Affinity determination of ricinus communis agglutinin ligands identified from combinatorial O- and S-,N-glycopeptide libraries

Two combinatorial glycopeptide libraries were synthesized on solid support via the "split-and-mix" method combined with the ladder synthesis strategy. The O-glycopeptide library contained Gal(beta1-O)Thr, whereas the S-,N-glycopeptide library contained both Gal(beta1-S)Cys and Gal(beta1-N)Asn. In this model study, the two libraries were screened against the fluorescently labeled lectin Ricinus communis agglutinin (RCA120). The screening results showed that both O- and S- or S-,N-glycopeptides were recognized by the lectin with similar amino acid recognition patterns. Surface plasmon resonance interaction studies demonstrated that both the selected S- or S-,N-glycopeptide hits and the O-glycopeptides bound to the lectin with a similar affinity. Structure 19, containing two glycosylated cysteine residues, bound to the receptor with the highest affinity (KA = 3.81 x 10(4) M(-1)), which is comparable to N-acetyllactosamine. Competition assays, in which some selected glycopeptides and methyl beta-d-galactopyranoside competed for the binding site of immobilized RCA120, showed that the glycopeptide-lectin interaction was carbohydrate-specific. Incubation of the O- and S-,N-glycopeptides with beta-galactosidase demonstrated the complete stability of S-,N-glycopeptides toward enzymatic degradation, whereas O-glycopeptides were not completely stable.     

Synthesis and Screening of Stereochemically Diverse Combinatorial Libraries of Peptide Tertiary Amides

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Highlights? A convenient synthesis of libraries of conformationally-constrained N-substituted oligoalanines ? A screen of such a library provided much higher affinity hits than a library of peptoids lacking c-alpha substitution ? Analysis of the isolated protein ligands demonstrated that the stereochemistry of the chiral centers was important ? These libraries should be a useful source of high affinity protein ligands     

Sialoside analogue arrays for rapid identification of high affinity siglec ligands

The siglec family of sialic acid binding proteins participates in diverse cell surface biology that includes regulation of immune cell signaling and the interaction of neuronal cells with glial cells. The weak intrinsic affinity of the natural sialoside ligands has hampered the development of synthetic ligand based probes needed to elucidate their roles in siglec function. In this report, we describe a glycan microarray comprising a library of 9-acyl-substituted sialic acids incorporated into sialosides containing the Neu5Acalpha2-3Gal and Neu5Acalpha-6Gal linkages commonly recognized by the siglecs. The array is demonstrated to exhibit utility for detecting 9-acyl substituents that increase the affinity of siglecs for their ligands. Substituents that increase affinity are anticipated to be useful for the design of high affinity ligand based probes of siglec function.     

Chemically defined sialoside scaffolds for investigation of multivalent interactions with sialic acid binding proteins

Four glycodendrons and a glycocluster were synthesized from carbohydrate building blocks to form paucivalent (di- to tetravalent) structures of controlled scaffold architectures. Enzymatic sialylation of the functionalized cluster and dendrons, terminated in lactose residues, generated a library of paucivalent synthetic sialosides displaying sialic acids with different dispositions. These newly constructed bioactive sialic acid-based structures were differentially recognized by sialoadhesin, a mammalian macrophage sialic acid binding protein. The binding of the sialosides to sialoadhesin was evaluated by an enzyme-linked immunosorbant assay to investigate the complementarity of scaffold structure and binding to sialoadhesin. Modulating the interaction between sialoadhesin and its sialic acid ligands has important implications in immunobiology.     

Steering Siglec–Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry

Sialic acid sugars that terminate cell‐surface glycans form the ligands for the sialic acid binding immunoglobulin‐like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross‐reactivity and led to the discovery of three selective Siglec‐5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec‐3 dampened the activation of Siglec‐3⁺ monocytic cells through the NF‐κB and IRF pathways.     

