Metabolomics and chemical library screening for antibacterial drug discovery

Abstract

There are twenty standard amino acids which are proteinogenic and can exist in two stereoisomeric forms, D- and L-enantiomers, except for glycine, which is achiral. The D-amino acids are not incorporated in proteins; however, they play important roles in many biological systems and processes. Amino acids are polar analytes and are difficult to separate on traditional silica based stationary phases in reverse phase chromatography to allow accurate quantification in their natural form. In part I of this dissertation, we investigated the use of a chiral derivatizing reagent known as Marfey’s reagent, coupled with liquid chromatography tandem mass spectrometry-based separation and quantitation for the common proteinogenic amino acid D- and L- stereoisomers. We compared standard formic acid and ammonium acetate based solvent systems in both positive and negative modes; and established baseline separation with superior sensitivity for all nineteen amino acid pairs and glycine with an analytical C8 column in reverse phase chromatography in negative ionization mode using an ammonium acetate based neutral solvent system. We optimized the derivatization reaction time for reaction completion and various parameters including linearity, matrix effect, stability, lower limit of detection and quantification to validate the analytical method. Finally, we showed the application of the developed method by determining the levels of proteinogenic amino acids DL-stereoisomers in mid-log phase cellular extracts of Methicillin-Resistant Staphylococcus aureus (MRSA F–182). This approach enables chromatographic resolution and MS/MS-based quantification of all twenty common proteinogenic D- and L- amino acids in complex matrices. We also used LC-MS/MS to separate and quantify deoxyribonucleic acid (DNA)/nucleosides to assess the activity of Ten-Eleven Translocation 2 (TET2) enzyme. Methylation of one of the DNA bases, cytosine at the C-5 position (5-methylcytosine, 5mC) plays a crucial role in epigenetic transcriptional regulation. TET-family dioxygenases (TET1, TET2 and TET3) initiate active demethylation of 5mC. TET2 oxidizes 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by iterative oxidation. Mutations in the TET2 gene are frequently detected in myeloid malignancies. Despite these critical emerging roles of TET dioxygenases in health and diseases, in vitro characterization of these enzymes and their mutants is still rudimentary. We compared both positive and negative ion based detection mode mass spectrometry and developed an ion-switching LC-MS/MS method for separation and quantification of four normal nucleosides (deoxyadenosine, A; deoxyguanosine, G; deoxycytidine, C; thymidine, T) and four cytosine modifications (5mC, 5hmC, 5fC, and 5caC). This approach enables analysis of these DNA nucleosides, specially 5hmC, 5fC and 5caC with the best sensitivity possible among different optimization efforts undertaken to elucidate the function of TET2 enzymes using analytical LC-MS/MS technology. This method can also be used to study effects of drugs on biosynthesis of DNA and can help in understanding the cellular biochemistry when organisms, such as bacteria are exposed to exogenous molecules interfering with DNA biosynthesis machinery.

Part II of this dissertation describes the development and optimization of a high throughput whole cell-based screening workflow that utilizes automated sample preparation and liquid chromatography tandem mass spectrometry technology. The newly established workflow was applied to screening of chemical compound drug libraries to identify novel antibacterial activities against MRSA by increasing the dimensionality of the screening effort. One dimension involved generating and screening a human liver microsome metabolized drug library in parallel with the original library to identify intrinsically active drugs and drugs with antibacterial metabolites. Several drugs demonstrated improved antibacterial activities against MRSA after microsomal metabolism e.g. capecitabine, balofloxacin, gatifloxacin. A second dimension involved screening in the presence and absence of cefoxitin to identify synergistic agents. Several drugs exhibited synergy with cefoxitin against MRSA that includes floxuridine, gemcitabine, novobiocin, rifaximin and 4-quinazolinediamin. This study demonstrates how a dimensionally enhanced comparative screening effort can identify new antibacterial agents and contribute to strategies for countering antibacterial resistance.