title: Synthesis and application of MeOSuc-Ala-Ala-Pro-Phe-CH2Cl as potent proteinase K inhibitor.
abstract: The synthesis and proteolytic inhibitor function of two new tetrapeptides, methoxysuccinyl-Ala-Ala-Pro-Phe-chloromethyl ketone (MeOSuc-AAPF-CH(2)Cl) and methoxysuccinyl-Ala-Pro-Ala-Phe-chloromethyl ketone (MeOSuc-APAF-CH(2)Cl) are described. The efficacy of these two new analogs in inhibiting the proteolytic activity of proteinase K has been compared with the previously-documented proteainase K inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MeOSuc-AAPV-CH(2)Cl). An examination of inhibitory activity using a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay in the presence of proteinase K reveals that the AAPF inhibitor (MeOSuc-AAPF-CH(2)Cl) at a concentration of 0.05mM allows a signal to be obtained for an exogenous target ("Xeno RNA") at 30cycles (i.e. Ct=30), whereas the MeOSuc-AAPV-CH(2)Cl control requires a 10-fold higher concentration (0.5mM) to produce the same Ct. Interestingly, the other new analog, with the rearranged amino acid sequence APAF (MeO Suc-APAF-CH(2)Cl), provides no proteinase K inhibition under the same experimental conditions. These results suggest that when P1 is phenylalanine, alanine at P2 and proline at P3 is not tolerated as a good proteinase K inhibitor. A plausible explanation for the higher efficiency of MeOSuc-AAPF-CH(2)Cl over control is proposed based on the molecular modeling studies.
authors.affiliation: Bioorganic Chemistry Division, Life Technologies Inc., 2130 Woodward Street, Austin, TX 78744-1832, USA.
authors.forename: Anilkumar R
authors.initials: AR
authors.lastname: Kore
authors.forename: Muthian
authors.initials: M
authors.lastname: Shanmugasundaram
authors.forename: Quoc
authors.initials: Q
authors.lastname: Hoang
authors.forename: Mack
authors.initials: M
authors.lastname: Kuo
authors.forename: Laura M
authors.initials: LM
authors.lastname: Chapman
authors.forename: Helen H
authors.initials: HH
authors.lastname: Chen
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000138:chemical synthesis* / Q000494:pharmacology*; D019286:Endopeptidase K / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D009842:Oligopeptides; D011480:Protease Inhibitors; C543155:methoxysuccinyl-Ala-Ala-Pro-Phe-chloromethyl ketone; D019286:Endopeptidase K