Chemical Biology Discussion Group Year-End Symposium

Agenda

* Presentation titles and times are subject to change.

Abstracts

Nitric Oxide Signaling: From Bugs to Humans
Michael Marletta, PhD, The Scripps Research Institute

Nitric oxide (NO) has long been known to be an intermediate in bacterial pathways of denitrification. It is only since the middle to late 1980s that it was found to play a central role in a much broader biology context. For example, it is now well established that NO acts as a signaling agent in higher organisms. Yet NO is toxic and reactive under biological conditions. How is the biology carried out by NO controlled? How is NO used and the inherent toxicity avoided? How do organisms tell the difference between NO and O2? What is the biological output? A molecular perspective on ligand discrimination in hemoproteins has emerged as has a further understanding and predictions about selective ligand sensing and function in biology.

A General Platform for Systematic Quantitative Evaluation of Small-molecule Permeability in Bacteria
Tony D. Davis, BS, Tan lab, Weill Cornell Graduate School of Medical Sciences

The chemical features that impact small-molecule permeability across bacterial membranes are poorly understood, and the resulting lack of tools to predict permeability presents a major obstacle to the discovery and development of novel antibacterials. Antibacterials are known to have vastly different structural and physicochemical properties compared to non antiinfective drugs, as illustrated herein by principal component analysis (PCA). To understand how these properties influence bacterial permeability, we have developed a systematic approach to evaluate penetration of diverse compounds into bacteria. Intracellular compound accumulation is quantitated using LC-MS/MS, then PCA and Pearson pairwise correlations are used to identify structural and physicochemical parameters that correlate with accumulation. An initial study using ten sulfonyladenosines in three bacteria with structurally distinct cellular envelopes has revealed that the impacts of several physicochemical parameters upon permeability and efflux sensitivity differ among the various bacteria in a non-obvious manner. This sets the stage for use of this platform in larger analyses to identify global relationships between chemical structure and bacterial permeability that would enable the development of predictive tools to accelerate antibiotic drug discovery.

Suppression of Aberrant Ras Activation in Cancer Cells with Synthetic Sos Mimics
Stephen Joy, MS, Arora lab, New York University

Aberrant activation of the small GTPase Ras by Sos-mediated nucleotide exchange can arise through diverse processes including oncogenic mutation of Ras or constitutive receptor tyrosine kinase (RTK) signaling. The resulting deregulation of cellular physiology impacts the growth and survival of ~30% of all human tumors. Recent evidence supports the therapeutic strategy of targeting aberrant Ras activity by blocking the Ras-Sos catalytic interaction, the rate-limiting step in canonical Ras activation. Here, we describe a synthetic mimic of the Ras-binding domain of Sos that inhibits Ras activation with high potency. Treatment of Ras-transformed cancer cells with the inhibitor suppressed the GTP loading of both wild-type and mutant oncogenic Ras proteins, and reduced the viability of a panel of cancer cell lines. Our studies with the direct inhibitor of Ras provide mechanistic insights regarding the role of the Ras-Sos interaction in maintaining oncogenic signaling. DNA content analysis reveals that the Ras inhibitor causes alterations to the cell cycle and engenders an apoptotic cell fate—a result that supports recent findings in Ras-transformed cancer cells where the wild-type Ras protein is post-transcriptionally silenced. Lastly, we show that the synthetic Sos mimics are transported into cells through macropinosomes, and demonstrate the exploitation of oncogene-stimulated macropinocytosis for specific targeting of cancer cells.

Synthesis of Dithioamide Peptides and Proteins and Their Application on Conformational Dynamic Study by Fluorescence Spectroscopy
Yun Huang, PhD, Petersson lab, University of Pennsylvania

Tracking protein conformational change is important to understand the folding and function of proteins. Förster resonant energy transfer (FRET) and photoinduced electron transfer (PET) are widely used to glean time-resolved structural information on protein motions. However, the relatively large size of fluorophores and quenchers may introduce significant perturbation on the protein structure. The thioamide bond, only one atom substitution to peptide bond, has recently been shown to represent the minimalist fluorescent quencher over various fluorophores by photo-induced electron transfer (PET) mechanism. Unlike commonly used fluorescence quenchers, thioamides are sufficiently small that they can be placed at nearly any position of the protein sequence without significantly altering the secondary structure. However, the moderate quenching efficiency may limit its sensitivity for application. Here, we show that two consecutive thioamide bonds could be incorporated into peptides and proteins, and the quenching effect is further strengthened compared with the mono-thioamide system. The dithioamide bonds thus provide increased sensitivity to detect the protein conformation changes.

Targeted Protein Destabilization in the Endoplasmic Reticulum Reveals an Estrogen-mediated Stress Response
Kanak Raina, MSc, Crews lab, Yale University

Accumulation of unfolded proteins within the endoplasmic reticulum of eukaryotic cells leads to an unfolded protein response (UPR) that either restores homeostasis or commits the cell to apoptosis. Tools traditionally used to study the UPR are unfortunately pro-apoptotic and thus confound analysis of long-term cellular responses to endoplasmic reticulum stress. Here, we describe an Endoplasmic Reticulum-localized HaloTag (ERHT) protein that can be conditionally destabilized using a small molecule hydrophobic tag (HyT36). Treatment of ERHT-expressing cells with HyT36 induces an acute, resolvable endoplasmic reticulum stress that results in transient UPR activation without induction of apoptosis. Transcriptome analysis of late-stage responses to this UPR stimulus revealed a link between UPR activity and estrogen signaling: UPR signaling induces estrogen receptor transcriptional activity, which modulates future sensitivity to endoplasmic reticulum stress. Estrogen receptor activation desensitizes MCF7 cells to endoplasmic reticulum stress, whereas, conversely, estrogen receptor inhibition sensitizes MCF7 cells to endoplasmic reticulum stress. Finally, estrogen receptor inhibition in multiple myeloma cell lines sensitized them to UPR activation and apoptosis induced by the proteasome inhibitor, epoxomicin. Our data suggest that estrogen signaling may play a role in the adaptive UPR, and manipulating this interaction may have therapeutic applications in the treatment of multiple myeloma.

Cellular Metabolism Regulates Sensitivity to GPX4-Dependent Ferroptosis
Kenichi Shimada, MA, MPhil, Stockwell lab, Columbia University

Abstract not available.

Nitrotyrosine as a Structural Switch in Designed Metalloproteins and Application as a Sensor for Tyrosine Nitration
Andrew Urmey, BS, Zondlo lab, University of Delaware

Tyrosine nitration is a specific, non-enzymatic post-translational modification associated with disease states and oxidative/nitrosative stress. Nitric oxide is produced as a signaling molecule and as a cytotoxic defense molecule in inflammation; this molecule generates other reactive nitrogen species (RNS) which can modify tyrosine to 3-nitrotyrosine via radical chemistry, potentially changing the structure and function of affected proteins. Nitrotyrosine is present in anionic and neutral states at physiological pH, with properties which vary significantly from native tyrosine and from each other. While it is a biomarker for various pathologies including Alzheimer's disease, methods for detection of nitrotyrosine are limited. To better understand tyrosine nitration, we have designed a series of 14- to 18-residue peptides which use tyrosine nitration as a structural switch upon metal binding. The designs are based on a Ca²⁺-binding EF-hand motif, in which a native glutamate essential for metal binding is changed to tyrosine; if nitrated, the resultant nitrotyrosine (pK_a~7) can mimic glutamate and coordinate metals in a bidentate fashion using the nitro group and phenolate oxygen. Compared to unmodified tyrosine, nitrotyrosine-containing peptides exhibited 20- to >100-fold increases in Tb³⁺ affinity. We concurrently designed a genetically encodable, "turn-off" fluorescent sensor of tyrosine nitration. In this sensor, tryptophan sensitizes Tb³⁺ emission via a FRET-like mechanism; if tyrosine is nitrated, however, it can both displace Glu16 to enhance binding and quench the energy transfer to completely abrogate emission by the bound metal, with nitration causing a 35-fold decrease in terbium luminescence versus unmodified peptide.

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