Towards targeted MRI: new MRI contrast agents for sialic acid detection

The detection of sialic acid in living systems is of importance for the diagnosis of several types of malignancy. We have designed and synthesized two new lanthanide ion ligands (L1 and L2) that are capable of molecular recognition of sialic acid residues. The basic structure of these ligands consists of a DTPA-bisamide (DTPA, diethylenetriamine pentaacetic acid) whose amide moieties each bear both a boronic function for interaction with the diol groups in the side chain of sialic acid, and a functional group that is positively charged at physiologic pH values and is designed to interact with the carboxylate anion of sialic acid. The relaxometric properties of the Gd3+ complexes of these two ligands were evaluated. The relaxivity of the GdL1 complex has a significant second-sphere contribution at pH values above the pKa of its phenylboronic acid moiety. The interaction of the Gd3+ complexes of L1 and L2 with each of several saccharides was investigated by means of a competitive fluorescent assay. The results show that both complexes recognize sialic acid with good selectivity in the presence of other sugars. The adduct formed by GdL2 with sialic acid has the higher conditional formation constant (50.43+/-4.61 M(-1) at pH 7.4). The ability of such complexes to recognize sialic acid was confirmed by the results of a study on the interaction of corresponding radiolabeled complexes (153SmL1 and 153SmL2) with C6 glioma rat cells. 153SmL2 in particular is retained on the cell surface in significant amounts.     

Alkyne Activation in the Diversity Oriented Synthesis of sp2-Rich Scaffolds: A Biased Library Approach for Targeting Polynucleotides (DNA/RNA)

Polynucleotides, DNA and RNA (mRNA and non-coding RNAs) are critically involved in the molecular pathways of disease. Small molecule binding interactions with polynucleotides can modify functional polynucleotide topologies and/or their interactions with proteins. Current approaches to library design (lead-like or fragment-like libraries) are based on protein-ligand interactions and often include careful consideration of the 3-dimensional orientation of binding motifs and exclude π-rich compounds (polyfused aromatics) to avoid off-target R/DNA interactions. In contrast to proteins, where π,π-interactions are weak, polynucleotides can form strong π,π-interactions with suitable π-rich ligands. To assist in designing a polynucleotide-biased library, a scaffold-divergent synthesis approach to polyfused aromatic scaffolds has been undertaken. Initial screening hits that form moderately stable polynucleotide-ligand-protein ternary complexes can be further optimized through judicious incorporation of substituents on the scaffold to increase protein-ligand interactions. An example of this approach is given for topoisomerase-1 (TOP1), generating a novel TOP1 inhibitory chemotype.     

New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes

Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg 114, and by the hydrophobic contacts made with Tyr 92 and Met 140. Molecular models were also produced for the binding of BM1 and BM3 (glutathione-substituted) to GSTI. In both cases the biomimetic dye forms multiple hydrophobic interactions with the enzyme through binding to a surface pocket. While the glutathioine moiety of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better purification results achieved with BM1.     

Tumor Carbohydrate Associated Antigen Analogs as Potential Binders for Siglec-7

We investigated two recently synthesized and characterized sialyl derivatives, bearing the Neu5Ac-α-(2-6)-Gal epitope, as promising binders for Siglec-7, an inhibitory Siglec mainly found on natural killer cells. A variety of sialoglycan structures can be recognized by Siglec-7 with implications in the modulation of immune responses. Notably, overexpression of sialylated glycans recognized by Siglec-7 can be associated with the progression of several tumors, including melanoma and renal cell carcinoma. NOE-based NMR techniques, including Saturation Transfer Difference and transferred-NOESY NMR, together with molecular docking and dynamic simulations were combined to shed light on the molecular basis of Siglec-7 recognition of two conformationally constrained Sialyl-Tn antigen analogs. We, therefore, identify the ligands epitope mapping and their conformational features and propose 3D models accurately describing the protein-ligand complexes. We found that the binding site of Siglec-7 can accommodate both synthetic analogs, with the sialic acid mainly involved in the interaction. Moreover, the flexibility of Siglec-7 loops allows a preferred accommodation of the more rigid compound bearing a biphenyl moiety at position 9 of the sialic acid that contributed to the interaction to a large extent. Our findings provided insights for developing potential novel high affinity ligands for Siglec-7 to hinder tumor evasion.     

