During spring quarter, UCSD ACS-SA partners with the Department of Chemistry and Biochemistry to host our annual Undergraduate Research Symposium, celebrating the research contribution of our student chemistry community! This event provides our student researchers with a great opportunity to practice science communication through a poster presentation! Presenting a poster is required for department honors, and the best presenters within each division will receive a symposium poster presentation award. More information on department honors and undergraduate student awards can be found below!
Elizabeth (Betsy) Komives grew up in a small town in Wisconsin. She did her undergraduate degree in Chemistry at MIT, her PhD in Pharmaceutical Chemistry at UC San Francisco, and after postdoctoral work at Harvard took a faculty position at UC San Diego in 1990. Her research is in protein-protein interactions and dynamics. She likes to explain that when the human genome was sequenced, we discovered that we have about the same number of genes as fruit-flies! The reason humans are so much more complicated is that we convert a single gene into many different proteins and then these proteins interact with one another forming complexes with varying functions. Throughout her career, Komives invented new methods for integrating biophysical tools to discover new principles, which have profoundly advanced our fundamental understanding of macromolecular recognition. She has trained over 30 PhDs who are now leaders in academia and the biotech industry. Throughout her career she has tried to integrate outreach activities, especially focusing on underprivileged high school student for whom she provides research opportunities and the confidence that they, too, can become scientific leaders. Her students learn to build a “family” based culture of honesty, trust, and mutual aid in what is typically a cut-throat and competitive environment.
Non-coding RNAs greater than 200-nucleotides are termed long non-coding RNAs (lncRNA) and have been shown to contribute to cellular regulation by controlling gene expression. Mis-regulation of lncRNAs is present in cancer. The lncRNA DUBR (DPPA2 Upstream Binding RNA), has been identified to be mis-regulated in several types of cancer including human colorectal carcinoma. However, the functions of DUBR in normal and diseased cells have not been well explored. High expression of DUBR in human colon cancer is predictive of poor patient outcome by Kaplan-Meier survival analysis. Previous research in the McHugh Lab has shown that DUBR is required for normal colon cancer cell growth. RNA Antisense Purification coupled with Mass Spectrometry (RAP-MS) experiments were conducted and showed that DUBR endogenously bound to epigenetic regulators – proteins in the nucleosome remodeling and deacetylase (NuRD) complex and DNA methyltransferase 1 (DNMT1). RNA immunoprecipitation experiments were conducted to confirm the protein binding partners of DUBR and In Vitro RNA pull-down experiments will be used to identify the region(s) of DUBR in which these epigenetic regulators are binding. qPCR analysis after DUBR KD showed upregulation of the neighboring gene B and T lymphocyte attenuator (BTLA). Furthermore, exogenous overexpression of BTLA caused cell death in HCT116 cells. We hypothesize that DUBR regulates colon cancer cell growth by fine tuning epigenetic protein function at nearby gene loci. This study will expand our understanding of this functional lncRNA DUBR.
New and emerging evidence has demonstrated the importance of long non-coding RNAs(lncRNA) and their involvement in cancer cell proliferation and metabolism. However, their specific biological functions as non-coding RNA are still poorly understood. Here, we report and characterize a lncRNA, termed Growth Regulator Antisense 1(GRAS1), in the lung carcinoma epithelial cell line A549. GRAS1 is determined to be critical for A549 cell viability based on the fact that knockdown of GRAS1 significantly lowers A549 cell survival rate. Upon knockdown of GRAS1, protein expression levels of the tumor suppressor gene p53 and the DNA damage marker γH2AX are significantly upregulated. Additionally, cell cycle phase distribution is affected as well. To understand the mechanism behind these observations, we used high-throughput RNA sequencing and uncovered that downregulation of GRAS1 impact multiple transcriptomic programs in lung cancer cells. Additionally, GRAS1 directly interacts with NF-κB activating protein (NKAP) and protects cells from ferroptosis. Interestingly, knockdown of GRAS1 in lung cancer cells causes a significant decrease in NKAP expression that induces a distinct type of programmed cell death called ferroptosis. Ferroptosis inhibitors, such as ferrostatin and α-tocopherol, can significantly rescue the effect of cell death caused by GRAS1 knockdown in A549. Overexpression of NKAP reverses the increased level of ferroptosis that is promoted by GRAS1 knockdown and leads to enhanced cell viability in A549.
The accumulation of aberrant protein splicing factors causes neurodegenerative diseases including frontotemporal dementia (FTD) in humans. TDP-43 binding to UG-rich RNAs has been shown to mediate protein stability and prevent aggregation, a main phenotype observed in FTD tissue samples. One of the most significant increases in TDP-43/RNA binding in FTD patient samples compared to healthy brain samples is to the long non-coding RNA MALAT-1, which the McHugh Lab has identified as a direct binder of TDP-43 in human cells. Cross-referencing common splice targets of MALAT1 and TDP-43 showed the Spermine/Spermidine Acetyltransferase 1 (SAT1) gene as an alternatively-spliced transcript, with a variant including an early stop codon to induce non-sense mediated decay. We hypothesize that MALAT1 is involved in the localization of TDP-43 around SAT1 in order for the protein to carry out its splicing function. Splicing assays conducted under MALAT-1 KD and TDP-43 KD conditions yielded a decrease in PSI, suggesting that less of the unstable mRNA that has the cryptic exon and undergoes nonsense mediated decay is produced. Knockdown in both MALAT1 and TDP43 both lead to an increase in the total gene expression of SAT1, affirming that more of the stable mRNA variant is produced. Overexpression of MALAT1 and TDP-43 resulted in opposite changes for PSI and gene expression of SAT1, further supporting the hypothesis. These findings are currently being validated in a disease-relevant model that simulates neurodegenerative Disease through MPP+ treatment, and overexpression of the SAT1 gene through excess polyamine addition.
Elucidating the mechanisms behind base editing outcomes through CRISPRi screens C to T base editors allow for the precise installation of point mutations in target DNA, making them useful for disease modelling and genetic therapeutic applications. However, they often suffer from low editing efficiency, and little is known about the biochemical pathways governing editing outcomes. Understanding which biochemical mechanisms base editors use to make an edit can inform development of more efficient DNA editing technologies. We used a CRISPR inhibition (CRISPRi) screen to repress gene expression of a pool of genes involved with DNA repair, and quantified which genes up- or down-regulate base editing. Our findings show it is possible to regulate gene expression of relevant genes to manipulate base editing outcomes.
