As part of a small multi-disciplinary research team (Mutabilis, Valvano, Burrows, Junop and Wright) with complementary expertise in microbial genetics, physiology, biochemistry, and structural biology we are tackling the problem of antimicrobial resistance using a novel approach. The approach centers on the identification and development of antibiotic adjuvants, which are molecules that overcome antibiotic resistance. Research of this group focuses on Gram-negative bacteria with special emphasis on those of environmental origin, which are increasingly being recognized as emerging, multi-drug resistant (MDR) pathogens impervious to the majority of the clinically available antimicrobial agents. The central idea is that molecules that do not intrinsically have antimicrobial activity per se can be utilized in combination with antibiotics as molecular adjuvants to overcome resistance. The research aims of this group are to identify and characterize specific molecules that will: (i) increase bacterial cell permeability, (ii) block the biosynthesis and assembly of the outer membrane, and (iii) and potentiate the effect of antibiotics across all bacterial cell growth states. We predict that this research will make current and future antibiotics more efficacious, and thus will have a major impact on the clinical treatment of infection and contribute to reduce the tremendous burden of antimicrobial resistance.
Although some bacteria may be genetically sensitive to a given antibiotic, they can also exhibit high-level resistance because of the physiological makeup of the organism. The defining characteristic of Gram-negative bacteria is that they are surrounded by an asymmetric lipid bilayer composed of lipopolysaccharide ( LPS ) and phospholipids. This outer membrane provides a highly effective barrier to many antibiotics, protects the organism from the host immune system, and provides chemical structures that enable the bacteria to adhere to surfaces and bind to host cells. Indeed, the primacy of species of Pseudomonas and Burkholderia in infectious disease associated with CF patients is due in large part to the broad resistance that these organisms exhibit against conventional antibiotics. This is primarily the result of the impenetrable LPS layer combined with a plethora of broad-spectrum efflux pumps spanning the cytoplasmic membrane and LPS. Blockade of the assembly of LPS therefore represents an attractive and relatively under explored target that can either directly impair cell growth (new antibiotics), decrease or eliminate microbial virulence, alter biofilm formation, or potentiate the activity of antibiotics (antibiotic adjuvants).
Our contribution to these efforts is focused on disruption of lipopolysaccharide ( LPS) biosynthesis. Work in the Valvano and Wright labs have already provided proof of principle that synthetic molecules can be isolated which block the synthesis of ADP-heptose, a key building block for the synthesis of LPS in Gram-negative bacteria and the overall stability of the outer membrane. The Junop laboratory is actively involved in elucidating the structure/function of all the enzymes involved in the ADP-heptose synthesis pathway. Therefore, with crystal structures and a high-throughput based screen for enzyme inhibitors, we are in a unique position to develop novel molecules that would increase the permeability of the Gram-negative bacterial outer membrane to host defense and currently less efficient antimicrobials.
Our lab has crystallized the majority of these enzymes and determined the atomic structure of two of these (PDB 2GMW , 2I22 , 2I2W ). We are now poised to approach this unique and essential Gram-negative biosynthetic pathway using modern target-based drug discovery methods. Thus, we will apply target-based drug discovery of ADP-heptose biosynthesis enzymes including high throughput screening of enzymes. We will complete and refine the atomic structures of all ADP-heptose biosynthetic enzymes and use mechanistic studies to link structure with function to inform inhibitor analysis. Sugar substrate binding site (and co-factor site where applicable) will be directly identified in each of the crystallized enzymes by soaking preformed crystals or co-crystallization of protein and substrate. We currently have a library of all the sugar substrate intermediates of this pathway, which were chemically synthesized and kindly provided by P. Kosma, University of Vienna. These experiments will permit us to construct a detailed structural map of this enzyme. Additional characterization will involve a detailed analysis of the kinetic parameters of each enzyme using the coupled assay we have recently developed . The structure/functional information gained on each of the enzymes from the pathway will allow us to use structure-based drug design based on lead structures and hits from screens to develop bioactive molecules that inhibit LPS biosynthesis.
Junop Lab 