Discovery and in Vivo Evaluation of the Potent and Selective PI3Kδ Inhibitors 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-6-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-0687) and 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-5-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-1430).

Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.

Discovery, Optimization, and in Vivo Evaluation of Benzimidazole Derivatives AM-8508 and AM-9635 as Potent and Selective PI3Kδ Inhibitors.

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

Off-target platelet activation in macaques unique to a therapeutic monoclonal antibody.

AMG X, a human neutralizing monoclonal antibody (mAb) against a soluble human protein, caused thrombocytopenia, platelet activation, reduced mean arterial pressure, and transient loss of consciousness in cynomolgus monkeys after first intravenous administration. In vitro, AMG X induced activation in platelets from macaque species but not from humans or baboons. Other similar mAbs against the same pharmacological target failed to induce these in vivo and in vitro effects. In addition, the target protein was known to not be expressed on platelets, suggesting that platelet activation occurred through an off-target mechanism. AMG X bound directly to cynomolgus platelets and required both the Fab and Fc portion of the mAb for platelet activation. Binding to platelets was inhibited by preincubation of AMG X with its pharmacological target or with anti-human Fc antibodies or by preincubation of platelets with AMG X F(ab')(2) or human immunoglobulin (IVIG). AMG X F(ab')(2) did not activate platelets. Thus, platelet activation required both recognition/binding of a platelet ligand with the Fab domain and interaction of platelet Fc receptors (i.e., FcγRIIa) with the Fc domain. These findings reflect the complexity of the mechanism of action of mAbs and the increasing awareness of potential for unintended effects in preclinical species.

Nuclear export of NBN is required for normal cellular responses to radiation.

Nijmegen breakage syndrome arises from hypomorphic mutations in the NBN gene encoding nibrin, a component of the MRE11/RAD50/nibrin (MRN) complex. In mammalian cells, the MRN complex localizes to the nucleus, where it plays multiple roles in the cellular response to DNA double-strand breaks. In the current study, sequences in mouse nibrin required to direct the nuclear localization of the MRN complex were identified by site-specific mutagenesis. Unexpectedly, nibrin was found to contain both nuclear localizing signal (NLS) sequences and a nuclear export signal (NES) sequence whose functions were confirmed by mutagenesis. Both nuclear import and export sequences were active in vivo. Disruption of either the NLS or NES sequences of nibrin significantly altered the cellular distribution of nibrin and Mre11 and impaired survival after exposure to ionizing radiation. Mutation of the NES sequence in nibrin slowed the turnover of phosphorylated nibrin after irradiation, indicating that nuclear export of nibrin may function, in part, to downregulate posttranslationally modified MRN complex components after DNA damage responses are complete.