RESUMO
Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor-related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose-response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.
Assuntos
Receptores Proteína Tirosina Quinases , Proteínas rab de Ligação ao GTP , Agrina/genética , Agrina/metabolismo , Animais , Camundongos , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. Thus, the LâR shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/química , Inibidores de Proteínas Quinases/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Modelos Biológicos , Nucleotídeos/química , Nucleotídeos/metabolismo , Fosforilação/efeitos dos fármacos , Estrutura Secundária de ProteínaRESUMO
The discovery of disease-modifying therapies for Parkinson's Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in LRRK2 are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. As such, research towards brain-permeable kinase inhibitors of LRRK2 has received much attention. In the course of a program to identify structurally diverse inhibitors of LRRK2 kinase activity, a 5-azaindazole series was optimized for potency, metabolic stability and brain penetration. A key design element involved the incorporation of an intramolecular hydrogen bond to increase permeability and potency against LRRK2. This communication will outline the structure-activity relationships of this matched pair series including the challenge of obtaining a desirable balance between metabolic stability and brain penetration.
Assuntos
Indazóis/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Descoberta de Drogas , Ligação de HidrogênioRESUMO
CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MWâ¯=â¯285 and TPSAâ¯=â¯66â¯Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKaâ¯=â¯8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uuâ¯=â¯0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Desenho de Fármacos , Glioblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Células MCF-7 , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Pim kinase inhibitors are promising cancer therapeutics. Pim-2, among the three Pim isoforms, plays a critical role in multiple myeloma yet inhibition of Pim-2 is challenging due to its high affinity for ATP. A co-crystal structure of a screening hit 1 bound to Pim-1 kinase revealed the key binding interactions of its indazole core within the ATP binding site. Screening of analogous core fragments afforded 1H-pyrazolo[3,4-c]pyridine (6-azaindazole) as a core for the development of pan-Pim inhibitors. Fragment and structure based drug design led to identification of the series with picomolar biochemical potency against all three Pim isoforms. Desirable cellular potency was also achieved.
Assuntos
Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Indazóis/síntese química , Indazóis/química , Indazóis/farmacologia , Camundongos , Modelos Moleculares , Proteínas Proto-Oncogênicas c-pim-1/química , Pirazóis/síntese química , Pirazóis/química , Piridinas/síntese química , Piridinas/química , Relação Estrutura-AtividadeRESUMO
Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named 'L' and 'R,' where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.
Assuntos
Trifosfato de Adenosina , Domínio Catalítico , Fosforilação , Conformação ProteicaRESUMO
Activation of the extracellular signal regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named "L" and "R", where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.
RESUMO
Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and are frequently altered in cancer cells, thereby leading to uncontrolled proliferation. In this context, CDK2 has emerged as an appealing target for anticancer drug development. Herein, we describe the discovery of a series of selective small molecule inhibitors of CDK2 beginning with historical compounds from our ERK2 program (e.g., compound 6). Structure-based drug design led to the potent and selective tool compound 32, where excellent selectivity against ERK2 and CDK4 was achieved by filling the lipophilic DFG-1 pocket and targeting interactions with CDK2-specific lower hinge binding residues, respectively. Compound 32 demonstrated 112% tumor growth inhibition in mice bearing OVCAR3 tumors with 50 mg/kg bis in die (BID) oral dosing.
RESUMO
VPS34 is a class III phosphoinositide 3-kinase involved in endosomal trafficking and autophagosome formation. Inhibitors of VPS34 were believed to have value as anticancer agents, but genetic and pharmacological data suggest that sustained inhibition of VPS34 kinase activity may not be well tolerated. Here we disclose the identification of a novel series of dihydropyrazolopyrazinone compounds represented by compound 5 as potent, selective, and orally bioavailable VPS34 inhibitors through a structure-based design strategy. A water-interacting hydrogen bond acceptor within an appropriate distance to a hinge-binding element was found to afford significant VPS34 potency across chemical scaffolds. The selectivity of compound 5 over PIK family kinases arises from interactions between the hinge-binding element and the pseudo-gatekeeper residue Met682. As recent in vivo pharmacology data suggests that sustained inhibition of VPS34 kinase activity may not be tolerated, structure-activity relationships leading to VPS34 inhibition may be helpful for avoiding this target in other ATP-competitive kinase programs.
Assuntos
Antineoplásicos , Classe III de Fosfatidilinositol 3-Quinases , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Autofagia , Endossomos , Fosfatidilinositol 3-Quinase/metabolismo , FosforilaçãoRESUMO
Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiro-azaindoline HPK1 inhibitors demonstrated improved in vitro ADME properties and the ability to induce cytokine production in primary human T-cells.
RESUMO
Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.
Assuntos
Neoplasias/tratamento farmacológico , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinonas/uso terapêutico , Quinases raf/antagonistas & inibidores , Animais , Azetidinas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Cães , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos Nus , Estrutura Molecular , Mutação , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Piperidinas/uso terapêutico , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Quinazolinonas/química , Quinazolinonas/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/genética , Quinases raf/metabolismoRESUMO
Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.
RESUMO
Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.
Assuntos
Domínio Catalítico , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Animais , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Células Sf9 , SpodopteraRESUMO
Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.
