Posters at a glance Alphabetical by last name 

Poster  Presenter  Department  Poster Title 

1  Ashkar, Shireen  Medicinal Chemistry 

Exploring Natural Products for Novel Inhibitors of Rifamycin-resistant RNA Polymerase of Mycobacterium tuberculosis 

2  Becerra, Jorge  Chemical Biology 

Elucidating NF-ϰB role in the inflammatory response through utilization of small stapled peptide inhibitors 

3  Birkle, Fabienne  Biological Chemistry 

A flexible serine loop in the ectodomain of tissue factor serves as potential allosteric linker between the phospholipid membrane and the tissue factor exosite 

4  Brixius-Anderko, Simone 

Medicinal Chemistry 

Structural differences in cytochrome P450 11B enzymes key for drugs controlling blood pressure and immune responses 

5  Cheung See Kit, Melanie 

Chemistry  Biochemical analysis of depolmitoylases 

6  Chun, Stephanie  Medicinal Chemistry 

Chemoenzymatic synthesis of α-amino ketones mediated by an 8-amino-7-oxononanoate synthase 

7  Crellin, John  Chemistry  Direct and site-specific profiling of native palmitoylation  

8  Foley, Hannah  Chemistry  Tertiary Structure Determination of OncomiR-1 using a Hybrid NMR/Cryo-EM Approach 

9  Franco, Monika  Chemical Biology 

Pseudouridylation of mRNA can alter translation elongation, fidelity and termination 

10  García-Torres Desirée 

Chemical Biology 

Differential effects of SmgGDS-607 on small GTPase prenylation 

11  Ge, Jian  Life Science Institute 

Physiological angiogenesis is independent of endothelial cell specific MMP14. 

12  Henley, Matthew  Chemical Biology 

Conserved Coactivator Binding Mechanisms Facilitate Discovery of Allosteric Small Molecules 

13  Hohlman, Robert  Medicinal Chemistry 

Synthesis and Biosynthesis of Novel Diketopiperazines and Naseseazines  

14  Howard, Christina  Chemical Biology 

Development of small molecule inhibitors of NSD3 histone methyltransferase 

15  Li, Hao  Pathology  Development of Chemical Probes Targeting ASH1L Histone Methyltransferase 

16  Montoya, Karen  Chemistry  Towards a post-PCR world: direct counting of microRNAs from single cells 

17  Olson, Keith  Pharmacology  The non-selective opioid diprenorphine produces delta-opioid receptor-mediated rapid antidepressant-like effects in mice 

18  Patel, Ayesha  Chemistry  Understanding the role of TRAF6 in the antiviral activity of viperin 

19  Pyser, Joshua  Chemistry  Chemoenzymatic, Enantiodivergent Synthesis of Biologically Active Azaphilones 

20  Ragazzone, Nicholas 

Medicinal Chemistry 

Structural Analysis of the DNA Binding Domain of VirF, the Key Transcriptional Activator in Shigella Pathogenesis 

21  Raiymbek, Gulzhan 

Biological Chemistry 

Non-enzymatic role of putative demethylase Epe1 

22  Rodríguez-Benítez, Attabey 

Chemical Biology 

Expanding the Synthetic Utility of the Flavin-dependent Monooxygenase TropB 

23  Rodríguez-Díaz, Jean 

Neuroscience  Kainate Induced Oscillations in Ex Vivo Hippocampal Sections 

24  Rosenblum, Sydney 

Chemical Biology 

Detecting miRNA-miRBP interactions with a cell-based split-enzyme assay 

25  Sandoval, Jorge  Chemical Biology 

The Discovery and Optimization of pre-microRNA Probes 

26  Song, Zhuoyi  Chemical Biology 

A Method for Detecting O2-Binding to Flavoproteins 

27  Thelen, Adam  Biological Chemistry 

Recognition of 1,N2-ethenoguanine by Human Alkyladenine DNA Glycosylase 

28  Vasquez, Rosa  Chemical Biology 

Structure and engineering of TamI, an iterative cytochrome P450 for late-stage C-H functionalization of tirandamycin antibiotics  

29  Wang, Wenjing  Life Science Institute 

A Light- and Calcium-Gated Transcription Factor for Tagging Activated Neurons 

30  Zetzsche, Lara  Chemical Biology 

Biocatalytic biaryl bond formation with cytochromes P450 

  

   

Poster Abstracts  Poster 1: Exploring Natural Products for Novel Inhibitors of Rifamycin-resistant RNA Polymerase of Mycobacterium tuberculosis  Shireen R. Ashkar, Ashootosh Tripathi, David Sherman, George Garcia  Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI  Tuberculosis (TB) is an infectious, pulmonary disease caused by the pathogen Mycobacterium tuberculosis (MTB) and is currently the leading cause of death worldwide from a single infectious agent. The current treatment regimen is 6-9 months long involving multiple drugs, often leading to patient noncompliance and allowing for the emergence of multi-drug and extensively drug resistant strains. Rifampin, the cornerstone of current TB treatment, works by binding to the β subunit of RNA polymerase, blocking the formation of RNA transcripts longer than 2-3 nucleotides. However, resistance to rifampin arises quickly and many drug-drug interactions also occur as a result of CYP450 over-induction via rifampin activation of hPXR. With their rich structural and chemical diversity, natural products have played a significant role in the development of novel therapeutics in recent decades. Rifampin itself is a semi-synthetic natural product derived from Amycolatopsis rifamycinica. In this study, a library of natural product extracts observed to be inhibitors of a coupled bacterial transcription/translation assay is being examined for potential inhibitors of MTB RNA polymerase. This is being done through a series of fractionations and purifications guided by in vitro biological activity, followed by characterization and structure elucidation of active components. Prediction and analysis of the biosynthetic gene clusters linked to production of those active secondary metabolites also serves as a means of better understanding those molecules. The goal is to identify natural product scaffolds which may be further pursued for the development of novel inhibitors of rifamycin-resistant MTB RNA polymerase with minimal off-target effects.     

