Exploration of bromodomain ligand-linker conjugation sites for efficient CBP/p300 heterobifunctional degrader activity.

Synthesis of proteolysis targeting chimeras (PROTACs) involves conjugation of an E3 ligase binding ligand to a ligand targeting a protein of interest via a rigid or flexible chemical linker. The choice of linker conjugation site on these ligands can be informed by structural analysis of ligand-target binding modes, the feasibility of synthetic procedures to access specific sites, and computational modeling of predicted ternary complex formations. Small molecules that target bromodomains - epigenetic readers of lysine acetylation - typically offer several potential options for linker conjugation sites. Here we describe how varying the linker attachment site (exit vector) on a CBP/p300 bromodomain ligand along with linker length affects PROTAC degradation activity and ternary complex formation. Using kinetic live cell assays of endogenous CBP and p300 protein abundance and bead-based proximity assays for ternary complexes, we describe the structure-activity relationships of a diverse library of CBP/p300 degraders (dCBPs).

Synthesis and Photothermal Effects of Intracellular Aggregating Nanodrugs Targeting Nasopharyngeal Carcinoma.

Chemotherapy for the treatment of nasopharyngeal carcinoma (NPC) is usually associated with many side effects; therefore, its treatment options have not yet been completely resolved. Improving distribution to the targeted tumor region and enhancing the cellular uptake of drugs can efficiently alleviate the above adverse medical effects. Near-infrared (NIR) laser light-mediated photothermal therapy (PTT) and photodynamic therapy (PDT) are promising strategies for cancer treatment. In the present study, we developed an efficient multifunctional nanocluster with enhanced targeting and aggregation efficiency for PTT and PDT that is composed of a biocompatible folic acid (FA), indocyanine green (ICG) and 2-cyanobenzothiazole (CBT)-functionalized peptide labeled with an aldehyde sodium alginate-modified magnetic iron oxide nanoparticle (ASA-MNP)-based nanocarrier. FA can bind to folate receptors on cancer cell membranes to enhance nanocluster uptake. CBT-modified peptide can react with glutathione (GSH), which is typically present at higher levels in cancer cells, to form intracellular aggregates and increase the local concentration of the nanodrug. In in vitro studies, these nanodrugs displayed the desired uptake capacity by NPC cells and the ability to suppress the growth of cancer cells under laser irradiation. Animal studies validated that these nanodrugs are safe and nontoxic, efficiently accumulate in NPC tumor sites following injection via the caudal vein, and shows superior inhibition of tumor growth in a tumor-bearing mouse model upon near-infrared laser irradiation. The results indicate the potential application of the multifunctional nanoparticles (NPs), which can be used as a new method for the treatment of folate receptor-positive NPC.

Enabling In Vivo Photocatalytic Activation of Rapid Bioorthogonal Chemistry by Repurposing Silicon-Rhodamine Fluorophores as Cytocompatible Far-Red Photocatalysts.

Chromophores that absorb in the tissue-penetrant far-red/near-infrared window have long served as photocatalysts to generate singlet oxygen for photodynamic therapy. However, the cytotoxicity and side reactions associated with singlet oxygen sensitization have posed a problem for using long-wavelength photocatalysis to initiate other types of chemical reactions in biological environments. Herein, silicon-Rhodamine compounds (SiRs) are described as photocatalysts for inducing rapid bioorthogonal chemistry using 660 nm light through the oxidation of a dihydrotetrazine to a tetrazine in the presence of trans-cyclooctene dienophiles. SiRs have been commonly used as fluorophores for bioimaging but have not been applied to catalyze chemical reactions. A series of SiR derivatives were evaluated, and the Janelia Fluor-SiR dyes were found to be especially effective in catalyzing photooxidation (typically 3%). A dihydrotetrazine/tetrazine pair is described that displays high stability in both oxidation states. A protein that was site-selectively modified by trans-cyclooctene was quantitatively conjugated upon exposure to 660 nm light and a dihydrotetrazine. By contrast, a previously described methylene blue catalyst was found to rapidly degrade the protein. SiR-red light photocatalysis was used to cross-link hyaluronic acid derivatives functionalized by dihydrotetrazine and trans-cyclooctenes, enabling 3D culture of human prostate cancer cells. Photoinducible hydrogel formation could also be carried out in live mice through subcutaneous injection of a Cy7-labeled hydrogel precursor solution, followed by brief irradiation to produce a stable hydrogel. This cytocompatible method for using red light photocatalysis to activate bioorthogonal chemistry is anticipated to find broad applications where spatiotemporal control is needed in biological environments.

Targeted degradation of the enhancer lysine acetyltransferases CBP and p300.

