RESUMO
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
To enable the clinical development of our CNS casein kinase 1 delta/epsilon (CK1δ/ε) inhibitor project, we investigated the possibility of developing a CNS positron emission tomography (PET) radioligand. For this effort, we focused our design and synthesis efforts on the initial CK1δ/ε inhibitor HTS hits with the goal of identifying a compound that would fulfill a set of recommended PET ligand criteria. We identified [3H]PF-5236216 (9) as a tool ligand that meets most of the key CNS PET attributes including high CNS MPO PET desirability score and kinase selectivity, CNS penetration, and low nonspecific binding. We further used [3H]-9 to determine the binding affinity for PF-670462, a literature CK1δ/ε inhibitor tool compound. Lastly, [3H]-9 was used to measure in vivo target occupancy (TO) of PF-670462 in mouse and correlated TO with CK1δ/ε in vivo pharmacology (circadian rhythm modulation).
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Caseína Quinase I/antagonistas & inibidores , Lactamas , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Animais , Células COS , Caseína Quinase I/metabolismo , Chlorocebus aethiops , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Desenho de Fármacos , Humanos , Lactamas/síntese química , Lactamas/farmacocinética , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição AleatóriaRESUMO
INTRODUCTION: Leucine-rich repeat kinase 2 (LRRK2) is a member of the Tyrosine Kinase-Like (TKL) branch of the kinome tree and is a multi-domain protein that includes GTPase and kinase activity. While genome-wide association studies (GWAS) has linked LRRK2 with Crohn's disease and leprosy, it has received the greatest attention due to it being implicated as one of the genetic loci associated with autosomal dominant inheritance in Parkinson's disease (PD). Areas covered: In this review, the small molecule patent literature from 2014-2016 with a focus on composition of matter and use patents was surveyed. Scifinder was primarily searched using 'LRRK2' as the query to identify all relevant literature and then triaged for small molecule patents. Expert opinion: The patent landscape around LRRK2 continues to develop. The early patents covered using existing kinase inhibitors for use against LRRK2. This evolved to compounds specifically designed for selectivity against LRRK2, but key exemplified compounds lacked sufficient brain exposure to affect sufficient efficacy. More recent compounds have addressed this deficiency and show greater potential for treating PD. While potency will be necessary to generate medicines with low human daily doses, brain penetration and safety will be the key differentiators for ultimately determining the most effective LRRK2 disease-modifying treatment for PD.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Doença de Parkinson/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Desenho de Fármacos , Estudo de Associação Genômica Ampla , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Patentes como Assunto , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Distribuição TecidualRESUMO
Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression.
Assuntos
Antiparkinsonianos/farmacologia , Doença de Parkinson/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , alfa-Sinucleína/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Injeções Intraventriculares , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/metabolismoRESUMO
Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.
Assuntos
Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteoma/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Sequência de Aminoácidos , Animais , Área Sob a Curva , Encéfalo/metabolismo , Cristalografia por Raios X , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação de Sentido Incorreto , Nitrilas/química , Nitrilas/farmacocinética , Doença de Parkinson/tratamento farmacológico , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteoma/química , Proteoma/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirróis/química , Pirróis/farmacocinética , RatosRESUMO
Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD). The most common mutant, G2019S, increases kinase activity, thus LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the structure, potential ligand-protein binding interactions, and pharmacological profiling of potent and highly selective kinase inhibitors based on a triazolopyridazine chemical scaffold.
Assuntos
Compostos Heterocíclicos com 2 Anéis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Piridazinas/farmacologia , Triazóis/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/química , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Piridazinas/síntese química , Piridazinas/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.
Assuntos
Pirazinas/síntese química , Pirazóis/síntese química , Receptor de Glutamato Metabotrópico 5/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Administração Oral , Regulação Alostérica , Animais , Antiparkinsonianos/efeitos adversos , Disponibilidade Biológica , Permeabilidade da Membrana Celular , Cães , Discinesia Induzida por Medicamentos/tratamento farmacológico , Células HEK293 , Humanos , Hipersensibilidade Tardia/induzido quimicamente , Levodopa/efeitos adversos , Macaca fascicularis , Células Madin Darby de Rim Canino , Masculino , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/etiologia , Doença de Parkinson/fisiopatologia , Pirazinas/farmacologia , Pirazinas/toxicidade , Pirazóis/farmacologia , Pirazóis/toxicidade , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Genetic mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to autosomal dominant Parkinson's disease. The most prevalent mutation, G2019S, results in enhanced LRRK2 kinase activity that potentially contributes to the etiology of Parkinson's disease. Consequently, disease progression is potentially mediated by poorly characterized phosphorylation-dependent LRRK2 substrate pathways. To address this gap in knowledge, we transduced SH-SY5Y neuroblastoma cells with LRRK2 G2019S via adenovirus, then determined quantitative changes in the phosphoproteome upon LRRK2 kinase inhibition (LRRK2-IN-1 treatment) using stable isotope labeling of amino acids in culture combined with phosphopeptide enrichment and LC-MS/MS analysis. We identified 776 phosphorylation sites that were increased or decreased at least 50% in response to LRRK2-IN-1 treatment, including sites on proteins previously known to associate with LRRK2. Bioinformatic analysis of those phosphoproteins suggested a potential role for LRRK2 kinase activity in regulating pro-inflammatory responses and neurite morphology, among other pathways. In follow-up experiments, LRRK2-IN-1 inhibited lipopolysaccharide-induced tumor necrosis factor alpha (TNFα) and C-X-C motif chemokine 10 (CXCL10) levels in astrocytes and also enhanced multiple neurite characteristics in primary neuronal cultures. However, LRRK2-IN-1 had almost identical effects in primary glial and neuronal cultures from LRRK2 knockout mice. These data suggest LRRK2-IN-1 may inhibit pathways of perceived LRRK2 pathophysiological function independently of LRRK2 highlighting the need to use multiple pharmacological tools and genetic approaches in studies determining LRRK2 function.
