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. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

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. A majority of mammalian TACAs are derived from what are referred to as mucin-type O-linked glycans on cell-surface proteins and are linked to the protein backbone through the hydroxyl group of either serine or threonine residues. A small group of structural studies that have compared mono- and oligosaccharides attached to each of these residues have shown that there are distinct differences in conformational preferences assumed by glycans attached to either "unmethylated" serine or ß-methylated threonine. This suggests that the linkage point of antigenic glycans will affect their presentation to the immune system as well as to various carbohydrate binding molecules (e.g., lectins). This short review, followed by our hypothesis, will examine this possibility and extend the concept to the presentation of glycans on surfaces and in assay systems where recognition of glycans by proteins and other binding partners can be defined by different attachment points that allow for a range of conformational presentations.

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

We have previously prepared gold nanoparticles (AuNPs) bearing the Thomsen-Friedenreich antigen disaccharide (TFag), 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. (R)-(+)-α-Lipoic acid (LA), a naturally occurring disulfide-containing organic acid that is used as a cofactor in many enzymatic reactions, has been used as a linker to conjugate various molecules to AuNPs through its branched di-thiol system to enhance nanoparticle stability. We sought to use a similar system to increase nanoparticle stability that was devoid of the chiral center in (R)-(+)-α-lipoic acid. Isolipoic acid, an isomer of LA, where the exocyclic pentanoic acid chain is shifted by one carbon on the dithiolane ring to produce an achiral acid, was thought to act similarly as LA without the risk of any contaminating (L)-(-) isomer. We synthesized AuNPs with ligands of both serine and threonine glycoamino acids bearing the TFag linked to isolipoic acid and examined their stability under various conditions. In addition, these particles were shown to bind to Galectin-3 and inhibit the interaction of Galectin-3 with a protein displaying copies of the TFag. These agents should prove useful in the design of potential antimetastatic therapeutics that would benefit from achiral linkers that are geometrically linear and achiral.

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. However, many of these therapies are very selective for specific patient populations and hence the search for improved therapeutics and refinement of techniques for delivery are ongoing and fervent research areas. Most of these agents are directed at protein/peptide epitopes, but glycans-based targets are gaining in popularity, and a handful of approved immunotherapies owe their activity to oligosaccharide targets. In addition, nanotechnology and nanoparticle-derived systems can help improve the delivery of these agents to specific organs and cell types based on tumor-selective approaches. This review will first outline some of the historical beginnings of this research area and subsequently concentrate on the last 5 years of work. Based on the progress in therapeutic design, predictions can be made as to what the future holds for increasing the percentage of positive patient outcomes for optimized systems.

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 ß-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. The glycosylated sequence contained the Thomsen-Friedenreich disaccharide, a pan-carcinoma, Tumor-Associated Carbohydrate Antigen (TACA), which has been a traditional target for antitumor vaccine design. These motifs were prepared with a cathepsin B protease cleavage site (Gly-Phe-Leu-Gly), loaded on the B13G-coated particles and these constructs were examined for Dectin-1 binding, APC processing and presentation in a model in vitro system and for immune responses in mice. We showed that these particles elicit strong in vivo immune responses through the production of both high-titer antibodies and priming of antigen-recognizing T-cells. Further examination showed that a favorable antitumor balance of expressed cytokines was generated, with limited expression of immunosuppressive Il-10. This system is modular in that any range of antigens can be conjugated to our particles and efficiently delivered to APCs expressing Dectin-1.

Synthetic ligands for PreQ1 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 PreQ1 riboswitch aptamers. These molecules bind specifically and reversibly to the aptamers with high affinity and induce a conformational change. Furthermore, the ligands modulate riboswitch activity through transcriptional termination despite no obvious chemical similarity to the cognate ligand. X-ray crystallographic studies reveal that the ligands share a binding site with the cognate ligand but make different contacts. Finally, alteration of the chemical structure of the ligand causes changes in the mode of RNA binding and affects regulatory function. Thus, target- and structure-based approaches can be used to identify and understand the mechanism of synthetic ligands that bind to and regulate complex, folded RNAs.

