ABSTRACT
Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by 8 (cell IC50: 571 nM). Systematic optimization of this series of analogs led to the discovery of 31, a potent (cell IC50: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat Kp,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser 422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation of this target may be relevant in the treatment of Alzheimer's disease and other tauopathies.
Subject(s)
Brain/metabolism , Drug Design , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , tau Proteins/metabolism , Animals , Humans , Indazoles/chemistry , Indazoles/metabolism , Indazoles/pharmacology , Mice , Molecular Targeted Therapy , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , RatsABSTRACT
Tau-tubulin kinase 1 (TTBK1) is a CNS-specific, kinase that has been implicated in the pathological phosphorylation of tau in Alzheimer's Disease (AD) and Frontotemporal Dementia (FTD). TTBK1 is a challenging therapeutic target because it shares a highly conserved catalytic domain with its homolog, TTBK2, a ubiquitously expressed kinase genetically linked to the disease spinocerebellar ataxia type 11. The present study attempts to elucidate the functional distinctions between the TTBK isoforms and increase our understanding of them as distinct targets for the treatment of neurodegenerative disease. We demonstrate that in cortical neurons, TTBK1, not TTBK2, is the isoform responsible for tau phosphorylation at epitopes enriched in tauopathies such as Serine 422. In addition, although our elucidation of the crystal structure of the TTBK2 kinase domain indicates almost identical structural similarity with TTBK1, biochemical and cellular assays demonstrate that the enzymatic activity of these two proteins is regulated by a combination of unique extra-catalytic sequences and autophosphorylation events. Finally, we have identified an unbiased list of neuronal interactors and phosphorylation substrates for TTBK1 and TTBK2 that highlight the unique cellular pathways and functional networks that each isoform is involved in. This data address an important gap in knowledge regarding the implications of targeting TTBK kinases and may prove valuable in the development of potential therapies for disease.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Cerebral Cortex/pathology , Epitopes/metabolism , HEK293 Cells , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Mice, Transgenic , Neurons/metabolism , Phosphopeptides/chemistry , Phosphopeptides/metabolism , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Domains , Protein Serine-Threonine Kinases/chemistry , Proteomics , Serine/metabolism , Structural Homology, Protein , tau Proteins/metabolismABSTRACT
Hyperphosphorylated tau protein is a pathological hallmark of numerous neurodegenerative diseases and the level of tau pathology is correlated with the degree of cognitive impairment. Tau hyper-phosphorylation is thought to be an early initiating event in the cascade leading to tau toxicity and neuronal death. Inhibition of tau phosphorylation therefore represents an attractive therapeutic strategy. However, the widespread expression of most kinases and promiscuity of their substrates, along with poor selectivity of most kinase inhibitors, have resulted in systemic toxicities that have limited the advancement of tau kinase inhibitors into the clinic. We therefore focused on the CNS-specific tau kinase, TTBK1, and investigated whether selective inhibition of this kinase could represent a viable approach to targeting tau phosphorylation in disease. In the current study, we demonstrate that TTBK1 regulates tau phosphorylation using overexpression or knockdown of this kinase in heterologous cells and primary neurons. Importantly, we find that TTBK1-specific phosphorylation of tau leads to a loss of normal protein function including a decrease in tau-tubulin binding and deficits in tubulin polymerization. We then describe the use of a novel, selective small molecule antagonist, BIIB-TTBK1i, to study the acute effects of TTBK1 inhibition on tau phosphorylation in vivo. We demonstrate substantial lowering of tau phosphorylation at multiple sites implicated in disease, suggesting that TTBK1 inhibitors may represent an exciting new approach in the search for neurodegenerative disease therapies.
Subject(s)
Central Nervous System Diseases/enzymology , Central Nervous System Diseases/pathology , Central Nervous System/enzymology , Central Nervous System/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , tau Proteins/metabolism , Animals , Cells, Cultured , Male , Mice, Inbred C57BL , Microtubules/drug effects , Microtubules/metabolism , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Organ Specificity , Phosphorylation/drug effects , Polymerization , Protein Binding/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , Small Molecule Libraries/pharmacology , Tubulin/metabolismABSTRACT
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.
Subject(s)
Nitriles/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proteome/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrroles/pharmacology , Amino Acid Sequence , Animals , Area Under Curve , Brain/metabolism , Crystallography, X-Ray , Drug Discovery , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice, Inbred C57BL , Mice, Transgenic , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutation, Missense , Nitriles/chemistry , Nitriles/pharmacokinetics , Parkinson Disease/drug therapy , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Proteome/chemistry , Proteome/metabolism , Pyrimidines/chemistry , Pyrimidines/pharmacokinetics , Pyrroles/chemistry , Pyrroles/pharmacokinetics , RatsABSTRACT
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.