Reverse transcriptases (RTs) are enzymes that are typically encoded in retroviruses and retroelements and are responsible for the synthesis of cDNA from RNA templates. The class of genetic retroelements known as group II introns encodes a maturase protein that contains an RT domain. The maturase binds to the group II intron RNA with picomolar affinity, and the resulting complex is able to integrate into double stranded DNA through a process known as retrotransposition, which involves cDNA synthesis. Although the mechanism of the maturase-aided forward and reverse splicing of the intron has been well studied, little is known about the RT catalyzed cDNA synthesis. In this work, we studied the RT activity of the T.el 4h group II intron ribonucleoprotein (RNP) complex. Our results show that the ribonucleoprotein can carry out cDNA synthesis using an external RNA template. These results, along with structural observations of RNP dimer formation by cryogenic electron microscopy, suggest that a protein dimer is involved in cDNA synthesis in vivo. Future work will focus on revealing mechanistic and structural aspects of the dimer formation and maturase mediated cDNA synthesis.
Metalloenzymes catalyze chemical reactions via their optimized coordination environments. However, natural metalloenzymes are restricted to biologically relevant reactions, and thus the goal of many protein design efforts is to expand the catalytic scope to include new-to-nature reactions. The Tezcan lab has previously designed a trimeric protein scaffold, Tet4, with a highly accurate Zinc binding site that can catalyze an abiological hydride transfer reaction. Inspired by natural multi-domain enzymes, I now aim to design a single-chain variant of Tet4 (scTet4) and use it to explore how the introduction of asymmetry can tune the properties of the metal center and the enzymatic activity of the protein. I have designed scTet4 variants with 15, 20 and 30 amino acid (aa) long linkers and validated their structure via AlphaFold2. The prediction for the 15 aa variant revealed structural deviations from Tet4. Circular dichroism spectroscopy and competitive metal binding titrations of the 15 aa variant showed that, though it largely maintained the same fold as Tet4, it was unable to bind Zn(II). Meanwhile, the AlphaFold2 predictions for the 20 and 30 aa variants agreed closely with the crystal structure of Tet4, suggesting that these longer linkers are needed to maintain the original Tet4 structure. I am currently in the process of experimentally characterizing the 20 and 30 aa variants. These scTET4 variants will set up future directed evolution experiments that will probe the effects of the protein scaffold on its catalytic properties and obtain a highly efficient artificial enzyme.
Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules provide advantages over target-focused approaches, including the ability to detect previously unknown targets, and the inclusion of chemical off-compete controls leading to high-confidence identifications. We describe the BioTAC system, a small-molecule guided proximity labelling platform, to rapidly identify both direct and complexed small molecule binding proteins. The BioTAC system overcomes a limitation of current approaches, and supports identification of both inhibitor bound and molecular glue bound complexes.
Previous research in our lab has shown a positive correlation between mRNA localization to the mitochondria and subsequent protein production of certain nuclear-encoded mRNAs in Saccharomyces Cerevisiae. We believe that the mechanism of localization is influenced by the metabolic state of the cell; we have seen that some mRNAs localize more efficiently in respiratory conditions compared to when in a fermentative state (termed “conditionally localized mRNAs”), while other constitutively localized mRNAs perform equally well regardless of metabolic state. The goal of my project has been to explore the mRNA localization mechanism further and investigate the role of different components in the mRNA sequence that might influence protein production for the conditionally localized mRNAs. Looking specifically at ATP Synthase, whose proteins are important for energy production of the cell, we have designed two types of fluorescently-labeled plasmids: one containing the ATP synthase promoter followed immediately by a green fluorescent label, and another containing the standard ATP synthase promoter and open reading frame sequence followed by a red fluorescent label. The magnitude of the change in protein expression between these constructs as the cell metabolism is manipulated will hopefully provide insight as to the extent of promoter and/or open reading frame contribution to mRNA-localization dependent protein expression. To induce changes in cell metabolism, we manipulate the translation elongation rate by two methods, first via an artificial, methionine-inhibitable technique, as well as more recently beginning trials with a more natural, nutrient-restricted based strategy.
Tyrosine kinase fusions have been identified as significant drivers in various cancer types, including glioblastoma, melanoma, and breast and lung carcinomas. The Neurotrophic Receptor Tyrosine Kinase genes NTRK1-3 encode the tropomyosin receptor kinase (TRK) proteins A-C, which play important roles in neuronal differentiation, proliferation, and migration. TRK ligands bind to the receptors, initiating dimerization, phosphorylation, and downstream activation. The NACC2:NTRK2 fusion contains the kinase domain of NTRK2 and a 5’ dimerization domain (BTB domain), which is predicted to lead to constitutive, ligand-independent downstream activation. Wei’s research has shown that the disruption of the BTB domain eliminates the transformation activities of NACC2:NTRK2 fusion. To further understand the oncogenic mechanism of NACC2:NTRK2 fusion, I investigated whether the BTB domain is the only domain in the NACC2 gene contributing to constitutive downstream activation. In this research, an “Extra Small Fusion” (ESFS) containing only the BTB domain from NACC2 and the same length of NTRK2 in the existing NACC2:NTRK2 fusion was created. Western blotting was performed to investigate the protein expression and phosphorylation of MAPK, PLC-γ, and STAT3 downstream signaling pathways in both the wildtype fusion and the ESFS. The results showed clear phosphorylation signals for all three downstream signaling pathways in the ESFS lane, which were similar in intensity to the signals detected in the wildtype fusion lane. Therefore, it can be concluded that the BTB domain alone in the NACC2:NTRK2 fusion is sufficient to activate the downstream signaling cascade.
Mitochondrial mRNA-protein complexes, which are encoded by the nucleus, require co-translational translocation to the mitochondria. Previous research has found that Tim50 is a mitochondrial protein in Saccharomyces cerevisiae whose open reading frame (ORF) contains a mitochondrial targeting sequence (MTS) followed by a short region of polyprolines. Polyprolines stall ribosome elongation, increasing the time the nascent MTS peptide is exposed and able to be recognized by the mitochondrial translocase of the outer membrane (TOM) complex. However, it is unclear whether mitochondrial localization relies on the duration of MTS exposure or if a stall is necessary. We have cloned two constructs, one which contains a long gene, and the other which contains a short gene that is immediately followed by a stalling sequence. The two constructs contain the Tim50 promoter and MTS followed by either a 3.1kb ORF (LacZ) or a shorter 0.9kb (iRFP) with a 3xCGA stall upstream of a nLuc reporter gene and twelve MS2 sequences. CGA is a strong stalling sequence. These two constructs distinguish whether a sustained exposure of the MTS or a stall is necessary for mitochondrial localization. To visualize mRNA localization in vivo, we have transformed these constructs into a yeast strain containing Su9-mCherry and MCP-GFP, which will fluorescently label the mitochondria and construct mRNA, respectively. We have used fluorescence microscopy to image the cells, and Matlab to quantify mRNA localization. mRNA localization appears higher for the stalling construct than the long ORF construct.
