Human factor Xa bound amidine inhibitor conformation by double rotational-echo double resonance nuclear magnetic resonance and molecular dynamics simulations.

Double rotational-echo double resonance (double REDOR) NMR was used to investigate the conformation of a (13)C-, (15)N-, and (19)F-labeled inhibitor (Berlex Biosciences compound no. ZK-806299) bound to human factor Xa. Conformationally dependent carbon-fluorine dipolar couplings were measured by (13)C[(19)F] REDOR. Natural abundance carbon signals in the full-echo spectra were removed by (13)C[(15)N] REDOR. Major and minor binding modes were suggested by the NMR data, but only the former had adequate signal to noise for distance determinations. Molecular dynamics simulations restrained by double-REDOR-determined intramolecular (13)C-(19)F distances revealed two models for the dominant binding mode that are consistent with the NMR data. We conclude that ZK-806299 binds similarly to both FXa. Moreover, it appears to bind to FXa in a fashion previously demonstrated for ZK-807834, a more selective FXa inhibitor.

Glycosaminoglycans in Human and Bovine Serum: Detection of Twenty-Four Heparan Sulfate and Chondroitin Sulfate Motifs Including a Novel Sialic Acid-modified Chondroitin Sulfate Linkage Hexasaccharide.

Heterogeneous heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) polysaccharides are important components of blood circulation. Changes in GAG quantity and structure in blood have been indicated in cancers and other human diseases. However, GAG quantities and structures have not been fully characterized due to lack of robust and sensitive analytical tools. To develop such tools, we isolated GAGs from serum and plasma. We employed liquid chromatography (LC) for GAG quantification and LC/mass spectrometry (MS) for GAG structural analysis. Twenty-four heparan and chondroitin sulfate motifs were identified, including linkage hexasaccharides, repeating disaccharide compositions, reducing, and non-reducing end mono-, di-, tri-, and tetrasaccharide structures. Disaccharides were detectable at picomolar level without radiolabeling or derivitization, so only a few ml of human and fetal bovine serum was required for this study. The detection of different reducing end structures distinct from GAG linkage hexasaccharides revealed that free GAG chains generated by GAG degradation enzymes co-existed with proteoglycans in serum. In addition, a novel sialic acid-modified linkage hexasaccharide was found conjugated to bikunin, the most abundant serum proteoglycan.

Activated contact system and abnormal glycosaminoglycans in lupus and other auto- and non-autoimmune diseases.

Systemic lupus erythematosus (SLE), heparin-induced thrombocytopenia (HIT), rheumatoid arthritis (RA) are marked by the presence of autoantibodies against negatively changed DNA, phospholipids, heparin, and chondroitin sulfate, respectively. Heparin/protein complexes induce contact system activation in HIT patient plasmas. The activated contact system generates thrombin. Thrombin is responsible for thrombosis, a common cause of death and disabilities for both HIT and SLE. In this chapter, we analyze plasma contact system proteins, thrombin- and kallikrein-like activities, glucosamine and galactosamine content from SLE-, RA-, osteoarthritis (OA)-, and psoriasis (Ps)-patient plasmas in addition to pooled 30+ healthy patient plasmas. We found that all SLE patient plasmas exhibited abnormal contact systems marked by the absence of high molecular weight kininogen, the presence of processed C1 inhibitor (C1inh), the display of abnormal thrombin- and kallikrein-like activities, and increased levels of plasma glucosamine and galactosamine. Different patterns of contact system activation distinguish SLE, RA, and Ps whereas no contact system activation is observed in normal and OA patient plasmas. The presence of paradoxical "lupus anticoagulants" in certain thrombosis-prone SLE patient plasmas, marked by delayed clotting in clinical plasma test, was explained by the consumption of contact system proteins, especially high molecular weight kininogen. Finally, we discovered that mouse and human SLE autoantibodies bind to cell surface GAGs with structural selectivity. In conclusion, markers of abnormal contact system activation represent potential new targets for autoimmune disease diagnosis, prevention, and treatment. These markers might also be useful in monitoring SLE activity/severity and in pinpointing patients with SLE-associated arthritis and psoriasis.

