Rebecca Adamek 

Chemistry and Biochemistry
Seth Cohen Lab

Rebecca Adamek

CBI Appointment Period: 2015-17
Grant Year: [1,2]

Thiohydroxypyridinones as a Scaffold for the Development of Potent New Delhi Metallo-ß-Lactamase-1 Inhibitors

Since their discovery in 1928 by Alexander Fleming, ß-lactam drugs have been the most successful and commonly prescribed class of antibiotics.  Unfortunately, the widespread use of this powerful drug class has created a natural selection pressure for bacteria to adapt and develop resistance mechanisms to these therapeutics; most notably the emergence of ß-lactamase enzymes.  ß-lactamases confer bacterial resistance through hydrolysis of the ß-lactam bond that is crucial for the activity of these drugs.  New Delhi Metallo-ß-lactamase (NDM-1) is a particularly worrisome metallo-ß-lactamase that utilizes two active site Zn2+ ions to achieve this hydrolytic activity.  NDM-1 has been shown to be capable of hydrolyzing all clinically relevant ß-lactams, including the carbapenems – which are considered a last resort drug against resistant infections.  Furthermore, NDM-1 is easily communicable between bacteria populations as it is encoded in blaNDM-1, a plasmid capable of horizontal transfer, and has already been detected in multiple strains of enterobacteriaceae.  Indeed, since its discovery in India in 2008, NDM-1 has rapidly spread among the general population worldwide and has not been limited to hospital-acquired infections.  In addition to this ease of transmission, there are currently no clinically approved drugs available against NDM-1, implicating that NDM-1 has a strong potential to lead to untreatable “superbug” type infections. The ultimate goal of this research is to develop potent inhibitors of NDM-1 to restore ß-lactam activity against resistant bacteria.  In order to meet this goal, we have synthesized and screened metal binding pharmacophore (MBP) libraries against NDM-1 to identify MBP fragments that selectively bind to the dinuclear Zn2+ NDM-1 active site.  This screen has revealed thiohydroxypyridinone-based compounds, 3-carboxy-1-hydroxypyridine-2-thione and 2-hydroxyisoquinoline-1-thione, as novel scaffolds for the development of NDM-1 inhibitors.  The use of a fragment growth strategy to derivatize these compounds has resulted in the discovery of a new, thiohydroxypyridinone-based NDM-1 inhibitor with a potent IC50 of 240 ± 10 nM.

 

Thomas Bartholow 

Chemistry and Biochemistry
Michael Burkart Lab

Thomas Bartholow

CBI Appointment Period: 2015-17
Grant Year: [1,2]

Understanding the Mechanisms of Protein-Protein Interactions in Fatty Acid Biosynthesis and its associated pathways

The Fatty acid biosynthetic (FAS) pathway functions through an iterative mechanism, using sequential conserved chemistries to efficiently and specifically build the fatty acids required for life. The acyl carrier protein (ACP) shuttles fatty acid chains as they are modified, facilitating each reaction and protecting the acyl chain. My research is focused on understanding the structure and dynamics of this pathway and ACPs interaction with its wide range of partner enzymes. Using NMR and crystallography I aim to characterize the interactions between ACP and partner enzymes which confer specific and efficient biosynthesis.

 

Patrick Brunson

Scripps Institute of Oceanography (SIO)
Moore/Allen Lab

Thomas Bartholow

CBI Appointment Period: 2016-18
Grant Year: [2,3]

Enzymatic Biosynthesis of Domoic Acid in the marine diatom Pseudo-nitzschia multiseries

Large blooms of Pseudo-nitzschia spp. diatoms produce high levels of domoic acid (DA), a neurotoxic glutamate analog.  Consumption of DA in the form of contaminated seafood can cause Amnesiac Shellfish Poisoning in humans, a life-threatening condition with chronic effects including short-term memory loss.  Despite decades of research, the genes encoding DA biosynthesis have remained elusive, precluding environmental genetic testing as a viable DA monitoring approach.  To address this lack of knowledge, we have constructed RNAseq libraries for P. multiseries cultures grown under nutritional stressors known to induce DA production in order to identify genes upregulated alongside increased toxin production.  Out of forty upregulated genes, we have identified three candidate genes which seem to encode enzyme activities relevant to DA biosynthesis.  Using heterologous protein expression in conjunction with in vitro biochemical assays, we have demonstrated clear biosynthetic roles for at least two of our candidate enzymes.  Biosynthesis begins with a terpene cyclase-like enzyme catalyzing N-prenylation of free L-glutamic acid using geranyl pyrophosphate, an unprecedented enzymatic reaction that is likely occurring within the diatom chloroplast.  In addition, an αKG-dependent iron dioxygenase catalyzes a cyclization reaction to form the pyrrolidine ring which is characteristic of domoic acid and its naturally-occurring isomers.  We are also investigating the enzymatic function of an upregulated cytochrome P450 which may introduce up to three successive rounds of oxidation on the terpene-derived side chain, forming the carboxylic acid residue found in mature DA.  These three genes are also found to be clustered alongside a transmembrane permease and a conserved hypothetical protein in both the P. multiseries genome and the P. multistriata genome, further implicating these genetic components in a common DA biosynthetic pathway.  Future directions will include full pathway reconstition in the model diatom Phaeodactylum tricornutum, further characterization of the transmembrane permease, and attempts to characterize DA biosynthetic gene knockouts in Pseudo-nitzschia diatoms.

