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Protein kinases comprise one of the most important group of targets for drug discovery research today. Methods to identify novel kinase inhibitors by high-throughput screening have evolved rapidly in recent years. An important aspect is the availability of fluorescent probes that can be applied in a homogeneous, or mix-and-measure, assay format. Here, we illustrate the application of fluorescence read-out technologies for kinase targets in light of our own experiences in assay development and high-throughput screening.

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[structure: see text]. The cyclooctenol derivative 1 can be transformed into the nine-membered ring lactone 3, as well as the amino-containing carbocycles 4 and 5. The corresponding ketone 2 gives access to the conformationally locked azasugar 6.

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Replacement of the highly basic benzamidine moiety with moderate basic amino-bicycloaryl moieties in a series of thrombin inhibitors related to NAPAMP provided potent enzyme inhibition and significant improvements in membrane transport and oral bioavailability.

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The synthesis of three heparin analogues (i.e. compounds VI-VIII) having perphosphorylated thrombin binding domains (TBDs) is reported. These compounds were tested in vitro for their antithrombin III (ATIII)-mediated anti-Xa and antithrombin activities. Conjugates VI and VIII show a remarkable increase in antithrombin activity compared to the structurally related conjugates with persulfated TBDs (i.e. compounds IV and V), whereas compound VII displays a diminished activity.

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The synthesis of a novel antithrombotic consisting of a heparin pentasaccharide conjugated to the active site inhibitor N-(2-naphtalenesulfonyl)-glycyl-(D)-4-aminophenyl-alanyl-piperidin e (NAPAP) (i.e. compound I) is reported. This conjugate shows a unique pharmacological profile both in vitro and in vivo having direct anti-thrombin and ATIII-mediated anti-Xa activity. Furthermore, conjugate I has a prolonged in vivo half-life compared to NAPAP (1.5 h vs 9 min.).

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Replacement of the highly basic benzamidine moiety of NAPAP by the moderately basic 1-aminoisoquinoline moiety resulted in thrombin inhibitors with improved selectivity towards trypsin and enhanced Caco-2 cell permeability.

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The syntheses of several heparin-like glycoconjugates (i.e., 16a-f) containing identical AT III binding domains (ABD) and spacers but different thrombin binding domains (TBDs) are described. Biological activities of conjugates 16a-f indicate that the thrombin inhibitory activity is mainly determined by the charge density of the TBD moiety.

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The in vitro antithrombotic activity of synthetic glycoconjugates I and II, comprising a flexible polyethylene glycol type and a rigid polyglucose type spacer, respectively, are compared to heparin.

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The synthesis of a series of analogues of the monosaccharide alpha-glucosidase I inhibitor N-decyl-1-deoxynojirimycin (1) is described. With the incorporation of a single oxygen atom particularly at position seven in the N-decyl side chain, i.e. to give N-7-oxadecyl-dNM (4), the therapeutic ratio (alpha-glucosidase I inhibitory activity over toxicity in HepG2 cells) increases considerably. N-7-Oxadecyl-dNM inhibits purified porcine liver alpha-glucosidase I with an IC50 value of 0.28 microM. The position of the oxygen atom in the N-decyl side chain is of importance since N-3-oxadecyl-dNM is less active and, moreover, is toxic to HepG2 cells at 3 mM. Subsequently, the synthesis of a disaccharide inhibitor of alpha-glucosidase I is described. The aminodisaccharide ManNH2 alpha 1,2Glc (12) inhibits alpha-glucosidase I with an IC50 value of 15.7 microM. Two closely related monosaccharide derivatives of 12 did not inhibit the enzyme at low microM concentrations (no inhibition at 5 microM), showing the additional effect of binding of the aglycon fragment of the molecule to the active site of alpha-glucosidase I. Next, the N-alkyl-dNM derivatives were analysed for antiviral and immunomodulatory activity in-vitro. It is found that the most potent alpha-glucosidase I inhibitor from this study, N-7-oxadecyl-dNM (4) inhibits HIV-1 induced syncytia formation and lymphocyte proliferation in-vitro. Finally, compound 4 was also investigated in-vivo. N-7-Oxadecyl-dNM (4) reduced adjuvant-induced arthritis in rats making this compound a potential candidate for treating autoimmune diseases like rheumatoid arthritis.

