Chemical Biology Discussion Group Year-End Symposium

Abstracts

Molecular Target Identification and Proposed Mechanistic Hypothesis for the Antidepressant and Neurorestorative Agent Tianeptine
Madalee M. Gassaway, MPhil¹

The pharmacological treatments for depression and other related disorders available today continue to suffer from major problems, including low response rate, slow onset of therapeutic effects, loss of efficacy over time, and serious side effects. Consequently the need to discover new therapeutic approaches that address these disorders is urgent. Interestingly, the atypical antidepressant tianeptine already meets in part these clinical goals. Though three decades of basic and clinical investigations have characterized the physiological effects, the molecular target of tianeptine and its mechanism of action remain unsolved. Herein, we report the characterization of tianeptine as a μ-opioid receptor (MOR) agonist. Using radioligand binding and cell-based functional assays, including bioluminescence resonance energy transfer (BRET)-based assays for G-protein activation and cAMP accumulation, we identified tianeptine as an efficacious MOR agonist (Ki-Human of 383±183 nM and EC50-Human of 194±70 nM for G-protein activation). Tianeptine was also a full δ-opioid receptor (DOR) agonist, although with much lower potency (EC50-Human of 37.4±11.2 μM for G-protein activation). In contrast, tianeptine was inactive at the κ-opioid receptor (KOR). On the basis of these pharmacological data, we propose that activation of MOR (or dual activation of MOR and DOR) could be the initial molecular event responsible for triggering many of the known acute and chronic effects of this agent, including its antidepressant and anxiolytic actions through modulation of the glutamatergic system, making MOR a “new” player in the battle against depression.
 
Coauthors: Marie-Laure Rives, PhD^2,3, Andrew C. Kruegel, MPhil¹, Jonathan A. Javitch, MD, PhD^2,3,4, and Dalibor Sames, PhD¹
1 Columbia University Department of Chemistry, New York, New York, United States
2 Columbia University Department of Psychiatry, New York, New York, United States
3 New York State Psychiatric Institute Division of Molecular Therapeutics, New York, New York, United States
4 Columbia University Department of Pharmacology, New York, New York, United States
 

O-GlcNAcylation Stabilizes Nod2, an Innate Immune Receptor Involved in Crohn’s Disease
Ching-Wen Hou, MS¹

Nucleotide-binding oligomerization domain2 (Nod2) is an intracellular receptor that can sense bacterial components, such as, Muramyl dipeptide (MDP). MDP is a peptidoglycan fragment from bacterial cell wall that can activate NF-kB, a transcriptional factor that induces the production of inflammatory molecules such as cytokines. In 2001, a genetic linkage analysis revealed three major mutations in Nod2 were linked to Crohn’s disease. Crohn’s-associated Nod2 variants have a loss of function phenotype where they display a decreased ability to turn on NF-kB. Recently, using the classic cycloheximide stabilization assay, we showed that Crohn’s-associated Nod2 variants have lower half-life compared with wild type Nod2 in cells. To further characterize this protein, the objective of this study is to determine if Nod2 is post translationally modified. GlcNAcylation is one of post translational modifications in which O-GlcNAc transferase (OGT) transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to selected serine and threonine residues of a target protein. As GlcNAc is a major component of peptidoglycan of bacterial cell wall and a large amount of GlcNAc is released from bacterial cell wall during cell wall remodeling, we hypothesized that Nod2 could be O-GlcNAcylated. Preliminary data show that wild type Nod2 and Nod2 variants are O-GlcNAcylated by using O-GlcNAc antibody (CTD110.6). In addition, increasing the O-GlcNAcylation level can increase the half- life of Nod2 in wild type Nod2 and affect Nod2 mediated NF-kB pathway .In future experiments, we will determine which residue of Nod2 is GlcNAcylated and investigate if this modification affects Nod2 mediated NF-kB pathway in Crohn’s-associated Nod2 variants.
 
Coauthors: Natasha E. Zachara, PhD², Catherine L. Grimes, PhD¹
1 University of Delaware, Newark, Delaware, United States
2 The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
 

Searching for Selective Reactions on Complex Molecular Scaffolds
Scott J. Miller, PhD, Department of Chemistry, Yale University

Natural products have provided perennial inspiration for the development of synthetic methods, and enzymes have provided an analogous platform for the conception of new catalysts. This lecture will recount an interplay of experiments stimulated by these two major classes of naturally occurring substances. Specifically, the discovery and use of peptides as catalysts for a variety of asymmetric bond formations will be presented. Likewise, applications of these catalysts to the synthesis and selective modification of complex molecules, including biologically active natural products, will be described. A particular emphasis will be placed on reactions that present unusual stereochemical challenges. An analysis of catalyst types that may be brought to bear on complex molecular environments will also be included.
 

Design and Synthesis of Cryptophane Biosensors for the Specific Detection of Cancer Cell Lines Using 129Xe NMR
Brittany A. Riggle, University of Pennsylvania, Department of Chemistry, Philadelphia, Pennsylvania, United States