Leucine-rich repeat kinase 2 (LRRK2) structural dynamics revealed by Gaussian-accelerated molecular dynamics simulations and Weighted Ensemble sampling
Parkinson’s disease (PD) is a neurodegenerative disease affecting millions worldwide, and one of the genes commonly mutated in PD is leucine-rich repeat kinase 2 (LRRK2). Previous research has shown that LRRK2 can bind to microtubules in vitro and block microtubule-based motors in single-molecule assays. The behavior of LRRK2 is both mutation-driven and conformation-dependent, with a closed conformation leading to increased kinase activity and oligomerization around microtubules. While the open structure of LRRK2 has been recently identified, its contribution to PD at a cellular and mechanistic level remains unclear. To investigate the impact of mutations on LRRK2 conformational dynamics, Gaussian-accelerated molecular dynamics (GaMD) is used in conjunction with Weighted Ensemble (WE) sampling. GaMD applies a boost potential to lower energy barriers to better sample conformational states, but it faces challenges in converging high-energy regions. In contrast, WE applies weights to trajectories to sample between states, but it can be challenging to achieve steady-state equilibrium. By combining GaMD and WE methods, the limitations of both approaches can be reduced, and thermodynamic and kinetic properties can be more accurately obtained. Analyzing common LRRK2 mutations using this hybrid approach can help to explain why specific mutations result in oligomerization and kinase activation, ultimately giving more insight into potential treatments for PD.
Mitochondria are an essential part to human life and mitochondrial dysfunction has been linked to many types of diseases and aging, but no definitive connection has been made. Therefore it is important to better understand how the composition of mitochondria is controlled. mRNA localization is one way in which gene expression can be affected post-transcriptionally, but there are many uncertainties in how mRNA localization to mitochondria is controlled. In order to get an idea of which genes have an effect, we will perform a genome-wide screening and turn off each gene in hopes of finding measurable differences in the localization. To accomplish this goal I am cloning nuclear-encoded mitochondrial mRNAs into a reporter that contains a library of barcoded CRISPR guide RNAs. Currently, I am focused on testing the effects of cyclohexamide on optimizing the localization of the mitochondrial outer membrane mRNA TOM70 and the oxidative phosphorylation mRNA (ATP3). The long term goal of this project is to use these reporter mRNA constructs to determine novel factors in the control of mRNA localization to the mitochondria.
Streptococcus pyogenes (Strep A) is a widespread gram-positive bacterial pathogen that causes severe and potentially lethal infections. Previous studies have reported that the major Strep A surface-associated virulence factor M protein binds CD46, a down regulator of the complement system, and thereby enables escape from immune killing. M proteins are antigenically sequence variables with >220 M types known. The long-term aim of this study was to understand the interaction between CD46 and the M protein at a structural level. As a first step, we sought to verify prior reports of interaction between the M6 protein, an antigenic sequence variant of the M protein, and CD46. We examined the interaction between intact M6 protein and a refolded version of the CD46 extracellular (eCD46) domain but observed no interaction in a co-precipitation assay. We hypothesized that glycosylation of CD46 was necessary for interaction, but still observed no interaction through ELISA when using a glycosylated and functionally validated form of eCD46. We next examined the interaction between eCD46 and whole Strep A strains (M1, M6, and M18) through ELISA. While binding to M1 and M6 strains was not evident, binding to the M18 strain was detectable albeit weak. This result suggests a protein besides M protein in the M18 strain binds CD46, but the weakness of the binding calls into question whether CD46 has functional relevance to Strep A virulence.
Mitochondria are dynamic organelles essential for regulating metabolism. Decreased function of mitochondria is related to disease states such as cancer and diabetes. Recent studies are elucidating the role of mRNA localization in gene expression and metabolism. It has been found in Saccharomyces cerevisiae that there is an upregulation of mitochondrial protein levels when nuclear-encoded mRNAs are localized to the mitochondrial surface and similarly when exogenous transcripts containing GFP are conditionally targeted to the mitochondrial surface. This reveals that localization to the mitochondria has a general function of upregulating protein synthesis. To test if this phenomenon is conserved in mammalian cells, transcripts of a luciferase reporter will be conditionally localized to the mitochondria using an MS2 system. We will attach MS2 coat proteins to a number of outer mitochondrial membrane proteins. This will allow a luciferase reporter with MS2 stem loops to be targeted to the mitochondrial surface. This reporter will then be expressed in HeLa and HEK293T cells followed by measuring mitochondrial targeting and protein production through luminescence readings as well as RT-qPCR to monitor mRNA levels and decay rates. If increased protein expression due to mRNA localization in Saccharomyces cerevisiae is conserved in mammalian cells, we can advance into discovering what mechanisms or factors along the mitochondrial surface affect protein expression. Understanding how the mitochondrial milieu impacts protein expression gives the opportunity to delve into how this phenomenon can be used as a tool to control expression of proteins in different disease models and cell types.
Lipopolysaccharides (LPS) are one of the major components of the cell membrane in gram-negative bacteria. They are heat stable molecules that provide structure to the bacterial membrane and form a line of defense against extracellular toxins. However, LPS acts as a toxin to most cells and is a major concern for research involving human or animal tissue. In the field of human milk oligosaccharides, pooled human milk samples (pHMOs) are often used in tissue and cell culture to study the effects of pHMOs on cell proliferation. To do this successfully, pHMO samples must have almost all of the naturally present LPS removed (>99% removed on average). The current method for this uses Detoxi-gel gravity columns that require researchers to manually separate the LPS over the course of 8 hours. This method, while effective, is mentally taxing and time inefficient for researchers. The goal of this project was to adapt and optimize the standard protocol for the EndoTrap HD FPLC Column for use with human milk oligosaccharides using the BioRad NGC Chromatography System. Over the course of 9 column applications, I was able to optimize the EndoTrap protocol to remove 80.34-99.995% of LPS from pHMO samples while retaining 87-92% of the pHMOs applied to the column. While this is close to current standards for LPS removal, further optimization is needed to decrease the time to run the protocol and improve LPS removal as the optimized protocol has not been successful in removing enough LPS for samples to be used in cell cultures.