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Computer modelling of the antithrombin III-heparin-thrombin complex inspired the synthesis of novel glycoconjugates, whose factor Xa and thrombin inhibitory activities can be adjusted in a rational way, leading to anticoagulants with unprecedented characteristics.

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In this paper, we report the synthesis of 'non-glycosamino' glycan analogues 5-10 of the antithrombin III binding pentasaccharide 1. Pentasaccharides 5-10 feature a pseudo-alternating EFGH tetrasaccharide sequence, that is, the disaccharide fragments EF and GH have the same substitution pattern. In the synthetic strategy applied for the synthesis of pentasaccharides 5-10, the properly protected EF disaccharide fragments 19 and 20 are obtained from their GH counterparts 17 and 18 by base-catalyzed epimerization. Series I, comprising pentasaccharides 5-7, has an invariable EFGH tetrasaccharide containing 2-O-sulfate 3-O-methyl uronic acid moieties. Series II, on the other hand, contains pentasaccharides 8-10 and has an invariable EFGH tetrasaccharide containing 2,3-di-O-methyl uronic acid moieties. Coupling disaccharides 17 with 25 and 18 with 26 exclusively afforded the alpha-coupled tetrasaccharides 27 and 28, respectively. Glycosylation of acceptor tetrasaccharides 29 and 30 with glucosyl donors 35, 36 and 39 provided, after deprotection and sulfation, the title-compounds 5-10. Biological data obtained with series I and II indicate that the in vivo half-life but not the intrinsic anti-Xa activity depends on the substitution pattern of the D-unit. In addition, the applicability of reversed UV capillary electrophoresis as an analytical tool to determine the purity of these 'non-glycosamino' glycans is demonstrated.

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The specific delivery of radioisotopes to a tumor at minimal radiation of normal tissue is the ultimate aim of radioimmunotherapy. In this respect a two-step pretargeting regimen generally leads to an improved tumor to normal tissue uptake ratio compared to direct administration of radioimmunoconjugates. In this paper, in vitro studies are described in which the specific hybridization of complementary DNA fragments is the recognition mechanism in a pretargeting regimen comprising tumor cell saturation with a monoclonal antibody (MoAb)-oligonucleotide conjugate, followed by administration of the radiolabeled complementary oligonucleotide. Complementary oligodeoxynucleotides (15-mers; melting temperature, 68 degrees C) were prepared on a DNA synthesizer. The 5'-end was derivatized with a functional group for labeling with iodine, and the 3'-end was substituted with an amino function suitable for conjugation to an antibody (or attachment of a biotin residue). Both terminal modifications ensure stability of the oligonucleotides against exonucleases because the unconjugated form is stable for 24 h and the conjugated form is stable for several days when incubated in human plasma at 37 degrees C. Antibody-DNA conjugates were prepared by introduction of sulfhydryl groups into the oligonucleotide, followed by conjugation to maleimide-substituted MoAbs. Typically, 3 oligonucleotides were conjugated to an IgG, and 4-6 were conjugated to an IgM with preservation of immunoreactivity. Histochemistry on fresh frozen sections of breast cancer tissue demonstrated qualitatively the specificity of our two-step procedure. In vitro experiments with human tumor cell lines and tumor-specific MoAbs showed that, after saturation with tumor-specific MoAb-DNA conjugates, quantitative hybridization of the tumor cell-bound oligonucleotides occurred at a 30-fold excess of the labeled complementary oligonucleotide: hybridization was complete after 30 min of incubation. No reaction was observed with an irrelevant MoAb-DNA conjugate. The oligonucleotide was neither taken up by tumor cells or endothelial cells nor hybridized to a significant extent with human genomic DNA. These data indicate the feasibility of this two-step approach in radioimmunotherapy.

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Recent advances in the molecular modelling of carbohydrates have brought this technique to a level comparable with that of protein and nucleic acid simulations. After a brief introduction to the techniques used in the computer simulation of carbohydrates and carbohydrate interactions, an overview of applications in the field of carbohydrate-related drug discovery is presented.

