Spotlights-Research at the CBI

Current CBI Fellows:

Joy Thames

As outbreaks of new viruses occur, the need for broad spectrum antiviral drugs has increased. In that regard, nucleos(t)ide analogues have a rich history as antivirals. One modification in nucleoside drug design that has proven successful is the use of acyclic sugars, such as that found in Acyclovir (ACV), an FDA-approved drug for herpes simplex virus. Research in the Seley-Radtke group has focused on the development of novel nucleos(t)ide analogues known as “fleximers”, which feature a “split” purine nucleobase, where a carbon-carbon single bond connects the pyrimidine and imidazole rings, thus introducing flexibility to the nucleobase scaffold. This endows the “fleximers” with potent activity not seen for the corresponding rigid analogues. Combining the flex-nucleobase with the acyclic sugar of ACV produced a series of doubly flexible Flex-ACV analogues. These novel analogues have exhibited low micromolar to nanomolar levels of activity against human coronaviruses (SARS-CoV-1, MERS, HuCoV-NL63) as well as Ebola, Yellow Fever, Dengue and TBEV, while ACV has no activity against those viruses. My project has focused on the design and synthesis of new Flex-ACV analogues, as well as the optimization of previously synthesized analogues for antiviral testing and mechanism of action studies. Recently Flex-ACV analogues have shown mid-micromolar activity against SARS-CoV-2. The biological window of the Flex-ACV compounds continues to be investigated, along with new fleximer analogues of cidofovir. Cidofovir is an FDA approved nucleoside drug for cytomegalovirus retinitis in aids patients and its lipid prodrug analogue Brincidofovir has exhibited activity against Ebola. Using modern synthetic methods I have been able to begin the synthesis of cidofovir analogues, as well as the synthesis of new Flex-ACV analogues.

Notable publications and presentations:

  1. Thames, J.E.; Waters III, C.D.; Valle, C.; Bassetto, M.; Aouadi, W.; Martin, B.; Selisko, B.; Falat, A.; Coutard, B.; Brancale, A.; Canard, B.; Decroly, E.; Seley-Radtke, K.L. Synthesis and biological evaluation of novel flexible nucleoside analogues that inhibit flavivirus replication in vitro. Bioorg. Med. Chem. 2020, 28(22), 115713.
  2. Thames, J.E. and Seley-Radtke, K.L. Comparison of the old and new- novel mechanisms of action for anti-coronavirus nucleoside analogues. Chimia. 2022, 76, 409-417.
  3. Thames, J.E., Correa-Sierra, C., Rege, A., Liu, S., Bieberich, C., Schang, L., Seley-Radtke, K.L. Synthesis and Biological Evaluation of Flex-Acyclovir Analogues Against SARS-CoV-2. Poster Presentation at ICAR Meeting (Online); March 2021.
  4. Thames, J.E. An Antiviral Safari Adventure. Oral Presentation, Pecha Kucha contest at ICAR Meeting (Seattle); March 2022.
  5. Thames, J.E., Lundberg, E.V., Esposito, F., Corona, A., Tramontano, E., Gentry, B., Seley-Radtke, K.L. Elucidation of the Mechanism of Action of Various Flex- Acyclovir Analogues. Poster Presentation at Chemistry Graduate Student Research Day (UMBC, Baltimore); March 2022.

