Search

Changes in bending of the DNA helix axis caused by the introduction of conformationally locked nucleotide analogs into the center region of the palindromic Dickerson dodecamer, d[(CGCGAATTCGCG).sub.2], have been studied by NMR measurement of residual one-bond [sup.13]C-[sup.1]H dipolar couplings. Thymidine analogs, in which the deoxyribose was substituted by bicyclo[3.1.0]hexane, were incorporated in the T7, T8, and T7T8 positions. These nucleotide analogs restrict the ring pucker to the C2'-exo or 'north' conformation, instead of C2'-endo or 'south,' which dominates in regular B-form DNA. For all three oligomers, bending toward the major groove is found relative to the native molecule. The effects are additive with bending of 5 [+ or -] 1[degrees] per locked nucleotide. Measurement of the change in bending is more accurate than measurement of the bending angle itself and requires far fewer experimental data. conformational change | liquid crystal | nucleic acid

Approximately 1 million people in the United States suffer from interstitial cystitis, a chronic painful urinary bladder disorder characterized by thinning or ulceration of the bladder epithelial lining; its etiology is unknown. We have identified a glycosylated frizzled-related peptide inhibitor of cell proliferation that is secreted specifically by bladder epithelial cells from patients with this disorder. This antiproliferative factor (APF) profoundly inhibits bladder cell proliferation by means of regulation of cell adhesion protein and growth factor production. The structure of APF was deduced by using ion trap mass spectrometry (MS), enzymatic digestion, lectin affinity chromatography, and total synthesis, and confirmed by coelution of native and synthetic APF derivatives on microcapillary reversed-phase liquid chromatography ([micro]RPLC)/MS. APF was determined to be an acidic, heat-stable sialoglycopeptide whose peptide chain has 100% homology to the putative sixth transmembrane domain of frizzled 8. Both synthetic and native APF had identical biological activity in normal bladder epithelial cells and T24 bladder cancer cells. Northern blot analysis indicated binding of a probe containing the sequence for the frizzled 8 segment with mRNA extracted from cells of patients with interstitial cystitis but not controls. APF is therefore a frizzled-related peptide growth inhibitor shown to contain exclusively a trans-membrane segment of a frizzled protein and is a potential biomarker for interstitial cystitis. frizzled-related peptide | growth inhibitor | bladder epithelium

Two isomers of methanocarba (MC) thymidine are used to determine whether kinases and polymerases discriminate between their substartes on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono suggests that kinases prefer substrates that adopt the S sugar conformation.

Results demonstrate that alpha-fluro-dideoxynucleosideATP in its north conformational form is the potent inhibitor of HIV-1 reverse transcriptase whereas the south conformational beta-fluro-dideoxynucleosideATP has to be converted to the alpha form before it can react with the enzyme.

Issues concerning the NMR spectroscopic examination in solution of the structures of beta-peptides derived from acids are presented. In solution these homooligomers display helixes containing twelve and fourteen membered hydrogen-bonded rings, which are similar to their solid state conformations.

Research has been conducted on the adenosine deaminase (ADA). The study of the ADA binding efficiencies and synthetic adenosine analogue deamination kinetics is presented.

The pulse labeling experiment with barnase has been re-done and no stable folding intermediate found. A native-state hydrogen exchange experiment has been carried out with barnase with no detection of any partially unfolded intermediate state, not consistent with results from kinetic deuterium hydrogen exchange pulse labeling and protein engineering studies. Data indicate that more investigation of the nature of the intermediate of barnase is needed.

Cyclin-dependent kinase 5 (CDK-5) phosphorylates the tail portions near the carboxy terminal of high molecular weight neurofilament proteins. CDK-5 specifically phosphorylates the serine residue within particular amino acid sequence. Circular dichroism studies of the reaction between CDK-5 and a range of synthetic peptides containing specific sequences were undertaken to determine the structural basis for substrate recognition. Results reveal that the specificity of CDK-5 is dependent on the structural dynamics undergone by the substrate.

A study was conducted to examine the ability of 5'-triphosphates to inhibit HIV reverse transcriptase (RT) to analyze the preference of 3'-azido-3'-deoxythimidine (AZT) for the extreme (sub 3)E conformation. Conformational preferences were corroborated by X-ray analysis. Results indicated that the inhibition of RT only occurred with the conformotionally locked (sub 2)E (N)-methano-carba-AZT 5'-triphosphate.

