Development of a fluorescent drug screening tool from the TEM-1 beta-lactamase

Abstract

Beta-lactamases are bacterial enzymes which can destroy beta-lactam antibiotics. These enzymes function by hydrolyzing the four-membered beta-lactam ring into clinically inactive carboxylic acid. The increasing emergence of new beta-lactamases has compromised the utility of many beta-lactam antibiotics. As such, extensive efforts have been made to discover new drugs, such as new-generation beta-lactam antibiotics and non-beta-lactam inhibitors. To this end, computational drug screening has been increasingly used to search for potential drug compounds capable of binding to the enzyme's active site. Despite its high efficiency, recent studies have shown that some compounds selected by computational drug screening can form aggregates in vitro to inhibit beta-lactamase activity via non-specific protein adsorption/absorption instead of blocking the enzyme's active site specifically. These 'false-positive' compounds may mislead drug discovery teams and therefore result in a huge waste of capital and resources. As such, it is highly desirable to develop a reliable drug screening tool that can screen for active-site-binding drugs in vitro. In this project, we developed a fluorescent drug screening tool from the TEM-1 beta-lactamase. This clinically significant enzyme is regarded as the ancestor from which various TEM variants have been derived. Thus, TEM-1 represents a good protein model for the development of in vitro drug screening tools from other clinically relevant TEM variants. The non-catalytic residue Val216 (at the top of the active site) of TEM-1 was first replaced by a cysteine and then labeled by thiol-reactive fluorescein. Despite these modifications, the labelled mutant remains similar in catalytic activity to the wild-type enzyme. The labelled mutant can distinguish compounds capable of binding to the active site from drug aggregates and non-binding compounds by giving characteristic fluorescence changes. Moreover, the labelled mutant can differentiate beta-lactam antibiotics with different potency by giving different fluorescence profiles. Mass spectrometric studies showed that the fluorescence changes are induced by the binding interactions of compounds to the active site. These findings indicate that the labelled V216C mutant can act as an in vitro drug screening tool to identify active-site-binding compounds and facilitate the drug development against TEM-type beta-lactamases.