Poster 2: Elucidating NF-κκB role in the inflammatory response through utilization of small stapled peptide inhibitors   Jorge Becerra1, Paul A. Bruno2, Andrew R. Henderson1, Anna K. Mapp1,2  

1Deparment of Chemical Biology, University of Michigan, Ann Arbor, MI 48109 2Department of Chemistry, University of Michigan, Ann Arbor, MI 48109  The regulation of transcription is an important molecular event that dictates the expression of many protein-coding genes. One transcriptional regulator involved in the regulation of cell proliferation and the inflammatory response is the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor. Any misregulation of this phosphorylated-dependent pathway can lead to cancer or a prolonged exposure to inflammation that leads to a physiological response associated with pain. Current attempts to target NF-κB activation include disrupting the interaction between its sensory protein NF-κB essential modulator (NEMO) and the catalytic subunits IKKα/IKKβ. Together these subunits form a heterotetramer responsible for activation of the pathway in response to external stimuli such as physiological, oxidative, and environmental stress. In this study we aim to expand on the classic peptide-based inhibitor to this protein-protein interaction (PPI), NBD, by making synthetic derivatives that are more potent and stable. Utilization of a synthetic loop replacement peptide, NBD2, has increased the stability of this interaction through a non-labile bond and increased the potency 10-fold as opposed to NBD. Our end goal is to find selective and potent inhibitors to this pathway by targeting the NEMO-IKKα/IKKβ interface PPI and how that regulates specific gene targets associated with the inflammatory response. These NBD stapled peptide derivates will give insights on how the NF-κB pathway regulates gene expression by utilizing tools such a qPCR and chemical crosslinking.       

Poster 3: A flexible serine loop in the ectodomain of tissue factor serves as potential allosteric linker between the phospholipid membrane and the tissue factor exosite  Fabienne Birkle1, James H. Morrissey1  

1Deparment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109  Interactions with anionic phospholipids, especially phosphatidylserine (PS), are essential for the procoagulant activity of the tissue factor - factor VIIa (TF-FVIIa) complex. Removal from the membrane decreases the activity of TF-FVIIa by several hundred-fold. Previous studies in our lab have shown that mutation of TF residues that are predicted to be located close to the membrane and the substrate-binding exosite results in decreased activation of FX and FIX.1,2 However, the exact role of membrane-adjacent TF residues in facilitating substrate activation is still unknown. In this study, we analyzed the membrane-adjacent TF residues Ser160-Ser161-Ser162-Ser163 via mutagenesis studies. These serines form a flexible, solvent-exposed loop that is poorly resolved in most crystal structures. Variations in the length of the serine loop, both by inserting one or deleting one to two serine residues, showed ten- to twentyfold decreased rates of FX activation compared to wild-type TF. This defect is made significantly worse by decreasing the amount of PS in the membrane. Deletion of three or all four serine residues resulted in poor TF expression, likely due to protein misfolding. In contrast, substituting specific serines in this loop to threonine had little to no effect on rates of FX activation. These results suggest that, while threonine mutations are tolerated, the exact length of the serine loop is essential for substrate activation by TF-FVIIa. Additionally, the serine loop and immediately adjacent TF residues seem to interact with PS head groups in the phospholipid membrane. We propose that this TF loop serves as an allosteric linker between the phospholipid membrane and the TF exosite to facilitate substrate activation.     

Poster 4: Structural differences in cytochrome P450 11B enzymes key for drugs controlling blood pressure and immune responses   Simone Brixius-Anderko1 and Emily E. Scott1,2  

1Deparment of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109 2Department of Pharmacology and Biophysics, University of Michigan, Ann Arbor, MI 48109  Human steroidogenic cytochromes P450 enzymes 11B1 (CYP11B1) and 11B2 (CYP11B2) generate cortisol and aldosterone, controlling stress/immune responses and blood pressure, respectively. Excessive cortisol production by CYP11B1 results in Cushing’s disease, while aldosterone overproduction by CYP11B2 causes hypertension and cardiac disease. Treatment of each disease by drugs inhibiting the respective enzyme is impeded because CYP11B1 and CYP11B2 have 93% identical amino acid sequences and no structures of CYP11B1 are available to identify structural differences that could be exploited clinically. We report the first X-ray structure of human CYP11B1, in complex with the inhibitor fadrozole. Comparison with the previously-available CYP11B2 structure with bound fadrozole reveals large differences in the shape of the drug-binding cavity and orientation of the bound inhibitor. In fact, the two CYP11B enzymes bind two chemically distinct enantiomers of fadrozole, with CYP11B1 binding (S)-fadrozole and CYP11B2 binding (R)-fadrozole. Knowledge of these distinct active site architectures provides a structural basis for the design and improvement of drugs that inhibit only CYP11B1 or CYP11B2 to treat Cushing’s disease or hypertension, respectively.     

Poster 5: Biochemical Analysis of Depalmitoylases  Melanie Cheung See Kit & Brent R Martin  Department of Chemistry, University of Michigan, Ann Arbor, MI 48109  S-palmitoylation describes the post-translational modification of proteins with fatty acyl groups at cysteine residues. This process is regulated by Palmitoyl Acyl Transferases (PATs), which append a 16-carbon fatty acyl group to specific cysteines to facilitate the trafficking and function of many membrane proteins. The reverse reaction is catalyzed by Acyl Protein Thioesterases (APTs), which can modulate the localization of Scribble, a scaffold protein which requires membrane localization to maintain cell polarity and growth suppression. In addition, ABHD17 proteins (A, B and C) are reported to catalyze protein de-palmitoylation. Each ABHD17 protein contains a cysteine-rich N-terminal domain important for S-palmitoylation and membrane localization. When overexpressed, ABHD17A is reported to mislocalize NRAS. In addition, a recent genome-wide CRISPR screen in AML cell lines identified ABHD17B as an essential gene in NRAS dependent cells. In addition to NRAS, ABHD17 modulates the S-palmitoylation of PSD-95, a scaffold protein involved in synapse development and function. We have expressed and purified ABHD17 enzymes for in vitro analysis, as well as established cell-based systems to profile depalmitoylase modulation of Ras isoform-specific signaling. Overall, this will further our understanding of ABHD17 substrates and the cellular role of depalmitoylases in regulating Ras-dependent cell growth.      

Poster 6: Chemoenzymatic synthesis of αα-amino ketones mediated by an 8-amino-7-oxononanoate synthase   Stephanie W. Chun1,2, Meagan E. Hinze2, Alison R. H. Narayan1,2  

1Deparment of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109 2Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109  The biosynthesis of the potent neurotoxin saxitoxin (STX) is initiated by SxtA, a four-domain type I polyketide-like synthase with unusual chemistry. Notably, the final domain, 8-amino-7-oxononanoate synthase (AONS), is a rare example of a polyketide synthase-associated α-oxoamine synthase. SxtA AONS is a pyridoxal phosphate-dependent enzyme that natively condenses the α-amino acid arginine and propionyl-acyl carrier protein to an α-amino ketone. Recently, we showed that SxtA AONS can also convert a panel of straight chain, branched and cyclic acyl-CoA molecules to the corresponding arginine-based ketone. Furthermore, we have been conducting studies to determine the minimum required activating thiol group and performing mutagenesis to expand the amino acid substrate tolerance. Our work will provide the initial steps toward chemoenzymatic synthesis of therapeutically relevant STX derivatives, and more broadly, will give access to α-amino ketones from α-amino acids in a single step.     

