Repurposing pyridoxamine for therapeutic intervention of intravascular cell-cell interactions in mouse models of sickle cell disease.

Adherent neutrophils on vascular endothelium positively contribute to cell-cell aggregation and vaso-occlusion in sickle cell disease. In the present study, we demonstrated that pyridoxamine, a derivative of vitamin B6, might be a therapeutic agent to alleviate intravascular cell-cell aggregation in sickle cell disease. Using real-time intravital microscopy, we found that one oral administration of pyridoxamine dose-dependently increased the rolling influx of neutrophils and reduced neutrophil adhesion to endothelial cells in cremaster microvessels of sickle cell disease mice challenged with hypoxia-reoxygenation. Short-term treatment also mitigated neutrophil-endothelial cell and neutrophil-platelet interactions in the microvessels and improved the survival of sickle cell disease mice challenged with tumor necrosis factor-α. The inhibitory effects of pyridoxamine on intravascular cell-cell interactions were potentiated by co-treatment with hydroxyurea. We observed that long-term (5.5 months) oral treatment with pyridoxamine significantly diminished the adhesive function of neutrophils and platelets and down-regulated the expression of E-selectin and intercellular adhesion molecule-1 on the vascular endothelium in tumor necrosis factor-α-challenged sickle cell disease mice. Ex vivo studies revealed that the surface amount of αMß2 integrin was significantly decreased in stimulated neutrophils isolated from sickle cell disease mice treated with pyridoxamine-containing water. Studies using platelets and neutrophils from sickle cell disease mice and patients suggested that treatment with pyridoxamine reduced the activation state of platelets and neutrophils. These results suggest that pyridoxamine may be a novel therapeutic and a supplement to hydroxyurea to prevent and treat vaco-occlusion events in sickle cell disease.

An Effective Strategy for Stabilizing Minimal Coiled Coil Mimetics.

Coiled coils are a major motif in proteins and orchestrate multimerization of various complexes important for biological processes. Inhibition of coiled coil-mediated interactions has significant biomedical potential. However, general approaches that afford short peptides with defined coiled coil conformation remain elusive. We evaluated several strategies to stabilize minimal helical bundles, with the dimer motif as the initial focus. A stable dimeric scaffold was realized in a synthetic sequence by replacing an interhelical ionic bond with a covalent bond. Application of this strategy to a more challenging native protein-protein interaction (PPI) suggested that an additional constraint, a disulfide bond at the internal a/d' position along with a linker at the e/e' position, is required for enhanced conformational stability. We anticipate the coiled coil stabilization methodology described herein to yield new classes of modulators for PPIs.

End-Capped α-Helices as Modulators of Protein Function.

Examination of complexes of proteins with other biomolecules reveals that proteins tend to interact with partners via folded sub-domains, in which the backbone possesses secondary structure. α-Helices, the largest class of protein secondary structures, play fundamental roles in a multitude of highly specific protein-protein and protein-nucleic acids interactions. Herein, we describe the potential of a helix nucleation strategy to afford modulators of protein-protein interactions.

Proflavine derivatives as fluorescent imaging agents of amyloid deposits.

A series of proflavine derivatives for use to further image Aß amyloid deposits were synthesized and characterized. Aged 3xTg-AD (23 months old) mice hippocampus sections incubated with these derivatives revealed preferential labeling of amyloid plaques. Furthermore an in vitro binding study showed an inhibitory effect, although moderate, of these compounds on Aß(40) fibril formation. This study highlights the potential of proflavine as a molecular scaffold for designing new Aß imaging agents, its native fluorescence allowing in vitro neuropathological staining in AD damaged brain sections.

5,11-Dimethyl-dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione.

In the mol-ecule of the title compound, C(16)H(14)N(2)O(2), an N,N'-dimethyl-dianthranilide, the two methyl groups are disordered over two positions; site occupation factors were kept fixed as 0.75:0.25 and 0.65:0.35. The dihedral angle between the two benzene rings is 75.57 (3)°.

Design, synthesis and protein-targeting properties of thioether-linked hydrogen bond surrogate helices.

Appropriately-placed hydrogen bond surrogates have been demonstrated to efficiently nucleate helical conformations. Herein we describe an efficient method for the synthesis of thioether-based hydrogen bond surrogate (teHBS) helices. A teHBS helix is shown to adopt a stable conformation and target its cognate protein receptor with high affinity.

Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach.

Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors. Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity. The hypothesis behind the design of stabilized helices and helix mimetics is that these medium-sized molecules may pursue their targets with higher specificity because of a larger number of contacts. This protocol describes an optimized synthetic strategy for the preparation of stabilized α-helices that feature a carbon-carbon linkage in place of the characteristic N-terminal main-chain hydrogen bond of canonical helices. Formation of the carbon-carbon bond is enabled by a microwave-assisted ring-closing metathesis reaction between two terminal olefins on the peptide chain. The outlined strategy allows the synthesis and purification of a hydrogen bond surrogate (HBS) α-helix in ∼ 1 week.