Discovery of Selective LRRK2 Inhibitors Guided by Computational Analysis and Molecular Modeling Huifen Chen,* ,† Bryan K. Chan, † Jason Drummond, ‡ Anthony A. Estrada, † Janet Gunzner-Toste, † Xingrong Liu, § Yichin Liu, ‡ John Moffat, ‡ Daniel Shore, † Zachary K. Sweeney, †,⊥ Thuy Tran, † Shumei Wang, † Guiling Zhao, † Haitao Zhu, ∥ and Daniel J. Burdick* ,† † Discovery Chemistry Department, ‡ Biochemical and Cellular Pharmacology Department, § Drug Metabolism and Pharmacokinetics Department, and ∥ Neuroscience Department, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States * S Supporting Information ABSTRACT: Mutations in the genetic sequence of leucine- rich repeat kinase 2 (LRRK2) have been linked to increased LRRK2 activity and risk for the development of Parkinson’s disease (PD). Potent and selective small molecules capable of inhibiting the kinase activity of LRRK2 will be important tools for establishing a link between the kinase activity of LRRK2 and PD. In the absence of LRRK2 kinase domain crystal structures, a LRRK2 homology model was developed that provided robust guidance in the hit-to-lead optimization of small molecule LRRK2 inhibitors. Through a combination of molecular modeling, sequence analysis, and matched molecular pair (MMP) activity cliff analysis, a potent and selective lead inhibitor was discovered. The selectivity of this compound could be understood using the LRRK2 homology model, and application of this learning to a series of 2,4-diaminopyrimidine inhibitors in a scaffold hopping exercise led to the identification of highly potent and selective LRRK2 inhibitors that were also brain penetrable. ■ INTRODUCTION Parkinson’s disease (PD) is a multisystem neurodegenerative disorder that is clinically characterized primarily by tremors, rigidity, and bradykinesia. 1 The current standard of care for PD patients is limited to symptomatic treatment, which only provides temporary attenuation of motor symptoms and does not affect the progression of neurodegeneration. There is, therefore, a strong demand for disease modifying or neuro- protective therapies. One of the more attractive targets for disease modification was identified in 2004 when genetic variations in the LRRK2 gene were linked to familial PD. 2,3 In particular, the specific G2019S mutation of LRRK2 has been associated with both familial and idiopathic PD. 4−6 The LRRK2 gene encodes a large protein with multiple domains, including a kinase domain. 7−9 The detailed physiological function and effectors of the LRRK2 kinase are largely unknown and remain to be determined. 10 Importantly, the G2019S mutation in the kinase domain is a dominant mutation that has been shown to increase LRRK2 kinase activity in vitro, suggesting that the kinase activity of LRRK2 is involved in Parkinson’s disease pathophysiogenesis. 11−14 Indeed, recent studies with non- specific LRRK2 small molecule inhibitors have suggested that inhibition of LRRK2 activity might ameliorate neurodegener- ative phenotypes in C. elegans and Drosophila Parkinson’s disease models and mouse models of LRRK2. 15,16 However, because of the lack of general kinase selectivity of compounds used in the efficacy studies, the biological effects of LRRK2 kinase inhibition remain to be elucidated. 17 A few selective inhibitors of LRRK2 kinase activity such as LRRK2-IN-1 18 and CZC-25146 19 have been described recently. However, they do not appear to have sufficient CNS exposure to be used in mammalian models of PD. A potent ALK/LRRK2 kinase inhibitor, TAE684, was recently reported to achieve significant brain exposure in mouse but did not inhibit LRRK2 phosphorylation in the brain. 20 The development of selective and brain penetrable LRRK2 inhibitors therefore remains a critical need for the LRRK2 field. 21 In this contribution, we describe our initial efforts to use structure-based design and computational approaches to identify useful LRRK2 chemical probes starting from a high-throughput screening effort. ■ RESULTS A high-throughput screening campaign using G2019S LRRK2 protein 22 yielded a number of interesting small molecule inhibitor scaffolds, including triazolopyridines and diaminopyr- imidines represented by compounds 1 and 2 (Figure 1). The triazolopyridine compounds were highly potent and had physical properties consistent with CNS penetration, 23 while the aminopyrimidine inhibitors had excellent ligand efficiency (LE). 24,25 It is well-known, however, that the diaminopyr- imidine motif is particularly well-represented in the kinase inhibition literature 26 and the potential selectivity of these compounds was a concern. We therefore focused our initial computer-aided design efforts on the development of models Received: March 31, 2012 Article pubs.acs.org/jmc © XXXX American Chemical Society A dx.doi.org/10.1021/jm300452p | J. Med. Chem. XXXX, XXX, XXX−XXX