Poster 7: Direct and Site-Specific Profiling of Native Palmitoylation  John Crellin , Brent Martin  Department of Chemistry, University of Michigan, Ann Arbor, MI 48109  Advances in high-resolution, LC-MS based, bottom-up proteomics allow us to sequence the proteome with site-specific resolution on abundant and stable PTMs. However, robust and sensitive methods for the direct detection and profiling of lipidated proteins remains elusive. A challenge of the LC-MS approach is the wide range in peptide solubility, and consequently, hydrophobic peptides lack appropriate solubility in typical sample preparation procedures and reverse-phase LC methods. This results in hydrophobic peptides being underrepresented in lists of identified peptides. Our goal is to develop methods to further elucidate the hydrophobic proteome and achieve greater resolution in the direct, site-specific detection of lipid modifications. Metabolic labeling with 2-bromopalmitate or 17-ODYA and subsequent proteomic runs have successfully yielded lists of identified proteins associated with palmitoylation events. However, the lack of charged residues in hydrophobic regions near palmitoylation sites can result in low ionization efficiency of palmitoylated peptides and poor detection sensitivity. Utilizing “click-chemistry” methods, we have designed competitive chemical probes that increase the ionization efficiency of labeled peptide ions. We expect that increasing the ion intensity should facilitate the detection of low-abundance modified peptides. In ion-mobility separation (IMS), peptide ions are separated in a time dimension based on cross-sectional surface area. Computational studies predict a significant effect of a bulky lipid on the flight-time of a peptide ion. This suggests that IMS may be a good candidate as resolving tool for detecting lipidated proteins. We plan to determine the effect of palmitoylation on the retention of peptide ions using IMS. 

 

   

Poster 8: Tertiary Structure Determination of OncomiR-1 using a Hybrid NMR/Cryo-EM Approach  

 

Hannah N. Foley1, Sarah C. Keane1,2 1Department of Chemistry, University of Michigan, Ann Arbor, MI 48109 2Department of Biophysics, University of Michigan, Ann Arbor, MI 48109 

 microRNAs (miRNAs) are short (~20-22 nucleotides) noncoding RNAs that play essential roles in the regulation of gene expression in higher eukaryotes. Dysregulation of gene expression can lead to disease; therefore the biogenesis of miRNAs is strictly regulated and includes several coordinated steps. Genes encoding miRNAs are first transcribed by RNA polymerase II or III. The resulting primary microRNA (pri-miRNA) transcript is then cleaved by the nuclear endoribonuclease, Drosha, forming a precursor-microRNA (pre-miR) hairpin. The pre-miR is exported out of the nucleus where it is further processed by a second endoribonuclease, Dicer, to generate a microRNA duplex. The passenger strand of the miRNA is degraded and the mature miRNA is loaded into the RNA-Induced Silencing Complex (RISC) to silence target mRNAs. Oncomir-1, or pri-miR-17-92a, is a primary microRNA transcript that encodes a cluster of six pre-miRNA hairpins. This cluster is highly expressed in numerous cancers including lung, breast, prostate, pancreatic, colorectal, and thyroid cancers. Interestingly, the individual pre-miRNAs are present in varying abundances in different tissues and at different stages in development, even having conflicting oncogenic and tumor suppressive activities in different cancers. We hypothesize that differential pre-miR expression in oncomiR-1 is a result of tertiary structure that restricts access to Drosha cleavage sites on the primary miRNA. Here, we describe progress toward sample preparation and tertiary structure determination of oncomiR-1 using a combination of cryo-electron microscopy and NMR spectroscopy. A high-resolution tertiary structure of oncomiR-1 will provide crucial insights into the structure-based regulation of pri-miRNA processing.     

Poster 9: Pseudouridylation of mRNA can alter translation elongation, fidelity and termination  Monika Franco1, Daniel Eyler2 and Kristin Koutmou1,2  

1Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109 2Department of Chemistry, University of Michigan, Ann Arbor, MI 48109  Chemical modifications of RNAs have long been appreciated as key modulators of non-coding RNA structure and function in cells. However, it has only recently become apparent that such modifications are also found in the coding sequences of mRNAs which direct protein synthesis. It is widely posited that the modification of mRNAs serves as a gene regulatory mechanism because the enzymatic incorporation of mRNA modifications has the potential to modulate mRNA stability, protein-recruitment, and translation in a programmed manner. We tested how one of the most common modifications present in mRNA coding sequences, pseudouridine (Ψ), impacts protein synthesis using a fully-reconstituted E. coli translation system. Our work reveals that replacing a single nucleotide with Ψ in an mRNA codon impedes amino acid addition. Additionally, we find that the incorporation of Ψ promotes the synthesis of multiple peptide products from a single mRNA sequence, and blocks translation termination by release factors. Our data further suggest that the ribosome does not always decode Ψ as a uridine, but instead sometimes preferentially reads the non-Watson Crick face of the base. These studies provide support for the provocative hypothesis that chemical modifications in mRNA could potentially provide a distinct way for cells to quickly and directly regulate and alter protein production.      

Poster 10: Differential effects of SmgGDS-607 on small GTPase prenylation   Desirée García-Torres1 and Carol A. Fierke1,2,3  

1Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109 2Department of Chemistry, University of Michigan, Ann Arbor, MI 48109 3Department of Chemistry, University of Texas A&M, College Station, TX 77843  Protein prenylation is an important post-translational modification. Proper prenylation of several small GTPases, including members of the Ras, Rho, and Rap families, ensures correct membrane localization and cellular function. During prenylation, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) catalyze the attachment of a 15-carbon farnesyl or a 20-carbon geranylgeranyl moiety, respectively, to the cysteine residue of the CAAX sequence located at the C-terminus of substrate proteins. Recent work in cells has demonstrated that SmgGDS (small GTP-binding protein GDP-dissociation stimulator) proteins can bind small GTPases and regulate their entry into the cellular prenylation pathway. To further characterize the potential role of SmgGDS proteins in regulating small GTPase prenylation, we directly tested whether SmgGDS-607 could inhibit the in vitro activity of GGTase-I or FTase for Ras and Ras-like proteins, KRas4B, HRas and DiRas1. We have shown that SmgGDS-607 doesn’t have a conserved role for regulation of prenylation. Instead, depending on the identity of the small GTPase, SmgGDS-607 can either inhibit or enhance prenylation. To investigate the factors that lead to the differential effects, we measured binding affinities and tested the kinetics of prenylation for the different GTPases. Our findings define a mechanism by which SmgGDS-607 can regulate prenylation and aid in the trafficking of prenylated proteins, offering new insights into inhibiting the prenylation of small GTPases and treating human diseases.     

