Inhibition of Human Cytomegalovirus Protease by Monocyclic β-Lactam Derivatives:  Kinetic Characterization Using a Fluorescent Probe

Abstract

Recent reports have demonstrated the potential of monocyclic β-lactam derivatives as inhibitors of human cytomegalovirus (HCMV) protease. Investigation of the mechanism of inhibition by NMR and mass spectrometry has revealed the presence of an acylenzyme intermediate suggesting that β-lactams are hydrolyzed by the enzyme and cause inhibition by competing with substrate. The potential of a fluorogenic β-lactam derivative for convenient kinetic characterization of this mechanism has been evaluated using 4S-(4-methylumbelliferone)-3R-methylazetidin-2-one-1-carboxylic acid (4-methylpyridyl) amide (1). Upon acylation of the enzyme, the fluorescent umbelliferone moiety is released, allowing for continuous monitoring of the hydrolytic process. Examination of a series of progress curves by numerical analysis has provided valuable information on acylation and deacylation rates which relate to the IC50 values observed for β-lactams. More importantly the potential of compound 1 as an active site titrating agent for HCMV protease has been exploited, and a simple protocol for rapid determination of active enzyme is described. The data are consistent with the HCMV protease dimer being composed of two functional active sites. This titrating agent represents an important tool that should significantly facilitate the characterization of this novel enzyme.

β-Lactone natural products and derivatives inactivate homoserine transacetylase, a target for antimicrobial agents

“…18 Several studies in the past have shown a time-dependent inhibition of b-lactam and b-lactone compounds against Ser/His/Asp catalytic triad enzymes such as penicillin-binding proteins, 34 b-lactamases 23,35 and several Ser proteases. [36][37][38] On the basis of this precedent for other Ser/His/Asp catalytic triad enzymes, we reasoned that HTA might be inhibited by similar compounds that have the potential to form a covalent bond with the catalytic Ser (Figure 4). Furthermore, the active site and substrate binding geometry of HTA is unique.…”

“…23 In contrast, b-lactams are reversible inhibitors of some Ser proteases. 37 In this case, the protease cleaves the b-lactams and the cleaved products compete with the substrate. HTA Hin is irreversibly inhibited by the three b-lactone compounds.…”

Synthesis of some new spirocyclic β-lactam and spirocyclic thiazolidin-4-one derivatives

“…β-Lactams act also as inhibitors for thrombin [5], human cytomegalovirus protease (HCMV) [6] ( a β-herpes virus) which is a serious pathogen in immunecompromised individuals [7], matrix metalloprotease [8], cysteine protease [9] and human leukocyte elastase (HLE) [10]. Spirocyclic β-lactams have attracted attention as they have been shown to be β-turn mimetics [11] and precursors for α,α-disubstituted β-amino acids [12].…”

Synthesis of 2‐Azetidinones Incorporating Carbenechromium(0) Moieties and Their Use in the Preparation of Penicillin‐ and Cephalosporin‐Containing Peptides

“…[7] Recently, the role of β-lactam antibiotics as neuroprotectors has also been reported as they increase the expression of glutamate carriers. [8] Following the developments in the synthesis of new 2-azetidinones, their use as intermediates in organic synthesis led to the term "β-lactam synthon method" being coined by Ojima and co-workers more than 20 years ago.…”