RNA binding proteins are heavily involved in regulating translation and gene expression. STAMP (Surveying Targets by APOBEC-Mediated Profiling) and TRIBE (Targets of RNA-binding proteins Identified By Editing) work in cohesion to identify where RBP’s are binding to RNA through the RNA-editing performance of APOBEC. STAMP, however, often has low yields and poor readings for various proteins. Possible RNA editing alternatives to APOBEC could increase the breadth of STAMP, but such alternatives have not yet been thoroughly analyzed in research yet. The goal of this experiment is to explore the best avenue for an improved version of STAMP by comparing the performance of various RNA editors when they are bound to an RNA binding protein. The proficiency of each combination is measured through the degree of editing of target mRNAs in HEK293T cells that serve as targets for RBP-RNA editor chimeras. In the end, a C-to-U editor and an A-to-G editor displayed 2-4 times more activity than the previously used APOBEC and ADAR enzymes in STAMP and TRIBE, respectively.
The production of inflammatory cytokines is triggered by the binding of ligands to cell-surface receptors, which triggers NF-B signaling. Numerous extracellular ligand-receptor interactions have been linked to the activation of both canonical and non-canonical NF-B signaling in Crohn’s disease. It may be possible to suppress the NF-B driven inflammation in Crohn’s disease by targeting a number of ligand-receptor interactions that are linked to protracted hyperinflammatory reactions. By interacting with the inducible cell-surface receptor Fn14, the tumor necrosis factor-like weak inducer of apoptosis (TWEAK) activates NF-B. The interplay between TWEAK and Fn14 has a significant impact on the persistence of inflammatory bowel disorders. TWEAK and Fn-14 have never been made in a lab before. For the purpose of producing the His(6)-tagged proteins, we acquired expression plasmids for each of these genes. We anticipated that because the proteins have cysteines and are secreted, they would not fold correctly in E. coli and would need to be refolded. We were successful in producing a good yield of TWEAK but not Fn-14. We are redesigning the plasmids using molecular biology techniques to express the proteins with just a C-terminal His(6) tag instead of the two His(6) tags that the plasmids originally linked to the proteins. We’ll discuss the findings from TWEAK’s preliminary refolding tests, which show that it expresses well in E. coli.
Inflammation involves the binding of ligands to cell-surface receptors which activates NFκB signaling subsequently causing the production of inflammatory cytokines. In Crohn’s disease, several extracellular ligand-receptor interactions have been implicated in activation of both canonical and non-canonical NFκB signaling. Several ligand-receptor interactions that are implicated in extended hyperinflammatory reactions could be excellent additional targets for inhibition of the NFκB mediated inflammation in Crohn’s disease. We are focusing on the tumor necrosis factor-like weak inducer of apoptosis (TWEAK) which activates NFκB via interactions with the inducible cell-surface receptor Fn14. The TWEAK-Fn14 interaction is strongly implicated in perpetuating inflammatory bowel diseases. Neither TWEAK nor Fn-14 have been produced in vitro before. We obtained expression plasmids of each of these genes to express the His(6)-tagged proteins. Since the proteins have cysteines and are secreted, we expected that they would not fold properly in E. coli and that they would require refolding. We were able to express TWEAK in good yield, but not Fn-14. The plasmids actually attached two His(6) tags onto the proteins, and we are using molecular biology techniques to redesign the plasmids to express the proteins with only a C-terminal His(6) tag. We will present results from initial refolding studies of TWEAK which expresses well in E. coli.
In bacteria, gene expression levels can be modulated by regulatory RNA elements. For example, bacteria have regulatory RNA elements called riboswitches, which are encoded in the 5’ untranslated region of mRNA transcripts. Riboswitches bind to ligands in the environment, such as metal ions and metabolites; this binding event prompts the RNA to rearrange its structure. Consequently, this structural rearrangement regulates transcription and translation of downstream mRNA, affecting the expression of the protein that the mRNA encodes. In E. coli, the mntP gene encodes a membrane protein that exports manganese out of the cytosol, and its expression is controlled by a riboswitch element. It has been found that this riboswitch specifically senses and responds to the manganese ion Mn2+. Manganese binding to the mntP riboswitch triggers the RNA to rearrange its structure to increase translation of the mntP gene, facilitating manganese efflux from the cytosol and helping to prevent manganese toxicity. Although responses to manganese in the mntP system have been explored, the specific mechanisms of ligand binding to the riboswitch and subsequent effects on transcription and folding of the mntP riboswitch remain unknown. To help fill this gap in knowledge, transcription of the sequence encoding the mntP riboswitch will be examined since the rate of transcription plays an important role in RNA structure formation. Specifically, I am reconstituting transcription on the mntP riboswitch sequence in vitro to analyze how the transcription rate is affected by (i) changing manganese concentration and (ii) the presence of protein factors that may assist the RNA folding and ultimate regulatory decision. Ultimately, these experiments will provide crucial insights that will help piece together the underlying regulatory mechanisms of the mntP riboswitch.
Nuclear factor-κB (NFκB) is a transcription factor that is essential for biological functions in the cell like immunity and inflammation response, and cell growth. NFκB contains a transcription activation domain (TAD) that helps activate and enhance the transcription activity. In previous studies in our lab, we identified a change in the binding affinity of NFκB to DNA in the presence of the TAD. As a result, the TAD facilitates the association of more NFκB dimers than the number of available κB sites. With the presence of the TAD, the ability of p50/RelA to bind to all the κB sequences was improved. At the same time, it had a higher impact on the binding affinity for nonspecific DNA sequences in which the binding affinity increased for more than 10 times. Our previous results suggest that the TAD could increase DNA binding affinity, and lower the sequence specificity. Currently, we are trying to identify which part of the protein is responsible for the increase in DNA binding affinity. In particular, specific site mutageneses, and different truncations of the TAD were tested for binding affinity to nonconsensus DNA. It seems that the connecting region between the RHD and the TAD may be the important part. The important function is improving binding to nonspecific DNA and I assay this by fluorescent anisotropy.
Schizophrenia is a polygenic mental illness that severely impacts the human quality of life due to abnormal reality interpretation. While certain rare genetic variants have high probabilities of causing schizophrenia, this study will focus on several common genetic variants involved in membrane trafficking and synapse function that may predispose individuals to this illness, especially when observed in combination. Endosomal or membrane trafficking is the neuron’s method of sorting cargo and recycling proteins/vesicles that occurs at both sides of the synapse. Base editing developed by Dr. Alexis Komor was used to install these point mutations to evaluate their impact on endosomal trafficking and synapse number and function. It is unknown, however, what causes these common variants to express the schizophrenia phenotype in some and not others. Base editing can be used to introduce these variants into an isogenic neuroblastoma cell line that can then be quantified and imaged by the Yu lab and Gupton lab, to determine the impact on the schizophrenia phenotype. The results from the data gathered at the Yu and Gupton labs will assess the impact of these genes on the function of the neurons, specifically the synaptic density and firing of action potentials. We anticipate our results to be a significant stepping stone in the direction of understanding schizophrenia as a polygenic disease and potentially open doors to developing more precise therapies depending on the specific variants causing the phenotype.