Glycosylated analogues of the main neutralizing determinant of gp120 were prepared and their conformations studied by NMR and circular dichroism spectroscopies. RP135, a 24-residue peptide from the HIV-1 IIIB isolate, was glycosylated with N- and O-linked sugars. It was revealed that covalently linking a carbohydrate to a peptide has significant effects on local conformation. The design of ordered and biologically relevant conformations of gp120 could help in designing more effective immunogens for development of HIV-1 vaccines.

Recent research has indicated that short beta-peptides of trans-APC very frequently adopt a 12-helical folding pattern. This has relevance to other research which shows the formation of 14-helix patterns by short oligomers of beta-substituted beta-amino acids. It seems likely that changing torsional preferences about the C-alpha-C-beta bonds of individual residues can have a significant impact on beta-peptide secondary structure. Beta-peptides offer predictable residue-based conformational control, and may therefore be well suited to molecular design efforts.

Carbocyclic ketohexose nucleosides is represented by psicoplanocin A and psico-cyclopenetenyladenosine. Geometrical characteristics of neplanocin A and the ketohexose nucleosides psicofuranine and decoynine are fused in the two steady compounds. Cyclopentenone is used to prepare both compounds in racemic form. D-ribonolactone yields the cyclopentenone required for the synthesis. An insulated hydroxymethyl segment is linked to the ketone carboxyl of the cyclopentenone to initiate the creation of surrogate glycon moiety, through nucleophilic incorporation of ((benzoyloxy)methyl)lithium to produce an intermediate.

L-ascorbic and D-isoascorbic acids are the precursors to four 2-deoxy-erythro-1,4-lactones in the D- and L-series and the four corresponding 2-deoxy-threo-1,4-lactones, with myristic acid acyl groups at either primary or secondary alcohol functions. The importance of the orientation of the fatty acid side chain is illustrated by the inhibition of these compounds of the binding of (3H)phorbol-12,13-dibutyrate to protein kinase C.

The presence of a free hydroxyl group at the 5 prime-position does not induce 1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) degradation in the presence of alkali media. The sensitivity to alkali attack of the ribose portion of zebularine may be due to the electron-withdrawing characteristic of the hemiaminal carbon 1. Analysis of the decomposition products of zebularine reveals the formation of the carcinogenic 1,3-propane dialdehyde or malondialdehyde, which may exert effect on the activity of the compound in the body.

The conformations of three 2',3'-difluoro uridine nucleosides are studied in order to define the roles that the two vicinal fluorine atoms has played in the puckering preferences of the furanose ring. The studies have shown that both gauche and antiperiplanar effects are operative to maintain a pseudodiaxial arrangement of the C2' and C3' vicinal fluorine atoms.

Carbohydrates substituted at the anomeric position with nitric oxide-releasing diazeniumdiolate ions that are a promising new class of prodrug, are reported to be stable at physiological pH but readily hydrolyzed by glycosidases. They are introduced as a means of selectively generating bioactive nitric oxide in cell types that possess the appropriate enzymatic activity.

The formation of short, stable helices in aqueous solution by beta-amino acid hexamers has been studied. An R,R,R-2,5-diaminocyclohexanecarboxylic acid derivative with differential protection on the amino groups and a series of hexa-beta-peptides with varying proportions of cyclohexyl and acyclic beta-amino acid residues have been synthesized. NMR spectroscopy could only be used with the hexamer with a 2:1 cyclohexyl:acyclic ratio since the analysis of all-cyclohexyl hexamers is hampered by poor spectral resolution.

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., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Barchi and Strain.)

We have previously prepared gold nanoparticles (AuNPs) bearing the Thomsen-Friedenreich antigen disaccharide (TF ag ), 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 TF ag 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 TF ag . These agents should prove useful in the design of potential antimetastatic therapeutics that would benefit from achiral linkers that are geometrically linear and achiral., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mondal and Barchi.)

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., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Barchi.)

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. 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., Competing Interests: Competing Interests: The authors declare no competing interests. Additional Information: Supporting Information accompanies this text.

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 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.

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 K d 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.

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 IC 50 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.