Current 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.

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

Mentor: Rollie Clem, Professor, Division of Biology, Kansas State University

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.

 

 

Past Research Projects

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.


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