Current Pilot Projects

StevenBloom

Steven Bloom, Assistant Professor, Dept. of Medicinal Chemistry;University of Kansas

Project TitleIlluminating Old Catalysts for the Synthesis of Anti-Infective HIV Peptides


Project Summary

The continual threat of resistance to HIV-1 treatments has fueled a need for developing new classes of antiretroviral inhibitors with novel mechanisms of action.i Antiretrovirals which target the HIV nucleocapsid (NC) protein have gained particular interests in recent years.ii NC is an essential protein involved in the recognition and packaging of viral RNA and the onset of early stage HIV infection. Disruption of NC function herefore provides a unique opportunity to arrest HIV viral replication at several points in the viral life cycle and to halt the progression of HIV infection altogether. Nevertheless, the highly flexible nature, high basicity, and the presence of zinc-coordinating zinc fingers have made NC a difficult target for traditional small molecule-based therapeutics.2 Synthetic peptides containing non-proteinogenic amino acids (NPAAs) represent an attractive alternative to small molecules for targeting complex proteins like NC.iii Despite this fact, the discovery of an NPAA-containing peptide inhibitor for NC has yet to occur. In 2008, Dietrich reported a short peptide sequence, His-Trp-Trp-Pro-Trp-Trp, which halted HIV-1 infectivity by competitively blocking the interaction between NC and y-RNA.iv Unfortunately, this peptide also displayed poor metabolic stability and bioavailability. As the incorporation of NPAAs into native peptides can enhance their receptor potency, selectivity, and metabolically stability, it stands to reason that the introduction of NPAAs into the Dietrich sequence could vastly improve its therapeutic potential in route to a new clinical candidate for HIV-1.

We propose here the use of a novel donor-acceptor platform to accelerate the discovery of an optimal NPAA-variant of the Dietrich sequence. In our approach, a single precursor peptide harboring a single dehydroalanine (Dha) "acceptor residue" in a defined position is made using standard solid phase peptide synthesis (SPPS). Then, visible light and a flavin photocatalyst are used to transfer the desired NPAA side chain to the Dha acceptor residue from a "donor" boronic acid. In this way, the product of a single SPPS procedure can be the progenitor of a multitude of peptides, each containing a different NPAA, at a defined location, in just one step per peptide. This strategy will enable NPAA-containing variants of the Dietrich sequence to be rapidly assembled and biologically evaluated.

Zarko Boskovic

Zarko Boskovic, Assistant Professor, Dept. of Medicinal Chemistry;University of Kansas

Project TitleSynthetic chemistry elaboration of fragments targeting the bacterial type III secretion


Project Summary

Type 3 secretion system is a molecular machine used by the bacteria of several species in the initial steps of invasion of human cells. We are at the critical point at which we have gathered a sufficient structural information about the proteins comprising this machine to begin designing small molecules capable of binding and potentially interfering with its intended function. This project builds on previous discoveries of weak fragment binders to the proteins forming the tip of this system made by DeGuzman lab. We will first build a model of binding of experimentally identified weak binders by considering electrostatic surface of IpaD and SipD proteins. This model will allow us to introduce changes to the known fragment with a particular emphasis on the introduction of three-dimensionality by incorporating stereocenters into the structures of newly-prepared analogs. Access to both enantiomers (mirror images) of these structures would allow us to test the stereospecificity of binding, which is an important indication for further elaboration of these fragments. Finally, we will develop a solution for the general problem of linking weak fragments identified in this and similar experiments by protein-templated linking of two fragment molecules. Taken together, successful completion of these aims would not only aid in "drugging" the type 3 secretion system, but would also solve a long-standing issue in fragment-based drug discovery - achieving successful linking of the weak binders into a more potent combination of them.