 

Tim Hufford

Oxygen is required for most organisms to produce the energy necessary to develop or maintain normal cell metabolism. Under hypoxic conditions, drastic changes in gene expression and metabolism must take place to facilitate an adaptive response. I am investigating the critical role of the N-myc Downstream Regulated Gene family in this hypoxia-induced metabolic suppression, using the hypoxia-tolerant zebrafish as a model organism. More specifically, my thesis research work addresses how members of this family, Ndrg1 in particular, are regulated at the transcriptional level to prolong hypoxia survival. We have found that Ndrg1a is the most robustly regulated family member, with a striking 9-fold up-regulation in response to prolonged anoxia, suggesting that this gene is critical for long term adaptation to hypoxia. We have further found that ndrg1a expands under prolonged anoxia to tissues where it is not expressed under normoxia, such as the muscle, inner ear and head vasculature, providing insight into novel protective functions that this gene may have. It is currently unknown to what degree Ndrg1a functions post-transcriptionally in zebrafish, yet there is evidence in cancer literature that a long non-coding RNA transcript variant carries out a specific hypoxia-induced role as well as Ndrg1 protein being reported to interact directly with transcription factors such as β-catenin. Ndrg1 protein also has a helix-turn-helix domain in its protein structure, hinting at a possible direct interaction with DNA. Our lab also has generated ndrg1a-/- CRISPR knockout mutants, allowing us to investigate ndrg1a function. I am currently performing RNA-seq analysis to identify genes/gene clusters across the zebrafish transcriptome that are modulated under hypoxia in an Ndrg1a-dependent manner, thus providing new and exciting insights into this adaptive response.

Notable publications and presentations:

  1. Le, N.*, Hufford, T.*, Brewster, R. Differential Expression and Hypoxia-mediated Regulation of the N-myc Downstream Regulated Gene Family. Submitted to FASEB Journal.
  2. Hufford T., Jong P., and Brewster R. Members of the NDRG family: Adapter proteins, oxygen sensors, and mediators of hypoxia tolerance (Co-first authorship). In preparation.
  3. Hufford, T..; Le, N.; Okafor, I.; Park, J.; Brewster, R. Investigating the Role of N-Myc Downstream Regulated Gene 1a (NDRG1a) in Anoxia-Induced Cell Cycle Arrest. Keystone Symposia, Hypoxia: Molecules, Mechanisms, and Disease, Keystone Resort, Keystone, Colorado; January 2020. Poster.
  4. Hufford, T.; Le, N.; Park, J.; Brewster, R. Unraveling Transcriptional Regulation Imparted by N-Myc Downstream Regulated Gene 1a (NDRG1a) Under Anoxia in Zebrafish. The 21st International Hypoxia Symposium 2019, Farimont Chateau Lake Louise, Alberta Canada; February 2019. Poster.

John Terrell

It has been well established that commonly used in vitro cell models, such as culture flasks or other flat-bottomed plastic systems, are insufficient for maintaining proper cell function. One clear piece of evidence regarding this is the loss of primary human hepatocyte (liver parenchymal cell) phenotype and function within 24-48 hours when seeded on a standard culture flask, but functions are retained for up to several weeks when cultured in a 3D environment. My research investigates multi-omics differences in hepatocytes grown in 2D and 3D environments to understand what effects surface topography has on the cell. Further on we will characterize how information from the cell culture surface is mechanistically transduced to the cell to cause these differences.

Notable publications and presentations:

  1. Terrell, J. A.; Jones, C. G.; Kabandana, G. K. M.; Chen, C. From cells-on-a-chip to organs-on-a-chip: scaffolding materials for 3D cell culture in microfluidics. Journal of Materials Chemistry B 2020, 8(31), 6667-6685.
  2. Huang, T.; Terrell, J. A.; Chung, J. H.; Chen, C. Electrospun Microfibers Modulate Intracellular Amino Acids in Liver Cells via Integrin β1. Bioengineering 20218(7).

