RESUMO
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
A series of α-aryl pyrrolidine sulfonamide TRPA1 antagonists were advanced from an HTS hit to compounds that were stable in liver microsomes with retention of TRPA1 potency. Metabolite identification studies and physicochemical properties were utilized as a strategy for compound design. These compounds serve as starting points for further compound optimization.
Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Pirrolidinas/farmacologia , Sulfonamidas/farmacologia , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Animais , Canais de Cálcio , Humanos , Microssomos Hepáticos/metabolismo , Pirrolidinas/síntese química , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Canal de Cátion TRPA1RESUMO
Eukaryotic translation initiation factor 2B (eIF2B) is a key component of the integrated stress response (ISR), which regulates protein synthesis and stress granule formation in response to cellular insult. Modulation of the ISR has been proposed as a therapeutic strategy for treatment of neurodegenerative diseases such as vanishing white matter (VWM) disease and amyotrophic lateral sclerosis (ALS) based on its ability to improve cellular homeostasis and prevent neuronal degeneration. Herein, we report the small-molecule discovery campaign that identified potent, selective, and CNS-penetrant eIF2B activators using both structure- and ligand-based drug design. These discovery efforts culminated in the identification of DNL343, which demonstrated a desirable preclinical drug profile, including a long half-life and high oral bioavailability across preclinical species. DNL343 was progressed into clinical studies and is currently undergoing evaluation in late-stage clinical trials for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Leucoencefalopatias , Doenças Neurodegenerativas , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Mutação , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Encéfalo/metabolismo , Leucoencefalopatias/metabolismoRESUMO
Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.
Assuntos
Barreira Hematoencefálica , Oligonucleotídeos Antissenso , RNA Longo não Codificante , Receptores da Transferrina , Animais , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/administração & dosagem , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/metabolismo , Humanos , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , Camundongos , Transporte Biológico , Macaca fascicularis , Técnicas de Silenciamento de Genes , Distribuição TecidualRESUMO
RIPK1 is a master regulator of inflammatory signaling and cell death and increased RIPK1 activity is observed in human diseases, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). RIPK1 inhibition has been shown to protect against cell death in a range of preclinical cellular and animal models of diseases. SAR443060 (previously DNL747) is a selective, orally bioavailable, central nervous system (CNS)-penetrant, small-molecule, reversible inhibitor of RIPK1. In three early-stage clinical trials in healthy subjects and patients with AD or ALS (NCT03757325 and NCT03757351), SAR443060 distributed into the cerebrospinal fluid (CSF) after oral administration and demonstrated robust peripheral target engagement as measured by a reduction in phosphorylation of RIPK1 at serine 166 (pRIPK1) in human peripheral blood mononuclear cells compared to baseline. RIPK1 inhibition was generally safe and well-tolerated in healthy volunteers and patients with AD or ALS. Taken together, the distribution into the CSF after oral administration, the peripheral proof-of-mechanism, and the safety profile of RIPK1 inhibition to date, suggest that therapeutic modulation of RIPK1 in the CNS is possible, conferring potential therapeutic promise for AD and ALS, as well as other neurodegenerative conditions. However, SAR443060 development was discontinued due to long-term nonclinical toxicology findings, although these nonclinical toxicology signals were not observed in the short duration dosing in any of the three early-stage clinical trials. The dose-limiting toxicities observed for SAR443060 preclinically have not been reported for other RIPK1-inhibitors, suggesting that these toxicities are compound-specific (related to SAR443060) rather than RIPK1 pathway-specific.
Assuntos
Doença de Alzheimer , Esclerose Lateral Amiotrófica , Proteína Serina-Treonina Quinases de Interação com Receptores , Doença de Alzheimer/tratamento farmacológico , Esclerose Lateral Amiotrófica/tratamento farmacológico , Método Duplo-Cego , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidoresRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic risk factors for Parkinson's disease (PD). Increased LRRK2 kinase activity is thought to impair lysosomal function and may contribute to the pathogenesis of PD. Thus, inhibition of LRRK2 is a potential disease-modifying therapeutic strategy for PD. DNL201 is an investigational, first-in-class, CNS-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor. In preclinical models, DNL201 inhibited LRRK2 kinase activity as evidenced by reduced phosphorylation of both LRRK2 at serine-935 (pS935) and Rab10 at threonine-73 (pT73), a direct substrate of LRRK2. Inhibition of LRRK2 by DNL201 demonstrated improved lysosomal function in cellular models of disease, including primary mouse astrocytes and fibroblasts from patients with Gaucher disease. Chronic administration of DNL201 to cynomolgus macaques at pharmacologically relevant doses was not associated with adverse findings. In phase 1 and phase 1b clinical trials in 122 healthy volunteers and in 28 patients with PD, respectively, DNL201 at single and multiple doses inhibited LRRK2 and was well tolerated at doses demonstrating LRRK2 pathway engagement and alteration of downstream lysosomal biomarkers. Robust cerebrospinal fluid penetration of DNL201 was observed in both healthy volunteers and patients with PD. These data support the hypothesis that LRRK2 inhibition has the potential to correct lysosomal dysfunction in patients with PD at doses that are generally safe and well tolerated, warranting further clinical development of LRRK2 inhibitors as a therapeutic modality for PD.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Animais , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Lisossomos/metabolismo , Camundongos , Mutação , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , FosforilaçãoRESUMO
The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson's disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.