Justin Hutchison

Justin Hutchison, Assistant Professor, Depts. of Civil, Environmental and Architectural Engineering, University of Kansas

Project Title: Phage-protein based removal of pathogenic non-tuberculous Mycobacterium from drinking water


Project Summary

The long-term goal of our work is to prioritize environmental reservoirs of pathogenic NTM, to understand NTM-mycobacteriophage relationships in drinking water treatment and distribution, and to utilize NTMspecific mycobacteriophage proteins to produce NTM-specific removal technologies for drinking water treatment. The overall objective of this proposal is to prioritize highly-pathogenic-NTM reservoirs to mine host-specific mycobacteriophages. These mycobacteriophages will be used to develop NTM-specific drinking water treatment technologies. Our focus in this proposal is on Mycobacterium abscessus (MABS). While MABS is the second most common cause of pulmonary infections in the U.S., MAB infections may have higher rates of adverse patient outcomes compared to more common NTM pathogens. The hypothesis of this study is MABS-specific mycobacteriophages sourced from MABS-rich samples can be used to identify novel, phage-based molecular targets for treatment or removal of MABS. The rationale for this project is that the development of MABS-specific mycobacteriophage tools will contribute to quantitative MABS risk assessments and promote alternative MABS treatment strategies. In order to achieve the objectives of this proposal and test our hypothesis, we will prioritize MABS-rich environments based on abundance and genetic similarity to pathogenic MABS strains using high-throughput sequencing data of environmental, human sputum, and clinically isolated samples. The basis of this comparison will use 16S rRNA for generic identification of the microbial community. To achieve a higher resolution of the Mycobacterium population, the RNA polymerase beta-subunit gene, rpoB, will be used. Once environmental MABS reservoirs have been prioritized, highly ranked environments will be mined for MABS-specific bacteriophages. Interactions between these mycobacteriophages and NTM organisms will be screened to identify novel NTM-molecular targets. The corresponding mycobacteriophage protein will be used to develop NTM-drinking water treatment technologies. Results from this study can inform immunocompromised patient behavior to avoid high-risk NTM reservoirs and determine if high-throughput sequencing of rpoB can be applied for species-level resolution of MABS in human sputum. The development of a high-throughput phage/host-protein screening platform would accelerate the identification of phage molecular targets, a potential source for the development of novel NTM antimicrobials.

Shyam Sathyamoorthi

Shyam Sathyamoorthi, Assistant Professor, Dept. of Medicinal Chemistry, University of Kansas

Project TitleFirst Enantioselective Syntheses of Bactobolins A, B, and Analogue Antiobiotics


Project Summary

Since the commercialization of penicillin in 1928, antibiotics have been hailed as “magic bullets”. In recent years, however, the number of bacterial strains resistant to clinically used antibiotics has sharply increased. The lack of viable first-line treatments of these bacterial infections has forced clinicians to consider secondline antibiotic options such as polymyxins and aminoglycosides, traditionally avoided because of significant toxicity. Thus, the development of antibiotics with new mechanisms of action for the control of pernicious bacterial infections is of vital importance. The synthesis of natural products and simplified derivatives has been a very successful strategy for the enrichment of the anti-infective armamentarium; a variety of clinically used antibacterial, antimalarial, and antifungal compounds have their origins in natural molecules. The specific aims of this proposal are: 1. Completion of the first enantioselective syntheses of Bactobolins A and B, two broad spectrum natural product antibiotics whose mechanism of action is the inhibition of protein translation via binding to an unprecedented site of the 50S ribosomal subunit and displacing tRNA bound at the P site. 2. Generation and evaluation of a plethora of functional analogues for future medicinal chemistry efforts. The rationale for this proposed research is that its success would allow for access to a diverse collection of antibacterial compounds with modes of action that are likely mechanistically distinct from FDA-approved antibiotics. The expected outcome of this research is the completion of several important steps towards the timely development of new, broad-spectrum, tolerable antibiotics. The successful execution of the research proposed herein is expected to have a significant positive impact by aiding the global effort to reduce human morbidity and mortality resulting from pernicious bacterial infections.

Past Pilot Projects

Brian Ackley

Brian D. Ackley, Associate Professor, Dept. of Molecular Biosciences;University of Kansas

Project TitleOn again/Off again: Is BLI-3 a ROS-activated pathogen killer?


