Browsing by Subject "Antibiotic"

Bistable switches are commonly observed in the regulation of critical processes such as cell cycles and differentiation. The switches possess two fundamental properties: memory and bimodality. Once switched ON, the switches can remember their ON state despite a drastic drop in stimulus levels. Furthermore, at intermediate stimulus levels with cellular noise, the switches can cause a population to exhibit bimodal distribution of cell states. Till date, experimental studies have focused primarily on cellular mechanisms that generate bistable switches and their impact on cellular dynamics.

Here, I study emergent bistability due to bacterial interactions with either synthetic gene circuits or antibiotics. A synthetic gene circuit is often engineered by considering the host cell as an invariable "chassis". Circuit activation, however, may modulate host physiology, which in turn can drastically impact circuit behavior. I illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear dilution of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further experimental measurements. The results reveal a novel mechanism of generating bistability and underscore the need to account for host physiology modulation when engineering gene circuits.

In the context of antibiotic treatment, I investigate bistability as the underlying mechanism of inoculum effect. The inoculum effect refers to the decreasing efficacy of an antibiotic with increasing bacterial density. Despite its implication for the design of antibiotic treatment strategies, its mechanism remains poorly understood. Here I show that, for antibiotics that target the core replication machinery, the inoculum effect can be explained by bistable bacterial growth. My results suggest that a critical requirement for this bistability is sufficiently fast turnover of the core machinery induced by the antibiotic via the heat shock response. I further show that antibiotics that exhibit the inoculum effect can cause a "band-pass" response of bacterial growth on the frequency of antibiotic treatment, whereby the treatment efficacy drastically diminishes at intermediate frequencies. The results have implications on optimal design of antibiotic treatment.

In the fight against antimicrobial resistance, chemotherapeutic agents derived from natural products have served as a first line of defense. However, widespread antibiotic resistance has created an urgency for the development of new therapeutics. Ramoplanins and enduracidins are first generation nonribosomally-encoded lipodepsipeptides with potent activities against a broad spectrum of Gram-positive pathogens, including those resistant to front line antibiotics. As antimicrobial agents with exciting therapeutic potentials, strategies are warranted to develop access to second generation derivatives to improve drug stability and tolerability. To this end, a targeted genome mining strategy was devised to identify peptide congeners from sequenced bacterial genomes. We identified six biosynthetic gene clusters predicted to produce unique antimicrobial congeners. Two such peptides have been isolated from their native producing strains, Micromonospora chersina strain DSM 44151 and Amycolatopsis orientalis strain DSM 40040, and characterized to expand this class of antibiotics for the first time since the discovery of ramoplanin 30 years ago. We additionally have pursued multidisciplinary strategies to define the activity and selectivity of biosynthetic machinery. Together, this work has provided access to novel peptide scaffolds for further therapeutic development and establishes a platform for antimicrobial discovery and biosynthetic engineering of complex peptide derivatives.

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.

Cancer is one of the most devastating diseases in modern society, with over 1.6 million new cancer cases occurring in the US alone each year. DNA-damaging agents are often the first line of defense against rapidly dividing cancer cells. However, cancer cells can become resistant to chemotherapy by up-regulating an error-prone DNA-repair process called translesion DNA synthesis (TLS). The Rev1 polymerase orchestrates this pathway by recruiting one of three inserter polymerases and the extender polymerase (Pol ζ) to bypass the lesion. Here we report the discovery and characterization of an inhibitor of the protein-protein interaction between Rev1 and Rev7, a subunit of Pol ζ, using biochemical and biophysical techniques. Our X-ray crystallographic structural analysis of the Rev1 and the inhibitor (JH-RE-06) complex reveals that the inhibitor blocks Rev7 binding by inducing Rev1 dimerization. Such an unexpected observation is confirmed by an in vitro crosslinking assay. In vitro cell-killing assays show that JH-RE-06 enhances sensitivity of a variety of cancer cell lines to a wide range of chemotherapeutic agents; furthermore, co-administration of JH-RE-06 with cisplatin significantly suppresses melanoma growth in mice and prolongs the survival time of tumor bearing mice, highlighting the therapeutic potential of translesion synthesis inhibitors as a novel class of cancer adjuvant therapeutics to enhance the outcome of chemotherapy currently available to cancer patients.

Due to their compromised immune systems, cancer patients are particularly susceptible to opportunistic bacterial infections, many of which are becoming rapidly resistant to current antibiotic therapies. We describe the combined use of X-ray crystallography and NMR spectroscopy to delineate a cryptic inhibitor envelope for optimization of a small molecule inhibitor of LpxC, an enzyme essential to the survival of Gram-negative bacteria. The resulting inhibitor shows vast improvement over its parent compound over a wide range of bacterial orthologs.

In summary, we demonstrate successful structural characterization and structure-guided design and optimization of lead compounds in two different systems. These studies have profound implications for drug discovery and lead optimization in other disease-relevant systems as well.

The incessant increase of antibiotic resistance among Gram-negative pathogens is a serious threat to public health worldwide. A lack of new antimicrobial agents, particularly those against multidrug-resistant Gram-negative bacteria further aggravates the situation, highlighting an urgent need for development of effective antibiotics to treat multidrug-resistant Gram-negative infections. Past efforts to improve existing classes of antimicrobial agents against drug-resistant Gram-negative bacteria have suffered from established (intrinsic or acquired) resistance mechanisms. Consequently, the essential LpxC enzyme in the lipid A biosynthesis, which has never been exploited by existing antibiotics, has emerged as a promising antibiotic target for developing novel therapeutics against multidrug-resistant Gram-negative pathogens.

In Chapter I, I survey the medically significant Gram-negative pathogens, the molecular basis of different resistance mechanisms and highlight the benefits of novel antibiotics targeting LpxC. In Chapter II, I discuss a structure-based strategy to optimize lead compounds for LpxC inhibition, revealing diacetylene-based compounds that potently inhibit a wide range of LpxC enzymes. The elastic diacetylene scaffold of the inhibitors overcomes the resistance mechanism caused by sequence and conformational heterogeneity in the LpxC substrate-binding passage that is largely defined by Insert II of LpxC. In Chapter III, I describe the structural basis of inhibitor specificity of first-generation LpxC inhibitors, including L-161,240 and BB-78485 and show that bulky moieties of early inhibitors create potential clashes with the a-b loop of Insert I of non-susceptible LpxC species such as P. aeruginosa LpxC, while these moieties are tolerated by E. coli LpxC containing long and flexible Insert I regions. These studies reveal large, inherent conformational variation of distinct LpxC enzymes, providing a molecular explanation for the limited efficacy of existing compounds and a rationale to exploit more flexible scaffolds for further optimization of LpxC-targeting antibiotics to treat a wide range of Gram-negative infections.

In Chapters IV and V, a fragment-based screening and structure-guided ligand optimization approach is presented, which has resulted in the discovery of a difluoro biphenyl diacetylene hydroxamate compound LPC-058 with superior activity in antibacterial spectrum and potency over all existing LpxC inhibitors. In Chapter VI, I describe our efforts to improve the cellular efficacy of LPC-058 by reducing its interaction with plasma proteins, such as human serum albumin (HSA). The binding mode of LPC-058 was captured in the crystal structure of HSA/LPC-058 complex. The acquired structural information facilitated the development of the dimethyl amine substituted compound LPC-088 that displays significantly improved cellular potency in presence of HSA.