Structure-based screening as applied to human FABP4: a highly efficient alternative to HTS for hit generation

The time-limiting step in HTS often is the development of an appropriate assay. In addition, hits from HTS fairly often turn out to be false positives and generally display unfavorable properties for further development. Here we describe an alternative process for hit generation, applied to the human adipocyte fatty acid binding protein FABP4. A small molecular ligand for FABP4 that blocks the binding of endogenous ligands may be developed into a drug for the treatment of type-2 diabetes. Using NMR spectroscopy, we screened FABP4 for low-affinity binders in a diversity library consisting of small soluble scaffolds, which yielded 52 initial hits in total. The potencies of these hits were ranked, and crystal structures of FABP4 complexes for two of the hits were obtained. The structural data were subsequently used to direct similarity searches for available analogues, as well as chemical synthesis of 12 novel analogues. In this way, a series of three selective FABP4 ligands with attractive pharmacochemical profiles and potencies of 10 microM or better was obtained.     

Point of attachment and sequence of immobilized peptide-acridine conjugates control affinity for nucleic acids

Screening of combinatorial libraries by spatial arraying strategies requires library members to be solid-phase immobilized. However, for nucleic acid ligands that bind via intercalation, immobilization may inhibit binding if the tethering functionality is present at the edge of the heterocyle that approaches the duplex during the binding reaction. We report here a method for immobilizing peptide-acridine conjugates (PACs) via either their C- or their N-terminus, corresponding to functionalization at either the 4- or the 9-position of acridine, respectively, and for assaying the nucleic acid binding properties of the resulting resins. We find that both the amino acid sequence of the PAC as well as its point of attachment to the solid support are important in determining affinity for duplex nucleic acids. These results have implications for the design of future on-bead and microarray-based selections and in understanding the nucleic acid binding of functionalized intercalators.     

Solid-phase synthesis of tetrasubstituted pyrazoles, novel ligands for the estrogen receptor

Most ligands for the estrogen receptor (ER) are not well suited for synthesis by combinatorial means, because their construction involves a series of carbon-carbon bond forming reactions that are not uniformly high yielding. In previous work directed to overcoming this limitation, we surveyed various phenol-substituted five-membered heterocycles, hoping to find a system that would afford both high ER binding affinity and whose synthesis could be adapted to solid-phase methods (Fink et al. Chem. Biol. 1999, 6, 206-219.) In this report, we have developed a reliable and efficient solid-phase method to prepare the best of these heterocycles, the tetrasubstituted pyrazoles, and we have used this methodology to produce small, discrete libraries of these novel ER ligands. We used a combination of FT-IR and nanoprobe (1)H NMR-MAS to characterize intermediates leading up to the final pyrazole products directly on the bead. We also developed a scavenging resin, which enabled us to obtain products free from inorganic contaminants. We prepared a 12-member test library, and then a 96-member library, and in both cases we determined product purity and ER binding affinity of all of the library members. Several interesting binding affinity patterns have emerged from these studies, and they have provided us with new directions for further exploration, which has led to pyrazoles having high affinity and potency as agonists and antagonists toward the ER alpha subtype.     