Project Summary

Bacterial pathogens are a significant hazard to human health. This is especially true given the rise of acquired resistance to many commonly used, and even last line, antibiotics. There is a critical need to better understand how hosts and pathogens interact, and how the host defends against the pathogen. The overarching goal of our research is to understand host-specific factors that are important for controlling pathogen infection and, ultimately, to use this information to develop novel approaches to treat infection. To do this we are using a genetically tractable infection model, the free-living soil nematode Caenorhabditis elegans. C. elegans are bacterivores, and, once ingested, bacterial pathogens induce an organismal response in the C. elegans intestine that overlaps with those observed in human cells, validating this as a model to understand infection response. Infections are known to stimulate the release of calcium from intracellular stores, which ultimately leads to innate immune activation. We recently discovered that C. elegans with mutations in a calcium binding protein, PBO-1 have increased susceptibility to pathogen infection. PBO-1 regulates Na+/H+ ion exchange pumps, including PBO-4, which regulates intestinal pH. pH can affect the activity of oxidases and peroxidases that can generate bactericidal reactive oxygen species (ROS), which is a critical component of innate immunity. Here we are seeking to generate new research tools that can enable us to visualize the ROS generated in infected C. elegans.We are also seeking research tools that can help us study the function of a critical enzyme in the clearance of infection, called BLI-3. Chemicals that inhibit BLI-3 will help us to better understand when during infection the enzyme functions. Activators of BLI-3 might help organisms to clear bacterial infections more quickly, and may serve as a lead for new antipathogenic compounds.

Brian Ackley

Brian D. Ackley, Associate Professor, Dept. of Molecular Biosciences;University of Kansas

Project TitleOn again, off again: Is pH a critical factor in pathogen killing?


Project Summary

Bacterial pathogens are a significant hazard to human health. This is especially true given the rise of acquired resistance to many commonly used, and even last line, antibiotics. There is a critical need to better understand how hosts and pathogens interact, and how the host defends against the pathogen. The overarching goal of our research is to understand host-specific factors that are important for controlling pathogen infection and, ultimately, to use this information to develop novel approaches to treat infection. To do this we are using a genetically tractable infection model, the free-living soil nematode Caenorhabditis elegans. C. elegans are bacterivores, and, once ingested, bacterial pathogens induce an organismal response in the C. elegans intestine that overlaps with those observed in human cells, validating this as a model to understand infection response. Infections are known to stimulate the release of calcium from intracellular stores, which ultimately leads to innate immune activation. We recently discovered that C. elegans with mutations in a calcium binding protein, PBO-1 have increased susceptibility to pathogen infection. PBO-1 regulates Na+/H+ ion exchange pumps, including PBO-4, which regulates intestinal pH. pH can affect the activity of oxidases and peroxidases that can generate bactericidal reactive oxygen species (ROS), which is a critical component of innate immunity. Here we are seeking to generate new research tools that can enable us to visualize the ROS generated in infected C. elegans. We are also seeking research tools that can help us study the function of a critical enzyme in the clearance of infection, called BLI-3. Chemicals that inhibit BLI-3 will help us to better understand when during infection the enzyme functions.

Cory J. Berkland

Cory J. Berkland, Solon E. Summerfield Distinguished Professor, Depts. of Pharmaceutical Chemistry and Chemical & Petroleum Engineering;University of Kansas

Project TitleModification of antibiotics to improve lung retention after inhalation


Project Summary

The pulmonary system is a common entry point for pathogens, unfortunately treating lung infections via oral or IV antibiotics can be challenging due to poor penetration into infected lung tissues, rapid systemic elimination of the therapy, and dose-limiting side effects. Inhaling novel antibiotics designed to persist in the lung compartment after inhalation may dramatically improve efficacy, minimize systemic side effects, allow for the delivery of much higher doses, and potentially eliminate the need for IV administration. Designing novel antibiotics for inhalation represents an untapped scheme, which we propose to advance though rational antibiotic design and a novel screening approach. Imipenem was selected as a core antibiotic structure due to its small size, potency against B. pseudomallei and lone carboxylic acid handle for modification. The literature and our experience indicates an increase in molecular size as well as structures that are hydrophilic (lung fluid retention) or hydrophobic (membrane retention), cleavable (prodrug) or not, offer promise and will be explored. We will synthesize this diverse chemical library and screen for minimum inhibitory concentration (MIC) in B. thailandensis (a surrogate for B. pseudomallei) and for retention in an in vitro lung compartment model. We hypothesize imipenem derivatives larger than 600 Da with a LogP higher than 3 will exhibit maximum retention in the lung compartment model. We propose two Specific Aims: Specific Aim #1: Design and synthesize derivatives of imipenem to improve lung retention after inhalation. Specific Aim #2: Identify antibiotic activity and permeability through model lung epithelium. We expect this Pilot Project will yield multiple imipenem derivatives that can ultimately be tested by our collaborators using mice infected by inhalation exposure to B. pseudomallei.

