Puromycin-sensitive aminopeptidase: An antiviral prodrug activating enzyme

Abstract

Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models (Eriksson et al. Molecular Pharmaceutics, 2008 vol 5 598-609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the LValine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycinsensitive aminopeptidase was generated and its substrate specificity investigated. The k cat for ValpNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher k cat for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design.

Prodrugs of Phosphonates and Phosphates: Crossing the Membrane Barrier

“…For example, the dipeptide prodrugs put forth by McKenna require removal of one of the amino acids by puromycin-sensitive aminopeptidase and spontaneous release of the remaining amino acid [155]. Modulating the enzymatic step that removes the first amino acid might provide an additional means to achieve tissue targeting depending on the expression of that enzyme.…”

Constrained H-Phe-Phe-NH2 Analogues with High Affinity to the Substance P 1–7 Binding Site and with Improved Metabolic Stability and Cell Permeability

“…The degradation is most probably a result of protease activity in the cell membrane in the apical region. It is well-known that brush border proteases are expressed in Caco-2-cells. , The set of sensitive peptides, all with a normal peptide backbone, are well in line with the structural preference for proteases. A majority of the peptides with a low recovery were also accompanied by a high clearance in the human liver microsomes (Table ), indicating also a contribution of protease mediated degradation here besides the normally dominating oxidative metabolism.…”

Gordon L. Amidon: Very Sustained Drug Absorption

“…By the late 1970s, Gordon was motivated to look at oral absorption much more mechanistically and turned his career interest in the direction of mechanistic mass transport analysis . This became a common theme for much of his later research work . Through the 1980s, he studied nutrient digestion and absorption, particularly for amino acids and peptides .…”