The enhancer factors CREB-binding protein (CBP) and p300 (also known as KAT3A and KAT3B) maintain gene expression programs through lysine acetylation of chromatin and transcriptional regulators and by scaffolding functions mediated by several protein-protein interaction domains. Small molecule inhibitors that target some of these domains have been developed; however, they cannot completely ablate p300/CBP function in cells. Here we describe a chemical degrader of p300/CBP, dCBP-1. Leveraging structures of ligand-bound p300/CBP domains, we use in silico modeling of ternary complex formation with the E3 ubiquitin ligase cereblon to enable degrader design. dCBP-1 is exceptionally potent at killing multiple myeloma cells and can abolish the enhancer that drives MYC oncogene expression. As an efficient degrader of this unique class of acetyltransferases, dCBP-1 is a useful tool alongside domain inhibitors for dissecting the mechanism by which these factors coordinate enhancer activity in normal and diseased cells.

Improving Tumor-to-Background Contrast through Hydrophilic Tetrazines: The Construction of 18 F-Labeled PET Agents Targeting Nonsmall Cell Lung Carcinoma.

Bioorthogonal reactions have been widely used in the biomedical field. 18 F-TCO/Tetrazine ligation is the most reactive radiolabelled inverse electron demand Diels-Alder reaction, but its application had been limited due to modest contrast ratios of the resulting conjugates. Herein, we describe the use of hydrophilic tetrazines to improve tumor-to-background contrast of neurotensin receptor targeted PET agents. PET agents were constructed using a rapid Diels-Alder reaction of the radiolabeled trans-cyclooctene (18 F-sTCO) with neurotensin (NT) conjugates of a 3,6-diaryltetrazine, 3-methyl-6-aryltetrazine, and a derivative of 3,6-di(2-hydroxyethyl)tetrazine. Although cell binding assays demonstrated all agents have comparable binding affinity, the conjugate derived from 3,6-di(2-hydroxyethyl)tetrazine demonstrated the highest tumor to muscle contrast, followed by conjugates of the 3-methyl-6-aryltetrazine and 3,6-diaryltetrazine.

Hydrophilic 18F-labeled trans-5-oxocene (oxoTCO) for efficient construction of PET agents with improved tumor-to-background ratios in neurotensin receptor (NTR) imaging.

An 18F-labeled trans-5-oxocene (oxoTCO) that is used to construct a PET probe for neurotensin receptor (NTR) imaging through tetrazine ligation is described here. PET probe construction proceeds with 70% RCY based on 18F-oxoTCO and is completed within seconds. The in vivo behaviour of the oxoTCO based PET probe was compared with those of analogous probes that were prepared from 18F-labeled s-TCO and d-TCO tracers. The hydrophilic 18F-oxoTCO probe showed a significantly higher tumor-to-background ratio while displaying comparable tumor uptake relative to the 18F-dTCO and 18F-sTCO derived probes.

The price of flexibility - a case study on septanoses as pyranose mimetics.

Seven-membered ring mimetics of mannose were studied as ligands for the mannose-specific bacterial lectin FimH, which plays an essential role in the first step of urinary tract infections (UTI). A competitive binding assay and isothermal titration calorimetry (ITC) experiments indicated an approximately ten-fold lower affinity for the seven-membered ring mannose mimetic 2-O-n-heptyl-1,6-anhydro-d-glycero-d-galactitol (7) compared to n-heptyl α-d-mannopyranoside (2), resulting exclusively from a loss of conformational entropy. Investigations by solution NMR, X-ray crystallography, and molecular modeling revealed that 7 establishes a superimposable H-bond network compared to mannoside 2, but at the price of a high entropic penalty due to the loss of its pronounced conformational flexibility. These results underscore the importance of having access to the complete thermodynamic profile of a molecular interaction to "rescue" ligands from entropic penalties with an otherwise perfect fit to the protein binding site.

Epitope-Resolved Detection of Peanut-Specific IgE Antibodies by Surface Plasmon Resonance Imaging.

Peanut allergy can be life-threatening and is mediated by allergen-specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes can identify specific interactions underlying the allergic response. Here, we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope-resolved detection. IgE antibodies were first captured by magnetic beads bearing IgE ϵ-chain-specific antibodies and then introduced into an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses were achieved by establishing a binding environment that minimized cross-reactivity while maximizing analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from patient serum samples (10 µL each) in a 45 min assay. Excellent correlation of Ara h2-specific IgE values was found between ImmunoCAP assays and the new SPRi method.

A practical and scalable synthesis of carbohydrate based oxepines.

An efficient, seven-step synthesis of carbohydrate based oxepines is reported using per-O-acetyl septanoses as key intermediates. The scope of the synthesis was evaluated by varying both the pyranose starting materials and protecting groups incorporated into the oxepine products. The practicality of the method make it amenable to scale up as demonstrated by the gram-scale synthesis of the d-glucose derived oxepine.

Synthesis of C-septanosides from pyranoses via vinyl addition and electrophilic cyclization.

A two-step synthesis of C-septanosides from pyranoses is reported. Vinyl addition to tetra-O-benzyl D-glucose, D-galactose, and D-mannose gave the corresponding allylic alcohols. Electrophilic cyclization followed by treatment with iodine gave iodomethyl C-septanosides suitable for substitution reactions. The cyclizations were diastereoselective, giving cis-1,2 configured C-septanosides. Selectivity is rationalized through a model for electrophilic additions that invokes the conformation of the allylic system. This new approach should be generally applicable to the synthesis of a variety of C-septanosides.