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. Glycan microarray analysis showed an intriguing binding pattern where the antiserum showed near complete specificity for MUC4 TR glycopeptides and peptides, relative to all components on the array. Tissue staining also showed distinct tumor specificity to pancreatic tumor tissue in relation to normal pancreatic tissue, with a preference for more aggressive tumor foci. Based on this data, we produced a monoclonal antibody whose binding and reactivity profile was similar to that of the polyclonal serum, with the added benefit of being more specific for the N-terminal glycosylated peptide domain. This epitope represents a novel immunogen to potentially develop diagnostic antibodies or immunotherapies against various MUC4-positive cancers.

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 (as-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 N-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. Unlike many previously published substitutions to APF, mono-4'-fluorination of the GalNAc residue did not affect the activity, whereas fluoro-derivatives of the galactose 4″-position or both 4' and 4″ hydroxyls showed a reduced potency relative to the monosubstituted GalNAc derivative. A fourth compound where the 4″ position of galactose was deoxygenated showed a lower potency than the parent and monosubstituted compounds. These results suggest that specific substitutions in the sugar moieties in the APF can be tolerated, and the glycomimetic design of APF analogues can include fluorine in the GalNAc sugar of the disaccharide.

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. Through a series of >150 microarray experiments, we assessed binding properties of a variety of plant lectins, monoclonal antibodies, and endogenous lectins. VVL, HPA, and SBA were all found to bind tightly to GalNAc-Tyr, and several Tn binding antibodies and blood group A antibodies were found to cross-react with GalNAc-Tyr. Thus, detection of GalNAc-Tyr modified proteins is an important consideration when analyzing results from these reagents. Additionally, we evaluated potential recognition by two mammalian lectins, human (hMGL) and murine (mMGL-2) macrophage galactose type C-type lectins. Both hMGL and mMGL-2 bound tightly to GalNAc-Tyr determinants. The apparent Kd values (∼1-40 nM) were on par with some of the best known ligands for MGL, such as the Tn antigen. hMGL also bound the natural beta-amyloid peptide containing a GalNAc-Tyr epitope. STD NMR experiments provided structural insights into the molecular basis of recognition. Finally, GalNAc-Tyr was selectively captured by mMGL-2 positive dendritic cells. These results provide the first evidence that GalNAc-Tyr modified proteins and/or peptides may be ligands for hMGL and mMGL-2 and offer unique structures for the design of MGL targeting agents.

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. Binding to Ubc9 was confirmed through the use of ligand-detected nuclear magnetic resonance, and inhibition of sumoylation in a reconstituted enzymatic cascade was found to occur with an IC50 of 75 µM. This work establishes the utility of the SMM approach for identifying inhibitors of E2 enzymes, targets with few known small-molecule modulators.

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. We purchased and analyzed 166 such tautomer pairs and triplets by 1H and 13C NMR to determine whether the CACTVS transforms accurately predicted what is the same "stuff in the bottle". Essentially all prototropic transforms with examples in the AMS were confirmed. Some of the ring-chain transforms were found to be too "aggressive", i.e. to equate structures with one another that were different compounds.

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 ß-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. We have previously prepared gold nanoparticles (AuNP) coated with the TFag in various presentations as potential anti-adhesive therapeutic tools or antitumor vaccine platforms. Here we describe the synthesis of TFag-glycoamino acid conjugates attached to gold nanoparticles through a combined alkane/PEG linker, where the TFag was attached to either a serine or threonine amino acid. Particles were fully characterized by a host of biophysical techniques, and along with a control particle carrying hydroxyl-terminated linker units, were evaluated in both Gal-3 positive and negative cell lines. We show that the particles bearing the saccharides selectively inhibited tumor cell growth of the Gal-3 positive cells significantly more than the Gal-3 negative cells. In addition, the threonine-attached TF particles were more potent than the serine-attached constructs. These results support the use of AuNP as antitumor therapeutic platforms, targeted against cell lines that express specific lectins that interact with TFag.