Assuntos
Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirazóis/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Cães , Feminino , Humanos , Macaca fascicularis , Células Madin Darby de Rim Canino , Masculino , Camundongos SCID , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this issue, Drawnel et al. (2017) introduce the concept of a "molecular phenotype" and demonstrate how "big data" coming from gene expression profiling, combined with signaling pathway information, small-molecule chemical information, preclinical animal models, and clinical samples can empower phenotypic discovery at several critical levels.
Assuntos
Descoberta de Drogas/métodos , Fenótipo , Biologia Computacional , Perfilação da Expressão Gênica , Transdução de SinaisRESUMO
Phenotypic drug discovery (PDD) approaches do not rely on knowledge of the identity of a specific drug target or a hypothesis about its role in disease, in contrast to the target-based strategies that have been widely used in the pharmaceutical industry in the past three decades. However, in recent years, there has been a resurgence in interest in PDD approaches based on their potential to address the incompletely understood complexity of diseases and their promise of delivering first-in-class drugs, as well as major advances in the tools for cell-based phenotypic screening. Nevertheless, PDD approaches also have considerable challenges, such as hit validation and target deconvolution. This article focuses on the lessons learned by researchers engaged in PDD in the pharmaceutical industry and considers the impact of 'omics' knowledge in defining a cellular disease phenotype in the era of precision medicine, introducing the concept of a chain of translatability. We particularly aim to identify features and areas in which PDD can best deliver value to drug discovery portfolios and can contribute to the identification and the development of novel medicines, and to illustrate the challenges and uncertainties that are associated with PDD in order to help set realistic expectations with regard to its benefits and costs.
Assuntos
Desenho de Fármacos , Descoberta de Drogas/métodos , Indústria Farmacêutica/métodos , Animais , Humanos , Terapia de Alvo Molecular , Fenótipo , Medicina de Precisão/métodosRESUMO
Assessment of synergistic effects of drug combinations in vitro is a critical part of anticancer drug research. However, the complexities of dosing and analyzing two drugs over the appropriate range of doses have generally led to compromises in experimental design that restrict the quality and robustness of the data. In particular, the use of a single dose response of combined drugs, rather than a full two-way matrix of varying doses, has predominated in higher-throughput studies. Acoustic dispensing unlocks the potential of high-throughput dose matrix analysis. We have developed acoustic dispensing protocols that enable compound synergy assays in a 384-well format. This experimental design is considerably more efficient and flexible with respect to time, reagent usage, and labware than is achievable using traditional serial-dilution approaches. Data analysis tools integrated in Genedata Screener were used to efficiently deconvolute the combination compound mapping scheme and calculate compound potency and synergy metrics. We have applied this workflow to evaluate interactions among drugs targeting different nodes of the mitogen-activated protein kinase pathway in a panel of cancer cell lines.
Assuntos
Antineoplásicos/farmacologia , Tecnologia Biomédica/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Acústica , Combinação de Medicamentos , Interações Medicamentosas , SoluçõesRESUMO
Using structure-based design, a novel series of pyridone ERK1/2 inhibitors was developed. Optimization led to the identification of (S)-14k, a potent, selective, and orally bioavailable agent that inhibited tumor growth in mouse xenograft models. On the basis of its in vivo efficacy and preliminary safety profiles, (S)-14k was selected for further preclinical evaluation.
Assuntos
Antineoplásicos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Descoberta de Drogas , Camundongos , Modelos Moleculares , Conformação Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Piridonas/administração & dosagem , Piridonas/química , Ratos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There has been a resurgence of interest in the use of phenotypic screens in drug discovery as an alternative to target-focused approaches. Given that oncology is currently the most active therapeutic area, and also one in which target-focused approaches have been particularly prominent in the past two decades, we investigated the contribution of phenotypic assays to oncology drug discovery by analysing the origins of all new small-molecule cancer drugs approved by the US Food and Drug Administration (FDA) over the past 15 years and those currently in clinical development. Although the majority of these drugs originated from target-based discovery, we identified a significant number whose discovery depended on phenotypic screening approaches. We postulate that the contribution of phenotypic screening to cancer drug discovery has been hampered by a reliance on 'classical' nonspecific drug effects such as cytotoxicity and mitotic arrest, exacerbated by a paucity of mechanistically defined cellular models for therapeutically translatable cancer phenotypes. However, technical and biological advances that enable such mechanistically informed phenotypic models have the potential to empower phenotypic drug discovery in oncology.
Assuntos
Antineoplásicos/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias/tratamento farmacológico , Descoberta de Drogas/história , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Ensaios de Seleção de Medicamentos Antitumorais/história , Ensaios de Seleção de Medicamentos Antitumorais/tendências , Previsões , História do Século XX , História do Século XXI , Humanos , FenótipoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) has drawn significant interest in the neuroscience research community because it is one of the most compelling targets for a potential disease-modifying Parkinson's disease therapy. Herein, we disclose structurally diverse small molecule inhibitors suitable for assessing the implications of sustained in vivo LRRK2 inhibition. Using previously reported aminopyrazole 2 as a lead molecule, we were able to engineer structural modifications in the solvent-exposed region of the ATP-binding site that significantly improve human hepatocyte stability, rat free brain exposure, and CYP inhibition and induction liabilities. Disciplined application of established optimal CNS design parameters culminated in the rapid identification of GNE-0877 (11) and GNE-9605 (20) as highly potent and selective LRRK2 inhibitors. The demonstrated metabolic stability, brain penetration across multiple species, and selectivity of these inhibitors support their use in preclinical efficacy and safety studies.