标签谷胱甘肽S-转移酶的二价亲和标记试剂的制备与应用

设计合成融合表达标签谷胱甘肽S-转移酶(GST)的二价亲和标记试剂,用于功能化磁珠后位点选择性固定化标签GST,为磁分离筛选配体混合物库提供固定化融合靶蛋白的候选方案。 为减少疏水配体在标签GST活性位点的结合,需同时占据标签GST双活性中心内疏水结合位点并发生共价修饰的二价亲和标记试剂。以双苯环为疏水定位基、溴乙酰基为巯基修饰基团、羧基为连接官能团得单价标记试剂,以二乙基三胺为连接臂将单价标记试剂与连接臂两端伯胺连接得标签GST的对称二价亲和标记试剂,再以线性三胺连接臂中间的氨基与羧基磁珠偶联得功能化磁珠。 表征目标化合物对标签GST的标记动力学、结合比;功能化磁珠对标签GST的不可逆固定化动力学和固载容量,及将磁珠表面二价亲和标记试剂转变成还原型谷胱甘肽(GSH)加合物后对标签GST可逆固定化的效果;以碱性磷酸酶及疏水荧光配体为模型考察磁珠固定化标签GST后的非特异结合。 目标化合物对标签GST半抑制浓度为(22±0.2) μmol/L,其与GSH的饱和加合物半抑制浓度为(0.41±0.06) μmol/L,二者与标签GST二聚体结合比接近1：1。 功能化磁珠对标签GST不可逆及可逆固定化的容量均接近25 mg/g磁珠。 偶联GST的磁珠对蛋白非特异吸附很弱,再进一步用单价亲和标记试剂和GSH加合物封闭固定化标签GST剩余的活性位点后对疏水小分子也无显著结合。 结果表明,所设计二价亲和标记试剂功能化磁珠适合用于标签GST及其融合表达蛋白的位点选择性固定化。     

A novel strategy for in vitro selection of peptide-drug conjugates

The chemical diversity of peptide and protein libraries generated from biological display systems is typically confined to the 20 naturally occurring amino acids. Here, we have developed a general strategy to introduce non-natural side chains into mRNA-display libraries via specific chemical derivatization. We constructed a mRNA-display library containing 3 x 10(12) different peptides bearing a pendant penicillin moiety in a fixed position. In vitro selection using this hybrid peptide-drug library resulted in novel inhibitors of the Staphylococcus aureus penicillin binding protein 2a (PBP2a). This strategy resulted in a penicillin-peptide conjugate that has at least 100-fold higher activity than the parent penicillin itself. Our approach provides a convenient way to enhance the efficacy of known drugs and facilitates the discovery of powerful new hybrid ligands with functionalities beyond those provided by the 20 naturally occurring residues.     

NMR structure elucidation of cyclic sialyl 6-sulfo Lewis x, a biologically dormant form of L-selectin ligand

The structure of cyclic sialyl 6-sulfo Lewis x, a new biologically dormant form of L-selectin ligand, was determined unambiguously by the accurate NMR analysis to have the lactamized ^{[5.], (a), (b), [2.], (a) and (b)}B conformation of its sialic acid. For the NMR structure elucidation were used various informations, such as chemical shifts values, appearance of amide proton, and NOE.     

A new class of arylpiperazine derivatives: the library synthesis on SynPhase lanterns and biological evaluation on serotonin 5-HT(1A) and 5-HT(2A) receptors

An efficient solid-supported method for the synthesis of a new class of arylpiperazine derivatives containing amino acid residues has been developed. A 72-membered library was synthesized on SynPhase Lanterns functionalized by a BAL linker. A one-pot cleavage/cyclization step of aspartic and glutamic acid derivatives yielded succinimide- and pyroglutamyl-containing ligands (chemsets 9 and 10). The library representatives under study showed different levels of affinity for 5-HT(1A) and 5-HT(2A) receptors (estimated K(i) = 24-4000 and 1-2130 nM, respectively). Several dual 5-HT(1A)/5-HT(2A) ligands were found, of which two (9(3,3) and 9(3,5)) displayed high 5-HT(2A) affinity comparable to that of the reference drug ritanserin. A set of individual fragment contributions for the prediction of 5-HT(1A) and 5-HT(2A) affinity of all the library members were defined on the basis of the Free-Wilson analysis of 26 compounds. An alkylarylpiperazine fragment had essentially the same impact on the affinity for both receptors, whereas different terminal amide fragments were preferred by 5-HT(1A) (chemset 17, R(2) = adamantyl) and 5-HT(2A) (chemset 9, R(2) = norborn-2-ylmethyl) binding sites.     