Indranil Biswas

Indranil Biswas, Professor, Dept. of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center

Project TitleDeveloping Assays to Identify Inhibitors of Hfq of Acinetobacter Baumannii


Project Summary

Acinetobacter baumannii, a gram-negative opportunistic pathogen, is becoming an important nosocomial causing a wide range of diseases and infections including ventilator-associated pneumonia and septicemia as well as urinary tract infections. The pathogen has emerged as one of the most highly antibiotic resistant in the US and elsewhere. Nearly, 70% of A. baumannii clinical isolates are now resistant to all drugs except collistin or tigecycline (known as extremely drug resistant or XDR). Furthermore, infections caused by A. baumannii that are resistant to all available antibiotics (known as pan-drug resistant or PDR) have already emerged and continue to increase since no new drug is in the pipeline that targets A. baumannii. The traditional antibiotics that target cell viability and growth perhaps are not the answer since they will drive the appearance of XDR or PDR further. The innovative approach would be the development of drugs that target the bacterial pathogenesis by inhibiting or controlling expressing of virulence factors. Hfq is a pleiotropic virulence regulator found in many pathogenic bacteria. It is a conserved protein that functions as a post-transcriptional regulator and displays RNA chaperone activity. Inactivation of hfq makes the cells sensitive to various environmental stresses, such as oxidative stress, displaying enhanced susceptibility to various antibiotics, and alteration of the synthesis of several proteins. Furthermore, pathogens lacking a functional Hfq protein are all attenuated for virulence. Therefore, Hfq is an ideal target for drug development to control a wide range of pathogens including A. baumannii.

The major goal of this study is to develop assays for a high-throughput screen (HTS) of small molecule inhibitors of Hfq using a heterologous reporter system and a native expression system. We expect that successful completion of this limited term study will establish an assay system than can be explored further for potential small molecule inhibitors of Hfq. Our long term goal is to stimulate new therapeutic strategies for A. baumannii infections by targeting Hfq and its regulatory mechanisms.

Jeffrey Bose

Jeffrey Bose, Assistant Professor, Dept. of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center

Project Title: Biochemical Characterization and Immunological Consequence of MRSA Fatty Acid Kinase


Project Summary

Previously, we identified the fatty acid kinase of methicillin-resistant Staphylococcus aureus (MRSA) as a contributor to the production of key virulence factors. Absence of the kinase leads to enhanced necrosis and altered immune signals in a murine model of skin infection. One observed cytokine difference was IL-17, a key immune response modulator that is produced by many cells types and has been shown to be important to S. aureus infection resolution. However, it is not known what immune cells are present and important for the observed changes in immune signals or whether this is due directly to altered IL-17 abundance. As a first step, we seek to identify the immune cells present at the site of infection and which cells make IL-17. This will be done using antibodies directed at different immune cell markers and flow cytometry from infected skin homogenates. In addition, we will use computational modeling and docking to develop analogs for proteinligand binding assays as well potential small molecule inhibitors. The results of these studies will shed light on the enhanced virulence of a fatty acid kinase mutant, provide a comprehensive view of immunological factors present during S. aureus skin infection, and be the first step toward developing novel molecules to target S. aureus fatty acid kinase.

