Current Research Projects

Mark P. Farrell

Mark P. Farrell, Assistant Professor, Dept. of Medicinal Chemistry, University of Kansas

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

Project TitleSynthetic Antibody Mimics as Antimalarial Agents


Project Summary

Infectious diseases have a detrimental effect on human health and are one of the leading causes of human deaths. While vaccines are the gold standard for the prevention of infectious disease, vaccines are not currently available for some of the most deadly disease causing pathogens (e.g., HIV and Malaria). As such, therapeutics are required for the treatment of such diseases, and in cases where vaccines prove ineffective. To date, these therapeutics have been extremely effective at quelling the effects of many infectious diseases, however, given the continued rise in the number of drug resistant pathogens, new therapies and therapeutic strategies are required to target these resistant pathogens in order to prevent catastrophic outbreaks. It is widely accepted that the selective pressures imposed by traditional therapeutics gives rise to these drug resistant pathogens, as such, pathogen virulence factors are now being targeted in order to prevent infectious diseases, as it is believed that this approach will limit the selective pressures that lead to the rise of drug resistant pathogens. While a number of these molecules have been shown to be efficacious for the treatment of infectious diseases, small molecules that target virulence factors are often applied prophylactically or in combination with traditional therapeutics. While antibody therapeutics that target virulence factors have been demonstrated to effectively act alone in the prevention and clearance of numerous infectious disease causing pathogens. Although these therapeutics hold great promise, the administration of antibodies in the regions that are predominantly affected by infectious diseases remains a cause of concern. As such, this project aims to develop virulence factor neutralizing small molecules and peptides that are also capable of eliciting an immune response against the pathogen. We aim to achieve this by preparing antivirulence molecules that can interact with the innate immune system, specifically with mannose recognizing C-type lectins, such as the mannose binding lectin, the mannose receptor and DC-Sign, in order to effect pathogen clearance. To test our hypothesis we aim to prepare molecules that target a virulence factor, namely the apical membrane antigen 1 (AMA1) protein, of the malaria causing parasite Plasmodium falciparum. We envision that the newly designed constructs will be capable of neutralizing the function of AMA1 while concomitantly eliciting an immune response toward the malaria causing pathogen that will facilitate immune induced clearance.

Anthony Fehr

Anthony R. Fehr, Assistant Professor, Dept. of Molecular Biosciences;University of Kansas

Mentor: David Davido, Professor of Molecular Biology;University of Kansas

Project TitleDeciphering the distinct roles of macrodomain ADP-ribose binding and hydrolysis in coronavirus replication


Project Summary

The highly-conserved coronavirus (CoV) macrodomain is essential for CoV pathogenesis in multiple animal models of infection, and thus it is a potential drug target for emerging CoVs that cause severe disease, such as Middle-East Respiratory Syndrome (MERS)-CoV or Porcine Epidemic Diarrhea Virus (PEDV). The macrodomain binds and hydrolyzes ADP-ribose from proteins, however, the contribution of specific residues in the ADP-ribose binding pocket to these activities are unknown. This gap in knowledge is a significant hurdle for identifying compounds that inhibit its activity. The long-term goal is to discern the mechanism by which the CoV macrodomain binds and cleaves ADP-ribose from proteins, determine its functions during infection, and identify compounds that inhibit its activity. The overall objective in this proposal is to determine the relative contribution of specific residues to ADP-ribose binding and hydrolysis, and correlate these results to their impact on virus replication. The central hypothesis is that specific residues in the macrodomain separately impact ADP-ribose binding vs catalysis, and also have distinct roles in virus replication. The rationale for this project is that a better understanding of the mechanism by which the CoV macrodomain functions both in vitro and in cell culture will provide a framework for the development of novel anti-viral therapeutics for highly pathogenic or emerging CoVs. The central hypothesis will be tested by pursuing the following two specific aims: 1) Determine the contribution of highly conserved macrodomain residues in ADP-ribose binding and hydrolysis; and 2) Correlate the level of macrodomain ADP-ribose binding and hydrolysis in vitro to virus replication in cell culture. Under the first aim, several recombinant macrodomain proteins with mutations in conserved residues will be tested in ADP-ribose binding and hydrolysis assays. These assays will determine the relative contribution of these residues to either binding or hydrolysis. Additionally, x-ray crystallography will be applied to select mutant proteins to determine how the mutation altered the structure of the protein to impact its activity. For the second aim, the same mutations made in aim 1 will be incorporated into recombinant MHV and MERS-CoV and test their roles in virus replication and ultimately correlate the biochemical activity of each residue to its effect on virus replication. The research proposed in this proposal is innovative, in the applicant’s opinion, because it studies the impact of specific macrodomain residues on macrodomain biochemistry and their role during infection in a cohesive manner, which has not previously been attempted. The proposed research is significant because they will provide unique insight into both the biochemistry and the function of the CoV macrodomain. These results will have a positive impact in the design and development of compounds that can inhibit this protein and potentially be developed into novel therapeutics to prevent CoV-induced disease.

