An immunoglobulin-like receptor, Allergin-1, inhibits immunoglobulin E-mediated immediate hypersensitivity reactions.

Anaphylaxis is a life-threatening immediate hypersensitivity reaction triggered by antigen capture by immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcvarepsilonRI) on mast cells. However, the regulatory mechanism of mast cell activation is not completely understood. Here we identify an immunoglobulin-like receptor, Allergin-1, that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain, and show it was preferentially expressed on mast cells. Mouse Allergin-1 recruited the tyrosine phosphatases SHP-1 and SHP-2 and the inositol phosphatase SHIP. Coligation of Allergin-1 and FcvarepsilonRI suppressed IgE-mediated degranulation of bone marrow-derived cultured mast cells. Moreover, mice deficient in Allergin-1 developed enhanced passive systemic and cutaneous anaphylaxis. Thus, Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice.

Cytotoxicity analysis of staphylococcal bi-component ß-pore forming toxins using the CHO cells expressing human lymphocyte receptor CCR5.

CCR5-mediated cytotoxicity of staphylococcal bi-component toxins was investigated using human CCR5-expressing CHO cells. Cytotoxicity of rim domain loop-exchange mutants between LukE and Hlg2 indicated that loop-4 of LukE is essential for cytotoxicity in combination with LukD. Interestingly, Hlg2 showed LukF-dependent CCR5-mediated cytotoxicity, suggesting that the F-components of toxins also play a role in the cell-specific cytotoxicity.

Spirodiketopiperazine-based CCR5 antagonist: discovery of an antiretroviral drug candidate.

Following the discovery that hydroxylated derivative 3 (Fig. 1) was one of the oxidative metabolites of the original lead 1, it was found that hydroxylated compound 4 possesses higher in vitro anti-HIV potency than the corresponding non-hydroxylated compound 2. Structural hybridation of 4 with the orally available analog 5 resulted in another orally-available spirodiketopiperazine CCR5 antagonist 6a that possesses more favorable pharmaceutical profile for use as a drug candidate.

Discovery of 4-[4-({(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxy)methyl]-2,5-dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenoxy]benzoic acid hydrochloride: a highly potent orally available CCR5 selective antagonist.

Based on the original spirodiketopiperazine design framework, further optimization of an orally available CCR5 antagonist was undertaken. Structural hybridization of the hydroxylated analog 4 derived from one of the oxidative metabolites and the new orally available non-hydroxylated benzoic acid analog 5 resulted in another potent orally available CCR5 antagonist 6a as a clinical candidate. Full details of a structure-activity relationship (SAR) study and ADME properties are presented.

Spirodiketopiperazine-based CCR5 antagonists: Improvement of their pharmacokinetic profiles.

Spirodiketopiperazine-based CCR5 antagonists, showing improved pharmacokinetic profiles without reduction in antagonist activity, were designed and synthesized. We also demonstrate the anti-HIV activity of a representative compound 12, as measured in a p24 assay.

Discovery of orally available spirodiketopiperazine-based CCR5 antagonists.

Using the previously reported novel spirodiketopiperazine scaffold, the design and synthesis of orally available CCR5 antagonists was undertaken. Compounds possessing a carboxylic acid function in the appropriate position showed improved oral exposure (AUC) relative to the initial chemical leads without reduction in the antagonist activity. The optimized compound 40 was found to show potent anti-HIV activity. Full details of structure-activity relationship (SAR) study are presented.

Rim domain loops of staphylococcal ß-pore forming bi-component toxin S-components recognize target human erythrocytes in a coordinated manner.

Staphylococcus aureus bi-component pore-forming toxins consist of S- and F-components, and form hetero-octameric beta-barrel pores on target blood cell membranes. Among them, γ-haemolysin (Hlg2 and F-component of Luk (LukF)) and LukED (LukE and LukD) possess haemolytic activity, whereas the Panton-Valentine leukocidin (LukS-PV and LukF-PV) does not lyse human erythrocytes. Here, we focussed on four loop structures in the rim domain of S-component, namely loops -1, -2, -3 and -4, and found that replacement of Loop-4 in both Hlg2 and LukE with that of LukS-PV abolished their haemolytic activity. Furthermore, LukS-PV gained haemolytic activity by Loop-4 exchange with Hlg2 or LukE, suggesting that Loop-4 of these S-components determined erythrocyte specificity. LOOP-1 and -2 enhanced the erythrocytes-binding ability of both components. Although Hlg2 and LukE recognize Duffy antigen receptor for chemokines on human erythrocytes, the ability of Loop-4 was not complementary between Hlg2 and LukE. Exchange of Hlg2 with LukE Loop-4 showed weaker activity than intact Hlg2, and LukE mutant with Hlg2 Loop-4 lost its haemolytic activity in combination of LukD. Interestingly, the haemolytic activities of these Loop-4 exchange mutants were affected by F-component, namely LukF enhanced haemolytic activities of these Hlg2 and LukE Loop-4 mutants, and also haemolytic activity of LukS-PV mutant with LukE Loop-4.

Design, synthesis, and biological evaluation of the combinatorial library with a new spirodiketopiperazine scaffold. Discovery of novel potent and selective low-molecular-weight CCR5 antagonists.

We previously reported the discovery of several spirodiketopiperazine derivatives as potent CCR5 antagonists with anti-HIV activity. Herein, we describe in detail the identification of these lead compounds using a combinatorial chemistry approach. A novel spirodiketopiperazine scaffold was designed on the basis of the concept of the privileged structure of G-protein-coupled receptors (GPCRs). This new framework was obtained in acceptable yield with high purity from the readily prepared isonitrile resin through the Ugi reaction, sequential transformations, and cyclative cleavage. By measuring the inhibitory activity of each compound in the initial library against the intracellular calcium mobilization stimulated by MIP-1alpha, several compounds were found to show modest but selective CCR5 antagonistic activity. After the rapid evaluation of these hit compounds, several single-digit nanomolar, low-molecular-weight CCR5 antagonists that can potently block the infectivity and replication of laboratory and clinical strains of HIV as well as those of highly drug-resistant HIV variants with minimal cytotoxicity have been identified.

Spirodiketopiperazine-based CCR5 antagonists: Lead optimization from biologically active metabolite.

Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds derived from beta-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of MIP-1alpha to human CCR5, compared with the non-hydroxylated derivatives and the other isomers.