Zach Nichols

Sample preparation is an essential step in nearly every analytical technique and is often the limiting factor in terms of costs and turnaround time. To minimize the time required and simplify the preparation process, many different sample preparation methods have been developed. One method that has been developed by the Geddes Lab is the use of devices called microwave lysing triangles (MLTs) which use thin metal films on a glass substrate to direct radiation from a microwave oven to a small area of high electric field intensity. When a biological sample is placed in this area and irradiated it undergoes rapid heating and the MLTs generate reactive oxygen species (ROS) from oxygen in the atmospheric environment which can inactivate pathogens, extract nucleic acids and proteins from cells, and fragment nucleic acids for downstream analysis or processing. My project focuses on further characterizing how MLTs generate ROS and  expanding their sample throughput via the modification of a common microplate. This is done by creating a periodic array of thin metal films on the bottom of the microplate, called a metasurface. Metasurfaces modulate electromagnetic radiation via the boundary conditions of different materials, in our case a metal and a dielectric, which are modified by changing the spatial arrangement of the materials. I have created and characterized several metasurfaces and microplate prototypes already and am currently working on comparing biological samples prepared with the microplates to those prepared with MLTs. Future work and cross-training will focus on assessing the utility of our microplates and MLTs in preparing samples for genomic sequencing assays as well as probing the ROS generation mechanism with fluorescent sensing probes.

Notable publications and presentations:

  1. Nichols, Z. E.; Saha, L.; Knoblauch, R.; Santaus, T. M.; Geddes, C. D. “Development of a Microplate Platform for High-Throughput Sample Preparation Based on Microwave Metasurfaces”
    IEEE Access. 2021, 9, 37823-37833.
  2. Nichols, Z. E.; Geddes, C. D. “Sample Preparation and Diagnostic Methods for a Variety of Settings: A Comprehensive Review” Molecules. 2021, 26 (18), 5666.
  3. Nichols, Z. E.; Geddes, C.D. Applying Metasurfaces to Biological Sample Preparation and Clinical Diagnosis. 2022 UMBC Graduate Research Conference. University of Maryland, Baltimore County. Baltimore, Maryland. March 2022. (Poster)

 

Alexandria Chan

Multiple myeloma is a cancer of the plasma cells which overproduce abnormal proteins that must be degraded by the malignant cells. Proteasome inhibitors, a first-line treatment for the disease, take advantage of this characteristic by blocking the main pathway of protein degradation, thereby resulting in malignant cell death. However, there is considerable resistance to these inhibitors, driving patients into the relapsed/refractory subgroup with poor prognosis. My research focuses on a polypharmacologic method to simultaneously inhibit both the proteasome and aggresome pathways in order to rescue therapeutic efficacy in resistant patients. Additional research implements a proteolysis targeting chimera (PROTAC) strategy to target myeloid cell leukeima-1 (MCL-1), an anti-apoptotic protein that is a long-sought after target for cancer therapeutics. This project aims to downregulate MCL-1 on a transcriptional, translational, or post-translational level through the degradation of related protein-targets. By targeting its downregulation, we are circumnavigating the negative side effects and difficulties associated with direct inhibition or degradation of MCL-1.

Notable publications and presentations:

  1. Chan, A. M. Goodis, C. G. Pommier, E. and Fletcher, S. Recent Applications of Covalent Chemistries in Protein-Protein Interaction Inhibitors. RSC Med. Chem. 2021, Just accepted.
  2. Chan, A. M. and Fletcher, S. Shifting the paradigm in treating multifactorial diseases: polypharmacological co-inhibitors of HDAC6. RSC Med. Chem. 2021, 12, 178-196.
  3. Chan, Alexandria. Cottingham, Andrea. Mitchell, Ashley. Haile Aytenfisu, Asaminew. MacKerell Jr., Alexander D. Fletcher, Steven. Polypharmacologic Approach to Relapsed/Refractory Multiple Myeloma: Dual Inhibition of the Proteasome and Aggresome Pathways. Medicinal Chemistry Gordon Research Conference. Poster Presentation. Mount Snow, Vermont. October 25-29, 2022.
  4. Chan, Alexandria. Cottingham, Andrea. Mitchell, Ashley. Fletcher, Steven. Paradigm Shift in Treating Multiple Myeloma: Dual Incapacitation of Proteasome and Aggresome Pathways with Polypharmacological Agents. American Chemical Society National Fall Conference 2021. Oral Presentation. Atlanta, Georgia. August 22, 2021.
  5. Chan, Alexandria and Fletcher, Steven. Indirect Downregulation of MCL-1 via Targeted PTORACs. American Chemical Society Mid-Atlantic Regional Meeting. Poster Presentation. Virtual. June 10, 2021.

