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Rational structure-based design has yielded highly potent inhibitors of cathepsin K (Cat K) with excellent physical properties, selectivity profiles, and pharmacokinetics. Compounds with a 3,4-(CH₃O)₂Ph motif, such as 31, were found to have excellent metabolic stability and absorption profiles. Through metabolite identification studies, a reactive metabolite risk was identified with this motif. Subsequent structure-based design of isoteres culminated in the discovery of an optimized and balanced inhibitor (indazole, 38).

Factor XI (FXI) is a key enzyme in the coagulation pathway and an attractive target for the development of anticoagulant drugs. A small number of high-resolution crystal structures of FXIa in complex with small synthetic inhibitors have been published to date. All of these ligands have a basic P1 group and bind exclusively in the nonprime side of the active site of FXIa. Here, two structures of FXIa in complex with nonbasic inhibitors that occupy both the prime and nonprime sides of the active site are presented. These new structures could be valuable in the design and optimization of new FXIa synthethic inhibitors.

Minimal sequence requirements for binding of substrate-derived statine peptides to the aspartyl enzyme were established on the basis of the X-ray cocrystal structure of the hydroxyethylene-octapeptide OM00-3 in complexation with BACE-1. With this information to hand, macrocyclic compounds that conformationally restrict and preorganize the peptide backbone for an entropically favoured binding to the enzyme active site cleft were designed. By means of a side chain-to-side chain ring closure between two aspartyl residues in the P2 and P3' positions through phenylene-1,3-dimethanamine, a 23-membered ring structure was obtained; this structure retained an extended conformation of the peptide backbone, including the transition state analogue statine for tight interactions with the two aspartyl residues of the active centre. The conformational preorganization of the inhibitor molecule was verified by NMR structural analysis and was then confirmed by the crystal structure of the BACE-1/inhibitor complex. Detailed insights into the binding mode of this macrocyclic inhibitor explained its moderate binding affinity in cell-free assays (K(i)=2.5 microM) and yielded precious information for possible structural optimization in view of the lack of steric clashes of the macrocycle with the flap domain of the enzyme.

Lenvatinib is an oral multikinase inhibitor that selectively inhibits vascular endothelial growth factor (VEGF) receptors 1 to 3 and other proangiogenic and oncogenic pathway-related receptor tyrosine kinases. To elucidate the origin of the potency of lenvatinib in VEGF receptor 2 (VEGFR2) inhibition, we conducted a kinetic interaction analysis of lenvatinib with VEGFR2 and X-ray analysis of the crystal structure of VEGFR2–lenvatinib complexes. Kinetic analysis revealed that lenvatinib had a rapid association rate constant and a relatively slow dissociation rate constant in complex with VEGFR2. Co-crystal structure analysis demonstrated that lenvatinib binds at its ATP mimetic quinoline moiety to the ATP binding site and to the neighboring region via a cyclopropane ring, adopting an Asp-Phe-Gly (DFG)-“in” conformation. These results suggest that lenvatinib is very distinct in its binding mode of interaction compared to the several approved VEGFR2 kinase inhibitors.

Directed screening of nitrile compounds revealed 3 as a highly potent cathepsin K inhibitor but with cathepsin S activity and very poor stability to microsomes. Synthesis of compounds with reduced molecular complexity, such as 7, revealed key SAR and demonstrated that baseline physical properties and in vitro stability were in fact excellent for this series. The tricycle carboline P3 unit was discovered by hypothesis-based design using existing structural information. Optimization using small substituents, knowledge from matched molecular pairs, and control of lipophilicity yielded compounds very close to the desired profile, of which 34 (AZD4996) was selected on the basis of pharmacokinetic profile.

The discovery of nitrile compound 4, a potent inhibitor of Cathepsin K (Cat K) with good bioavailability in dog is described. The compound was used to demonstrate target engagement and inhibition of Cat K in an in vivo dog PD model. The margin to hERG ion channel inhibition was deemed too low for a clinical candidate and an optimisation program to find isosteres or substitutions on benzothiazole group led to the discovery of 20, 24 and 27; all three free from hERG inhibition.

Background: Lenvatinib mesilate (lenvatinib) selectively inhibits vascular endothelial growth factor receptors (VEGFR1-3), and other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib is currently evaluated in several clinical trials, including two phase 3 clinical trials in patients with thyroid cancers and hepatocellular carcinoma. Recently, lenvatinib achieved the primary endpoint in a phase 3 clinical trial in patients with differentiated thyroid cancer. To gain insight into the origin of the potency of lenvatinib in VEGFR2 inhibition, crystal structure analysis of VEGFR2 complexed with lenvatinib and kinetics interaction analysis of lenvatinib against VEGFR2 were conducted in comparison with other VEGFR2 inhibitors. Experimental procedures: Recombinant human VEGFR2 was crystalized with lenvatinib or sorafenib, respectively. The resulting crystals of kinase-inhibitor complexes were subjected to x-ray structural analysis and the three dimensional structures were solved. The reporter displacement assay was conducted by using recombinant human VEGFR2 and a synthetic reporter probe. Reporter displacement was measured after addition of compounds and the kinetic parameters were determined. Results: In crystal structure analysis, both lenvatinib and sorafenib bind to the ATP-binding site and the neighboring allosteric region. However there was a notable difference in the conformation of the DFG motif in the activation loop. While DFG motif assumed a DFG-out conformation in the complex with sorafenib as reported, it was maintained in a DFG-in conformation in the complex with lenvatinib. In the reporter displacement assay, lenvatinib showed intermediate binding kinetics of those of sorafenib and sunitinib. Its association rate constant (kon) and dissociation rate constant (koff) values were 4.8 × 105 s−1 × M−1 (61 times of sorafenib, a representative type 2 inhibitor) and 9.9 × 10−4 s−1 (3.8 times), respectively. Sunitinib, a representative type 1 VEGFR2 inhibitor had a much faster binding kinetics and we could not determine kinetics parameters (kon>1.9 × 105, koff >57 × 10−4). Conclusions: Lenvatinib was suggested to have interaction with not only ATP region but also the neighboring allosteric region of VEGFR2, which is a typical feature of type 2 inhibitors. However, VEGFR2 adopted DFG-in conformation in complex with lenvatinib, which is a characteristic of type 1 inhibitors. Kinetics analysis revealed that lenvatinib had intermediate characteristics among these three types of VEGFR2 inhibitors. These results suggest lenvatinib is very distinct in its ability in binding with VEGFR2. Citation Format: Kiyoshi Okamoto, Megumi Ikemori Kawada, Anja Jestel, Konstanze von König, Yasuhiro Funahashi, Tomohiro Matsushima, Akihiko Tsuruoka, Atsushi Inoue, Junji Matsui. Distinct binding mode of lenvatinib to VEGFR2 revealed by biochemical characterization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1362. doi:10.1158/1538-7445.AM2015-1362