Magnetic resonance imaging (MRI) is a versatile and commonly employed technique for the diagnosis of disease. However, because MRI typically utilizes endogenous proton signals, it suffers from poor contrast. As such, approximately half of all MRI procedures in humans are performed using gadolinium chelates, which effectively enhance contrast but do not readily identify biomolecular targets at sub-mM concentrations. Non-endogenous, hyperpolarized (HP) nuclei, such as 129Xe offer potential advantages in sensitivity and molecular specificity. Our lab has developed xenon biosensors exploiting the favorable association between 129Xe and water-soluble cryptophane cages. We have developed the highest xenon affinity cryptophanes measured to date; with association constants up to 42,000 M-1 at 298 K. By employing hyperpolarized chemical exchange saturation transfer (hyperCEST) we are able to reach picomolar detection limits for our cryptophane biosensors. Xenon biosensors also exploit the considerable NMR chemical shift sensitivity of 129Xe bound inside the cryptophane. This affords the possibility of designing a multitude of cryptophane conjugates that identify different cancer biomarkers in a biological specimen using 129Xe NMR spectroscopy. We have previously designed cryptophane conjugates that specifically target folate, integrin, and carbonic anhydrase receptors as well as matrix metalloproteinases that are over-expressed in many cancers. Here we describe a pH-responsive biosensor, which was designed to preferentially label cancer cells. Additionally, we are exploring the use of cryptophane as a biophysical probe for protein-protein interactions and conformational changes.
 
Coauthors: Yanfei Wang, Ivan J. Dmochowski, PhD
University of Pennsylvania, Department of Chemistry, Philadelphia, Pennsylvania, United States
 

A Radical SAM Enzyme that Crosslinks Unactivated Amino Acids
Kelsey R. Schramma, MA, Princeton University, Princeton, NJ, United States

Nature is very adept at transforming polypeptides into bioactive molecules employing a wide variety of strategies. These strategies, often seen in the biosynthetic pathways of bioactive secondary metabolites, are frequently utilized in the generation of macrocyclic peptides. One such cyclic peptide, streptide, was recently discovered in streptococcal bacteria and has been identified as a quorum sensing regulated secondary metabolite. Our lab has determined the structure of streptide, and identified the presence of an unprecedented carbon-carbon crosslink between the β-carbon of a lysine residue and the indole-C7 of a tryptophan side chain. Here we show that the formation of this novel carbon-carbon bond is catalyzed by a radical SAM enzyme, which we have termed StrB. We find that StrB contains two [4Fe-4S] clusters, one that activates SAM to generate the 5’-deoxyadenosyl radical, which initiates catalysis, and a second cluster, which is essential for catalytic activity. We further show that StrB installs the unusual Lys-to-Trp crosslink in a single step, thus providing a new route for peptide cyclization.  A mechanism for this unusual reaction is proposed.
 
Coauthors: Leah B. Bushin, BA, and Mohammad R. Seyedsayamdost, PhD
Princeton University, Princeton, NJ, United States
 

Application of New Technologies to Profile Substrates for Protein Methyltransferases
Chamara Senevirathne, PhD, Memorial Sloan-Kettering Cancer Center, New York, New York, United States

Protein Methyltransferases (PMTs) catalyzed methylation play a critical role in disease formation. Development of methods to identify methylation event in normal and diseased states is critical in order to fully characterize cell biology and treat many diseases including cancer. However, the current approaches are incapable of unambiguous profiling of the role of PMTs-catalyzed methylation event. Hence, new approaches are needed to explicitly characterize PMTs activities and substrates in cells, which will lead to understand the mode of action of methylation. Recently our lab has developed new biochemical tool, a Bioorthoganal Profiling of Protein Methylation (BPPM) methodology to unambiguously characterize the methylation events and targets in cells. In the current study, we developed a new method for substrate profiling of individual PMTs using BPPM coupled protein microarray technology. One of key findings demonstrates that the system is compatible with cell lysetes. This means that methyltransferases in their native state, which is important to maintain their activity in normal or disease state. We tested variety of engineered enzyme including PRMT1, PRMT2, G9a, SYMD3, and EZH2. On the other hand, we have developed another method on fluorous based separation for targets of methyltransferases. In this application fluorous tagged small molecule used to identify the methylation sites on peptides. The method will have great advantages over commonly used biotin-avidin based approaches. These methods thus allows us to map the methylation sites of many biologically-relevant targets and thus access the unprecedented accuracy to understand the downstream functions of these events.
 
Coauthor: Minkui Luo, PhD, Memorial Sloan-Kettering Cancer Center, New York, New York, United States
 

Controlling Sulfuryl-Transfer In Vivo One Compound at a Time
Ting Wang¹

Sulfonation is a reversible modification that regulates the binding of endogenous and xenobiotic small-molecules to receptors, controls their half-lives and is intimately linked to human disease and drug efficacy. In humans, these reactions are catalyzed by a small (13 member) family of broad-specificity enzymes, the cytosolic sulfotransferases (SULTs). Here, molecular principles of SULT and nuclear-receptor ligand-specificity are used to create, for the first time, a design strategy that is capable of preventing the sulfation of a single compound in vivo without inhibiting SULTs or significantly altering the receptor-binding functions of the compound. Unlike classical inhibition approaches, this new strategy prevents sulfonation without inhibiting SULTs and thus maintains their homeostatic functions. The strategy is demonstrated for the nuclear-receptor family in an ex-vivo structure-activity study based on the scaffold of raloxifene (Evista®) - an FDA-approved nuclear-receptor agonist – and is shown to dramatically enhance the efficacy of apomorphine (an FDA-approved dopamine-receptor agonist) in zebrafish. The results indicate that sulfuryl-transfer and its attendant biology can now be controlled in vivo on a compound-by-compound basis. These concepts are expected to lead to new highly specific in vivo probes of sulfuryl-transfer biology, improvements in drug design and substantial enhancements in the efficacy of numerous FDA approved drugs.
 
Coauthors: Ian Cook¹, Lei Feng³, Hua Wang², Felix Kopp², Peng Wu², Florence Marlow³ and Thomas Leyh¹
1 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, United States
2 Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, United States
3 Department of Developmental & Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, United States