Nanomolar E-selectin antagonists with prolonged half-lives by a fragment-based approach.

Selectins, a family of C-type lectins, play a key role in inflammatory diseases (e.g., asthma and arthritis). However, the only millimolar affinity of sialyl Lewis(x) (sLe(x)), which is the common tetrasaccharide epitope of all physiological selectin ligands, has been a major obstacle to the development of selectin antagonists for therapeutic applications. In a fragment-based approach guided by NMR, ligands binding to a second site in close proximity to a sLe(x) mimic were identified. A library of antagonists obtained by connecting the sLe(x) mimic to the best second-site ligand via triazole linkers of different lengths was evaluated by surface plasmon resonance. Detailed analysis of the five most promising candidates revealed antagonists with K(D) values ranging from 30 to 89 nM. In contrast to carbohydrate-lectin complexes with typical half-lives (t(1/2)) in the range of one second or even less, these fragment-based selectin antagonists show t1/2 of several minutes. They exhibit a promising starting point for the development of novel anti-inflammatory drugs.

Pre-organization of the core structure of E-selectin antagonists.

A new class of N-acetyl-D-glucosamine (GlcNAc) mimics for E-selectin antagonists was designed and synthesized. The mimic consists of a cyclohexane ring substituted with alkyl substituents adjacent to the linking position of the fucose moiety. Incorporation into E-selectin antagonists led to the test compounds 8 and the 2'-benzoylated analogues 21, which exhibit affinities in the low micromolar range. By using saturation transfer difference (STD)-NMR it could be shown that the increase in affinity does not result from an additional hydrophobic contact of the alkyl substituent with the target protein E-selectin, but rather from a steric effect stabilizing the antagonist in its bioactive conformation. The loss of affinity found for antagonists 10 and 35 containing a methyl substituent in a remote position (and therefore unable to support to the stabilization of the core) further supports this hypothesis. Finally, when a GlcNAc mimetic containing two methyl substituents (52 and 53) was used, in which one methyl was positioned adjacent to the fucose linking position and the other was in a remote position, the affinity was regained.

High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-x(L) and Bcl-2 as TolAIII-fusion proteins.

A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.