Structural and Biochemical Studies of LpxH in Lipid A Biosynthesis

Abstract

Due to the lack of effective treatment, death from infectious diseases was the leading cause of mortality worldwide up until early 1900. Since the discovery of penicillin by Alexander Fleming in 1929, a plethora of novel antibiotics were identified. More than 20 novel classes of antibiotics have been developed between 1930 and 1962. These discoveries drastically decreased the fatalities due to bacterial infections. However, resistance to antibiotics began to arise, and resistance to all of the available antibiotics have been reported. As abundance of the resistant strains are spreading at an alarming rate, there is an urgent need for novel antibiotics.Lipid A biosynthesis is essential in nearly all Gram-negative bacteria. Therefore, the enzymes involved in the essential steps of the pathway are attractive targets for novel antibiotic development. LpxH, an enzyme involved in the fourth step of the pathway, is particularly attractive as studies have shown that inhibition of LpxH leads to toxic accumulation of lipid intermediates, offering an additional mechanism of killing bacteria. This study is aimed at providing key biochemical and structural information needed to understand the mechanism of LpxH and to target this enzyme for inhibition.In chapter I, the urgent need for novel antibiotics especially against Gram-negatives is highlighted. Importance of lipid A biosynthesis reveals the enzymes of the pathway, such as LpxH, as attractive targets. In chapter II, the first structure of LpxH is described. The structure elucidates the key structural information about the mechanism of LpxH and binding of its product, lipid X. A potential inhibitor of LpxH, AZ1, has been recently reported, and chapter III focuses on characterizing the inhibition by AZ1 both biochemically and structurally. Several LpxH orthologs were tested against AZ1, and the first inhibitor-bound structure of LpxH is described. The structure reveals the key interactions between the AZ1 compound and LpxH, setting the foundation for optimization of the compound. The structure also revealed that the active site is unoccupied by the compound. Thus fragment-based drug design may become a viable strategy upon identification of additional LpxH inhibitors that target the active site. Chapter IV focuses on the efforts to design LpxH inhibitor with better potency through identification of new compounds. In chapter V, future studies that will lead to development of more potent antibiotics targeting LpxH are outlined.Collectively, the work contained in this thesis substantially broadens the knowledge of the LpxH mechanism and suggests possible strategies to effectively inhibit this enzyme. Structural and biochemical knowledge on AZ1 inhibition of LpxH and identification of a new compound as LpxH inhibitor sets the foundation on novel antibiotic development targeting LpxH.