Kenji Sagawa

Spirodiketopiperazine-Based CCR5 Inhibitor Which Preserves CC-Chemokine/CCR5 Interactions and Exerts Potent Activity against R5 Human Immunodeficiency Virus Type 1 In Vitro

ABSTRACT We identified a novel spirodiketopiperazine (SDP) derivative, AK602/ONO4128/GW873140, which specifically blocked the binding of macrophage inflammatory protein 1α (MIP-1α) to CCR5 with a high affinity (Kd of ≈3 nM), potently blocked human immunodeficiency virus type 1 (HIV-1) gp120/CCR5 binding and exerted potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates, including multidrug-resistant HIV-1 (HIV-1MDR) (50% inhibitory concentration values of 0.1 to 0.6 nM) in vitro. AK602 competitively blocked the binding to CCR5 expressed on Chinese hamster ovary cells of two monoclonal antibodies, 45523, directed against multidomain epitopes of CCR5, and 45531, specific against the C-terminal half of the second extracellular loop (ECL2B) of CCR5. AK602, despite its much greater anti-HIV-1 activity than other previously published CCR5 inhibitors, including TAK-779 and SCH-C, preserved RANTES (regulated on activation normal T-cell expressed and secreted) and MIP-1β binding to CCR5+ cells and their functions, including CC-chemokine-induced chemotaxis and CCR5 internalization, while TAK-779 and SCH-C fully blocked the CC-chemokine/CCR5 interactions. Pharmacokinetic studies revealed favorable oral bioavailability in rodents. These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection.

THE PROTEIN-TYROSINE PHOSPHATASE SHP-2 ASSOCIATES WITH TYROSINE-PHOSPHORYLATED ADHESION MOLECULE PECAM-1 (CD31)

Aggregation of many cell-surface receptors results in tyrosine phosphorylation of numerous proteins. We previously observed the tyrosine phosphorylation of the platelet/endothelial cell adhesion molecule, PECAM-1 (CD31), after FcεRI stimulation in rat basophilic leukemia RBL-2H3 cells. Here we found that PECAM-1 was also transiently tyrosine-phosphoryated after adherence of these cells to fibronectin. Similarly aggregation of the T cell receptor on Jurkat cells also induced this tyrosine phosphorylation. The protein-tyrosine phosphatase SHP-2 is a widely expressed cytosolic enzyme with two Src homology 2 (SH2) domains. SHP-2, but not the related protein-tyrosine phosphatase SHP-1, associated with PECAM-1. This association of the two proteins correlated with the extent of the tyrosine phosphorylation of PECAM-1. A fusion protein containing the two SH2 domains of SHP-2 precipitated PECAM-1 from cell lysates and also directly bound to phosphorylated PECAM-1. In immune precipitate phosphatase assays, there was tyrosine dephosphorylation of PECAM-1. Therefore, integrin and immune receptor activation results in tyrosine phosphorylation of PECAM-1 and the binding of the protein-tyrosine phosphatase SHP-2, which could regulate receptor-mediated signaling in cells.

Aggregation of the High Affinity IgE Receptor Results in the Tyrosine Phosphorylation of the Surface Adhesion Protein PECAM-1 (CD31)

One of the earliest events after aggregation of the high affinity receptor for IgE (FcεRI) on mast cells is the activation of protein tyrosine kinases resulting in tyrosine phosphorylation of numerous proteins. Using a monoclonal antibody raised against the rat basophilic leukemia RBL-2H3 cells, we identified that platelet/endothelial cell adhesion molecule 1 (PECAM-1 or CD31) was tyrosine phosphorylated in these cells. Aggregation of PECAM-1 did not induce a detectable increase in its tyrosine phosphorylation, nor did it result in degranulation. However, the minimal tyrosine phosphorylation of PECAM-1 in nonstimulated cells was dramatically increased after FcεRI aggregation. This receptor-induced tyrosine phosphorylation of PECAM-1 was an early event, independent of Ca2+ influx or of the activation of protein kinase C and of cell adhesion. PECAM-1 is an adhesion molecule that is required for the transmigration of leukocytes across the endothelium into sites of inflammation. Therefore tyrosine phosphorylation of PECAM-1 may modulate its interaction with other molecules, thereby regulating the migration of basophils into inflammatory sites.

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.

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.