Assuntos
Doença de Parkinson , Animais , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Pulmão , Morfolinas , Mutação , Primatas , Pirimidinas , PirróisRESUMO
Ever since the world-shaping discovery of penicillin, nature's molecular diversity has been extensively screened for new medications and lead compounds in drug discovery. The search for agents intended to combat infectious diseases has been of particular interest and has enjoyed a high degree of success. Indeed, the history of antibiotics is marked with impressive discoveries and drug-development stories, the overwhelming majority of which have their origin in natural products. Chemistry, and in particular chemical synthesis, has played a major role in bringing naturally occurring antibiotics and their derivatives to the clinic, and no doubt these disciplines will continue to be key enabling technologies. In this review article, we highlight a number of recent discoveries and advances in the chemistry, biology, and medicine of naturally occurring antibiotics, with particular emphasis on total synthesis, analogue design, and biological evaluation of molecules with novel mechanisms of action.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Antibacterianos/química , Desenho de FármacosRESUMO
Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.
Assuntos
Pró-Fármacos/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , Sulfonamidas/farmacologia , Canal de Cátion TRPA1/antagonistas & inibidores , Animais , Cães , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Ligantes , Células Madin Darby de Rim Canino , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Prolina/síntese química , Prolina/farmacocinética , Ratos , Solubilidade , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Sulfonamidas/farmacocinética , Canal de Cátion TRPA1/químicaRESUMO
A new synthetic method providing expedient access to a wide range of polyfunctionalized N-hydroxyindoles (IV) is reported. These unique constructs are assembled by nucleophilic additions to in situ generated α,ß-unsaturated nitrones (III) through carbon-carbon and carbon-heteroatom bond formation. The new synthetic technology was applied to the synthesis of nocathiacin I (1) model systems (2 and 3a-c) containing the N-hydroxyindole structural motif.
RESUMO
Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor 14, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Desenho de Fármacos , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
There is an urgent need for the development of Parkinson's disease (PD) treatments that can slow disease progression. The leucine-rich repeat kinase 2 (LRRK2) protein has been genetically and functionally linked to PD, and modulation of LRRK2 enzymatic activity has been proposed as a novel therapeutic strategy. In this review, we describe the bioactivity of selected small molecules that have been used to inhibit LRRK2 kinase activity in vitro or in vivo. These compounds are important tools for understanding the cellular biology of LRRK2 and for evaluating the potential of LRRK2 inhibitors as disease-modifying PD therapies.
Assuntos
Antiparkinsonianos/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Antiparkinsonianos/farmacocinética , Antiparkinsonianos/farmacologia , Encéfalo/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Permeabilidade , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.
Assuntos
Pulmão/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Animais , Biomarcadores/sangue , Biomarcadores/urina , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Rim/anormalidades , Rim/efeitos dos fármacos , Rim/patologia , Rim/ultraestrutura , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Pulmão/anormalidades , Pulmão/patologia , Pulmão/ultraestrutura , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/química , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.
Assuntos
MAP Quinase Quinase Quinases/antagonistas & inibidores , Degeneração Neural/prevenção & controle , Doenças Neurodegenerativas/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Animais , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Descoberta de Drogas , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Modelos Químicos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , RatosRESUMO
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.
Assuntos
Encéfalo/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Pirimidinas/química , Animais , Linhagem Celular , Hepatócitos/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Macaca fascicularis , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Pirazóis/farmacocinética , Pirazóis/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We have recently reported a series of tetrahydroquinazoline (THQ) mTOR inhibitors that produced a clinical candidate 1 (GDC-0349). Through insightful design, we hoped to discover and synthesize a new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable, and efficacious mTOR inhibitor.
Assuntos
Inibidores Enzimáticos/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tropanos/farmacologia , Cromatografia Líquida , Inibidores Enzimáticos/química , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Tropanos/químicaRESUMO
The modulation of LRRK2 kinase activity by a selective small molecule inhibitor has been proposed as a potentially viable treatment for Parkinson's disease. By using aminopyrazoles as aniline bioisosteres, we discovered a novel series of LRRK2 inhibitors. Herein, we describe our optimization effort that resulted in the identification of a highly potent, brain-penetrant aminopyrazole LRRK2 inhibitor (18) that addressed the liabilities (e.g., poor solubility and metabolic soft spots) of our previously disclosed anilino-aminopyrimidine inhibitors. In in vivo rodent PKPD studies, 18 demonstrated good brain exposure and engendered significant reduction in brain pLRRK2 levels post-ip administration. The strategies of bioisosteric substitution of aminopyrazoles for anilines and attenuation of CYP1A2 inhibition described herein have potential applications to other drug discovery programs.
RESUMO
Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.
RESUMO
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.
Assuntos
Modelos Moleculares , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Biologia Computacional , Humanos , Janus Quinase 2/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Morfolinas/química , Morfolinas/farmacocinética , Morfolinas/farmacologia , Permeabilidade , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacocinética , Tiazóis/farmacologiaRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser(1292) occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser(1292) autophosphorylation. Mutation of Ser(1292) to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser(1292) as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser(1292) autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser(1292) autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.