NF-κB family of inducible transcription factors bind to consensus NF-κB motifs at the promoter or enhancer regions to regulate inflammatory transcription. We found TNFα- dependent rapidly activated genes displayed an abundance of low affinity NF-κB motifs distributed around the consensus NF-κB binding site. These low affinity NF-κB motifs enhance overall occupancy NF-κB transcription factors on DNA to enhance transcription level. In the context of the Cxcl2 promoter, a low affinity κB site located downstream of a core κB site impacted RelA binding and RelA-dependent transcriptional activation. Further, Mass-spec analysis identified the NFATc1, and other cofactors are important transcriptional regulators at the Cxcl2 promoter. NFATc1 is associated with NF-κB at the Cxcl2 promoter site to bind the core and low affinity κB elements. Overall, we proposed that NFATc1 and other cofactors are important in the recruitment of NF-κB DNA binding affinity.
Glycosaminoglycans (GAGs) are extracellular polysaccharides that participate in many cellular processes including cell protection, signal propagation, growth factor binding, and stem cell differentiation. GAGs are displayed on protein scaffolds in structures called proteoglycans (PGs) which are a component of the extracellular glycocalyx. While PGs are central to many cellular processes, they are challenging to study in vitro as they are difficult both to isolate and to synthesize in their native forms. To overcome this obstacle, we have developed an improved method to approximate PG structures via chemoenzymatic synthesis; these conjugates are defined as neoProteoglycans (neoPGs). We have designed a tunable strategy which permits conjugation of a GAG to a distinct point on a modified protein. Our novel bifunctional linker employs reductive amination to introduce a tyrosine-like handle to the GAG chain reducing end. The reactive handle is then linked to the protein core by Tyrosinase enzyme to produce the neoPG reagent of a defined structure and valency. This efficient method utilizes a protein core to mimic natural PG structures. These neoPGs can broadly be used to study PGs in vitro by immobilizing them on a microarray, for example, as well as soluble reagents for cell based assays.
Chemistry Education Research seeks to understand and improve learning experiences in chemistry fields by investigating different aspects of learning activities for their effects on problem-solving skills, comprehension outcomes, and engagement levels. This study investigates learning experiences in chirality, which has been shown to be a difficult topic for students because understanding chirality includes a complex interplay between spatial abilities and superimposability. A majority of current lessons on chirality lack explicit instruction on spatial abilities, but significant work has been done to explore new teaching techniques meant to improve student spatial skills, including interactive workshops, use of 3D, 2D, and virtual molecular modeling softwares, critical thinking worksheets, and guided lessons. Similar to 3D printing, 3D pens melt PLA filament so that users can build 3D shapes in a hands-on manner. This study proposes an interactive 3D pen workshop as a tool to enhance students’ understanding of chirality. The working hypothesis was that working with 3D pens will increase student engagement, enhance problem-solving skills in chirality, and increase confidence in their own skills and understanding. Positive changes in engagement were observed in a majority of the participants during initial testing, while negative changes were observed with problem-solving skills and confidence. After revisions were made, further tests revealed notable positive changes in problem-solving abilities, confidence levels, and attitudes about Organic Chemistry. Further improvements can be made to increase problem-solving and deeper levels of chirality understanding rather than related or adjacent topics.
A Functional 3D Printed Model for the Walden Inversion
Ball-and-Stick (BS) molecular models are common aids used by students to help understand the structure in Organic Chemistry. However, BS models are rigid and unable to convey the changes in connectivity and stereochemistry that occur during chemical reactions. Traditionally, students often struggle to visualize the Walden Inversion – commonly presented to support the stereochemical changes of a concerted SN2 mechanism. Computer graphics can visually present similar visual information but lack tactile feedback and, more importantly, are consumed passively as non-interactive videos. Here we present a functional 3D-printed model that physically demonstrates the Walden inversion in an SN2 reaction. This low-cost model moves freely between configurations, show each stage of the reaction coordinate, including the transition state, and can be printed out on any consumer-grade 3D printer for student use and in-class demonstration.
Oxygen minimum zones (OMZs) and overall deoxygenation of marine environments are expanding due to global warming caused by anthropogenic climate change. Increasing OMZs impact which marine microbial communities are present, their metabolisms, and the biogeochemical cycles these microbes facilitate, including phosphorus (P) nutrient cycling. To investigate connections between microbial community metabolisms and P cycling across OMZs, we sampled along a longitudinal transect in the North Pacific Ocean on the RV Sally Ride in December 2022. The 5 stations along the transect consistently crossed hypoxic conditions (<2 mL O2 L-1) representative of OMZs. Our main question was if phosphorus cycling can be predicted by community structure in OMZs. We hypothesized that alkaline phosphatase activity (APA) would be greater where dissolved inorganic phosphorus (DIP) concentrations were a smaller proportion of total dissolved phosphorus (TDP) as well as that there would be distinct shifts in P-uptake rates and DIP turnover times between the hypoxic and oxic conditions. At each station, we collected whole seawater at 13 depths to be analyzed for dissolved inorganic nutrients and cell counts. Additionally, seawater was filtered onto GF-75 (0.3 μm pore size) glass fiber filters for total particulate phosphorus (TPP), carbon, nitrogen, and adenylates, including adenosine triphosphate (ATP). Supor filters (0.2 μm pore size) were used to collect samples for DNA amplicon sequencing. Finally, microbial activity was quantified through MUF-P assays for APA and using P32-labeled radioactive incubations to measure microbial total P uptake rates and ATP production rates. DIP turnover rates, TPP turnover rates, and ATP turnover rates could all be calculated using concentration and radioactive data. I will present our preliminary findings from this work that highlight links between phosphorus cycling and the microbial community structure and metabolisms across depth and oxygen concentrations.
Recent experimental advances in miniaturization of thermoelectric devices necessitate corresponding thermodynamic theory. Standard quantum thermodynamics (quantum statistical mechanics) assumes negligible system-bath coupling, but this assumption breaks down at nanoscale, where energy of the system is of the same order as the coupling. Thermodynamic formulation for nonequilibrium quantum systems strongly coupled to baths has not been fully developed. Several suggestions are available in recent literature. Here, we use a formulation utilizing the von Neumann expression for reduced density matrix as system thermodynamic entropy to analyze the Carnot cycle in a nanoscale device. The formulation is the only one available today consistent with microscopic dynamics. We check consistency of the formulation by evaluating efficiency and entropy production of a single level model of the device coupled to macroscopic hot and cold fermionic reservoirs for weak (idealized) and strong (realistic) system-bath couplings at reversible driving. We show that the lack of energy resolution in the formulation results in efficiency lower than the Carnot efficiency even at reversible driving and negative entropy production for some processes. To restore intuitively expected reversible behavior at adiabatic driving and consistency with the second law of thermodynamics, an energy-resolved thermodynamic formulation has to be developed.
HONO Formation in The Marine Boundary Layer
Formation mechanisms for atmospheric nitrous acid (HONO) is essential to understand as HONO photodissociates at solar wavelengths to yield hydroxyl and nitric oxide radicals. Hydroxyl and nitric oxide radicals are contributors to the atmosphere’s oxidative capacity and reactive nitrogen cycle, respectively. Despite its importance, different HONO sources and sinks still need more investigation to understand fully. In this research, the formation mechanisms of HONO in the marine boundary layer (MBL) are being explored as oceans cover over 70% of the Earth. More specifically, HONO forms upon nitrate photolysis during the daytime in the MBL, occurring mainly in nitrate-containing, aged sea spray aerosols (SSA). SSA is also enriched in organics due to bubble-bursting at the ocean surface. Recently it was shown that marine dissolved organic matter (m-DOM), a complex organic mixture, has been found to enhance HONO formation in irradiated nitrate solutions. The enhancement is proposed to be due to the formation of superoxide and hydroxyl radicals from the aliphatic and chromophoric compounds within m-DOM. Due to the complexity of the m-DOM sample, this study aims to use molecular proxies to understand better the mechanisms for enhanced HONO formation in the presence of organics compounds. In this research, mixtures of ethylene glycol (EG) and 4-benzoyl benzoic acid (4-BBA), an aliphatic and a chromophoric component of m-DOM, respectively, were used in HONO enhancement studies. A home-build incoherent broadband cavity enhanced absorption spectrometer (IBBCEAS) measures HONO formation down to ppb levels. This work aims to provide scaling factors for HONO formation representative of the enhanced amounts formed from nitrate photolysis in the presence of organic compounds for atmospheric chemistry models. Ultimately, these data will be used to understand global sources of gas-phase HONO.
Minerals, which are essential to environmental systems including the atmosphere and geosphere, participate in a myriad of chemical reactions that impact air quality, water quality and climate. Therefore, a deeper understanding of geochemical thin films and particles is necessary to better understand the heterogeneity and impacts. Recently, these systems have been probed with micro-spectroscopic probes. The goal of this project is to investigate the chemical and physical properties of different minerals utilizing four different vibrational spectroscopic techniques including Attenuated Total Reflection – Fourier Transform Infrared (ATR-FTIR), Optical Photothermal Infrared (O-PTIR), Atomic Force Microscopy – Photothermal Infrared (AFM-PTIR), and Raman spectroscopy. The integration of data analysis across all dimensions, from the macro- to micro- to the nanoscale, is made possible by these four techniques. Each one analyzes various chemical and physical properties that should be taken into consideration for environmental studies. A comprehensive understanding of the heterogeneities of mineral samples is provided by the combination of these techniques, which will prove to be extremely useful for future studies in this field.
Plants produce a diverse array of primary and specialized metabolites that include highly conserved biochemicals essential for cellular metabolism alongside defense-related specialized metabolites that are commonly unique within a given genus or species. Gas chromatography-mass spectrometry (GC-MS) is a powerful analytical method for analysis of diverse small metabolites with wide-ranging polarities from sugars and amino acids to sterols (cholesterol) and hydrocarbon waxes1, 2. Benefits of GC-MS include reliable isomer separation, standardized ionization, and large existing spectral libraries. However, broad GC-MS metabolomics of complex lipophilic samples cannot be effectively analyzed3 and many potential GC-MS applications are prevented by co-existing non-volatile contaminants incompatible with the GC inlets and columns. Vapor Phase Extraction (VPE) is a sample preparation technique that was developed two decades ago for selectively obtaining various small molecule acids (as methyl ester derivatives) and natural volatile organic compounds from non-volatile matrixes.4-6 and can also selectively acquire small molecule trimethylsilyl (TMS) derivatives of analytes containing carboxylic acids, alcohols, and amines6. Our research aims to create an improved simple analytical method that combines well-established TMS derivatization approaches1,3 along with VPE protocols to generate a clean sample with a maximum diversity of primary and specialized metabolites.
Single-molecule magnets (SMMs) and single-ion magnets (SIMs) are a class of compounds characterized by their ability to retain magnetization after biasing with an external magnetic field. SMMs and SIMs have potential applications in technology, most notably in high-density information storage, quantum computing and molecular spintronic devices. These compounds undergo a process known as slow magnetic relaxation, whereby magnetic spins equilibrate sometime after an external field has been applied and then removed. In this work we present a method for controlling the magnetic relaxation of a given SIM by targeting the intermolecular dipolar coupling between magnetic units. To this end, we have synthesized a series of three SIMs, all featuring the same magnetic unit (bis(η8-cyclooctatetraenyl)erbium(III) or Er(COT)2-) and varying only the charge-balancing cation with the intent of altering the distances and angles between magnetic units. With the magnetic unit held constant in these compounds, variations in magnetic measurements can be attributed to differences in intermolecular dipolar coupling facilitated by each compound’s distinct crystal packing. Magnetic measurements were collected with a superconducting quantum interference device (SQUID). In our systems of choice, we show a suppression of magnetic relaxation via quantum tunneling of magnetization in a series of SIMs via targeted alteration of crystal structure parameters.
Group 7 transition metals are novel in that they supply an uneven electron count which finds itself easily susceptible to radical chemistry. Nonetheless, Manganese is interesting because it is evenly separated between early and late transition metals; along with being the 5th most abundant metal on Earth’s core, Manganese possesses an interesting platform for developing a catalytic complex. Herein, we will discuss the synthesis, reactivity and crystal structures of the first Manganese trianion complex, Mn(CNAr(Tripp)2))2(CO)2 -2. Particular interest on it’s interaction with phosphorus fragments, white phosphorous (P4) and Di-phosphorous (P2) will be emphasized. Activation of coordination sites via reductive elimination opens a large reactive face on the metal that has the likelihood of housing the first mononuclear P4 complex on a group 7 metal. The ultimate goal is to characterize new binding motifs for passivating pyrophoric phosphorus and releasing it in quantitative yields for industrial applications. Additionally, this complex’s reactivity with BF3 and ability to form a terminal BF, isoelectronic to “CO” and subsequently a Mn – Boron triple bond will be studied. Unlike CO, which is a stable molecule at room temperature and readily serves as both a bridging and terminal ligand to transition metals, BF is unstable below 1800°C in the gas phase, and its coordination chemistry is substantially limited.
