Selective Recognition of Carbohydrate Antigens by Germline Antibodies Isolated from AID Knockout Mice.

Germline antibodies, the initial set of antibodies produced by the immune system, are critical for host defense, and information about their binding properties can be useful for designing vaccines, understanding the origins of autoantibodies, and developing monoclonal antibodies. Numerous studies have found that germline antibodies are polyreactive with malleable, flexible binding pockets. While insightful, it remains unclear how broadly this model applies, as there are many families of antibodies that have not yet been studied. In addition, the methods used to obtain germline antibodies typically rely on assumptions and do not work well for many antibodies. Herein, we present a distinct approach for isolating germline antibodies that involves immunizing activation-induced cytidine deaminase (AID) knockout mice. This strategy amplifies antigen-specific B cells, but somatic hypermutation does not occur because AID is absent. Using synthetic haptens, glycoproteins, and whole cells, we obtained germline antibodies to an assortment of clinically important tumor-associated carbohydrate antigens, including Lewis Y, the Tn antigen, sialyl Lewis C, and Lewis X (CD15/SSEA-1). Through glycan microarray profiling and cell binding, we demonstrate that all but one of these germline antibodies had high selectivity for their glycan targets. Using molecular dynamics simulations, we provide insights into the structural basis of glycan recognition. The results have important implications for designing carbohydrate-based vaccines, developing anti-glycan monoclonal antibodies, and understanding antibody evolution within the immune system.

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. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to nonglycosylated amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization. The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and vaccine antigens.

Cortical bone thickness at common miniscrew implant placement sites.

INTRODUCTION: The purpose of this study was to assess age, sex, and regional differences in the cortical bone thickness of commonly used maxillary and mandibular miniscrew implant placement sites. METHODS: Cone-beam computed tomography images, taken at 0.39-mm voxel size, of 52 patients, including 26 adolescents (13 girls, ages 11-13; 13 boys, ages 14-16) and 26 adults (13 men and 13 women, ages 20-45), were evaluated. The cone-beam computed tomography data were imported into 3-dimensional software (version 10.5, Dolphin Imaging Systems, Chatsworth, Calif); standardized orientations were used to measure cortical bone thickness at 16 sites representing the following regions: 3 paramedian palate sites, 1 infrazygomatic crest site, 4 buccal interradicular sites of the mandible, and 4 buccal and 4 lingual interradicular sites in the maxilla. RESULTS: Multivariate analysis of variance (MANOVA) showed no significant differences in cortical bone thickness between the sexes. There were significant (P <0.05) differences between adolescents and adults, with adult cortices significantly thicker in all areas except the infrazygomatic crest, the mandibular buccal first molar-second molar site, and the posterior palate site. Cortical bone was thicker in the posterior than in the anterior mandibular sites. In the adults, interradicular bone in the maxillary first premolar-second premolar, and second premolar-first molar sites was thicker than bone at the lateral incisor-canine and first molar-second molar sites. Anterior paramedian palatal bone was significantly thicker than bone located more posteriorly. The mandibular buccal and infrazygomatic crest regions had the thickest cortical bone; differences between the maxillary buccal, the maxillary lingual, and the palatal regions were small. CONCLUSIONS: Maxillary and mandibular cortical bones at commonly used miniscrew implant placement sites are thicker in adults than in adolescents. There are also differences in cortical bone thickness between and within regions of the jaws that must be considered when placing miniscrew implants.

Effects of hapten density on the induced antibody repertoire.

Small peptides and oligosaccharides are important antigens for the development of vaccines and the production of monoclonal antibodies. Because of their small size, peptides and oligosaccharides are non-immunogenic on their own and typically must be conjugated to a larger carrier protein to elicit an immune response. Selection of a suitable carrier protein, conjugation method, and hapten density are critical for generating an optimal immune response. We used a glycan array to compare the repertoire of antibodies induced after immunizing with either low or high-density conjugates of the tumor-associated Tn antigen. At high hapten density, a broader range of antibodies was induced, and reactivity to the clustered Tn antigen was observed. In contrast, antibodies induced by the low-density conjugate had narrower reactivity and did not bind the clustered Tn antigen.

Development of antiproliferative phenylmaleimides that activate the unfolded protein response.

The current paper presents the synthesis and evaluation of a series of maleimides that were designed to inhibit the Cdc25 phosphatase by alkylation of catalytically essential cysteine residues. Although in HepB3 cell culture assays the analogues did exhibit antiproliferative IC(50) values ranging from sub-micromolar to greater than 100 microM, inhibition of Cdc25 through cysteine alkylation could not be demonstrated. It was also found that analysis using fluorescence activated cell sorting (FACS) following treatment with the most potent analogue (1t) did not provide data consistent with inhibition at one specific point in the cell cycle, as would be expected if Cdc25A were inhibited. Further studies with a subset of analogues resulted in a correlation of antiproliferative potencies with activation of the unfolded protein response (UPR). The UPR is a regulatory pathway that temporarily suspends protein production when misfolding of proteins occurs within the endoplastic reticulum (ER). In addition, ER chaperones that promote proper refolding become up-regulated. If cellular damage cannot be resolved by these mechanisms, then the UPR can initiate apoptosis. The current study indicates that these maleimide analogues lead to UPR activation, which is predictive of the selective antiproliferative activity of the series.

