Enzyme Inhibition and Kinetics by Substrate-based Peptide Inhibitors Targeting SARS-CoV-2
Disciplines
Biochemistry | Medicinal-Pharmaceutical Chemistry | Organic Chemistry
Abstract (300 words maximum)
Peptide therapeutics are increasingly important in drug development due to their high specificity and potential as antiviral agents. This study focuses on substrate-based peptide inhibitors targeting the SARS-CoV-2 chymotrypsin-like protease (3CLpro), a critical enzyme in viral replication. Substrate-based peptide inhibitors were synthesized using CEM Liberty Blue peptide synthesizer with automated Fmoc solid phase synthesis protocols. The peptide was synthesized using Rink-amide resin with a loading capacity of 0.58 mmol/g under high-swelling conditions. The process involved repeated cycles of deprotection and coupling. After synthesis, the peptide-resin complex was cleaved with a 95% trifluoroacetic acid (TFA) solution, followed by precipitation and purification using diethyl ester. Mass spectrometry confirmed successful synthesis, showing a prominent peak at m/z 1393.75 corresponding to the expected [M+H]+ ion. To evaluate the peptide’s inhibitory activity, a selected ion monitoring (SIM) assay was performed using liquid chromatography-mass spectrometry (LCMS). The peptide demonstrated a consistent inhibitory effect across three trials, yielding an IC50 value of 4.10 micromolar. Moreover, LCMS based enzymatic kinetics assay was developed by synthesizing the N-terminal product of the NSP8/9 substrate SFVKLF/NNELSP. Initially, the velocity of the N-terminal product was obtained without adding the peptide inhibitor then various concentrations of the peptide inhibitors were used to monitor the reduction of the product velocity. Our results highlight promising antiviral candidates that effectively disrupt SARS-CoV-2 protease activity and inhibit viral replication, as evidenced by the observed decrease in the product velocity. Once consistent trends are established, we will determine key enzyme kinetic parameters—Km, Vmax, and Ki—to characterize the inhibitor’s potency and mechanism of action, including whether peptide inhibitors work competitively or noncompetitively.
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Academic department under which the project should be listed
CSM – Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad Halim
Enzyme Inhibition and Kinetics by Substrate-based Peptide Inhibitors Targeting SARS-CoV-2
Peptide therapeutics are increasingly important in drug development due to their high specificity and potential as antiviral agents. This study focuses on substrate-based peptide inhibitors targeting the SARS-CoV-2 chymotrypsin-like protease (3CLpro), a critical enzyme in viral replication. Substrate-based peptide inhibitors were synthesized using CEM Liberty Blue peptide synthesizer with automated Fmoc solid phase synthesis protocols. The peptide was synthesized using Rink-amide resin with a loading capacity of 0.58 mmol/g under high-swelling conditions. The process involved repeated cycles of deprotection and coupling. After synthesis, the peptide-resin complex was cleaved with a 95% trifluoroacetic acid (TFA) solution, followed by precipitation and purification using diethyl ester. Mass spectrometry confirmed successful synthesis, showing a prominent peak at m/z 1393.75 corresponding to the expected [M+H]+ ion. To evaluate the peptide’s inhibitory activity, a selected ion monitoring (SIM) assay was performed using liquid chromatography-mass spectrometry (LCMS). The peptide demonstrated a consistent inhibitory effect across three trials, yielding an IC50 value of 4.10 micromolar. Moreover, LCMS based enzymatic kinetics assay was developed by synthesizing the N-terminal product of the NSP8/9 substrate SFVKLF/NNELSP. Initially, the velocity of the N-terminal product was obtained without adding the peptide inhibitor then various concentrations of the peptide inhibitors were used to monitor the reduction of the product velocity. Our results highlight promising antiviral candidates that effectively disrupt SARS-CoV-2 protease activity and inhibit viral replication, as evidenced by the observed decrease in the product velocity. Once consistent trends are established, we will determine key enzyme kinetic parameters—Km, Vmax, and Ki—to characterize the inhibitor’s potency and mechanism of action, including whether peptide inhibitors work competitively or noncompetitively.