Jens Breinholt

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Iminosugars: potential inhibitors of liver glycogen phosphorylase

The first synthesis of the single isomers (3R,4R,5R); (3S,4S,5S): (3R,4R,5S) and (3S,4S,5R) of 5-hydroxymethyl-piperidine-3,4-diol from Arecolin is reported, including the synthesis of a series of N-substituted derivatives of the (3R,4R,5R)-isomer (Isofagomine). The inhibitory effect of these isomers as well as of a series of N-substituted derivatives of the (3R,4R,5R)-isomer and selected hydroxypiperidine analogues on liver glycogen phosphorylase (GP) showed that the (3R,4R,5R) configuration was essential for obtaining an inhibitory effect at submicromolar concentration. The results also showed that all three hydroxy groups should be present and could not be substituted, nor were extra OH groups allowed if sub-micromolar inhibition should be obtained. Some inhibitory effect was retained for N-substituted derivatives of Isofagomine; however, N-substitution always resulted in a loss of activity compared to the parent compound, IC50 values ranging from 1 to 100 microM were obtained for simple alkyl, arylalkyl and benzoylmethyl substituents. Furthermore, we found that it was not enough to assure inhibitory effect to have the (R,R,R) configuration. Fagomine, the (2R,3R,4R)-2-hydroxymethylpiperidine-3,4-diol analogue, showed an IC50 value of 200 microM compared to 0.7 microM for Isofagomine. In addition, Isofagomine was able to prevent basal and glucagon stimulated glycogen degradation in cultured hepatocytes with IC50 values of 2-3 microM.

The existence of multiple conformers of interleukin-21 directs engineering of a superpotent analogue.

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, α-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay.

A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes.

A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed.

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5Å resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131 ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.

Evidence against Glycogen Cycling of Gluconeogenic Substrates in Various Liver Preparations

The effect of inhibition of glycogen phosphorylase by 1,4-dideoxy-1,4-imino-d-arabinitol on rates of gluconeogenesis, gluconeogenic deposition into glycogen, and glycogen recycling was investigated in primary cultured hepatocytes, in perfused rat liver, and in fed or fasted rats in vivo clamped at high physiological levels of plasma lactate. 1,4-Dideoxy-1,4-imino-d-arabinitol did not alter the synthesis of glycerol-derived glucose in hepatocytes or lactate-derived glucose in perfused liver or fed or fasted rats in vivo. Thus, 1,4-dideoxy-1,4-imino-d-arabinitol inhibited hepatic glucose output in the perfused rat liver (0.77 ± 0.19versus 0.33 ± 0.09, p < 0.05), whereas the rate of lactate-derived gluconeogenesis was unaltered (0.22 ± 0.09 versus 0.18 ± 0.08,p = not significant) (1,4-dideoxy-1,4-imino-d-arabinitol versusvehicle, μmol/min * g). Overall, the data suggest that 1,4-dideoxy-1,4-imino-d-arabinitol inhibited glycogen breakdown with no direct or indirect effects on the rates of gluconeogenesis. Total end point glycogen content (μmol of glycosyl units/g of wet liver) were similar in fed (235 ± 19versus 217 ± 22, p = not significant) or fasted rats (10 ± 2 versus 7 ± 2, p = not significant) with or without 1,4-dideoxy-1,4-imino-d-arabinitol, respectively. The data demonstrate no glycogen cycling under the investigated conditions and no effect of 1,4-dideoxy-1,4-imino-d-arabinitol on gluconeogenic deposition into glycogen. Taken together, these data also suggest that inhibition of glycogen phosphorylase may prove beneficial in the treatment of type 2 diabetes.

Enantiomer separation of 7‐des‐methyl‐ormeloxifene using sulfated β‐cyclodextrin in countercurrent chromatography

The enantiomers of 7-des-methyl-ormeloxifene were separated by countercurrent chromatography (CCC) using sulfated beta-cyclodextrin as chiral selector, representing the first reported successful application of a cyclodextrin derivative in CCC-based resolutions. The choice of chiral selector relies on extreme separation factors observed in chiral capillary electrophoresis, and suitable CCC conditions were developed employing an analytical toroidal coil countercurrent chromatograph. Preparative separation of the enantiomers was performed using a conventional, preparative CCC-instrument. Copyright 1999 Wiley-Liss, Inc.

Synthesis of symmetrical dimeric N,N′-linked peptides on solid support by olefin metathesis

Abstract A method has been developed for the synthesis of dimeric ligands of biological relevance on solid support using olefin metathesis as a key step. With the ruthenium catalyst used, the size of the peptide fragment did not influence the reaction. If the double bond involved was separated by at least ∼2 methylene groups from an amide group, products of good purity could be recovered.

Design and synthesis of triglyceride analogue biotinylated suicide inhibitors for directed molecular evolution of lipolytic enzymes

The design, synthesis, and inhibition properties of two new triglyceride analogue biotinylated suicide inhibitors (2) and (3) for directed molecular evolution of lipolytic enzymes by phage-display is described.

Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen–Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation

Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering.