Novel Antibiotic Targeting: Bacterial DNA Polymerases

The objective of this project is to develop novel antibiotics to treat antibiotic-resistant gram-positive (Gr+) and gram-negative (Gr-) bacterial infections. The work utilizes the class III bacterial DNA polymerase (pol III), an unexploited target. The bacterial pol IIIs are highly conserved enzymes that are required for the synthesis of DNA during chromosomal replication. When an inhibitor of pol III is applied to a growing bacterium, it stops replication, and, thus, like the replication-specific quinolone antibiotics, it is bactericidal. Furthermore, the pol III-selective inhibitors are equally effective against clinically relevant antibiotic-resistant and antibiotic-sensitive pathogens. Using our proprietary antibiotic discovery platforms (Bacto-III and Replix) , we have developed a novel class of inhibitor that is active against DNA polymerase (pol) IIIC and E. The new family of compounds has displayed favorable properties (i.e. inhibition of Gr+ DNA polymerases as well as bacteria, low in vitro mammalian toxicity) and we are optimizing the most promising structure utilizing a rational drug design approach to develop antibacterial lead compounds.

Bacterial DNA Helicases: Targets for Novel Antibiotics

We are developing new chemical classes of anti-bacterial agents that will target replicative DNA helicase (RDH), an essential target in the DNA replication pathway, and will use them to treat resistant organisms. For this purpose, we have developed a high throughput screen to identify inhibitors of Staphylococcus aureus RDH. This screen will detect inhibitors of any of the multiple essential helicase functions, including strand unwinding, ATPase-coupled translocation, DNA binding and protein-protein interactions.

Novel Therapies for Staphylococcal BioFilm-Related Infections

Biofilms are surface-attached bacterial communities encased in a hydrated matrix of exopolysaccharide. In the body, infecting bacteria form biofilms on medical implants, such as indwelling catheters. In this biofilm mode of growth, they are resistant to antibiotics and attack by the body's immune system. Staphylococcal biofilms are the leading cause of hospital acquired implant-based infections, which result in approximately 30,000 deaths per year. S. epidermidis is the leading cause of these infections. The overall goal of this project is to discover drugs that selectively block the formation of staphylococcal biofilms. These drugs will be used to coat the surfaces of medical implants to prevent biofilm development when implants are placed in patients.

Type III Secretion Inhibitors for Anti-Infective Therapy

Pseudomonas aeruginosa is a common and extremely virulent cause of serious infections in immune compromised/suppressed patients (e.g., HIV and cancer), cystic fibrosis patients, and those on mechanical ventilation or with burn wounds. Frequent antibiotic resistance and the highly virulent nature of P. aeruginosa make it deadlier than most other bacteria. New chemical classes of antibiotics acting on novel accessible targets are crucial for continued effective therapy against P. aeruginosa. The strategy of this project is to develop new drugs by screening a diverse collection of synthetic and natural product compounds against extra-cellular targets that are critical for virulence. The type III secretion system (TTSS), dedicated to the secretion of toxins and their translocation into the cytoplasm of human cells has been validated as a clinically important target in P. aeruginosa. The goal of this project is to identify specific inhibitors of TTSS and to develop them into novel antibiotics for P. aeruginosa infections.

Inhibitors of Fatty Acid Biosynthesis for Anti-Infective Therapy

Prior discoveries of compounds which inhibit FAB pathway enzymes in other species have validated this pathway as useful for drug discovery. However, the existence of fabK, a structurally unrelated paralog of fabI in P. aeruginosa, and the permeability and efflux obstacles of that species have rendered these compounds ineffective against P. aeruginosa. Our approach to identifying effective new anti-Pseudomonal drugs is to develop and utilize FAB pathway-specific cellular reporter screens. These bioluminescent cell-based screens are sensitive and inexpensive, and they identify inhibitors which gain access to the cytoplasm and avoid efflux sufficiently to generate a report. Drugs targeting FAB will also block the production of acyl-homoserine lactone (AHL) based quorum-sensing signals, resulting in inhibition of virulence as well as growth. Furthermore, hits may have broad efficacy among Gram(-) species because of sequence similarity among FAB targets; such hits will have higher priority for drug development.

TLR5 Antagonists for Anti-Infective Therapy

The goal of this research is to develop therapeutic agents that antagonize the pro-inflammatory signaling pathways resulting from the interaction between TLR5 and flagellins of P. aeruginosa and/or B. pseudomallei. This innovative approach aims to disrupt the mechanism by which these species induce an excessive debilitating inflammatory response by the host. New drugs with this capability will be used to combat Pseudomonas and Burkholderia infections directly or in combination with traditional antibacterial therapies. Bacterial flagellin, the monomeric subunit of flagella, is the primary target of the innate immune response of the host during infection by these species. In most mucosal tissues, flagellin interacts with toll-like receptor 5 (TLR5) and leads to the generation of a pro-inflammatory response through N F-kB regulated gene expression. Normally an early protective reaction to infection, this response is detrimental to the host causing shock, sepsis, pneumonia or chronic inflammatory disease in the case of Pseudomonas and Burkholderia infections.

Novel Antibiotic Targeting: Initiation of Bacterial DNA Synthesis

The continuing erosion of the efficacy of current antibiotics demands the creation of new antibacterials that are not subject to existing mechanisms of resistance. Our strategy is to focus on under-exploited targets in drug-validated pathways. The goal of this project is to discover small molecule inhibitors of the essential E. coli DnaA protein, a novel and unexploited target for antibacterial agents. In preliminary studies, we designed a cell-based DnaA assay that depends on a positive outcome, (i.e., increased growth and survival of a mutant cell that is unable to grow otherwise) and is exquisitely sensitive to inhibition of DnaA.

Novel Antibiotic Targeting: Sensitizing Bacteria to Fluoroquinolones.

Bacterial stress responses represent an important mechanism of intrinsic antibiotic resistance and for the generation of resistance mutations. Antibiotics that interfere with DNA replication, such as quinolones, fluoroquinolones, and trimethoprim, as well as ß-lactam antibiotics, strongly induce the SOS response, a generalized stress response for mitigating cellular damage produced by genotoxic compounds. Observations that SOS-defective bacteria are significantly more sensitive to quinolone, fluoroquinolone, and ß-lactam antibiotics than are wild type bacteria confirm the importance of this pathway for reducing antibiotic damage to bacteria. In addition, SOS-defective mutations greatly reduce the rate of generation of antibiotic resistance as well as the mobilization of virulence factors. Our strategy is to identify small molecules that inhibit SOS induction using a cell-based reporter assay and to develop them into drugs that sensitize bacteria to the bactericidal effects of fluoroquinolone antibiotics.