Endothelial integrin α3ß1 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. Here, using Western blot and LC tandem mass spectrometry analyses of pull-downs utilizing TF-Ag loaded gold nanoparticles, we identified Gal-3, endothelial integrin α3ß1, Src kinase, as well as 5 additional molecules mapping onto focal adhesion pathway as parts of the macromolecular complexes formed at the endothelial cell membranes downstream of TF-Ag/Gal-3 interactions. In a modified parallel flow chamber assay, inhibiting α3ß1 integrin greatly reduced the strength of tumor/endothelial cell interactions without affecting the initial cancer cell adhesion. Further, the macromolecular complex induced by TF-Ag/Gal-3/α3ß1 interactions activates Src kinase, p38, and ERK1/2, pathways in endothelial cells in a time- and α3ß1-dependent manner. We conclude that, following the initial metastatic cell attachment to endothelial cells mediated by TF-Ag/Gal-3 interactions, endothelial integrin α3ß1 stabilizes tumor/endothelial cell adhesion and induces the formation of macromolecular signaling complex activating several major signaling pathways in endothelial cells.

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; Galß1-3GalNAcα), with a selectivity rivaling or exceeding that of lectins and antibodies. To understand the basis for TFα recognition by one such VLR (VLRB.aGPA.23), we measured thermodynamic parameters for the binding interaction and determined the structure of the VLRB.aGPA.23-TFα complex to 2.2 Å resolution. In the structure, four tryptophan residues form a tight hydrophobic cage encasing the TFα disaccharide that completely excludes buried water molecules. This cage together with hydrogen bonding of sugar hydroxyls to polar side chains explains the exquisite selectivity of VLRB.aGPA.23. The topology of the glycan-binding site of VLRB.aGPA.23 differs markedly from those of lectins or antibodies, which typically consist of long, convex grooves for accommodating the oligosaccharide. Instead, the TFα disaccharide is sandwiched between a variable loop and the concave surface of the VLR formed by the ß-strands of the leucine-rich repeat modules. Longer oligosaccharides are predicted to extend perpendicularly across the ß-strands, requiring them to bend to match the concavity of the VLR solenoid.

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. High resolution structural information on mucins is lacking due to their complexity, in particular their large size and the many variants of O-linked glycans produced in their biosynthesis. This review discusses the structures of glycopeptides that contain "mucin-type" glycosylation, and concentrates primarily on data obtained by NMR spectroscopy. The effect of the glycan on the peptide backbone, the features that have shown to be common to this type of glycosylation and the differences of glycosylation at serine and threonine residues are the major topics of discussion.

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. However, this SAR model could not explain the change in activity associated with minor alterations in the peptide sequence. Presented is computational analysis of 14 APF derivatives to identify structural trends from which a more detailed SAR is obtained. The APF activity is found to be dictated by the close interplay between carbohydrate-peptide and peptide-peptide interactions. The former involves hydrogen bond and hydrophobic interactions, and the latter is dominated by hydrophobic interactions. The highly flexible hydrophobic peptide adopts collapsed conformations separated by low energy barriers. APF activity correlates with hydrophobic clustering associated with amino acids 4A, 6V, and 8V. Peptide conformations are highly sensitive to single point mutations, which explain the experimental trends. The presented SAR will act as a guide for lead optimization of more potent APF analogues of potential therapeutic utility.

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. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2-5 nm in diameter), performed using membranes of mammalian cells stably expressing human A1, A2A, and A3ARs, showed that the desired selectivity was retained with K(i) values (nanomolar) of A3AR agonist 21b and A2AAR antagonists 24 and 26a of 14 (A3), 34 (A2A), and 69 (A2A), respectively. The corresponding monomers displayed K i values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A3 and A2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.

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. We report a comparison of the reactivity of several known precursor gold nanoparticles (citrate-stabilized, pentanethiol-stabilized, tetraoctylammonium bromide-stabilized, and 4-dimethylaminopyridine-stabilized) to several biologically relevant ligands, including amino acids, peptides, and carbohydrates. We found that citrate-stabilized and 4-dimethylaminopyridine-stabilized gold nanoparticles have broader reactivities than the other precursors studied. Citrate-stabilized gold nanoparticles are more versatile precursors because they can be prepared in a wide range of sizes and are very stable. The hydrophobic pentane-stabilized gold nanoparticles made them "inert" toward highly water-soluble ligands. Tetraoctylammonium bromide-stabilized gold nanoparticles exhibited selective reactivity, especially for small, unhindered and amphiphilic ligands. Depending on the desired ligand and size of AuNPs, a judicious selection of the available precursors can be made for use in place-exchange reactions. In preparing water-soluble AuNPs with biologically relevant ligands, the nature of the incoming ligand and the size of the AuNP should be taken into account in order to choose the most suitable place-exchange procedure.