Poster 11: Physiological angiogenesis is independent of endothelial cell specific MMP14.   Jian Ge1, Stephen J. Weiss1  

1Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109  Mmp14 is a transmembrane enzyme which functions in digesting extracellular matrix during normal physiological processes. Mmp14 distinguishes itself from all other MMPs as the only family member whose global deletion in mice results in postnatal death. As the global knockout mmp14 mice have defects in different tissues and organs. Mmp14 was supposed to be important in angiogenesis as if it is specifically deleted in endothelial cells. However, we find that there is no defect in the vascular structure using Tie2-Cre to delete mmp14 in endothelial cells. We detect the vasculature in the brain, peritoneal wall, intestinal villi, and mesentery, which origin from three germ layers. The extracellular matrix deposit is identical between control and conditional knockout mice under normal development. We further evaluate the vascular function, permeability, via intravenous (i.v.) injection and measurement of the Evans Blue in the perfused mesentery and other tissues, there is still no difference between wildtype and conditional knockout mice. Finally, we isolate the endothelial cells from both wildtype and conditional knockout mice by endothelial cell surface marker, CD31. RNAseq is performed using these cells, there is very little variation in gene profiling. We think the endothelial mmp14 does not contribute to the angiogenesis in physiological state, while the change of gene expression leads us to study the role of mmp14 under pathological conditions.     

Poster 12: Conserved Coactivator Binding Mechanisms Facilitate Discovery of Allosteric Small Molecules   Matthew J. Henley1, Amanda L. Peiffer1, Andrew R. Henderson1, Nicholas J. Foster1, Brian M. Linhares2, Zachary B. Hill3, James A. Wells3, Tomasz Cierpicki1,2, Charles L. Brooks III1,2,4, Carol A. Fierke1,4 and Anna K. Mapp1,4  

1Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109 2Department of Biophysics, University of Michigan, Ann Arbor, MI 48109 3Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158 4Department of Chemistry, University of Michigan, Ann Arbor, MI 48109  Molecular recognition in protein-protein interactions is commonly understood in terms of the structure-function paradigm: complementary structural elements interact by forming a defined set of intermolecular interactions. However, interactions between transcriptional activators and the transcriptional machinery eschew this paradigm; a single activator-binding domain (ABD) within a transcriptional coactivator can interact with >10 activators that may share no clear molecular recognition elements. Further one ABD accommodates diverse activator sequences with similar binding affinity. Without clear recognition principles, it has proven nearly impossible to develop small-molecule modulators. Here, we seek to develop a molecular recognition model by characterizing the ABD of Mediator subunit Med25, a structurally unique ABD that is formed from a rigid seven-stranded β-barrel core flanked by conformationally dynamic loops and helices. We use transient kinetics and protein NMR to demonstrate that, when bound to an activator, Med25 shifts between 2-3 conformational states in a manner dependent on the specific binding partner. Thus, conformational lability is a common mechanism to accommodate diverse binding partners. The plasticity of the Med25 ABD also results in allosteric communication between its two binding sites, and targeting a dynamic region with a covalent small molecule recapitulates the allosteric effects of the natural activators. Our data implicate common mechanisms for activator engagement in structurally divergent ABDs. Further, our evidence suggests that small-molecule targeting strategies aimed at conformationally mobile structural elements can take advantage of the plasticity of ABDs to provide chemical probes.     

Poster 13: SYNTHESIS AND BIOSYNTHESIS OF NOVEL DIKETOPIPERAZINES AND NASESEAZINES  Robert M. Hohlman1, Vikram V. Shende2 and David H. Sherman1,2 

1Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Mi 48109 2Department of Chemical Biology, University of Michigan, Ann Arbor, Mi 48109 

The diketopiperazine (DKP) ring structure is a privileged chemical structure found in numerous natural products such as spirotryprostatin B, mycocyclosin and naseseazine B. Some of these natural products arise from dimerization of two monomers connected by a carbon-carbon bond arising from C–H functionalization. The naseseazines, in particular, has been examined for total chemical synthesis but regioselectivty and C-H functionalization have been challenges faced by these groups. Current methods to overcome these problems involve pre-functionalization with electron withdrawing groups on the indole ring of the donor monomer and a halogen on the acceptor carbon of the other monomer. The biosynthetic gene cluster responsible for the biosynthesis of (–)-naseseazine C was recently identified, and the cytochrome P450 (NasD) that catalyzes the dimerization reaction has been isolated and purified. Using NasD and a homolog, NasD’, novel naseseazines have been synthesized via biocatalysis but limitations in substrate scope have been observed. Using an obtained crystal structure, specific residues of the active site have been targeted for site directed mutagenesis to broaden the substrate scope of the NasD’. Furthermore, novel DKP substrates, arising from known amino acids and their derivatives, have been synthesized using both known and new chemical synthetic strategies and subjected to the dimerization reaction. Naseseazine C has shown moderate anti-plasmodial activity and these new naseseazines could provide new leads for anti-plasmodial drugs. 

   

Poster 14: Development of small molecule inhibitors of NSD3 histone methyltransferase 

Christina Howard,1,2,3 Eungi Kim,3 Sergei Zari,3 Caroline Nikolaidis,3 Trupta Purhohit,3 Hongzhi Miao,3 Hao Li,1,2,3 Juliano Ndoj,3 Hyoje Cho,3 Tomasz Cierpicki,1,3 Jolanta Grembecka1,2,3  

1 Program in Chemical Biology, University of Michigan, Ann Arbor, MI, 48109 2Chemical Biology Interface Training Program, University of Michigan, Ann Arbor, MI, 48109 3Department of Pathology, University of Michigan, Ann Arbor, MI, 48109 

Nuclear SET Domain containing protein 3 (NSD3) is a histone lysine methyltransferase that mono and dimethylates histone 3 at lysine 36 (H3K36) to affect gene expression. NSD3 is a driver gene in the 8p11-12 amplicon, which is present in 10-15% of breast cancers and associated with poor prognosis. Breast cancer cell lines containing this amplicon show decreased viability upon siRNA knockdown of NSD3, indicating its important role in cancer cell survival. However, the function of NSD3 in breast cancer is not fully elucidated. NSD3 is a complex protein consisting of several protein-protein interaction domains as well as the catalytic SET domain, and it is unclear which domains are relevant to its role in breast cancer. Our goal is to develop selective small molecule inhibitors of NSD3 that can be utilized to investigate the role of NSD3 in breast cancer and other cancers, as well as evaluate the therapeutic potential of targeting NSD3. Towards that end, we have performed NMR-based fragment screening and identified a hit that binds to the catalytic SET domain of NSD3 with millimolar binding affinity. This fragment-like compound has been extensively optimized into a series of inhibitors which block the histone methyltransferase activity of NSD3 with low micromolar activity. These compounds exhibit strong effects on the growth of triple negative breast cancer cells with some selectivity. Current work is being focused on developing inhibitors with increased selectivity and potency, and on further characterization of the role of NSD3 in triple negative breast cancer. 