Josephine R. Chandler

Josephine R. Chandler, Assistant Professor, Dept. of Molecular Biosciences;University of Kansas

Project TitleChemical biology studies of malleilactone, a small-molecule toxin produced by Burkholderia pseudomallei


Project Summary

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a human disease that is quite difficult to treat and contributes to about 90,000 deaths worldwide per year. Despite the increasing incidence of melioidosis, this pathogen is poorly understood in terms of its basic biology and this deficit remains a barrier to developing new therapies to treat melioidosis. Our long-term goal is to define the underlying mechanisms of B. pseudomallei virulence, and use this information to identify novel therapeutic interventions to treat this challenging human disease. This proposal is focused on a B. pseudomallei small-molecule toxin, malleilactone, which plays an important role in B. pseudomallei pathogenesis. The particular function of malleilactone has remained elusive, in part because it has been difficult to produce in standard laboratory conditions and also because its purification has remained problematic. However, recent approaches involving genetic and chemical means to elicit production of malleilactone, and production of highly pure malleilactone through the KU COBRE SCB Core, facilitated a new line of experimentation to understand exactly how malleilactone functions in the cell. Preliminary studies indicate malleilactone coordinates with iron, and promotes growth in iron-depleted conditions such as that encountered during host infections. The mechanism appears to be unique from that of other known siderophores that scavenge iron. Our studies also show malleilactone can be self-toxic under certain conditions, and that this toxicity is abrogated by an antibiotic efflux pump that contributes to the export of malleilactone. The central hypothesis of this proposal is that malleilactone benefits the cell under conditions where iron is limited and causes self-toxicity in conditions where resistance is impaired. The pilot experiments proposed here aim to 1) elucidate the role of malleilactone in scavenging iron and 2) establish the role of efflux pumps in preventing self-toxicity. These results are essential to gain a mechanistic understanding of how malleilactone contributes to B. pseudomallei infections in the host, and might ultimately lead to development of new therapeutics to treat melioidosis.

David Davido

David Davido, Associate Professor, Dept. of Molecular Biosciences, University of Kansas

Project Title: The Chemical Biology of HSV Gene Expression


Project Summary

The specific events that dictate herpes simplex virus type 1 (HSV-1)-cell interactions critically affect the outcome leading to either lytic or latent infection. An HSV-1 immediate-early (IE) regulatory protein that plays a key role in this process is infected cell protein 0 (ICP0). The ICP0 gene encodes a 775 amino acid (aa) protein that is a phosphorylated, nuclear E3 ubiquitin (Ub) ligase with the capacity to activate transcription of all classes of HSV-1 genes. ICP0 transactivates viral genes via its E3 ubiquitin ligase activity, ubiquitin being a post-translational modification typically associated with protein stability. As HSV-1 is an obligate intracellular pathogen that requires host factors to replicate, host cell factors are likely to play important roles in the ability of ICP0 to stimulate HSV gene expression. While insights as to how ICP0 and its interactions with cellular factors enhance HSV-1 replication has been primarily performed through cell biological and genetic based assays, a chemical biological approach using bioactives and natural compounds to understand ICP0 function and HSV-1 replication remain largely unstudied. Until potential inhibitors and pathways ICP0 interacts with are identified, it will be unclear as to the exact mechanisms ICP0 plays in the switch between the lytic and latent or quiescent phases of infection. Our long-term research goal is to elucidate the molecular interactions between HSV-1 and its host that modulate the HSV-1 life cycle and use this knowledge to ultimately develop therapeutic interventions for treating patients with HSV-1 diseases. The objective in this proposal is to initially identify novel mechanisms by which ICP0 stimulates viral gene expression to enhance HSV-1 productive infection using a chemical biology approach. Our central hypothesis is that inhibition of ICP0 function with small compounds will lead to the discovery of novel ICP0 interactions or pathways (with viral and/or cellular factors) that promote HSV-1 gene expression. Given the time frame of this pilot project grant, one specific aim is proposed: Specific Aim #1: Identify novel inhibitors of HSV-1 ICP0 and viral replication using chemical libraries that include compounds that recognize specific cellular targets/pathways.