 

Mark Lee

Across all bacteria, the ferrous iron (Fe2+) uptake (Feo) system is the most prevalent and the most well-distributed system dedicated to the acquisition of Fe2+. However, despite Feo’s nearly ubiquitous presence across the prokaryotic domain, this system remains poorly understood. The Feo system consists canonically of three proteins: FeoA, FeoB, and FeoC. FeoB is a complex, transmembrane G protein and is arguably the most important component of the Feo system. The function of FeoB is to transport iron across a lipid bilayer to within the cytosol, but the structural and mechanistic details of this process are lacking. My research focuses on the incorporation of FeoB into lipid-protein and copolymer nanodiscs for functional and structural characterizations of FeoB. In addition, recent studies have suggested that not all FeoBs are GTP-specific, but rather may be NTPases. To probe this hypothesis, I am working to determine the structure of the N-terminal domain of FeoB (NFeoB) in the presence of adenosine nucleotides. Finally, recent bioinformatics data from my lab have identified genes encoding a small transmembrane protein (designated FeoD) of unknown function adjacent to FeoB in the feo operon. This protein is predicted to bear a single transmembrane helix with a C-terminal cysteine-rich tail. Because FeoD is present in many bacterial genomes that lack FeoC (a soluble, [Fe-S] cluster-binding protein) I predict that FeoD may function similarly. Thus, I am also working to clone, to express, to purify, and to characterize (structurally and functionally) the newly-discovered FeoD protein. Combined, this work helps elucidate the mechanistic details of this essential iron acquisition pathway.

Notable publications and presentations:

  1. Brown, J. B.; Lee, M. A.; and Smith, A. T. The structure of Vibrio cholerae FeoC reveals conservation of the helix-turn-helix motif but not the cluster-binding domain. bioRxiv. 2022, DOI: 10.1101/2022.02.26.482101
  2. Sestok, A. E.; Lee, M. A.; and Smith, A. T. Prokaryotic ferrous iron uptake: exploiting pools of reduced iron across multiple microbial environments. In: Hurst, C.J. (eds) Microbial Metabolism of Metals and Metalloids. Advances in Environmental Microbiology,2022, 10, 299-357. Springer, Cham.
  3. Brown JB, Lee MA, Smith AT. Ins and Outs: Recent Advancements in Membrane Protein-Mediated Prokaryotic Ferrous Iron Transport. Biochemistry. 2021 Nov 9;60(44):3277-3291.

Past CBI Fellows and Members:

Dr. Evgenia Barannikova

Patricia Boyd

Dr. Joel Brandis

Dr. Luke K. Brewer

Dr. Janae Brown (Baptiste)

Dr. Richard Brown

Dr. Brian M. Cawrse

Dr. Kenneth C. Childers

Dr. Emily E Chea 

Dr. Alecia T. Dent

Dr. Lance Dockery

Dr. Brandon J. Drennen

Dr. Amy Defnet

Dr. Nopondo N. Esemoto

Dr. Miji Jeon

Dr. Erin L. Kennedy

Dr. Rachael Knoblauch

Ramon Martinez III

Dr. Ryan McDonald

Maraki Negesse

Dr. Kiwon Ok

Dr. Jong Park

Dr. Jordan D. Pritts

Dr. Elizabeth Robinson

Dr. Tonya M. Santaus

Dr. Danielle Schmitt

Dr. Alex E. Sestok

Dr. Michael White

Dr. Denise N. Williams

Dr. Tyree Wilson

Dr. Mary K. Yates

Stephanie Zalesak