 

Kayla Busby 

Chemistry and Biochemistry
Neal Devaraj Lab

Thomas Bartholow

CBI Appointment Period: 2017-18
Grant Year: [3]

Enzymatic Site-Specific Labeling of RNA for Affinity Isolation of RNA-Protein Complexes

Recent efforts by the Encyclopedia of DNA Elements (ENCODE) to characterize the transcriptome have uncovered that while only 1-2% of the genome codes for proteins, a majority of the genome (~62%) is transcribed into RNA. Despite the rapid identification of noncoding RNAs, the characterization of these RNAs has lagged behind, in part due to the difficulty of identifying the proteins that interact with an RNA of interest. My research aims to develop an efficient method for the isolation of RNA-protein complexes via direct labeling of a specific cellular RNA with a purification handle such as biotin. Our lab’s recently developed methodology, RNA Transglycosylation at Guanosine (RNA-TAG) covalently modifies RNA by facilitating an enzymatic reaction in which a guanine nucleobase is replaced with a derivative of the nucleobase preQ1. Through further development of this method, we aim to characterize disease relevant RNA-protein interactions.

 

Naneki Collins  

Chemistry and Biochemistry
Nathan Gianneschi Lab

Naneki Collins

CBI Appointment Period: 2015-17
Grant Year: [1,2]

A polymer-based strategy for the targeted delivery of cancer drugs via degradable nanomaterials

My research is focused on using ring-opening metathesis polymerization (ROMP) to create self-assembled polymeric nanoparticles for the delivery of peptide targeted, platinum cancer therapeutics. By employing a hydrophobic, covalently linked, directly polymerizable platinum drug monomer in tandem with a hydrophilic oligoethylene glycol (OEG) monomer, amphiphilic block copolymers can be synthesized which self-assemble into micelles upon dialysis from organic to aqueous media. These nanoparticles are size-tunable in the 10-200 nm range based on the polymer block sizes and are very stable with little release of platinum over time, yet show IC50 values comparable with those of the parent drugs in vitro. By decorating the micelles with targeting antibodies which are directly polymerized onto the hydrophilic ends of the polymer chains, the particles can be directed selectively to cancerous tissues. We have begun in vivo studies to determine targeting efficiency and tumor shrinkage in subcutaneous mouse tumor models using these particles and preliminary results are promising.

Currently, most ROMP-based polymers are not degradable, and none have yet been shown to degrade in vivo, so I am also interested in developing degradable versions of these polymeric materials for drug delivery as well as other applications.

 

 

Ashley Kroll 

NanoEngineering
Liangfang Zhang Lab

Ashley Kroll

CBI Appointment Period: 2015-16
Grant Year: [1]

Cancer Cell Membrane-Coated Nanoparticles for Anticancer Vaccination

This project utilizes the cell membrane coated nanoparticle technology for anticancer vaccination. PLGA particles can be loaded with adjuvant and coated with cancer cell derived membrane as an antigen. These particles take advantage of their small size by releasing adjuvant to its endosomal receptors when endocytosed. The cancer cell membrane coated nanoparticle (CCNP) also improves upon current vaccination formulations by using the membrane as a purified and multivalent antigen source compared to whole cell lysate formulations, which include intracellular components that can dilute immune system training, and single antigen formulations which can be evaded by antigen shedding by tumor cells.