Porous silicon (pSi) is a promising material for various applications due to its large surface area, biocompatibility, biodegradability, and tunable optical properties. One of the useful properties of pSi is its ability to be oxidized by water, making it suitable for oxidative trapping-based drug loading methods and for biosensor applications. This study aimed to investigate the effect of varying light intensities and aqueous solution composition on the rate of photo-oxidation of pSi to better understand the factors that impact the photo-oxidation process. The Spectroscopic Liquid Infiltration Method (SLIM) was used as the primary method to quantify the results through different parameters, such as porosity and percent filled. By comparing porosity over a 5-hour period in the same water condition but at different values of light intensity, our results indicated that higher light intensity correlates with faster oxidation rates. Moreover, we used the SLIM method to study the impacts of experimental conditions on other optical properties of the mesoporous materials, such as refractive index. These findings contribute to a better fundamental understanding of the aqueous oxidation of pSi, which can aid in the development of new applications for this material.
Multivalency is the principle of establishing strong, yet reversible non-covalent chemical bonds, which are present in nature. This principle is important in biological interactions such as protein-saccharide interactions. The goal of this work is to replicate multivalent properties that are dominated in nature to either promote or inhibit biological molecular recognition events from occurring. Our work is designed to mimic these interactions by developing a modular and tunable molecular platform that provides atomic-level structural precision. Self-assembled supramolecular metallcages are tunable systems that can have varying size, shape, and topology and have been studied thoroughly over the last several decades. Functionalizing these metallcages with saccharides allows us to control the size, shape, electronics, and multivalent properties of these systems. We have thus far built a library of functionalized metallcages through subcomponent self-assembly of various multitopic amines with glycosylated formyl pyridine units in the presence of iron(II) ions. The resulting tetrahedral, helical and mononuclear complexes have been shown to bind to a model protein (Concanavalin A) with high affinity. We now present a method of modulating the overall charge of the resulting complexes through incorporation of precisely designed organic building blocks, which allows us to evaluate the effect of charge on the binding strength of multivalent constructs. New glycosylated complexes using a tri-amino organic subcomponent that caps the face of a tetrahedral metallcage with an 8+ overall charge will be presented in addition to protein-binding data.
Multivalency is a key component in protein binding, allowing for strong, yet reversible, interactions that dictate biological processes. We have been developing synthetic metal organic cages (MOCs) that are designed to bind to protein targets to mimic or inhibit these important types of biological interactions. MOCs are highly tunable supramolecular structures, which provide us with precise control over their size, morphology and valency through rational molecular design. Furthermore, lectins, which are carbohydrate-binding proteins that are highly specific for sugar groups, are crucial to many biological processes, including the agglutination of cells. Functionalizing this new class of MOCs with various saccharides is a modular approach to understanding molecular recognition processes of this class of proteins. Here, we present new MOCs with peripheral lactose and galactose groups. We propose these cages bind to Galectin-3, a protein target within a class of lactose- and galactose-binding lectins involved in malignant cell adhesion, to study this protein’s inhibitory effects on metastasis and cell growth. Due to their cage-like nature, the MOCs are also capable of encapsulating guest molecules within their internal cavities, creating potential new class of drug delivery vehicles. To couple with the anticancer properties of inhibiting Galectin-3, 5-Fluorouracil and its uptake/release from the cage will be studied. Multivalency, coupled with the host-guest binding, presents these compounds with a wide variety of medicinal applications. Presented here are low-toxicity lactose- and galactose- functionalized Fe(II) complexes prepared through the coordination-driven self-assembly of simple building blocks to test their inhibitory properties on Galectin-3.
Catalytically synthesizing disilane compounds (RH2Si—SiH2R) from primary silanes (RH3Si) has been a long-standing challenge, with currently known systems largely only capable of dehydrocoupling silanes to a mixture of disilanes, trisilanes, and other polysilanes. Other known examples of catalytic methods have been restricted to use of tertiary and secondary silanes to observe disilane formation1. The development therein of an effective catalytic system to selectively homocouple primary silanes to disilanes is inherently desirable. Currently, literature2 provides for the synthesis of a narrow range of these compounds by a Wurtz-type coupling but is limited in use due to a multistep pathway that goes through the chlorosilane before reducing with lithium to produce the disilane with moderate to low yields. Moreover, the reduction step also reverts some back to the primary silane, accounting for the lowered yields. To selectively homocouple monosilanes to disilanes, we plan on utilizing the low valent paramagnetic MoI2(CO)2(CNArDipp2)2 (1) complex, which features the sterically encumbering m-terphenyl isocyanide ligand CNArDipp2 (ArDipp2 = 2,6-(2,6-(i-Pr)2C6H3)2C6H3). We anticipate this bulk to block further coupling and successive polymerization, favoring the formation of the disilane. Preliminary experiments have displayed novel reactivity, showing the formation of a Mo—Mo dimer when 1 is placed in the presence of excess phenylsilane. From single crystal X-ray diffraction, the dimer Mo2(µ2-I2)(µ2-(SiHPh)2)(CO)4(CNArDipp2)2 (2) was observed and features two bridging silanes and two bridging iodine atoms. The structure of 2 serves as a possible intermediate along the pathway towards the silane homocoupling. NMR, IR, and GCMS instruments will be used to analyze and screen reaction conditions and procedures. Reactivity will be further expanded to explore other monosilanes after the optimization of the catalytic system with phenylsilane.
Ferrocene has the potential to undergo photooxidation to form stable ferrocenium ion. Thus, surface grafted porous silicon with vinyl ferrocene may have light-regulated hydrophobicity. Hydrosilylation, the addition of Si-H bond across alkene or vinyl group, is suitable for connecting vinyl ferrocene and silicon surface via Si-C bond. The usual thermal hydrosilylation of alkene or vinyl group with surface Si-H group requires catalyst or light promotion, and the reactants usually need to be purged using a glove box or Schlenk line to prevent oxidation of porous silicon. However, the operation of the glove box and Schlenk line is relatively complicated, and the efficiency of hydrosilylation is highly variable. Here, we aim to explore the thermolytic grafting of polyvinyl ferrocene to porous silicon photonic crystals. This technique does not need extra molecular or light input or complicated operation. The success of thermolytic grafting can be reflected directly by the color change of the photonic crystals. In addition, the surface grafted photonic crystals with vinyl ferrocene will be characterized using infrared spectroscopy, contact angle, reflection spectrum, porosity, and thickness.