Exploration of structure--activity relationships among foldamer ligands for a specific protein binding site via parallel and split-and-mix library synthesis

We describe the use of parallel and split-and-mix library synthesis strategies for exploration of structure-activity relationships among peptidic foldamer ligands for the BH3-recognition cleft of the anti-apoptotic protein Bcl-xL. This effort began with a chimeric (alpha/beta+alpha)-peptide oligomer (composed of an alpha/beta-peptide segment and an alpha-peptide segment) that we previously identified to bind tightly to the target cleft on Bcl-xL. The side chains that interact with Bcl-xL were varied in a 1000-member one-bead-one-compound library. Fluorescence polarization (FP) screening identified four new analogues with binding affinities similar to that of the lead compound but no analogues with enhanced affinity. These results suggested that significant improvements in affinity were unlikely in this series. We then used library synthesis to examine backbone variations in the C-terminal alpha-peptide segment of the lead compound. These studies provided an opportunity for direct comparison of parallel and split-and-mix synthesis formats for foldamer libraries with respect to synthetic variability and assay sensitivity. We found that compounds from both the parallel and one-bead-one-compound libraries could be reliably screened in a competition FP assay without purification of library members. Our findings should facilitate the use of combinatorial library synthesis for exploration of foldamers as inhibitors of protein-protein interactions.     

Use of catalyst pharmacophore models for screening of large combinatorial libraries

Using a data set comprised of literature compounds and structure-activity data for cyclin dependent kinase 2, several pharmacophore hypotheses were generated using Catalyst and evaluated using several criteria. The two best were used in retrospective searches of 10 three-dimensional databases containing over 1,000,000 proprietary compounds. The results were then analyzed for the efficiency with which the hypotheses performed in the areas of compound prioritization, library prioritization, and library design. First as a test of their compound prioritization capabilities, the pharmacophore models were used to search combinatorial libraries that were known to contain CDK active compounds to see if the pharmacophore models could selectively choose the active compounds over the inactive compounds. Second as a test of their utility in library design again the pharmacophore models were used to search the active combinatorial libraries to see if the key synthons were over represented in the hits from the pharmacophore searches. Finally as a test of their ability to prioritize combinatorial libraries, several inactive libraries were searched in addition to the active libraries in order to see if the active libraries produced significantly more hits than the inactive libraries. For this study the pharmacophore models showed potential in all three areas. For compound prioritization, one of the models selected active compounds at a rate nearly 11 times that of random compound selection though in other cases models missed the active compounds entirely. For library design, most of the key fragments were over represented in the hits from at least one of the searches though again some key fragments were missed. Finally, for library prioritization, the two active libraries both produced a significant number of hits with both pharmacophore models, whereas none of the eight inactive libraries produced a significant number of hits for both models.     

Generation of a dynamic combinatorial library using sialic acid aldolase and in situ screening against wheat germ agglutinin

This paper describes the generation of a dynamic combinatorial library of sialic acid analogues using sialic acid aldolase. Addition of wheat germ agglutinin to the equilibrating libraries results in selective amplification of one or more members.     

Novel affinity ligands for chromatography using combinatorial chemistry

Spatially addressable combinatorial libraries were synthesized by solution phase chemistry and screened for binding to human serum albumin. Members of arylidene diamide libraries were among the best hits found, having submicromolar binding affinities. The results were analyzed by the frequency with which particular substituents appeared among the most potent compounds. After immobilization of the ligands either through the oxazolone or the amine substituent, characterization by surface plasmon resonance showed that ibuprofen affected the binding kinetics, but phenylbutazone did not. It is therefore likely that these compounds bind to Site 2 in sub domain IIIA of human serum albumin (HSA).