Eric J. Deeds

Eric J. Deeds, Associate Professor, Dept. of Molecular Biosciences, University of Kansas

Project Title: Novel Strategies for Proteasome Inhibition in Mycobacterium Tuberculosis


Project Summary

The proteasome is a large macromolecular machine that serves as the proteolytic component of the primary protein degradation pathway in eukaryotes, archaea, and actinomycete bacteria. It has emerged as a major drug target in a number of diseases, particularly in the treatment of cancer and tuberculosis infections. Currently-available active-site inhibitors of the tuberculosis proteasome tend to significantly inhibit the human proteasome or other human proteases, and to our knowledge there are no proteasome inhibitors that are being actively investigated to target tuberculosis infections. The goal of this Pilot Project is to pursue two independent approaches for developing more specific tuberculosis proteasome inhibitors. Cells do not synthesize the proteasome as a single, active unit, but rather as a set of protein subunits that must be assembled into a specific structure in order to function. In particular, the proteasome Core Particle (CP), which is the catalytic component of the complex, is not active until fully assembled. While the overall architecture of the human and tuberculosis proteasomes are similar, the tuberculosis CP has protein interaction interfaces that are very different from those present in the human proteasome, and assembles via a completely different pathway. It has thus been suggested that an assembly inhibitor might provide greater specificity for the tuberculosis proteasome. Working with the Computational Chemical Biology (CCB) core of the CBID COBRE, we have developed the first known assembly inhibitor of the bacterial CP. While this molecule shows clear inhibition of assembly, it lacks both solubility and potency, which has hindered further characterization and testing. A major goal of this proposal is to work with the CCB and Synthetic Chemical Biology (SCB) cores to improve the solubility and efficacy of this inhibitor to support future validation and development. The second approach that we are pursuing focuses on a natural product called fellutamide B, which shows very high activity against both the tuberculosis and human CPs. Crystal structures indicate that fellutamide B binds these two CP’s in very different ways, and the second goal of this proposal is to work with the CCB and SCB cores to leverage this difference and generate derivatives that are specific for tuberculosis. If successful, these studies will provide two separate classes of tool compounds for probing proteasome function and assembly in bacterial cells. Compounds discovered through this work should also serve as the starting point for the eventual development of novel classes of therapeutics targeting CP assembly.

Brandon DeKosky

Brandon DeKosky, Assistant Professor, Depts. of Pharmaceutical Chemistry and Chemical Engineering, University of Kansas

Project Title: A New Experimental Platform to Analyze anti-gB Antibodies in Human B Cells


Project Summary

This project will establish a new experimental pipeline for rapid analysis of antibody immune pressure against viral pathogens, which holds the potential to accelerate the discovery of therapeutic and vaccine candidates against persistent viruses. This project will develop new research tools to investigate adaptive immune pressure against human cytomegalovirus (HCMV), which is a highly prevalent pathogen infecting the majority of individuals in the world and causes significant morbidity and mortality in immunocompromised patients and in congenital infections with prevalence of around one in 200 births. Here we will develop a new antibody isolation assay to identify and express anti-HCMV glycoprotein B (gB) antibodies that have potential HCMV neutralization capacity among human antibody repertoires. We will also transfer established HCMV neutralization assays into a new high-throughput robotic format to rapidly screen our isolated antibodies for HCMV neutralization. This work will leverage established high-throughput immune profiling techniques recently invented by the PI to interrogate anti-HCMV antibody-based immunity and will greatly extend our capabilities in high-throughput sequencing and analysis of antiviral antibody repertoires. This pilot project will establish the protocols, research environment, and expertise to attract external funding and begin clinical research studies regarding the features of effective and ineffective adaptive immune pressure against HCMV infections. In particular, this project will establish a new research environment for follow-up collaborative studies investigating effective vs. ineffective adaptive immune pressure against HCMV in a prospective cohort of matched mother and infant pairs. In the long-term, the research catalyzed by this pilot project will accelerate growth of technologies for rapid analysis of adaptive immunity against persistent viruses. These new technologies will enable discovery of potent antibodies to prevent and treat viral infections in vulnerable populations, beginning with HCMV.