Glycosaminoglycans and activated contact system in cancer patient plasmas.

Oncogenic mutations create cancer cells. Cancer cells require thrombin for growth, angiogenesis, and metastasis. All cancer patients display a hypercoagulable state, which includes platelet activation, blood coagulation, complement activation, vasodilatation, and inflammation. This often results in thrombosis, the second leading cause of death in cancer patients. It is established that chemically oversulfated glycosaminoglycans (GAGs) induce thrombin generation through contact system activation in human plasma. Thrombin is responsible for thrombosis. In this chapter, we show that plasmas from lung cancer patients contain activated contact systems apparent by the absence of high molecular weight kininogen and processed C1inh, by abnormal kallikrein and thrombin activities, and by increased glucosamine, galactosamine, and GAG levels. Activated contact systems were also evident in plasmas from breast, colon, and pancreatic cancer patients. These data suggest that GAGs or other molecules produced by tumors induce abnormal thrombin generation through contact system activation. Therefore, the contact system and glycans represent new targets for cancer diagnosis, prevention, and treatment.

High affinity glycosaminoglycan and autoantigen interaction explains joint specificity in a mouse model of rheumatoid arthritis.

In the K/BxN mouse model of rheumatoid arthritis, autoantibodies specific for glucose-6-phosphate isomerase (GPI) can transfer joint-specific inflammation to most strains of normal mice. Binding of GPI and autoantibody to the joint surface is a prerequisite for joint-specific inflammation. However, how GPI localizes to the joint remains unclear. We show that glycosaminoglycans (GAGs) are the high affinity (83 nm) joint receptors for GPI. The binding affinity and structural differences between mouse paw/ankle GAGs and elbows/knee GAGs correlated with the distal to proximal disease severity in these joints. We found that cartilage surface GPI binding was greatly reduced by either chondroitinase ABC or beta-glucuronidase treatment. We also identified several inhibitors that inhibit both GPI/GAG interaction and GPI enzymatic activities, which suggests that the GPI GAG-binding domain overlaps with the active site of GPI enzyme. Our studies raise the possibility that GAGs are the receptors for other autoantigens involved in joint-specific inflammatory responses.

Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives.

Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.

Inhibition or activation of Apert syndrome FGFR2 (S252W) signaling by specific glycosaminoglycans.

Most Apert syndrome patients harbor a single amino acid mutation (S252W) in fibroblast growth factor (FGF) receptor 2 (FGFR2), which leads to abnormal FGF/FGFR2 signaling. Here we show that specific combinations of FGFs and glycosaminoglycans activate both alternative splice forms of the mutant but not of the wild-type FGF receptors. More importantly, 2-O- and N-sulfated heparan sulfate, prepared by a combined chemical and enzymatic synthesis, antagonized the over-activated FGFR2b (S252W) to basal levels at nanomolar concentrations. These studies demonstrated that specific glycosaminoglycans could be useful in treating ligand-dependent FGFR signaling-related diseases, such as Apert syndrome and cancer.

Characterization of the complex of a trifluoromethyl-substituted shikimate-based bisubstrate inhibitor and 5-enolpyruvylshikimate-3-phosphate synthase by REDOR NMR.

A combination of (15)N[(19)F], (31)P[(15)N], and (31)P[(19)F] rotational-echo double-resonance NMR has been used to characterize the conformation of a bound trifluoromethylketal, shikimate-based bisubstrate inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase. The solid-state NMR experiments were performed on the complex formed in solution and then lyophilized at low temperatures in the presence of stabilizing lyoprotectants. The results of these experiments indicate that none of the side chains of the six arginines that surround the active site forms a compact salt bridge with the phosphate groups of the bound inhibitor.

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase.

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase-S3P-Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native (31)P in S3P and Glp, biosynthetically (13)C-labeled S3P, specifically (13)C and (15)N labeled Glp, and a variety of protein-(15)N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

Investigation of the binding of epimer A of the covalent hydrate of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine to a recombinant F22W Bacillus subtilis lumazine synthase mutant by (15)N[(19)F] REDOR NMR.

