Evaluation of steroidal amines as lipid raft modulators and potential anti-influenza agents.

The influenza A virus (IFV) possesses a highly ordered cholesterol-rich lipid envelope. A specific composition and structure of this membrane raft envelope are essential for viral entry into cells and virus budding. Several steroidal amines were investigated for antiviral activity against IFV. Both, a positively charged amino function and the highly hydrophobic (ClogP≥5.9) ring system are required for IC50 values in the low µM range. An amino substituent is preferential to an azacyclic A-ring. We showed that these compounds either disrupt or augment membrane rafts and in some cases inactivate the free virus. Some of the compounds also interfere with virus budding. The antiviral selectivity improved in the series 3-amino, 3-aminomethyl, 3-aminoethyl, or by introducing an OH function in the A-ring. Steroidal amines show a new mode of antiviral action in directly targeting the virus envelope and its biological functions.

Lipid-like sulfoxides and amine oxides as inhibitors of mast cell activation.

The drug miltefosine is a prototypic lipid-like compound thought to modulate membrane environments and thereby indirectly prevent receptor-mediated signaling events. In addition to its primary therapeutic indications in cancer and leishmaniasis, miltefosine has also been shown to block immunoglobulin E receptor-dependent mast cell activation. Miltefosine and other alkylphospholipids that are active in mast cell degranulation assays contain a positively charged nitrogen and a phosphate group that are important for activity. In addition to alkylphospholipids, ceramides are also known to act on membrane environments and inhibit mast cell activation. We have systematically searched a very large compound collection for other lipid-like inhibitors of mast cell activation. Analogs of an initially identified screening hit were synthesized and preliminary SAR information was collected, leading to the identification of sulfoxide and amine oxide containing lipid-like compounds as new inhibitors of mast cell activation. Sulfoxide and amine oxide derivatives were found to be only slightly less active than miltefosine.

Structural design, solid-phase synthesis and activity of membrane-anchored ß-secretase inhibitors on Aß generation from wild-type and Swedish-mutant APP.

Covalent coupling of ß-secretase inhibitors to a raftophilic lipid anchor via a suitable spacer by using solid-phase peptide synthesis leads to tripartite structures displaying substantially improved inhibition of cellular secretion of the ß-amyloid peptide (Aß). Herein, we describe a series of novel tripartite structures, their full characterization by NMR spectroscopy and mass spectrometry, and the analysis of their biological activity in cell-based assays. The tripartite structure concept is applicable to different pharmacophores, and the potency in terms of ß-secretase inhibition can be optimized by adjusting the spacer length to achieve an optimal distance of the inhibitor from the lipid bilayer. A tripartite structure containing a transition-state mimic inhibitor was found to be less potent on Aß generation from Swedish-mutant amyloid precursor protein (APP) than from the wild-type protein. Moreover, our observations suggest that specific variants of Aß are generated from wild-type APP but not from Swedish-mutant APP and are resistant to ß-secretase inhibition. Efficient inhibition of Aß secretion by tripartite structures in the absence of appreciable neurotoxicity was confirmed in a primary neuronal cell culture, thus further supporting the concept.

Computational screening for membrane-directed inhibitors of mast cell activation.

Receptor-mediated signaling events frequently depend on the integrity of their membrane environments. Only a limited number of compounds are currently available that are known or thought to modulate membrane environments and affect signaling events without disrupting membrane structure. Among these are alkylphospholipids including the drug miltefosine that is approved for the treatment of breast cancer and leishmaniasis. In addition, miltefosine has recently been shown to block immunoglobulin E receptor-dependent mast cell activation. On the basis of these findings, we have explored other alkylphospholipids as potential inhibitors of mast cell activation and confirmed the inhibitory activity of five molecules. By comparing the head groups of these alkylphospolipids common pharmacophore features were determined. Through computational screening utilizing this pharmacophore information a new lipid-like inhibitory chemotype was identified that blocked mast cell activation with potency comparable to miltefosine.

Efficient inhibition of the Alzheimer's disease beta-secretase by membrane targeting.

beta-Secretase plays a critical role in beta-amyloid formation and thus provides a therapeutic target for Alzheimer's disease. Inhibitor design has usually focused on active-site binding, neglecting the subcellular localization of active enzyme. We have addressed this issue by synthesizing a membrane-anchored version of a beta-secretase transition-state inhibitor by linking it to a sterol moiety. Thus, we targeted the inhibitor to active beta-secretase found in endosomes and also reduced the dimensionality of the inhibitor, increasing its local membrane concentration. This inhibitor reduced enzyme activity much more efficiently than did the free inhibitor in cultured cells and in vivo. In addition to effectively targeting beta-secretase, this strategy could also be used in designing potent drugs against other membrane protein targets.

Structure-activity relationships of dynorphin a analogues modified in the address sequence.

The peptide [Pro3]Dyn A(1-11)-NH2 2 exhibits high affinity (K(i) = 2.4 nM) and over 2000-fold selectivity for the opioid receptor. Stepwise removal of the C-terminal residues from this ligand demonstrated that its positively charged Arg residues, particularly Arg6 and Arg7, were crucial for binding to the kappa receptor. Analogues shorter than seven amino acids lacked significant affinity for opioid receptors. Comparison with a series of truncated analogues of Dyn A showed that the relative losses in binding potency differed only slightly between the two series. The neutral residues Ile8 and Pro10 could be removed without significant loss in affinity for the kappa receptor. Their replacement, in the Pro3 analogue, with additional Arg residues led to analogues with improved kappa affinity (e.g., [Pro3,Arg8]Dyn A(1-11)-NH2 20: K(i)(kappa) = 0.44 nM). This type of modification did not compromise the high kappa selectivity of the Pro3 analogues. These findings support the view that a negatively charged domain in the putative second extracellular loop of the kappa receptor selectively recognizes residues 6-11 of dynorphin through electrostatic interactions. As with parent compound 2, analogue 20 and related compounds displayed kappa antagonist properties.

Structure-activity relationships of dynorphin analogs substituted in positions 2 and 3.

Following up on the observation that the dynorphin analog [Pro(3)]Dyn A(1-11)-NH(2) 2 possesses high affinity and selectivity for the kappa opioid receptor, a number of related peptides were prepared and characterized by radioligand binding and [(35)S]GTPgammaS assays. While incorporation of 2-azetidine carboxylic acid in position 3 led to the equally potent analog 3, the corresponding analog containing piperidine-2-carboxylic acid showed a nearly 90-fold reduction in kappa affinity. Differential preferred bond angles phi in the three building blocks might account for these observations. Compounds 2 and 3 were kappa antagonists with IC(50) values of 380 and 350 nM, respectively. The Sar(3) analog 7 and the Sar(2) analog 8 were kappa agonists, with greater selectivity than Dyn A(1-11)-NH(2) 1. In view of their high kappa affinities (8: K(i) = 1.5 nM; 2: K(i) = 2.4 nM), the new analogs were surprisingly weak kappa agonists or antagonists, e.g., the EC(50) value for the agonist 8 was 280 nM. Different kappa receptor subtypes in binding vs functional assays can not account for these results, since both assays were performed using the same membrane preparation.