Diisocyanates are critical building blocks for the synthesis of thermoplastic polyurethanes (TPUs), a class of industrially significant polymers. Currently, diisocyanates are primarily synthesized from petrochemical feedstocks under toxic phosgenation conditions. Terminal, aliphatic diacids can be biologically sourced and serve as a starting material for a sustainable synthetic route to diisocyanates. Our group previously developed a flow chemistry approach to the final two steps of this route that proceeds through an acyl azide intermediate, affording the isocyanate moiety through the Curtius rearrangement. This work details the scaling of this operation and efforts to optimize the reaction conditions. Factors experimented with include effective stoichiometry, substrate chain length, anhydrous control strategies, recycling of solvent, and reaction temperature. Using NMR spectroscopy, we were able to analyze the formation of impurities, directing our experimentation. We engineered the flow system to run autonomously and discovered that the liquid product can be isolated via precipitation of impurities. The current optimized conditions have provided two-step yields of up to 81% at a 50-gram scale. This represents a production rate of 181 mg/min of 1,7-heptamethylene diisocyanate (HepMDI) at peak operation.
Synthesis of Potential Epoxyketone-Based Plasmodium Proteasome Inhibitors
Malaria, caused by various Plasmodium species, sickens nearly 250 million people globally and leads to 619,000 deaths yearly. Of these deaths, 77 percent are children under 5 years of age. Thus, new and more efficacious agents to treat this devastating disease are urgently needed. Our laboratory has previously discovered that epoxyketone-based proteasome inhibitors, based on the marine cyanobacterial natural products carmaphycin A and B, are potent inhibitors of Plasmodium. Here, the total chemical synthesis of two potentially potent and highly-selective epoxyketone-based inhibitors of the Plasmodium parasite proteasome are reported. Synthesis of these compounds was accomplished through chemical deprotection and subsequent coupling of amino acids and other small molecules. The final products consist of dipeptide scaffolds whose N-termini were coupled to different carboxylic acids and whose C-termini were joined to an epoxyketone-based warhead. Synthetic schemes, expectations, outcomes, and future plans will be discussed.
Pinacolborates (PinB-OR) are common waste products of chemical reactions and have little demonstrated synthetic value. The primary goal of this project is to design ways to valorize these borate waste molecules, which can be synthesized via direct dehydrogenative coupling of pinacolborane (HBPin) with an alcohol (HOR) reagent. This process is currently being explored using procedures published by Dr. Erik Romero and Dr. Guy Bertrand in 2016 that successfully proved the formation of an O-B bond between these two reactants without the need for a catalyst. Lewis base binding studies using 11B NMR spectroscopy showed that 1,8-Diazabicyclo[5.4. 0]undec-7-ene (DBU) and 4-dimethylaminopyridine (DMAP), among others, bind to the boron atom of PinB-OR. We predict that the B–O bond in these adducts should be considered “activated” and might react with ambiphilic molecules, which have both electrophilic and nucleophilic sites. The products of these reactions are expected to be synthetically valuable boryl ethers made by reforming a C-B bond that recombines fragments of the starting borate with the ambiphile. Discovering conditions enabling these transformations is the subject of our ongoing investigations, which includes examining the reactivity of halogenated and nonhalogenated allylic compounds and ynones.
Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that facilitate novel protein-protein interactions with a target protein and the E3 ubiquitin ligase to result in polyubiquitination and subsequent proteasomal degradation. PROTACs have three main parts: a target protein ligand, a linker, and an E3 ligase ligand. PROTACs have gained attention in recent years due to the ability to target and degrade classically undruggable targets, such as kinases, at sub-stoichiometric amounts. However due to their high molecular weight (>1000dA), they tend to suffer from low cell permeability: this extends to the blood brain barrier (BBB) which prevents access to the central nervous system (CNS) proteome. A prodrug approach, in which a labile protecting group that is cleaved once it reaches the targeted area is attached to a drug active site, has previously improved oral bioavailability, but has not yet been used to address CNS permeance. By modifying the E3 ligase ligand, which is conserved across multiple PROTACs, a generalized approach utilizing passive diffusion, active transport, and other mechanisms to improve BBB permeance can be created. This study necessitates a potent scaffold on which to test effectiveness of the various promoieties: dAURK-4 is one such PROTAC that has been shown to potently and selectively degrade Aurora kinase A, a serine/threonine kinase that is overexpressed in cancer tissues. Synthesis of dAURK-4 necessitates Alisertib, a potent aurora kinase A inhibitor used as the target ligand in dAURK-4. To date, an efficient and scalable synthesis for Alisertib has been developed to facilitate this goal.
The RET (REarranged during Transfection) gene, which encodes for a receptor tyrosine kinase, is an established oncogene associated with neuroblastoma as well as several forms of adult cancer. In the context of neuroblastoma, overexpression of RET has been determined to be associated with poor prognosis prompting interest in RET inhibition as a form of therapy. While multikinase inhibitors have been shown to display activity against RET, they lack selectivity resulting in unwanted side effects that limit their efficacy, safety, and thus clinical applications. Therefore, recent efforts have been focused on designing selective RET inhibitors, but due to high degrees of conservation between kinase binding pockets, this remains a challenge. Free rotation around aryl-aryl bonds commonly occurs in drug scaffolds; however, most small-molecules bind to their target within only a narrow range of these available conformations. Leveraging atropisomerism to restrict accessible low energy dihedral conformations, a highly selective RET inhibitor, R-getretinib, was synthesized by successfully locking a promiscuous scaffold into a stable atroposiomer favored by RET. Here, we present data confirming the high potency and selectivity of the novel RET inhibitor, R-getretinib, in vitro.
Non-fouling substances are vital in their ability to increase the safety and longevity of certain products by preventing the adhesion of substances to surfaces, thereby preserving their physical and chemical properties. These substances are common in many industries, including biomedical, industrial, and imaging. Common non-fouling materials include fluorous organic materials, many of which can be categorized as polyfluoroalkyl substances (PFAs). However, PFAs pose a significant impact to human and environmental health due to their resistance to degradation and harmful longevity in the environment. To reduce the negative effects of PFAs, we propose that NHC-PF5-containing polymers offer a useful alternative to PFAs, as NHC-PF5 adducts exhibit similar non-fouling properties, are stable, and contain high fluorine content while lacking in fluorocarbons, making them environmentally healthier than PFAs. The objective of the project is to optimize the generation of NHC-PF5 adducts based on imidazolium and triazolium model systems through silyl transfer methodology. In order to lower the temperature of the reaction, reaction conditions will be varied and the process will be monitored using 19F NMR. The non-fouling properties of the resulting NHC-PF5 material will be tested through the use of atomic force microscopy (AFM) for protein adsorption and a goniometer for contact angle measurements.