Assuntos
Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteômica , Adenoviridae/genética , Animais , Astrócitos/metabolismo , Células Cultivadas , Quimiocina CXCL10/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Lipopolissacarídeos/farmacologia , Espectrometria de Massas , Camundongos , Camundongos Knockout , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Fosforilação , Plasmídeos/genética , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Titânio/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.
Assuntos
Caseína Quinase Idelta/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas/química , Ligantes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos MolecularesRESUMO
A series of acyloxyalkyl and amidooxyalkyl ketones appended to a carbobenzyloxy aspartic acid core have been prepared. The most potent of these new inhibitors was 4i with a K(i) of 0.5 microM. These two series provide an improved understanding of the binding requirements for the hydrophobic prime side of ICE.
Assuntos
Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Cetonas/farmacologia , Humanos , Modelos Moleculares , Monócitos/efeitos dos fármacosRESUMO
Succinic acid amides have been found to be effective P2-P3 scaffold replacements for peptidic ICE inhibitors. Heteroarylalkyl fragments occupying the P4 position provided access to compounds with nM affinities. Utilization of an acylal prodrug moiety was required to overcome biopharmaceutical issues which led to the identification of 17f, a potential clinical candidate.
Assuntos
Amidas/química , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Ácido Succínico/química , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacocinética , Meia-Vida , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We describe three novel regioisomeric series of aryl naphthyridine analogs, which are potent antagonists of the Class III GPCR mGlu5 receptor. The synthesis and in vitro and in vivo pharmacological activities of these analogs are discussed.
Assuntos
Naftiridinas/síntese química , Naftiridinas/farmacologia , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Cricetulus , Ratos , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/fisiologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Rational replacement of the alkyne linker of mGluR5 antagonist MPEP gave 7-arylquinolines. SAR optimization gave an orally active compound with high affinity for the MPEP binding site.
Assuntos
Desenho de Fármacos , Antagonistas de Aminoácidos Excitatórios/química , Antagonistas de Aminoácidos Excitatórios/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Modelos Moleculares , Estrutura Molecular , Piridinas/química , Piridinas/farmacologia , Receptor de Glutamato Metabotrópico 5 , Relação Estrutura-AtividadeRESUMO
Several beta-amino tetrazole analogs of gabapentin 1 and pregabalin 2 were prepared by one of two convergent, highly efficient routes, and their affinity for the alpha(2)-delta protein examined. Two select compounds with potent affinity for alpha(2)-delta, 8a and 16a, were subsequently tested in vivo in an audiogenic seizure model and found to elicit protective effects.
Assuntos
Anticonvulsivantes/síntese química , Anticonvulsivantes/farmacologia , Ácidos Carboxílicos/química , Epilepsia Reflexa/prevenção & controle , Ácido gama-Aminobutírico/análogos & derivados , Aminas/síntese química , Aminas/química , Aminas/farmacologia , Animais , Anticonvulsivantes/química , Sítios de Ligação , Ácidos Cicloexanocarboxílicos/síntese química , Ácidos Cicloexanocarboxílicos/química , Ácidos Cicloexanocarboxílicos/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Gabapentina , Camundongos , Camundongos Endogâmicos DBA , Estrutura Molecular , Pregabalina , Subunidades Proteicas/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade , Ácido gama-Aminobutírico/síntese química , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/farmacologiaRESUMO
The formation of reactive metabolites from a number of compounds was studied in vitro using a mixture of non-labeled and stable isotope labeled glutathione (GSH) as a trapping agent. GSH was labeled by incorporating [1,2-(13)C(2),(15)N]glycine into the tripeptide to give an overall increase of 3 Da over the naturally occurring substance. Detection and characterization of reactive metabolites was greatly facilitated by using the data-dependent scanning features of the linear ion trap mass spectrometers to give complimentary and confirmatory data in a single analytical run. A comparison was made by analyzing the samples simultaneously on a triple-stage quadrupole mass spectrometer operated in the constant neutral loss mode. The compounds studied included 2-acetamidophenol, 3-acetamidophenol, 4-acetamidophenol (acetaminophen), and flufenamic acid. GSH adducts for each of these compounds produced a characteristic pattern of 'twin ions' separated by 3 Da in the mass spectral data. This greatly facilitated the detection and characterization of any GSH-related adducts present in the microsomal extracts. Furthermore, characterization of these adducts was greatly facilitated by the rapid scanning capability of linear ion trap instruments that provided full-scan, MS/MS and MS(3) data in one single analysis. This method of detecting and characterizing reactive metabolites generated in vitro was found to be far superior to any of the existing methods previously employed in this laboratory. The combination of two techniques, stable isotope labeled glutathione and linear ion traps, provided a very sensitive and specific method of identifying compounds capable of producing reactive metabolites in a discovery setting. The complimentary set of mass spectral data (including full-scan, MS/MS and MS(3) mass spectra), obtained rapidly in a single analysis with the linear ion trap instruments, greatly accelerated identification of metabolically bioactivated soft spots on the molecules. This in turn enabled chemists to rapidly design out the potential metabolic liability from the back-up compounds by making appropriate structural modifications.