KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets.

The effect of a methyl group on structure and function: Serine vs. threonine glycosylation and phosphorylation.

A variety of glycan structures cover the surface of all cells and are involved in myriad biological processes, including but not limited to, cell adhesion and communication, protein quality control, signal transduction and metabolism, while also being intimately involved in innate and adaptive immune functions. Immune surveillance and responses to foreign carbohydrate antigens, such as capsular polysaccharides on bacteria and surface protein glycosylation of viruses, are the basis of microbial clearance, and most antimicrobial vaccines target these structures. In addition, aberrant glycans on tumors called Tumor-Associated Carbohydrate Antigens (TACAs) elicit immune responses to cancer, and TACAs have been used in the design of many antitumor vaccine constructs.

Isolipoic acid-linked gold nanoparticles bearing the thomsen friedenreich tumor-associated carbohydrate antigen: Stability and studies.

We have previously prepared gold nanoparticles (AuNPs) bearing the Thomsen-Friedenreich antigen disaccharide (TF), a pan-carcinoma, Tumor-Associated Carbohydrate Antigen (TACA), as tools for various assays and biological applications. Conjugation to AuNPs typically involves the use of thiols due to the affinity of sulfur for the gold surface of the nanoparticle. While a use of a single thiol-containing ligand bound to the gold surface is standard practice, several studies have shown that ligands bearing multiple thiols can enhance the strength of the conjugation in a nearly linear fashion.

Glycoconjugate Nanoparticle-Based Systems in Cancer Immunotherapy: Novel Designs and Recent Updates.

For many years, cell-surface glycans (in particular, Tumor-Associated Carbohydrate Antigens, TACAs) have been the target of both passive and active anticancer immunotherapeutic design. Recent advances in immunotherapy as a treatment for a variety of malignancies has revolutionized anti-tumor treatment regimens. Checkpoint inhibitors, Chimeric Antigen Receptor T-cells, Oncolytic virus therapy, monoclonal antibodies and vaccines have been developed and many approvals have led to remarkable outcomes in a subset of patients.

A Stable Gold Nanoparticle-Based Vaccine for the Targeted Delivery of Tumor-Associated Glycopeptide Antigens.

We have developed a novel antigen delivery system based on polysaccharide-coated gold nanoparticles (AuNPs) targeted to antigen presenting cells (APCs) expressing Dectin-1. AuNPs were synthesized de-novo using yeast-derived β-1,3-glucans (B13G) as the reductant and passivating agent in a microwave-catalyzed procedure yielding highly uniform and serum-stable particles. These were further functionalized with both a peptide and a specific glycosylated form from the tandem repeat sequence of mucin 4 (MUC4), a glycoprotein overexpressed in pancreatic tumors.

A Tumor-Selective Monoclonal Antibody from Immunization with a Tumor-Associated Mucin Glycopeptide.

We have previously studied the generation of immune responses after vaccination with tumor-associated carbohydrate antigen (TACA)-containing glycopeptides from the tandem repeat (TR) sequence of MUC4, an aberrantly expressed mucin in pancreatic adenocarcinomas. A specific lead antigen from that study containing the Thomsen-Friedenreich TACA disaccharide facilitated the pursuit of a monoclonal antibody to this synthetic hapten. Initial evaluation of polyclonal antiserum resulting from immunization with a KLH conjugate of this glycopeptide into rabbits showed high titer antibodies by ELISA assays, and selective immunoreactivity with MUC4 cells by western blot and flow cytometry techniques.

Synthetic ligands for PreQ riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure.

Riboswitches are naturally occurring RNA aptamers that regulate gene expression by binding to specific small molecules. Riboswitches control the expression of essential bacterial genes and are important models for RNA-small molecule recognition. Here, we report the discovery of a class of synthetic small molecules that bind to PreQ riboswitch aptamers.

Glycoamino Acid Analogues of the Thomsen-Friedenreich Tumor-Associated Carbohydrate Antigen: Synthesis and Evaluation of Novel Antiproliferative Factor Glycopeptides.

Glycoamino acid analogues of the Thomsen-Friedenreich antigen disaccharide, where the 4' and 4″ hydroxyl groups were substituted with fluorine or hydrogen, were synthesized and incorporated into the asialylated antiproliferative factor (-APF), a biologically active form of APF, a glycopeptide found in the urine of patients with interstitial cystitis. Various strategies were employed to incorporate the fluorine atom at the 4-positions of either the galactose or -acetylgalactosamine unit of the disaccharide antigen, based on stereochemistry and reactivity. These glycopeptides were evaluated in antiproliferative assays on both primary normal bladder epithelial cells and T24 bladder carcinoma cells.

GalNAc-Tyrosine Is a Ligand of Plant Lectins, Antibodies, and Human and Murine Macrophage Galactose-Type Lectins.

In 2011, a new type of protein O-glycosylation was discovered in which N-acetylgalactosamine is attached to the side chain of tyrosine (GalNAc-Tyr). While present on dozens of proteins, the biological roles of GalNAc-Tyr are unknown. To gain insight into this new type of modification, we synthesized a group of GalNAc-Tyr glycopeptides, constructed microarrays, and evaluated potential recognition of GalNAc-Tyr by a series of glycan-binding proteins.

A Small-Molecule Microarray Approach for the Identification of E2 Enzyme Inhibitors in Ubiquitin-Like Conjugation Pathways.

