TLP-1 and TLP-3. The Michaelis-Menten kinetics results showed that the velocity of the product formation significantly decreased at the concentration of 6.25, 10 and 12 μM for the TLP3 with a Ki value of 2.31 µM. Lineweaver-Burk plots were used to obtain Vmax and Km. The analysis of the Lineweaver-Burk Plot revealed a steadily decreasing Vmax value as the concentrations of TLP3 increased. However, Km values remained unchanged. This result showed that TLP3 peptide can act as a noncompetitive inhibitor which can bind to free enzyme 3CLpro as well as the 3CLpro-substrate complex. In either case, the binding of peptide prevents the formation of product. Moreover, the noncompetitive inhibition of the peptide toward 3CLpro enzyme cannot be overcome by increasing substrate concentration.