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Partially methylphosphonate-modified oligodeoxynucleotides were synthesized on solid-phase by employing the easily removable 2-(acetoxymethyl)benzoyl (AMB) group as base-protecting group. Although a rapid AMB deprotection can be accomplished in methanolic potassium carbonate, the lability of the methylphosphonate linkage towards potassium carbonate/methanol excludes the use of this deprotection reagent. Thus, saturated ammonia solution in methanol was investigated as an alternative reagent for AMB removal. It is demonstrated that the combination of the AMB protective group and ammonia/methanol as deprotection reagent significantly improves the synthesis of methylphosphonate-modified DNA fragments. A mild overnight treatment at room temperature is sufficient for complete removal of the AMB group, whereas deprotection of conventionally protected oligonucleotides requires much longer exposure to basic conditions at elevated temperatures.

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One of the major challenges in radioimmunotherapy is the specific delivery of radioisotopes to tumor cells while minimizing normal tissue radiation. In this respect, the application of two-step pretargeting schemes generally leads to more favorable tumor to normal tissue uptake ratios than direct administration of radioimmunoconjugates. In this study, we present the specific hybridization of complementary DNA fragments as a novel recognition mechanism in pretargeting. Briefly, our strategy involves first administration of antibody-DNA conjugate, followed by targeting with radiolabeled complementary DNA (antisense DNA). Complementary oligodeoxynucleotides (14-mers, Tm = 57 degrees C), in which part of the phosphodiesters has been replaced by methylphosphonates (to ensure stability against nucleases), were prepared on a DNA synthesizer. The oligonucleotides were further derivatized via a uridine moiety at their 5'-end in such a way that radiolabeling or conjugation with antibodies could be accomplished. Both a murine IgG (anti-hCG) and the human anti-tumor IgM 16.88 were conjugated with one to three oligonucleotides via the heterobifunctional cross-linker SMCC. Incubation of these immunoconjugates with the radiolabeled antisense DNA revealed specific hybridization with the antibody-linked oligonucleotides. Antigen binding studies performed with antigen-coated matrices showed that the immunoreactivity of the antibody-DNA conjugate is preserved. Moreover, it is demonstrated that the radiolabeled DNA is still capable of hybridizing selectively with the oligonucleotides of the immunoconjugate, when the latter is bound to its antigen.

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The syntheses are described of four isosteric racemic myo-inositol 1,4,5-trisphosphate (1) analogues with the phosphate groups replaced by sulfonamide (2), sulfate (3), methylphosphonate (4), and carboxymethyl (5). None of these compounds had any affinity for the IP3 receptor or induced platelet aggregation.

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Synthetically prepared ribosylribitol phosphate dimer and trimer analogous to fragments of the capsular polysaccharide of Haemophilus influenzae type b, containing either an amino- or a (masked) thiol-functionalized spacer, were conjugated to protein by two different methods. The thiol-containing carbohydrates were conjugated to tetanus toxoid or H. influenzae outer membrane protein using N-succinimidyl 3-(2-pyridyldithio)propionate. Glutaric dialdehyde was used to conjugate the amino-containing carbohydrates with tetanus toxoid. All conjugates were able to bind antibodies raised against the native bacterial polysaccharide, as determined by competition ELISA. The glutaric dialdehyde conjugate prepared from ribosylribitol phosphate trimer and tetanus toxoid induced a strong polysaccharide-specific antibody response in mice and rabbits.

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Synthetic oligosaccharides derived from the capsular polysaccharide (PRP) of Haemophilus influenzae type b were conjugated to carrier proteins via a thioether linkage. Conjugates were made of trimeric and tetrameric ribose-ribitol-phosphate and tetanus toxoid or diphtheria toxin. All conjugates elicited anti-PRP antibody responses with an increasing immunoglobulin G/immunoglobulin M ratio in adult mice and monkeys. Trimer conjugates elicited lower anti-PRP antibody responses compared with tetramer conjugates. Adult monkeys responded equally well to the tetrameric oligosaccharide-tetanus toxoid conjugate as to the oligosaccharide-CRM197 conjugate (HbOC), which elicits protective levels of serum antibodies in human infants after two or three injections.

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