Management of chronic nonmalignant pain with nonsteroidal antiinflammatory drugs. Joint opinion statement of the Ambulatory Care, Cardiology, and Pain and Palliative Care Practice and Research Networks of the American College of Clinical Pharmacy.

Chronic nonmalignant pain is a major burden on the health care system in the United States. Frequently, nonsteroidal antiinflammatory drugs (NSAIDs) are used to assist in the management of various chronic pain syndromes. Although evidence is accumulating on the potential toxicities associated with NSAIDs, clear recommendations are lacking to guide the appropriate use of these drugs. Equivocal data, especially with respect to cardiovascular risk, further confuse a clear treatment pathway when assessing pharmacotherapy. Originally, cyclooxygenase selectivity appeared to be a determining factor in choosing an agent because of the presumed lack of effect on the cardiovascular and gastrointestinal renal systems. This theory, however, was recently dispelled. To provide guidance on the selection of an NSAID for various chronic pain syndromes, members of the Ambulatory Care, Cardiology, and Pain and Palliative Care Practice and Research Networks of the American College of Clinical Pharmacy evaluated evidence-based use of NSAIDs for frequently encountered pain syndromes, with special focus on the adverse effects of this class of agents.

Synthesis, biological activity, and crystal structure of potent nonnucleoside inhibitors of HIV-1 reverse transcriptase that retain activity against mutant forms of the enzyme.

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 microM, EC50 = 0.44 microM, and TC50 >/= 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants.

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.

Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth.

Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in solution and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have especially strong binding (apparent Kd  = 0.5-1.0 nm) to the tumor-associated Tn antigen. High-resolution crystal structures were determined for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive analysis of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the molecular recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. DATABASE: Structural data are available in the PDB under the accession numbers 5T50, 5T52, 5T55, 5T54, 5T5L, 5T5J, 5T5P, and 5T5O.

Molecular modeling calculations of HIV-1 reverse transcriptase nonnucleoside inhibitors: correlation of binding energy with biological activity for novel 2-aryl-substituted benzimidazole analogues.

The energies and physical descriptors for the binding of 20 novel 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole analogues (BPBIs) to HIV-1 reverse transcriptase (RT) have been determined using Monte Carlo (MC) simulations. The crystallographic structure of the lead compound, 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole, was used as a starting point to model the inhibitors in both the bound and the unbound states. The energy terms and physical descriptors obtained from the calculations were correlated with their respective experimental EC(50) values, resulting in an r(2) value of 0.70 and a root-mean-square deviation (rms) of 0.53 kcal/mol. The terms in the correlation include the change in total Coulombic energy and solvent-accessible surface area. Structural analysis of the data files from the BPBI calculations reveals that all of the analogues with good biological activity show the formation of a hydrogen bond between the ligand and the backbone nitrogen atom of lysine 103. By use of the structural results, two novel BPBI inhibitors have been designed and calculations have been carried out. The results show the formation of the desired hydrogen bonds, and the DeltaG(binding) values predict the compounds to be excellent RT inhibitors. Subsequent synthesis and biological activity testing of these analogues have shown the validity of the predictive calculations. If the BPBIs are modeled in a site constructed from the crystal coordinates of a member of another class of nonnucleoside inhibitors (the 4,5,6,7-tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepine-2(1H)-thione and -one (TIBO) compounds), the correlation with the same terms drops slightly, giving an r(2) value of 0.61 with an associated root-mean-square value of 0.53 kcal/mol. Conversely, if the TIBO compounds are modeled in a site constructed from the BPBI complex crystal coordinates, a correlation can be obtained using the drug-protein interaction energy and change in the total number of hydrogen bonds, giving an r(2) value of 0.63. These are the same descriptors that were used for the TIBO compounds modeled in their own sites, where the r(2) value was 0.72. These data suggest that it may be possible, in some cases, to design novel inhibitors utilizing structural data from related, but not identical, inhibitors.

Microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies.

Antigen arrays have become important tools for profiling complex mixtures of proteins such as serum antibodies. These arrays can be used to better understand immune responses, discover new biomarkers, and guide the development of vaccines. Nevertheless, they are not perfect and improved array designs would enhance the information derived from this technology. In this study, we describe and evaluate a strategy for varying antigen density on an array and then use the array to study binding of lectins, monoclonal antibodies, and serum antibodies. To vary density, neoglycoproteins containing differing amounts of carbohydrate were synthesized and used to make a carbohydrate microarray with variations in both structure and density. We demonstrate that this method provides variations in density on the array surface within a range that is relevant for biological recognition events. The array was used to evaluate density dependent binding properties of three lectins (Vicia villosa lectin B4, Helix pomatia agglutinin, and soybean agglutinin) and three monoclonal antibodies (HBTn-1, B1.1, and Bric111) that bind the tumor-associated Tn antigen. In addition, serum antibodies were profiled from 30 healthy donors. The results show that variations in antigen density are required to detect the full spectrum of antibodies that bind a particular antigen and can be used to reveal differences in antibody populations between individuals that are not detectable using a single antigen density.