Stephanie Shames

Stephanie R. Shames, Assistant Professor, Division of Biology, Kansas State University

Mentor: Philip R. Hardwidge, Professor of Diagnostic Medicine/Pathobiology, Kansas State University

Project TitleChemical Inhibition of Legionella pneumophila Metaeffector Function


Project Summary

Intracellular bacterial pathogens utilize highly evolved virulence factors to replicate in eukaryotic cells. Legionella pneumophila (Lpn) naturally parasitizes unicellular fresh water protozoa but can cause a severe pneumonia in humans called Legionnaires’ disease. Human infection results from inhalation of Lpn originating from anthropomorphic fresh water environments containing the bacteria and subsequent bacterial replication in alveolar macrophages. Lpn replication within phagocytes is facilitated by a large arsenal of virulence factors termed effector proteins that are translocated directly into infected host cells. The long-term objectives of this proposal are to develop chemical inhibitors of effector function as therapeutics to treat infectious diseases and enhance understanding of bacterial pathogenesis mechanisms. The Lpn effector Lpg2505 is a ‘metaeffector’ that interacts directly with another Lpn effector called SidI. SidI function is toxic to eukaryotic cells; however, this toxicity is suppressed by Lpg2505 to promote Lpn replication. Consequently, expression of sidI in the absence of Lpg2505 is detrimental to Lpn replication in protozoa and macrophages. Thus, the central hypothesis of this proposal is that chemical inhibition of Lpg2505-mediated regulation of SidI will impair Lpn intracellular replication. To test the central hypothesis, the following specific aims will be pursued. Aim 1 is to use genetics, structural biology, and chemical biology to define the Lpg2505-SidI protein-protein interaction interface. Aim 2 is to collaborate with the CBID IDAD Core at the University of Kansas to perform a high-throughput screen to identify chemical inhibitors of Lpg2505-mediated regulation of SidI. Identified compounds will be evaluated for their ability to control Lpn replication in protozoan and mammalian hosts. The results of this work will improve human health by through the development of compounds that can utilized to (1) impair Lpn intracellular replication; (2) enhance understanding of metaeffector function; and (3) reveal molecular mechanisms of bacterial pathogenesis that enable replication within eukaryotic phagocytes. Thus, this work will provide a foundation for use of chemical biology to study effector function and development of chemical inhibitors of virulence factor function as a means to combat a variety of infectious diseases.

 

 

Past Research Projects

Michael Clift

Michael Clift, Assistant Professor, Dept. of Chemistry, University of Kansas

Mentor:  Paul R. Hanson, Professor of Chemistry; University of Kansas

Project TitleSynthesis and Biological Evaluation of Benzophenanthridine Alkaloid Natural Products and Derivatives


Project Summary

The discovery of new antibiotic agents that operate through novel modes of action is regarded as potential solution to combat growing bacterial resistance. Recently, inhibition of FtsZ polymerization has emerged as a promising new target for the development of antibiotics. FtsZ is a highly conserved, prokaryotic, tubulin-like protein that undergoes self-polymerization to enable bacterial cell division. Berberine and tetrahydroprotoberberine (THPB) possess modest affinity for FtsZ that imbues these alkaloid natural products with antibacterial properties. Semi-synthesis has enabled the preparation of relatively potent (MIC = 1 μg/mL) berberine derivatives; however, semi-synthesis is inherently limited by the natural reactivity of berberine itself and the lack of a flexible and concise synthetic approach to access berberine analogs has left significant gaps in what is known about the structure−activity relationships that exist between berberine and FtsZ. The overall objective of this proposal is to design, synthesize and test previously inaccessible berberine-like compounds to discover new antibiotics that target bacterial cell division. Three aims are proposed to pursue this objective:

Specific Aim #1: Prioritize synthetic targets by using synthesis in silico to generate a custom compound library that will be used for virtual screening against FtsZ polymerization.