The two epimeric covalent hydrates A and B of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine are metabolically stable analogues of hypothetical intermediates proposed in the reactions catalyzed by riboflavin synthase and lumazine synthase. To confirm the stereochemical assignments previously based solely on results for epimer B, a (15)N[(19)F] REDOR NMR study was performed on the complex formed from epimer A and a recombinant, uniformly (15)N-labeled F22W mutant of Bacillus subtilis lumazine synthase. The results indicate that the fluorines of the ligands are closer to the side chain nitrogens of Arg127 and farther away from the side chain nitrogens of Lys135 in epimer B than in epimer A. These results are consistent with the assignment of the earlier 7R configuration of epimer A and the 7S configuration of epimer B.

Rotational-echo double resonance characterization of vancomycin binding sites in Staphylococcus aureus.

Solid-state NMR experiments with stable isotope-labeled Staphylococcus aureus have provided insight into the structure of the peptidoglycan binding site of a potent fluorobiphenyl derivative of chloroeremomycin (Eli Lilly LY329332). Rotational-echo double resonance (REDOR) NMR provided internuclear distances from the 19F of this glycopeptide antibiotic to natural-abundance 31P and to specific 13C and 15N labels biosynthetically incorporated into the bacteria from labeled alanine, glycine, or lysine in the growth medium. Results from experiments with intact late log phase bacteria and cell walls indicated homogeneous drug-peptidoglycan binding. Drug dimers were not detected in situ, and the hydrophobic fluorobiphenyl group of LY329332 did not insert into the bilayer membrane. A model of the binding site consistent with the REDOR results positions the vancomycin cleft around an un-cross-linked D-Ala-D-Ala peptide stem with the fluorobiphenyl moiety of the antibiotic near the base of a second, proximate stem in a locally ordered peptidoglycan matrix.

Rotational-echo double resonance characterization of the effects of vancomycin on cell wall synthesis in Staphylococcus aureus.

Cross-polarization magic-angle spinning and rotational-echo double resonance 13C and 15N NMR experiments have been performed on intact cells of Staphylococcus aureus labeled with D-[1-13C]alanine and [15N]glycine or with [1-13C]glycine and L-[epsilon-15N]lysine. The cells were harvested during stationary or exponential growth conditions, the latter in media with and without the addition of vancomycin. The results of these experiments allowed the in situ determination of the relative concentrations of peptidoglycan cross-links (the number of peptide-stem D-alanines covalently linked to a pentaglycyl bridge) and bridge-links (the number of peptide-stem lysines covalently linked to a pentaglycyl bridge). The concentration of cross-links remained constant in the presence of vancomycin, whereas the number of bridge-links decreased. These changes suggest that vancomycin (at therapeutic levels) interrupts peptidoglycan synthesis in S. aureus by interference with transglycosylation.

Recognition of an unnatural difluorophenyl nucleotide by uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognize and remove unwanted uracil bases from the genome but does not react with its structural congener, thymine, which differs by a single methyl group. Two factors that determine whether an enzyme flips a base from the duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracil recognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotide analogue that is an excellent isostere of uracil but possesses no hydrogen bond donor or acceptor groups. By using binding affinity measurements, solution (19)F NMR, and solid state (31)P[(19)F] rotational-echo double-resonance (REDOR) NMR measurements, we establish that UDG partially unstacks F from the duplex. However, due to the lack of hydrogen bonding groups that are required to support an open-to-closed conformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway and suggest structural models of the metastable state that are consistent with the REDOR NMR measurements.

Conformation of a bound inhibitor of blood coagulant factor Xa.

13C[(15)N] and (13)C[(19)F] rotational-echo double-resonance NMR have been used to characterize the enzyme-bound structure of ZK-816042, an amidine-imidazoline inhibitor of human factor Xa (FXa). The NMR experiments were performed on a lyophilized FXa-inhibitor complex. The complex was formed in solution in the presence of stabilizing excipients and frozen after gradual supercooling prior to lyophilization. The results indicate that the inhibitor binds with a distribution of orientations of the imidazoline ring.