 

Taryn Lucas

Chemistry and Biochemistry
Kamil Godula Lab

Thomas Bartholow

CBI Appointment Period: 2016-18
Grant Year: [2,3]

Determining the Effect of Receptor Presentation on Viral Binding

By implementing a glycan microarray format, glycopolymers of various lengths and containing several sugar valencies of a2-6/a2-3 sialyllactose can be printed on a glass slide at different concentrations to achieve an assortment of receptor presentations for the influenza virus. Glycopolymers offer many advantages over the heterogeneous mixture of glycoconjugates obtained from biological sources. First, the exact carbohydrate composition and linkage type can be known with certainty, a feat that is not possible with isolated glycoconjugates due to the non-templated post-translational glycosylation machinery within cells.  Secondly, our polymer backbone allows for the incorporation of a tagging molecule with ease. We incorporate a TAMRA fluorophore that allows us to determine the concentration of polymer in each printing solution. Furthermore, synthesis of the polymer can be altered in a way that permits the systematic investigation of sugar presentation on viral binding.  Microarrays are a high-throughput means to evaluate protein-sugar interactions. Whole virus can be incubated on the array at a range of concentrations to uncover which configuration of glycan receptors affords optimal binding of the hemagglutinin (HA) protein on the viral coat. This protein acts as a lectin to bind sialic acid-containing receptors on the host cell surface, constituting one of the first steps for viral entry and infection. Neuraminidase (NA) is another important viral coat protein, responsible for enzymatically releasing bound virus from receptors. This protein is essential for viral movement through mucosal barriers that contain “decoy” receptors.  The activity of NA and the affinity of HA must be balanced to afford optimal infectivity. Our microarrays will be used to study the dynamic nature of HA and NA.  After and initial reading of viral binding at 0 °C, the arrays will be incubated at 37 °C, where NA is active and will result in release of the virus from the glycopolymers. A time-course study will reveal the activity of NA. This platform provides a convenient method to investigate these viral proteins in a controlled manner. The information gained in these studies will be useful in determining how HA and NA behavior influences infection in vivo.

 

Rohit Subramanian

Chemistry and Biochemistry
Akif Tezcan Lab

Rohit Subramanian

CBI Appointment Period: 2015-16
Grant Year: [1]

Enzyme-directed addition of functional molecules to protein biomaterials

Our research uses enzymes to selectively assemble functional semi-synthetic, crystalline protein and peptide arrays. The foundation of this research is built using a novel technological platform for self-propagation demonstrated by our laboratory. This platform has been developed utilizing the self-assembly of the RIDC3 protein, a four-helix bundle cytochrome variant, in the presence of zinc ions to induce the formation of 2D sheets or tubular structures. Here we present progress toward the functionalization of RIDC3 sheets to create new biomaterials with potential for uses in biosensing and diagnostics. Toward this goal, the eleven amino acid peptide ‘ybbR’ (DSLEFIAKSLA) was chosen to insert into the monomeric RIDC3 protein. A chemical route to enzymatically-driven functionalization has been studied extensively using the ybbR peptide as a substrate for phosphopantatheinyl transferases (PPTases). YbbR labeling by PPTase offers a highly versatile tool for site-selective covalent protein modification. The transfer of 4’-phosphopantetheine, which is derived from coenzyme A (CoA), to an active site serine residue on the ybbR peptide allow for the addition of virtually any moiety to the peptide. As a proof of principle, coenzyme A covalently attached to a rhodamine dye has been used as a means of visualizing the successful transfer of a phosphopantetheine to protein materials displaying ybbR. Incorporating ybbR into the RIDC3 system has been undertaken in a variety of approaches to explore functional protein arrays.

Thus far, we have developed two routes for the surface modification of RIDC3-based protein arrays. The first is a post-assembly chemical modification of pre-formed RIDC3 sheets with a functional peptide onto surface exposed lysine residues. As a proof-of-principle, enzymatic labeling was accomplished using a TAMRA-modified CoA analog as a colored, fluorescent readout. The second route to chemical modification of RIDC3 arrays is in efforts to use the enzymatic transfer to covalently link large biomolecules to arrays. In contrast to TAMRA-CoA labeling of RIDC-ybbR arrays, we sought to covalently link the CoA substrate onto protein surfaces. We tested the effectiveness of the new reaction by enzymatically attaching a GFP-ybbR construct to visualize the reaction by confocal microscopy.

In lieu of chemically modifying array surfaces to incorporate ybbR, we looked toward genetic incorporation of ybbR onto protein biomaterials. Initial studies with a RIDC3-ybbR construct showed that the protein was very unstable upon the genetic incorporation of the peptide. Our recent report on the robust assembly of unsupported 2D lattices makes use of the C4 symmetric protein Rhamnulose-1-phosphate aldolase (RhuA) containing precisely-placed cysteine residues at its corners (C98RhuA). Thus far, we have successfully formed 2D arrays with the C98RhuA-ybbR construct, bearing an identical arrangement of proteins to that of the parent C98RhuA construct. The peptide is appended to the C-terminus of C98RhuA such that the peptide is displayed at the surface of an array and interference with disulfide formation is minimized. We have also observed the successful enzymatic transfer of TAMRA onto C98RhuA-ybbR tetramers using TAMRA-CoA as the enzyme substrate.