Specific Aim #2: Develop a concise and flexible total synthesis of berberine and THPB, and use this synthetic route to access a wide range of previously inaccessible analogs.

Specific Aim #3: Evaluate fully synthetic berberine analogs in bacterial growth inhibition assays, FtsZ GTPase activity assays, and FtsZ polymerization assays to identify antibiotic compounds that operate through inhibition of cell division.

The major innovations include 1) the development of a concise and flexible synthetic route that will facilitate the rapid preparation of previously inaccessible berberine and THPB analogs, and 2) the use of an in silico synthesis/virtual screening approach to prioritize target compounds. The proposed work is significant because the development of novel small molecule inhibitors of cell division has the potential to deliver 1) chemical probes that will enable future studies on the therapeutic potential of FtsZ inhibitors, and 2) hit compounds that define a new class of antibiotic therapeutics.

Maria Kalamvoki

Maria Kalamvoki, Assistant Professor, Dept. of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center

Mentor: Edward B. Stephens, Professor of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center

Project TitleDeveloping chemical inhibitors of essential ICP0 functions in Herpes Viruses


Project Summary

Herpes simplex virus (HSV) causes diseases ranking in severity from annoying labialis and genital infections, to blinding keratitis, risk of developing encephalitis, risk of transmission to newborns, and increased risk of acquiring HIV-1 infection. Following lytic infection in epithelial cells at the portal of entry in the body, HSV establishes a latent infection in sensory neurons. Occasionally the virus is reactivated, generally as a result of a weakened immune system causing recurrent diseases. The current antiviral used to treat herpesvirus infections, acyclovir, although effective at blocking viral DNA synthesis, has limited bioavailability and acts late during the replication when many viral products are already present. Due to low lipophilicity it does not cross the blood brain barrier to prevent encephalitis. Drug resistance has been reported in immunocompromised individuals.

To infect and persist in the human body, HSV must overcome strong innate and adaptive immune responses. The infected cell protein 0 (ICP0), an immediate early protein of the virus, plays fundamental roles in this process. Its two most prominent functions are to render the infected cells resistant to the antiviral activity of interferons and to block the silencing of viral DNA and initiate transcription. ICP0 acts as an E3 ubiquitin ligase to degrade the innate immune components PML and SP100 that are constituents of the ND10 nuclear bodies where the viral genome is deposit and it is silenced. Following their degradation the ND10 bodies are dispersed and this is essential for viral gene expression. In tandem, ICP0 blocks the silencing of viral DNA through dissociation of repressor complexes. Subsequently, ICP0 recruits chromatin remodeling enzymes such as the histone acetyl transferase CLOCK (Circadian Locomotor Output Cycles Kaput), along with its partner BMAL-1, to activate viral gene expression. CLOCK is recruited to the viral genome via the direct interaction of ICP0 with BMAL-1. Failure of ICP0 to execute any of these functions impairs virus replication. ICP0 is essential in vivo and the ICP0 E3 ligase and the ICP0 null mutants fail to counteract IFN responses. This results in a failure to spread from the initial site of infection and less efficient reactivation. Given that ICP0 executes its functions immediately after the entry of the virus into the cell and before the onset of proteins synthesis, we hypothesize that small compounds interfering with these essential ICP0 functions will impede the viral infection and attenuate HSV reactivation.

To test our hypothesis we have formulated two Specific Aims: In Aim 1, we propose to identify compounds that block the HSV ICP0 E3 ligase activity in vitro. In Aim 2, we will identify compounds that block the interaction of ICP0 with BMAL-1 and thereby will block viral gene expression. The University of Kansas (KU)-High Throughput Screening collection (HTSC) of over 300,000 compounds will be utilized with the support of the KU High Throughput Screening Laboratory (HTSL). The results are expected to identify novel compounds with antiviral activity. Additionally, these compounds will serve as tools to characterize the ICP0 functions.

