Discovery of a Celecoxib Binding Site on Prostaglandin E Synthase (PTGES) with a Cleavable Chelation-Assisted Biotin Probe.

The coxibs are a subset of nonsteroidal anti-inflammatory drugs (NSAIDs) that primarily target cyclooxygenase-2 (COX-2) to inhibit prostaglandin signaling and reduce inflammation. However, mechanisms to inhibit other members of the prostaglandin signaling pathway may improve selectivity and reduce off-target toxicity. Here, we report a novel binding site for celecoxib on prostaglandin E synthase (PTGES), which is an enzyme downstream of COX-2 in the prostaglandin signaling pathway, using a cleavable chelation-assisted biotin probe 6. Evaluation of the multifunctional probe 6 revealed significantly improved tagging efficiencies attributable to the embedded picolyl functional group. Application of the probe 6 within the small molecule interactome mapping by photoaffinity labeling (SIM-PAL) platform using photo-celecoxib as a reporter for celecoxib identified PTGES and other membrane proteins in the top eight enriched proteins from A549 cells. Four binding sites to photo-celecoxib were mapped by the probe 6, including a binding site with PTGES. The binding interaction with PTGES was validated by competitive displacement with celecoxib and licofelone, which is a known PTGES inhibitor, and was used to generate a structural model of the interaction. The identification of photo-celecoxib interactions with membrane proteins, including the direct binding site on the membrane protein PTGES, will inform further functional followup and the design of new selective inhibitors of the prostaglandin signaling pathway.

The Metabolic Chemical Reporter 6-Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O-GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O-Glucose.

Metabolic chemical reporters of glycosylation in combination with bioorthogonal reactions have been known for two decades and have been used by many different research laboratories for the identification and visualization of glycoconjugates. More recently, however, they have begun to see utility for the investigation of cellular metabolism and the tolerance of biosynthetic enzymes and glycosyltransferases to different sugars. Here, we take this concept one step further by using the metabolic chemical reporter 6-azido-6-deoxy-glucose (6AzGlc). We show that treatment of mammalian cells with the per- O-acetylated version of 6AzGlc results in robust labeling of a variety of proteins. Notably, the pattern of this labeling was consistent with O-GlcNAc modifications, suggesting that the enzyme O-GlcNAc transferase is quite promiscuous for its donor sugar substrates. To confirm this possibility, we show that 6AzGlc-treatment results in the labeling of known O-GlcNAcylated proteins, that the UDP-6AzGlc donor sugar is indeed produced in living cells, and that recombinant OGT will accept UDP-6AzGlc as a substrate in vitro. Finally, we use proteomics to first identify several bona fide 6AzGlc-modifications in mammalian cells and then an endogenous O-glucose modification on host cell factor. These results support the conclusion that OGT can endogenously modify proteins with both N-acetyl-glucosamine and glucose, raising the possibility that intracellular O-glucose modification may be a widespread modification under certain conditions or in particular tissues.

Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs.

Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.

Convergent in situ assembly of injectable lipogel for enzymatically controlled and targeted delivery of hydrophilic molecules.

Applications of liposomes are limited due to their rapid blood clearance and non-specific biodistribution. Surface modification of liposomes could overcome these disadvantages. However, direct coating of liposome surface may cause disruption of liposomes. Herein we present a "top-down" method to coat liposomes in situ with tumor (CD44 receptor) targeting polymer, hyaluronan (HA), by taking advantages of "click" type chemistries and enzymatic degradation. Liposomes entrapped within HA gel were stable without leaking of small cargo molecules from the interior of the liposomes. This injectable liposome-in-hydrogel (lipogel) drug delivery system can achieve sequential two-step release: (1) liposomes release from lipogel after HA degradation; (2) small molecules release from liposomes after the liposomes disruption (either before or after cellular uptake). Similarly to HA coating, this strategy could be used for in situ "top-down" modification of liposomes with other targeting biopolymers. Additionally, it provides the possibility to deliver different types of molecules from two compartments of the lipogel, i.e. large biomacromolecules from the exterior of liposomes and small hydrophilic molecules from the interior of liposomes, locally and systemically.