   

Poster 15: Development of Chemical Probes Targeting ASH1L Histone Methyltransferase 

Hao Li1,2 Jing Deng1, David Rogawski1, Dmitry Borkin1, Trupta Purohit1, Szymon Klossowski1, Katarzyna Kempinska1, EunGi Kim1, Zhuang Jin1, Juliano Ndoj1, Marta Szewczyk1, Deanna Montgomery3, Hyo Je Cho1, Hongzhi Miao1, Tomasz Cierpicki1,2, Jolanta Grembecka1,2,3  

1Department of Pathology, University of Michigan, Ann Arbor, MI 48109  2Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109  3Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109 

ASH1L (absent, small, or homeotic-like 1) is a SET domain-containing histone lysine methyltransferase. It is shown to mono- and di-methylate lysine 36 on histone H3 (H3K36), which is associated with transcriptional activation. Emerging data link ASH1L to multiple cancers. ASH1L gene is amplified in 27% of aggressive, basal-like breast cancers, and high levels of ASH1L mRNA are associated with shorter survival in breast cancer patients. ASH1L activates clusters of HOX genes, including HOXA9, which is overexpressed in acute myeloid leukemias (AML) and leukemias with Mixed Lineage Leukemia (MLL) rearrangements. Therefore, well- characterized chemical probes with inhibitory activity against ASH1L would be extremely valuable for elucidating the biological functions of ASH1L in cancers. To develop ASH1L inhibitors, we performed an NMR-based fragment screening using 15N- labelled ASH1L SET domain. We identified compounds that bind to ASH1L with millimolar binding affinity and performed extensive optimization. Through these efforts, we developed compounds that bind to ASH1L with nanomolar binding affinity. Biochemical analyses demonstrated that these compounds effectively inhibit ASH1L histone methyltransferase activity through a noncompetitive inhibition mechanism with nucleosome substrate. We also obtained high resolution crystal structures of ASH1L-SET in complex with inhibitors, revealing that compounds bind to a well-defined pocket surrounded by the autoinhibitory loop. The activity of ASH1L inhibitors was further tested in MLL leukemia cells, resulting in selective inhibition of cell proliferation and differentiation in these cells. Our study demonstrates that development of ASH1L inhibitors is feasible, resulting in compounds with potent activity in leukemia cells. To our knowledge, these compounds represent the first-in-class chemical probes targeting ASH1L to investigate its role in leukemia and other diseases. 

 

   

Poster 16: Towards a post-PCR world: direct counting of microRNAs from single cells  

Karen Montoya1, Greg Shelley2, Evan Keller2, Nils G. Walter1  

1Deparment of Chemistry, University of Michigan, Ann Arbor, MI 48109 2Department of Urology and Pathology, University of Michigan, Ann Arbor, MI 48109  Cell-to-cell heterogeneity is always present in a cell population, and bulk observations may not always represent all behaviors of a single cell. Therefore, analyzing single cells permits discovery of mechanisms not seen when studying a bulk population of cells. Current techniques for single-cell analysis usually lack adequate sensitivity and quantitative accuracy for rare or hard-to-amplify species. However, there is a new technique that promises to bypass these challenges: an amplification-free kinetic fingerprinting approach for digital single-molecule detection, called Single-Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS). This method allows for highly specific, sensitive, and rapid detection of molecular species, but has not been tested on single cells and will require optimization and coupling with other methods, such as single-cell extraction and lysis. In this study, we investigate the detection of miRNA panels in single cells by combining microfluidics and single-molecule microscopy. Our end goal is to develop a platform for parallel detection of multiple microRNA (miRNA) biomarkers, to be able to detect their abundance in single cells to help better understand human health and predict the likelihood of human disease, including cancer progression.      

Poster 17: The non-selective opioid diprenorphine produces delta-opioid receptor-mediated rapid antidepressant-like effects in mice  Keith M. Olson1, Todd M. Hillhouse1, James E. Hallahan1, Evan Schramm1 and John Traynor1  

1Department of Pharmacology, University of Michigan, Ann Arbor, MI., USA  

 Major depressive disorder (MDD) is the most common mood disorder worldwide with a lifetime prevalence of ~15%. However, current FDA approved treatments are limited by a multi-week delayed onset of action and minimal efficacy in some patients. Preclinical evidence indicates delta-opioid receptor (DOR) agonists and kappa-opioid receptor (KOR) antagonists may produce rapid onset antidepressant effects and provide a novel depression target in treatment resistant patients [3H]-diprenorphine saturation binding experiments in membranes expressing the mu-opioid receptor (MOR); (KDµ = 0.31 +/- 0.04); DOR (KDδ = 1.1 nM +/- 0.16); and KOR (KOR KDκ = 0.36 +/- 0.09) revealed non-selective affinity, as expected. In vitro [35S]GTPγS assays in the same cell lines revealed diprenorphine is a DOR and KOR partial agonist (EC50δ = 4.1 nM +/- 2.0, EMAXδ = 40 % +/- 5.0; EC50κ = 0.71 nM +/- 0.21, EMAXκ = 41 % +/- 5.3); it also has potent MOR antagonist against DAMGO (KB = 0.18 +/- 0.05). In vivo diprenorphine produced anti-depressive-like effects in the tail suspension test and the novelty-induced hypophagia test that were blocked by the DOR-selective antagonist naltrindole. While classical DOR agonists, such as SNC80, produce seizure activity, diprenorphine did not produce seizures and blocked SNC80 mediated seizures indicating that higher DOR efficacy is needed to produce seizures than anti-depressant-like effects. Alternatively, the DOR/KOR partial agonist and MOR antagonist profile of diprenorphine may mitigate against seizures. Future directions include the development of diprenorphine analogues to determine if modulating DOR/KOR agonist activity improves diprenorphine anti-depressant-like efficacy.     