Subject(s)
Heterocyclic Compounds, 2-Ring/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Pyridazines/pharmacology , Triazoles/pharmacology , Crystallography, X-Ray , Dose-Response Relationship, Drug , Heterocyclic Compounds, 2-Ring/chemical synthesis , Heterocyclic Compounds, 2-Ring/chemistry , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary/drug effects , Pyridazines/chemical synthesis , Pyridazines/chemistry , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/chemistryABSTRACT
A novel transformation of 2-(trimethylsilyl)phenyl trifluoromethanesulfonate aryne precursors is described.
ABSTRACT
Protein kinase C θ (PKCθ) has a central role in T cell activation and survival; however, the dependency of T cell responses to the inhibition of this enzyme appears to be dictated by the nature of the antigen and by the inflammatory environment. Studies in PKCθ-deficient mice have demonstrated that while antiviral responses are PKCθ-independent, T cell responses associated with autoimmune diseases are PKCθ-dependent. Thus, potent and selective inhibition of PKCθ is expected to block autoimmune T cell responses without compromising antiviral immunity. Herein, we describe the development of potent and selective PKCθ inhibitors, which show exceptional potency in cells and in vivo. By use of a structure based rational design approach, a 1000-fold improvement in potency and 76-fold improvement in selectivity over closely related PKC isoforms such as PKCδ were obtained from the initial HTS hit, together with a big improvement in lipophilic efficiency (LiPE).
Subject(s)
Autoimmune Diseases/drug therapy , Isoenzymes/antagonists & inhibitors , Piperazines/chemical synthesis , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Pyridines/chemical synthesis , Animals , Drug Design , Humans , Inhibitory Concentration 50 , Interleukin-2/antagonists & inhibitors , Lymphocyte Activation/drug effects , Mice , Piperazines/pharmacokinetics , Protein Kinase C-theta , Pyridines/pharmacokinetics , T-Lymphocytes/immunologyABSTRACT
A method for the Pd-catalyzed coupling of 2-aminothiazole derivatives with aryl bromides and triflates is described. Significantly, for this class of nucleophiles, the coupling exhibits a broad substrate scope and proceeds with a reasonable catalyst loading. Furthermore, an interesting effect of acetic acid as an additive is uncovered that facilitates catalyst activation.
Subject(s)
Palladium/chemistry , Thiazoles/chemistry , Catalysis , Hydrocarbons, Brominated/chemistry , Mesylates/chemistry , Molecular StructureSubject(s)
Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Drug Interactions , Pharmacokinetics , Animals , Apoproteins/metabolism , Cytochrome P-450 Enzyme System/chemistry , Drug Discovery , Heme/metabolism , Humans , Models, Biological , Structure-Activity Relationship , Time FactorsABSTRACT
We report our studies on the use of two catalyst systems, based on the ligands BrettPhos (1) and RuPhos (2), which provide the widest scope for Pd-catalyzed C-N cross-coupling reactions to date. Often low catalyst loadings and short reaction times can be used with functionalized aryl and heteroaryl coupling partners. The reactions are highly robust and can be set up and performed without the use of a glovebox. These catalysts should find wide application in the synthesis of complex molecules including pharmaceuticals, natural products and functional materials.
ABSTRACT
Simple and efficient procedures for the Pd-catalyzed cross-coupling of primary and secondary amines with halo-7-azaindoles(pyrrolo[2,3-b]pyridine) are presented. Previously, no general method was available to ensure the highly selective reaction of the heteroaryl halide in the presence of the unprotected azaindole N-H. Using palladium precatalysts recently reported by our group, such reactions are easily accomplished under mild conditions that can be applied to cross-coupling reactions with a wide array of aliphatic and aromatic amines.
Subject(s)
Indoles/chemistry , Palladium/chemistry , Amination , Catalysis , Ligands , Molecular StructureABSTRACT
The Pd-catalyzed amination of unprotected benzo-fused heterocycles is reported, which allows for greater flexibility and efficiency in the modification of this important class of molecules. The generality of these simple and efficient procedures is demonstrated through the synthesis of a wide variety of structural types.
Subject(s)
Heterocyclic Compounds/chemistry , Palladium/chemistry , Amination , Catalysis , Ligands , Molecular StructureABSTRACT
Adding an aryne to a tertiary allylamine affords o-allylaniline products of an aza-Claisen rearrangement. The aryne simultaneously provides the pi component for the rearrangement and the quaternization event that lowers the activation energy for the sigmatropic shift. The reaction was applied to the synthesis of medium-ring benzannulated amines (see scheme).
Subject(s)
Aza Compounds/chemistry , Benzene Derivatives/chemistry , Amines/chemistry , Catalysis , Piperidines/chemical synthesis , Piperidines/chemistryABSTRACT
Aryl iodides have been introduced as electrophiles in the three-component Heck coupling of arynes. Following optimization studies to favor three- versus two-component coupling, the reaction proceeds in good yield to afford a variety of functionalized biaryls.
Subject(s)
Palladium/chemistry , Catalysis , Iodides/chemistryABSTRACT
A new three-component coupling reaction of benzyne is described that uses two intermolecular carbopalladation reactions to produce 1,2-functionalized benzene derivatives.