Joanna Slusky, Assistant Professor, Depts. of Computational Biology and Molecular Biosciences, University of Kansas

Mentor: Lynn Hancock, Associate Professor, Dept. of Molecular Biosciences, University of Kansas 

Project Title: Targeting Oligomerization to Potentiate a Broad Spectrum of Antibiotics


Project Summary

Antibiotic resistance is correlated with overexpression of the acridine efflux pump. This pump is the preeminent efflux pump in gram-negative bacteria and is responsible for shuttling out most classes of antibiotics. Previous efforts have led to compounds that disable pumps by inhibiting one of the drug binding sites in the inner membrane component of the pump, but such compounds have proven toxic and overly specific. Here, we propose to create peptides and peptidomimetics that prevent oligomerization of the outer membrane component of the acridine pump. The outer membrane component of the acridine pump is a trimeric β-barrel called TolC. Targeting the outer membrane portion of the pump reduces concerns over toxicity because the target complex is unique to outer membranes and human cells do not possess an outer membrane. Moreover, targeting oligomerization instead of targeting one of the two binding sites broadens the applicability of the inhibitor. Specifically, by targeting oligomerization we can stop all efflux through the pump, not just the antibiotics that interact with one of the multiple acridine pump binding sites. Our long-term goal is to develop compound that resensitize gram-negative antibiotic resistant bacteria to a variety of antibiotics. Our central hypothesis is that we can disrupt assembly of the outer membrane β-barrel component of efflux pumps, by binding their interface strands with β-hairpin peptides or β-hairpin mimetics similar to the interfacial strands’ native binding partners. This will be significant because it represents a step towards enable a revival of existing antibiotics for resistant superbugs, by using helper drugs that would be less likely to suffer from toxicity or over specificity. This work is innovative because it introduces a new type of inhibitor for β-barrels, extending the method of dominant negative fragment inhibition for use in the outer membrane. We plan to carry out this project through pursuit of two aims. In the first aim we will create a screen to find peptides that disrupt TolC drug efflux. We will carry this out by creating a β-hairpin library modeled after the β-strands at the interface of the TolC trimeric interaction. Successful folding and binding of these peptides will be identified through FACS and replica plating, then we will test successful peptides on a broad range of antibiotics. In the second aim we will design peptidomimetics that disrupt TolC oligomerization. We do this by designing β-hairpin mimetics, synthesizing these, and testing their activity against several gram-negative bacteria and antibiotics.

Zhilong Yang, Assistant Professor, Division of Biology, Kansas State University

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

Project Title: Chemical approaches towards understanding and preventing poxvirus infection


Project Summary

Poxviruses remain to have significant impacts on the public health after the eradiation of smallpox, the deadliest disease in human history, as they comprise highly dangerous emerging and re-emerging pathogens of humans and other vertebrates. Poxviruses are also being utilized as vectors to treat various infectious diseases and multiple cancers. Like all viruses, poxviruses rely on host cell factors to complete their lifecycles. Consequently, there is a significant need to identify and characterize cellular functions required for poxvirus replication. However, the roles of most cellular functions in poxvirus replication are poorly understood. The objective of this project is to identify and characterize specific cellular functions that are important for poxvirus replication, using vaccinia virus as our model poxvirus. The approach is to first identify bioactives and chemical compounds with known cellular targets that inhibit vaccinia virus replication, followed by genetic and biochemical characterization to determine the underlying viral and cellular mechanisms. The project will be implemented through two parallel Specific Aims. In the Specific Aim 1, a class III protein deacetylase SIRT1 inhibitor Ex-527 has been identified to potently inhibit vaccinia virus replication in a small-scale screening. The viral and cellular mechanisms by which Ex-527 inhibition of vaccinia virus replication will be determined. Upon completion of this aim, it is anticipated to uncover the role of SIRT1 in VACV replication. In the Specific Aim 2, a high-throughput screening will be carried out to identify cellular functions important for vaccinia virus replication through screening a collection of bioactive and FDA-approved compounds. Upon accomplishing the aim 2, it is anticipated to understand the roles of cellular functions in vaccinia virus replication in a more comprehensive manner and to determine the most prominent candidates for follow-up mechanistic studies. Taken together, this project will provide novel insights into specific cellular functions in vaccinia virus replication through a chemical approach combined with genetic and biochemical characterization.


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