Poster 18: Understanding the role of TRAF6 in the antiviral activity of viperin  Ayesha M. Patel, Soumi Ghosh, and E. Neil. G. Marsh  Department of Chemistry, University of Michigan, Ann Arbor, MI 48109  Viperin is a recently identified putative radical S-adenosylmethionine (SAM)-dependent enzyme that is involved in the cellular antiviral response. It is one of the interferon- stimulated genes (ISGs), activated as a part of the innate immune system. However, the biochemical role of viperin in the innate immunity signaling pathway is still unclear. Because Toll-like receptor 7 (TLR7) and Toll-like receptor 9 (TLR9) sense viral nucleic acids and promote type I interferon production during viral infection, viperin is predicted to be a key component in these signaling pathways. It recruits the signaling proteins to lipid bodies, and thereby facilitates the downstream activation process. Here, we studied the interaction of viperin with tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), one of the key proteins in the signaling complex. TRAF6 mediates the signal through the ubiquitination of interleukin 1 receptor associated kinase 1 (IRAK1). We present studies on the interaction of viperin with TRAF6, and examine how this interaction affects the ubiquitination activity of TRAF6 and the radical SAM activity of viperin.     

Poster 19: Chemoenzymatic, Enantiodivergent Synthesis of Biologically Active Azaphilones 

 Joshua Pyser1, Summer Baker Dockery1, and Alison R. H. Narayan1,2  

1Department of Chemistry, University of Michigan, 2Department of Chemistry and Life Sciences Institute, University of Michigan 

 Abstract: Azaphilones are a class of fungal natural products that contain a highly oxygenated pyranoquinone bicyclic core. Many of these fungal pigments posses potent biologically activity (exhibiting anticancer, anti-inflammatory, and antimicrobial properties), making them interesting synthetic targets for the development of new pharmaceutical agents. However, their construction has proven challenging due to their nonaromatic, bicyclic structure and the presence of a stereocenter. The classical approach toward this class of molecules relies on the oxidative dearomatization of prefunctionalized arene building blocks. These reactions are often mediated by copper or lead complexes, as well as hypervalent iodine species, and typically require harsh reaction conditions. Nature has evolved efficient enzyme catalysts to perform oxidative dearomatization with exquisite site- and stereoselectivity, making them an exciting alternative to the current state of the art. These enzymes can operate on a number of substrates under mild conditions, using O2 as the terminal oxidant. Therefore, this work seeks to develop an enantiodivergent, chemoenzymatic route to azaphilone natural products through the use of these enzymatic biocatalysts. Thus far, progress has been made towards these compounds by the production of a common substrate that can be dearomatized by two homologous flavin-dependent monooxygenases, giving each C3 epimer of a general azaphilone scaffold.   

   

Poster 20: Structural Analysis of the DNA Binding Domain of VirF, the Key Transcriptional Activator in Shigella Pathogenesis 

  Nicholas J. Ragazzone1, George A. Garcia1  

1Deparment of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109  Shigella flexneri is the main cause of bacterial dysentery in humans known as shigellosis. Infections by Shigella lead to over one million deaths worldwide each year, the majority of the victims being children under five years of age. The bacterium relies on various virulence factors that are essential to pathogenesis. These processes rely on an AraC family main transcriptional regulator, VirF, to activate transcription of the virulence genes. We hypothesize that antimicrobial agents can be developed to target VirF and inhibit virulence of Shigella . Targeting virulence factors required for infections is a relatively new strategy for developing antimicrobials. Since virulence factors such as VirF are not required for bacterial viability, inhibition of virulence pathways is expected to result in less selective pressure to develop resistance.  We conducted a high-throughput screen that identified five lead compounds to further pursue in hit-to-lead development. Using an electrophoretic mobility shift assay, we determined that one of the compounds, 19615, inhibits VirF activity by disrupting the ability of the protein to bind to the promoter DNA. Using predictive modeling of the VirF amino acid sequence, we found many similarities between VirF and other AraC family transcriptional activators, particularly in the carboxyl-terminus that contain the helix-turn-helix motifs that interact with the promoter. With this information, we plan on evaluating 19615 for activity against a number of other protein family members. We also hope to obtain a co-crystal structure of the c-terminal DNA binding domain of VirF in complex with 19615.  

   

Poster 21: Non-enzymatic role of putative demethylase Epe1 1Raiymbek, G., 1Khurana, N., 1An, S.J., 1Gopinath, S., 1Biswas, S., 1Larkin, A., 1Ragunathan, K. 1Deparment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109. ragunath@umich.edu.com  

 Heterochromatin is characterized by histone hypoacetylation and an enrichment of methylation marks on histones which are associated with repression. In fission yeast, a single H3 methyltransferase Clr4 (Suv39h) directs all of the methylation of H3K9 i.e., H3K9me2/3 which enforces the establishment of transcriptionally silent regions in the genome referred to as heterochromatin. A putative histone demethylase, Epe1, is recruited to sites of heterochromatin via interaction with a chromo-domain containing protein Swi6, homolog of HP1. The loss of Epe1 has been associated with the propagation of silent epigenetic states in a DNA or RNA sequence independent manner. How Epe1 carries out its function as an anti-silencing factor remains unknown. Despite having sequence homology with known histone demethylases, no detectable demethylase activity has been observed for Epe1 in vitro. Here, we demonstrate that mutations within the catalytic JmjC domain of Epe1 disrupt its interaction with Swi6. Using a combination of genetic and biochemical assays, we show that the interaction between Epe1 and Swi6 is direct, and is independent of the co-factors: Fe and 2-OG. Intriguingly, the interaction between these two proteins is sensitive to the mutations that affect the structure of the JmjC domain. Furthermore, our immunoblot and in vitro assays showed that heterochromatin formation or the addition of an H3K9 tri-methyl peptide enhances the interaction between the two proteins. Finally, we demonstrate that this ability of Epe1 to selectively sequester Swi6 prevents the recruitment of histone deacetylases. Our work supports models where histone turnover, rather than enzymatic histone demethylation is the primary determinant of epigenetic stability and inheritance.     

mailto:ragunath@umich.edu.com

Poster 22: Expanding the Synthetic Utility of the Flavin-dependent Monooxygenase TropB 