E2 enzymes in ubiquitin-like conjugation pathways are important, highly challenging pharmacological targets, and despite significant efforts, few noncovalent modulators have been discovered. Small-molecule microarray (SMM)-based screening was employed to identify an inhibitor of the "undruggable" small ubiquitin-like modifier (SUMO) E2 enzyme Ubc9. The inhibitor, a degradation product from a commercial screening collection, was chemically synthesized and evaluated in biochemical, mechanistic, and structure-activity relationship studies.

Experimental and Chemoinformatics Study of Tautomerism in a Database of Commercially Available Screening Samples.

We investigated how many cases of the same chemical sold as different products (at possibly different prices) occurred in a prototypical large aggregated database and simultaneously tested the tautomerism definitions in the chemoinformatics toolkit CACTVS. We applied the standard CACTVS tautomeric transforms plus a set of recently developed ring-chain transforms to the Aldrich Market Select (AMS) database of 6 million screening samples and building blocks. In 30 000 cases, two or more AMS products were found to be just different tautomeric forms of the same compound.

Synthesis and cell-selective antitumor properties of amino acid conjugated tumor-associated carbohydrate antigen-coated gold nanoparticles.

The Thomsen Friedenreich antigen (TFag) disaccharide is a tumor-associated carbohydrate antigen (TACA) found primarily on carcinoma cells and rarely expressed in normal tissue. The TFag has been shown to interact with Galectin-3 (Gal-3), one in a family of β-galactoside binding proteins. Galectins have a variety of cellular functions, and Gal-3 has been shown to be the sole galectin with anti-apoptotic activity.

Endothelial integrin α3β1 stabilizes carbohydrate-mediated tumor/endothelial cell adhesion and induces macromolecular signaling complex formation at the endothelial cell membrane.

Blood borne metastatic tumor cell adhesion to endothelial cells constitutes a critical rate-limiting step in hematogenous cancer metastasis. Interactions between cancer associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag) and endothelium-expressed galectin-3 (Gal-3) have been identified as the leading molecular mechanism initiating tumor/endothelial cell adhesion in several types of cancer. However, it is unknown how these rather weak and transient carbohydrate/lectin mediated interactions are stabilized.

Recognition of the Thomsen-Friedenreich pancarcinoma carbohydrate antigen by a lamprey variable lymphocyte receptor.

Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs were recently shown to recognize glycans, such as the tumor-associated Thomsen-Friedenreich antigen (TFα; Galβ1-3GalNAcα), with a selectivity rivaling or exceeding that of lectins and antibodies. To understand the basis for TFα recognition by one such VLR (VLRB.

Mucin-type glycopeptide structure in solution: past, present, and future.

Mucins are very high molecular weight glycoproteins that form a "mucus" barrier at the surface of epithelial cells. They are heavily glycosylated with O-linked glycans that are involved in myriad cellular functions, including protection from external changes in pH, ion flux and reactive oxygen species. Aberrations in mucin expression and their glycan constitution have been associated with many disease states including gastritis, pulmonary disorders and cancer.

Conformational determinants of the activity of antiproliferative factor glycopeptide.

The antiproliferative factor (APF) involved in interstitial cystitis is a glycosylated nonapeptide (TVPAAVVVA) containing a sialylated core 1 α-O-disaccharide linked to the N-terminal threonine. The chemical structure of APF was deduced using spectroscopic techniques and confirmed using total synthesis. The synthetic APF provided a platform to study amino acid modifications and their effect on APF activity, based on which a structure-activity relationship (SAR) for APF activity was previously proposed.

Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles.

Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand.

A survey of place-exchange reaction for the preparation of water-soluble gold nanoparticles.

Water-soluble gold nanoparticles (AuNPs) have gained considerable attention because they offer a myriad of potential applications, especially in the fields of biology and medicine. One method to prepare such gold nanoparticles is through the well-known Murray place-exchange reaction. In this method, precursor gold nanoparticles, bearing labile ligands and with very good size distribution, are synthesized first, and then reacted with a large excess of the desired ligand.

Sugar-binding proteins from fish: selection of high affinity "lambodies" that recognize biomedically relevant glycans.

Glycan-binding proteins are important for a wide variety of basic research and clinical applications, but proteins with high affinity and selectivity for carbohydrates are difficult to obtain. Here we describe a facile and cost-effective strategy to generate monoclonal lamprey antibodies, called lambodies, that target glycan determinants. We screened a library of yeast surface-displayed (YSD) lamprey variable lymphocyte receptors (VLR) for clones that can selectively bind various biomedically important glycotopes.

Divergent behavior of glycosylated threonine and serine derivatives in solid phase peptide synthesis.

Solid phase peptide coupling of glycosylated threonine derivatives was systematically evaluated. In contrast to glycosylated serine derivatives which are highly prone to epimerization, glycosylated threonine derivatives produce only negligible amounts of epimerization. Under forcing conditions, glycosylated threonine analogs undergo β-elimination, rather than epimerization.

Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines.

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant".

Specific fluorine labeling of the HyHEL10 antibody affects antigen binding and dynamics.

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding.

Enhanced epimerization of glycosylated amino acids during solid-phase peptide synthesis.

Glycopeptides are extremely useful for basic research and clinical applications, but access to structurally defined glycopeptides is limited by the difficulties in synthesizing this class of compounds. In this study, we demonstrate that many common peptide coupling conditions used to prepare O-linked glycopeptides result in substantial amounts of epimerization at the α position. In fact, epimerization resulted in up to 80% of the non-natural epimer, indicating that it can be the major product in some reactions.