Kyle Powell

Nucleation-dependent polymerization is an essential component of amyloid-mediated neuronal cell death

Accumulating evidence suggests that amyloid protein aggregation is pathogenic in many diseases, including Alzheimers disease. However, the mechanisms by which protein aggregation mediates cellular dysfunction and overt cell death are unknown. Recent reports have focused on the potential role of amyloid oligomers or protofibrils as a neurotoxic form of amyloid-β (Aβ) and related amyloid aggregates. Here we describe studies indicating that overt neuronal cell death mediated by Aβ1-40 is critically dependent on ongoing Aβ1-40 polymerization and is not mediated by a single stable species of neurotoxic aggregate. The extent and rate of neuronal cell death can be controlled by conditions that alter the rate of Aβ polymerization. The results presented here indicate that protofibrils and oligomeric forms of Aβ most likely generate neuronal cell death through a nucleation-dependent process rather than acting as direct neurotoxic ligands. These findings bring into question the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide formazan assay (MTT assay) as a reporter of Aβ-mediated neuronal cell death and suggest that diffusible Aβ protofibrils and oligomers more likely mediate subtle alterations of synaptic function and long-term potentiation rather than overt neuronal cell death. These results have been extended to Aβ1-42, the non-Aβ component of Alzheimers disease amyloid plaques, and human amylin, suggesting that nucleation-dependent polymerization is a common mechanism of amyloid-mediated neuronal cell death. Our findings indicate that ongoing amyloid fibrillogenesis may be an essential mechanistic process underlying the pathogenesis associated with protein aggregation in amyloid disorders.

Polo-like Kinase 2 (PLK2) Phosphorylates α-Synuclein at Serine 129 in Central Nervous System

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system.

α2β1 and αVβ1 integrin signaling pathways mediate amyloid-β-induced neurotoxicity

Pathological hallmarks of Alzheimers disease are the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles, and neurodegeneration. The principal component of amyloid plaques is the amyloid-β peptide (Aβ). Accumulating evidence indicates that Aβ may play a causal role in Alzheimers disease. In this report, we demonstrate that Aβ deposition and neurotoxicity in human cortical primary neurons are mediated through α2β1 and αVβ1 integrins using specific integrin-blocking antibodies. An aberrant integrin signaling pathway causing the neurotoxicity is mediated through Pyk2. The role of α2β1 and αVβ1 integrins can be extended to another amyloidosis using an amylin in vitro neurotoxicity model. These results indicate that the α2β1 and αVβ1 integrin signaling pathway may be critical components of neurodegeneration in Alzheimers disease and that integrins may recognize and be activated by a shared structural motif of polymerizing amyloidogenic proteins.

Highly selective c-Jun N-terminal kinase (JNK) 2 and 3 inhibitors with in vitro CNS-like pharmacokinetic properties prevent neurodegeneration.

In this Letter, we describe the discovery of selective JNK2 and JNK3 inhibitors, such as 10, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs, p38α and ERK2. Substitution of the naphthalene ring affords an isoform selective JNK3 inhibitor, 30, with approximately 10-fold selectivity over both JNK1 and JNK2. A naphthalene ring penetrates deep into the selectivity pocket accounting for the differentiation amongst the kinases. Interestingly, the gatekeeper Met146 sulfide interacts with the naphthalene ring in a sulfur-π stacking interaction. Compound 38 ameliorates neurotoxicity induced by amyloid-β in human cortical neurons. Lastly, we demonstrate how to install propitious in vitro CNS-like properties into these selective inhibitors.

Design and synthesis of a novel, orally active, brain penetrant, tri-substituted thiophene based JNK inhibitor.

The SAR of a series of tri-substituted thiophene JNK3 inhibitors is described. By optimizing both the N-aryl acetamide region of the inhibitor and the 4-position of the thiophene we obtained single digit nanomolar compounds, such as 47, which demonstrated an in vivo effect on JNK activity when dosed orally in our kainic acid mouse model as measured by phospho-c-jun reduction.

Design and synthesis of brain penetrant selective JNK inhibitors with improved pharmacokinetic properties for the prevention of neurodegeneration.

The SAR of a series of brain penetrant, trisubstituted thiophene based JNK inhibitors with improved pharmacokinetic properties is described. These compounds were designed based on information derived from metabolite identification studies which led to compounds such as 42 with lower clearance, greater brain exposure and longer half life compared to earlier analogs.

Design and synthesis of disubstituted thiophene and thiazole based inhibitors of JNK.

From high throughput screening, we discovered compound 1, the prototype for a series of disubstituted thiophene inhibitors of JNK which is selective towards closely related MAP kinases p38 and Erk2. Herein we describe the evolution of these compounds to a novel class of thiophene and thiazole JNK inhibitors that retain favorable solubility, permeability, and P-gp properties for development as CNS agents for treatment of neurodegeneration. Compound 61 demonstrated JNK3 IC(50)=77 nM and retained the excellent broad kinase selectivity observed for the series.

Highly selective c-Jun N-terminal kinase (JNK) 3 inhibitors with in vitro CNS-like pharmacokinetic properties II. Central core replacement.

In this Letter, we describe the evolution of selective JNK3 inhibitors from 1, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs. Strong SAR was found for substitution of the naphthalene ring, as well as for inhibitors adopting different central scaffolds. Significant potency gains were appreciated by inverting the polarity of the thione of the parent triazolothione 1, resulting in potent compounds with attractive pharmacokinetic profiles.