  Attabey Rodríguez Benítez1, Sara Tweedy1, Summer A. Baker Dockrey2, April L. Lukowski1, Dr. Troy Wymore, Dr. Dheeraj Khare4, Charles L. Brooks III4, Janet L. Smith1,3,4, Alison R. H. Narayan1,2,4*  1Program of Chemical Biology, University of Michigan, Ann Arbor, MI 48109 2Department of Chemistry, University of Michigan, Ann Arbor, MI 48109 3Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109 4Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 5Department of Biophysics, University of Michigan, Ann Arbor, MI 48109 

 Oxidative dearomatization is a powerful transformation; however, a limited number of highly enantioselective catalytic methods have been reported. Nature performs this reaction catalytically with excellent site- and stereoselectivity under mild conditions. For example, direct hydroxylation is performed seamlessly by the flavin-dependent monooxygenase TropB. This enzyme can achieve complete conversion of phenol substrates to dearomatized products in 1 h using molecular oxygen as the stoichiometric oxidant. TropB-catalyzed hydroxylation provides an efficient route to chiral intermediates valuable for the synthesis of complex bioactive molecules without the drawbacks of conventional chemical methods. To expand the substrate scope of TropB, a structure-guided protein engineering effort was employed. Overall, these efforts have enhanced the synthetic utility of this biocatalyst. Structural information has also informed a set of mechanistic hypotheses, which have been probed through mutagenesis experiments.     

Poster 23: Kainate Induced Oscillations in Ex Vivo Hippocampal Sections  J.C Rodríguez Díaz1,2 , K.S. Jones1,2  

1 Neuroscience Graduate Program, University of Michigan 2 Department of Pharmacology, University of Michigan Ann Arbor MI  Coordinated and synchronized activity in the brain is known to result in different types of oscillatory activity. Oscillations play crucial roles in many cognitive processes such as memory formation and attention. GABAergic interneurons are crucial for synchronizing neuronal activity leading to gamma oscillations (30-60 Hz). Abnormalities in oscillatory activity in the prefrontal cortex and hippocampus have been implicated in the pathology of some mental disorders including schizophrenia, however the neurobiological mechanism underlying these abnormal oscillations are not yet fully understood. We set out to develop an assay that would allow for the study of gamma oscillations in hippocampal sections using microelectrode arrays. Extracellular electrophysiological recordings were performed using 64- channel perforated micro electrode arrays (pMEAs). Oscillatory activity in the gamma band was induced using kainate bath application. Established oscillations were inhibited by bath application of the GABAA receptor antagonist bicuculline. Oscillatory activity in the CA1 and CA3 regions of the ippocampus were induced and maintained by kainate. In CA1, oscillations had a narrow band with a peak at around 30 Hz while in CA3 the oscillations were broader. Kainate-induced oscillations in CA1 and CA3 were abolished by bicuculline application. These studies suggest that kainate-induced oscillatory activity can serve as a model of in vivo GABA- dependent gamma oscillations in the hippocampus. Furthermore, pMEAs provide a tool to study oscillations in nearby regions during chemically induced oscillations. Future studies will focus on determining the oscillations sensitivity to other drugs such as dopamine and glutamate receptor antagonist.     

Poster 24: Detecting miRNA-miRBP interactions with a cell-based split-enzyme assay  Sydney L. Rosenblum1, Daniel A. Lorenz1, Amanda L. Garner1,2  

1Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109 2Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109  

 Greater than one-thousand human miRNAs are believed to control the translation of greater than 60% of all protein-coding genes. Accordingly, miRNA dysregulation is a common hallmark of countless human diseases, including cancers, neurological diseases, and cardiac diseases, making miRNA and miRNA binding proteins (miRBPs) attractive drug targets. Recent advances in high-throughput technology have enabled large-scale screening of small molecules and natural products against RNA targets. However, subsequent characterization of promising chemical scaffolds is hindered by the lack of reliable assays designed to detect target engagement of RNA-targeted compounds or protein-RNA interaction inhibitors. Most existing methods of determining target engagement successfully measure ligand engagement of protein targets, but such systems cannot be easily adapted to detect RNA target engagement or the disruption of protein-RNA interactions. The development of novel methods of detecting RNA-ligand and RNA-protein interactions in cells will not only provide tools for studying miRNA-miRBP interactions, but will also facilitate the discovery of miRNA-targeted ligands and miRNA-miRBP inhibitors. Here I describe a cellular assay that utilizes the split-enzyme NanoBiT® (Promega) system to detect the miR-miRBP interaction between the pre-let7 class of miRNA and Lin28. This strategy is currently being expanded to enable the detection and study of various miR-miRBP interactions as well as measure the cellular activity of small molecule inhibitors of such interactions.     

Poster 25: The Discovery and Optimization of pre-microRNA Probes  Jorge Sandoval  Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109  MicroRNAs (miRNAs or miRs) are a highly conserved non-coding class of RNAs that have been found to regulate more than 60% of protein expression via RNA Induced Silencing Complex (RISC). Over 2,500 miRNAs have been catalogued and each one has conserved targets and even more non- conserved targets. Dysregulation of miRNA expression has been linked to cancer and other diseases, making miRNAs an attractive target for therapeutic innovation. However, targeting miRNAs has remained a challenge due to: (1) their negatively charged backbone; (2) their largely dynamic structures, and (3) localization and relative abundance within a cell. MiRNAs must undergo several maturation steps; of note is the Dicer mediated maturation of precursor-miRNA (pre-miRNA or pre-miR) to mature miRNA. We hypothesized that: (1) we could target pre-miRNA and disrupt its interactions with Dicer to inhibit Dicer mediated maturation; and (2) natural product extracts (NPEs) would allow us to screen several compounds at once and allow us to more quickly identify potential pre-miRNA probes. The specific aims of this proposal are to: (1) to screen and validate pre- miRNA binders; (2) to optimize their activity and confirm their mechanism of action. The long term goal of this project is to elucidate the mechanisms by which miRNA are matured and how they carry out their gene silencing activity. Furthering our understanding of miRNAs will allow us to develop a new therapeutic approach to prevent and cure many human diseases.     

Poster 26: A Method for Detecting O2-Binding to Flavoproteins  Zhuoyi Song, Caroline Crosby, Bruce A. Palfey   Program in Chemical Biology and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, 48109  

The controversial topic of whether flavoenzymes bind oxygen has long been discussed, but there are few methods to actually detect this. A sensitive spectrophotometric method has been developed for determining high concentrations of oxygen in aqueous solution. O2 is reduced to H2O2 enzymatically using glucose oxidase. Then 4-aminoantipyrine and phenol are oxidized by horseradish peroxidase and H2O2, producing quinoeimine, which is pink (absorbance maximum at 505 nm). Therefore, the oxygen concentration can be measured based on the increase in absorbance at 505 nm. The method has been preliminary proved using 40 mM phosphate buffer equilibrated with different concentrations of oxygen; a linear relationship between the oxygen concentration and A505 was found. This method enables us to measure total O2 in a solution. If O2 actually does bind to a flavoenzyme, an amount of O2 exceeding that in buffer alone will be measured. We have already proved that the method is viable by proteins such as Lysozyme and hemoglobin. We are in the process of testing a number of oxidases, monooxygenases, and other flavoproteins. 

