title: Cytomegalovirus protease targeted prodrug development.
abstract: Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 40850, USA.
authors.forename: Hairat
authors.initials: H
authors.lastname: Sabit
authors.forename: Arik
authors.initials: A
authors.lastname: Dahan
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.forename: Chester J
authors.initials: CJ
authors.lastname: Provoda
authors.forename: Kyung-Dall
authors.initials: KD
authors.lastname: Lee
authors.forename: John H
authors.initials: JH
authors.lastname: Hilfinger
authors.forename: Gordon L
authors.initials: GL
authors.lastname: Amidon
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D018938:Caco-2 Cells; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000201:enzymology*; D016503:Drug Delivery Systems; D015195:Drug Design; D004952:Esters / Q000737:chemistry; D015774:Ganciclovir / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D008956:Models, Chemical; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry*; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013696:Temperature
chemical_list: D000998:Antiviral Agents; D004952:Esters; D011355:Prodrugs; D010447:Peptide Hydrolases; D015774:Ganciclovir