Revathi Govind

Revathi Govind, Assistant Professor of the Division of Biology, Kansas State University

Project TitleCurtailing Clostridium Difficile Virulence


Project Summary

Clostridium difficile is the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-infections (CDI) because it disrupts normal protective gut flora and enables C. difficile to colonize the colon. The current treatment for CDI, administration of additional antibiotics, is increasingly ineffective and often results in relapse of the disease. New strategies to treat this important pathogen are urgently needed and one such approach is to target its virulence. Toxigenic C. difficile strains produce two toxins, toxin A and toxin B that are considered to be the major virulence factors. The toxins encoding genes, tcdA and tcdB are part of a pathogenicity locus, which also carry the gene encodes for the toxin genes positive regulator tcdR. TcdR is an alternate sigma factor that is specifically required for expression of tcdA and tcdB. In a preliminary study we found that tcdR in C. difficile to affect both toxin production and sporulation. It is hypothesized that a small molecule that inhibits TcdR would block toxin production along with sporulation and will serve as an anti-pathogenic agent against C. difficile. Different TcdR activated promoter-reporter fusions were developed and have been shown in E. coli to be appropriate for high-throughput screening. In the first specific aim, we propose to develop series of recombinanat TcdR activated promoter reporter fusions in E. coli and in B. subtilis for high throughput screening. Employing these reporter fusions, we will conduct a pilot screening of small molecule libraries from the KU-HTS core for compounds that inhibit TcdR activity. Second aim of the grant will focus on understanding the influence of TcdR on sporulation. We propose to study the influence on TcdR on sin (sporulation inhibition) locus transcription in C. difficile.

Alternate sigma factors are known to regulate virulence and virulence associated genes in many pathogenic bacteria. Including toxin genes, TcdR may regulate other virulence-associated genes in C. difficile. We have created and characterized, tcdR mutant in two different C. difficile strains. Mutation in tcdR affected both toxin production and sporulation in C. difficile. Microarray analysis revealed many differentially expressed sporulation-associated genes in tcdR mutant. In this project in our first aim, we propose to test the role of TcdR in C. difficile sporulation. In our second aim, we are proposing to monitor TcdR dependent promoter expression at cellular level, using a novel reporter system. During the current decade there has been a dramatic increase in the incidence and severity of C. difficile infections due to the emergence of hypertoxinogenic C. difficile strains. Our long- term goal is to unravel pathogenic mechanisms of C. difficile, thus new strategies to prevent, treat and manage C. difficile infection can be developed.

Phillip R. Hardwidge

Philip R. Hardwidge, Professor, Depts. of Diagnostic Medicine and Pathobiology, Kansas State University

Project Title: Bacterial glycosyltransferase inhibitors as anti-virulence compounds


Project Summary

Gram-negative bacterial pathogens interact with mammalian cells by using ‘type III secretion systems (T3SS)’ to inject proteins directly into infected host cells. Many of these injected protein ‘effectors’ are enzymes that modify the structure and function of human proteins by catalyzing the addition of unusual post-translational modifications. T3SS effectors play essential roles in bacterial virulence and are important targets for anti-virulence compounds that can be used to replace or augment traditional antibiotic regimens. The NleB (E. coli) and SseK (Salmonella enterica) T3SS effectors are glycosyltransferases that modify protein substrates on arginine residues. This modification is especially interesting because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. These enzymes are extremely important to pathogen virulence. NleB-deficient Citrobacter rodentium (a mouse pathogen used as a model organism for studying pathogenic E. coli) do not cause mortality to mice. NleB is also a signature of enterohemorrhagic E. coli (EHEC) strains with the ability to cause foodborne outbreaks and the often-fatal hemolytic uremic syndrome (HUS) in humans. Salmonella strains lacking SseK are defective for replication in macrophages and colonization of mice. Preliminary data show: 1) crystallization of multiple NleB/SseK orthologs; 2) determination of the mechanism by which these proteins glycosylate host substrates; 3) development of a preliminary high-throughput screening (HTS) assay to identify EHEC NleB1 inhibitors; 4) characterization of two compounds (100066N and 102644N) that inhibit NleB1 with IC50s of ~200 nM; and 5) validation that neither inhibitor blocks the activity of the essential human O-GlcNAc-transferase (OGT). The following specific aims are proposed: 1) Characterize the mechanisms by which 100066N and 102644N inhibit NleB1 activity. 2) Conduct a larger HTS assay to identify NleB/SseK inhibitors with increased potency. The proposed experiments will provide novel insight into how NleB/SseK modify the poorly nucleophilic guanidinium group of arginines, will provide novel probes to monitor the activity of these enzymes, and will also advance the development of anti-virulence compounds targeting important human pathogens.