    

Poster 27: Recognition of 1,N2-ethenoguanine by Human Alkyladenine DNA Glycosylase  Adam Thelen and Patrick O’Brien  Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109  Alkylated DNA bases can be both mutagenic and toxic to living cells. The base excision repair pathway removes a variety of single-nucleotide DNA lesions from the genome and incorporates new nucleotides to properly complement the opposing strand. Glycosylase enzymes catalyze the first step of base excision repair, wherein a lesion is detected and the damaged base is cleaved at the N-glycosidic bond. This is generally accomplished via a base flipping mechanism which positions the substrate into the enzyme active site. The human alkyladenine DNA glycosylase (AAG) acts by such a mechanism, excising a broad range of alkylated purine lesions. AAG was initially reported to recognize all of the etheno adducts that are formed during lipid peroxidation and through exposure to vinyl chlorides. However, 3,N4-ethenocytosine was later discovered not to be a substrate of AAG. Conflicting results remain for AAG-catalyzed excision of 1,N 2-ethenoguanine (εG). This work characterizes the steady state and transient kinetics of εG excision by human AAG. Gel-based glycosylase activity assays reveal that εG is excised with far less efficiency than other substrates of AAG such as 1,N6-ethenoadenine (εA). εG excision is also shown to be strongly inhibited by the presence of undamaged DNA. Mutation of asparagine residue 169 of AAG, which partially defines the active site pocket, increases the excision of εG by 9-fold, demonstrating that the active site discriminates against εG as a substrate. Together, these findings make a strong case against εG as a primary target of AAG.     

Poster 28: Structure and engineering of TamI, an iterative cytochrome P450 for late-stage C-H functionalization of tirandamycin antibiotics  Rosa Vasquez1,2, Kinshuk Srivastava1, Kersti Haatveit3, Sean Newmister1, Jessica L. Stachowski2, Yogan Khatri1, K. N. Houk, John Montgomery2 and David H. Sherman1*.   1Life Sciences Institute and 2Departments of Chemistry, Medicinal Chemistry, and Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109, USA, 3Departments of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095, USA.   Tirandamycin antibiotics are structurally intriguing molecules that show a vast array of antibacterial, antiparasitic and antifungal activities. The heavily oxidized bicyclic ketal moiety of tirandamycin is key to biological activity. Elucidating the tirandamycin biosynthetic pathway provided access to unique enzymes involved in the late-stage C-H functionalization of tirandamycin. The multi-step oxidative cascade responsible for the tailoring of the bicycling ketal moiety is mediated by two co-dependent oxidative enzymes, TamI, an iterative cytochrome P450 monooxygenase, and TamL, a flavin adenine dinucleotide-dependent oxidase. Here, we characterized the P450 TamI and performed Molecular Dynamics (MD) in tandem with site-directed mutagenesis to gain insights into the substrate binding mechanism and exquisite selectivity displayed by this remarkable enzyme. Substrate engineering and MD-guided mutagenesis efforts are undergoing to modulate the enzyme's regio- and stereoselectivity as well as expand its substrate scope to include a variety of cyclic and bicyclic scaffolds.     

Poster 29: A Light- and Calcium-Gated Transcription Factor for Tagging Activated Neurons   Wenjing Wang1,2, Tanyaporn Pattarabanjird3, Craig Wilds4, Mateo Sanchez5  

1Department of Chemistry, University of Michigan, Ann Arbor, MI 48109 2Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 3School of Medicine, University of Virginia, Charlottesville, VA 22903 4Picower Institute, Massachusetts Institute of Technology, MA 02139 5Department of Genetics, Stanford University, CA 94305  It remains a major challenge in neuroscience to search for the neuronal ensembles underlying specific behaviors. Real time imaging of neuronal activity using activity based fluorescent sensors only allows one to look at a small field of view at one time and does not enable further characterization of those neurons. To circumvent these limitations, we designed a light- and calcium-gated transcription factor that can label neurons that are activated during the user-defined time window (via light irradiation) with the expression of any chosen transgene. We used directed evolution to optimize the light/dark signal noise ratio of the tool. Preliminary results in mice indicates the possibility of utilizing this tool to tag activated neurons in a short time window. Further optimization and engineering are currently underway to generate the next generation, more robust activity based tools for tagging functionally defined neurons.      

Poster 30: Biocatalytic biaryl bond formation with cytochromes P450  Lara E. Zetzsche1,2, Meagan E. Hinze2, Jessica A. Yazarians2,3, and Alison R. H. Narayan1,2,3  

1Program in Chemical Biology, 2Life Sciences Institute, University of Michigan, 3Department of Chemistry, University of Michigan  Axially chiral biaryl scaffolds are present in many synthetically valuable organometallic ligands and natural products with potent bioactivities, including compounds with antimicrobial and anticancer properties. Despite their utility, achieving site- and atroposelectivity in these reactions using traditional synthetic chemistry remains a challenge. Current strategies often rely on blocking groups to limit the available sites of bond formation or tethering approaches to help direct the selectivity of the oxidative coupling reaction; nevertheless, both strategies often suffer from low yields, require harsh reaction conditions, and lack atroposelectivity. Conversely, Nature has evolved enzymes capable of forming these bonds with excellent site- and atroposelectivity. KtnC and DesC are fungal cytochrome P450 enzymes that catalyze the dimerization of a coumarin substrate to form complementarily axially chiral 8,8’- and 8,6’-bicoumarins, respectively. Heterologous expression of these enzymes in yeast allows for whole-cell biotransformations with high conversions to their corresponding biaryl products with site- and atroposelectivity. Initial characterization of the substrate scope of the wild type P450s show that they can catalyze the oxidative coupling of a wide range of phenolic substrates outside of the native reaction with varying levels of efficiency. Current efforts are focused on (1) engineering of a soluble, catalytically self-sufficient chimeric enzyme and (2) expansion of natively accessible chemistry through directed protein evolution. Together these endeavors will create a platform for a versatile, robust method for site- and atroposelective biocatalytic formation of diverse biaryl scaffolds.