Michael Zhuo Wang

Michael Zhuo Wang, Associate Professor, Dept. of Pharmaceutical Chemistry, University of Kansas

Project TitleIdentification of CYP5122A1 inhibitors as therapeutic agents for leishmaniasis


Project Summary

Human leishmaniasis is a devastating infectious disease caused by protozoan parasites belonging to the genus of Leishmania. The disease is found in more than 90 countries and responsible for an estimated 1-2 million new infections each year worldwide. Leishmaniasis is also common in returning U.S. military personnel from Iraq and Afghanistan, with more than two thousand cases reported since 2001. Leishmania parasites cause disfiguring skin sores (cutaneous leishmaniasis or CL) and life-threatening infection of vital internal organs (visceral leishmaniasis or VL). VL (~20% of all leishmaniasis cases) is the most dangerous and fatal form of the disease, with a mortality rate close to 100% if left untreated. It is the second most deadly parasitic disease in the world (after malaria). Despite being a serious public health problem in many endemic regions, there are no vaccines or preventative chemotherapies available for leishmaniasis control. Current antileishmanial drugs have limited efficacy, serious side effects and high cost. Hence, there is a major unmet medical need for safe and effective drugs against leishmaniasis. Leishmanial CYP5122A1 is a novel cytochrome P450 (CYP) enzyme that is essential for the survival of L. donovani, a major causative agent for VL. Independent and our own studies have led us to hypothesize that a two-tier screening cascade, consisting of primary high-throughput screening (HTS) assays and secondary function and activity assays will identify novel CYP5122A1 inhibitors as therapeutic agents for leishmaniasis control. To test the hypothesis, two specific aims are proposed to answer two main questions: 1) Can the fluorescence-based inhibition assay be translated into a robust HTS assay? 2) Will the proposed two-tier screening cascade lead to discovery of novel antileishmanial compounds? Innovative experimental approaches will be employed in the proposed project, e.g., a new HPLC-MS/MS-based sterol assay, fluorescence-based CYP5122A1 and CYP51 inhibition assays, and cutting-edge high content imaging-based intracellular antileishmanial assays. If successfully completed, the proposed project will establish robust primary HTS assays and validate a two-tier screening cascade. Both are critically important for a future NIH R01 application that aims to identify novel CYP5122A1 inhibitors as therapeutic agents for leishmaniasis control.

Wolfram R. Zückert

Wolfram R. Zückert, Professor, Dept. of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center

Project TitleSmall Molecule Inhibitors of Bacterial Surface Lipoprotein Secretion


Project Summary

The overall goal of the proposal is to identify novel small molecule inhibitors of bacterial envelope biogenesis that will yield novel molecular tools to dissect biogenesis pathways and may lead to a new generation of antimicrobials. In addition to providing novel intervention strategies for infectious diseases, the work will shed further light on the secretion mechanisms of major bacterial envelope proteins. Our model organism, the Lyme disease spirochete Borrelia burgdorferi, lacks lipopolysaccharide (LPS), a major surface component of the outer membrane of other diderm bacteria such as the gram-negative Escherichia coli. Instead, B. burgdorferi expresses an extraordinary number of outer membrane lipoproteins with various crucial functions in transmission, colonization and persistence. Yet, our understanding of lipoprotein secretion pathways remains limited. We have identified several components of the B. burgdorferi lipoprotein secretion pathway, including components of a partial Lol (Lipoprotein outer membrane localization) pathway. We have solved the structure of the B. burgdorferi periplasmic lipoprotein carrier/chaperone LolA and are now beginning a detailed structure-function analysis. In this 1-year collaborative pilot project, we will initiate a screen for inhibitors of lipoprotein secretion in B. burgdorferi by adapting an already established 96-well-plate protein localization screen using fluorescent B. burgdorferi lipoprotein fusions. Initial lead compounds will be analyzed for their cognate targets and their spatiotemporal action affecting lipoprotein secretion by genomic sequencing and through established B. burgdorferi protein localization assays. Overall, the proposal will combine synergistic approaches to yield novel information that will significantly impact our understanding of bacterial protein secretion in a medically important model organism.


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