Alzheimer s disease (AD) is a neurodegenerative disease whose most common symptom is a progressive loss of cognitive functions and intellectual

Transcription

1 Maryam Farrokhnia 1

2 Alzheimer s disease (AD) is a neurodegenerative disease whose most common symptom is a progressive loss of cognitive functions and intellectual abilities, resulting in serious impairment of daily life activities. AD accounts for 70% of dementia cases globally and it is estimated that more than 20 million people are currently affected by it worldwide. 2

3 pharmacological treatments of AD are limited to only two groups of drugs: acetylcholine esterase (ACE) inhibitors and memantine. While ACE inhibitors increase the neuronal levels of acetylcholine, memantine acts as an antagonist of the neuronal N-methyl- d -aspartate (NMDA) receptor, reducing the neuronal overstimulation caused by glutamic acid. Unfortunately, these drugs are not entirely effective and their benefits are limited to the early stages of the disease. 3

4 The cholinergic hypothesis The β-amyloid hypothesis The tau hypothesis 4

5 In the continuous quest for novel small molecules therapeutics as potential treatments for AD, medicinal chemists have recently turned their efforts toward the inhibition of several protein kinases involved in tau phosphorylation. Targets such as GSK-3β, cdk5, p38, Erk1 and 2, JNK, CK1δ and CK1e directly contribute to tau phosphorylation and several small molecule inhibitors of these proteins have been reported over the last two decades. 5

6 DYRK1A is a eukaryotic serinethreonine protein kinase which belongs to the super family of the CMGC group of proline-argininedirected serinethreonine kinases. The DYRK family is highly conserved and comprises five mammalian subtypes including 1A, 1B, 2, 3 and 4. Dualspecificity kinases phosphorylate a single tyrosine residue in their activation loop (Tyr321 in DYRK1A), thus catalyzing their auto-activation. 6

7 The DYRK1A gene is located in the Down syndrome critical region (DSCR) of chromosome 21 (21q22.2) and was identified as the candidate gene responsible for the effects of Down syndrome (DS), such as intellectual disability, microcephaly and age associated neurodegeneration. 7

8 Increased levels of DYRK1A have been reported in various areas of the brain of AD patients. In particular, the percentage of DYRK1A-positive nuclei in the frontal cortex of AD patients is approximately 10% compared with 0.5% in normal brains. 8

9 DYRK1A is involved in two main neurodegenerative processes of AD: the formation of APs and NFTs. DYRK1A directly phosphorylates tau proteins at 11 serine and threonine residues, some of which have been detected to be phosphorylated in tau aggregates of NFTs of AD brains. 9

10 The observations would suggest a role of DYRK1A in the development of AD and inhibition of this target may be beneficial for AD patients, for example, by resulting in reduced symptomatology and improved life conditions. 10

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13 The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. 13

14 Screening for Pharmacological Inhibitors of DYRKs and CLKs: Leucettamine B as an Inhibitory Scaffold. Selectivity of Leucettine L41. (1) Protein Kinase Screens. Structural Insight into the Binding of Leucettine L41 with Its Kinase Targets. Inhibition of Endogenous Kinase Targets in Cells by Leucettine L41. Leucettine L41 Provides Protection against Glutamate- Induced Cell Death. Leucettine L41 Reduces Neurodegeneration in Amyloid Precursor Protein Transfected Brain Slices 14

15 To screen for pharmacological inhibitors of DYRKs and CLKs, DYRK1A and CLK1 were selected as the primary screening targets, while the other DYRKs and CLKs were used in secondary selectivity assays. 15

16 Results showed that a few well characterized kinase inhibitors also inhibited DYRK1A and CLK1 and suggested that selective DYRK1A and CLK1 inhibitors might be derived from some of these scaffolds which included indirubins, meriolins, and purines. In addition, most but not all DYRK1A inhibitors were efficient on CLK1 and many were active on PIM1. Harmine, a reference inhibitor for DYRK1A, also inhibited CLK1, while TG003, a reference CLK1 inhibitor, inhibited DYRK1A 16

17 A library of 40,000+ purified, low molecular weight compounds with high chemical diversity was screened in the DYRK1A/CLK1 medium throughput screen. This allowed the identification of a small number of inhibitory scaffolds including the expected harmine and some of its analogues. Among these scaffolds was leucettamine B, a natural product initially identified from marine calcareous sponges of the genus Leucetta. 17

18 To investigate the selectivity of leucettine L41, we first screened the three compounds against a panel of 27 purified kinases. 18

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22 To better understand these kinase/ inhibitor selectivities and to generate models for structure based optimization, we cocrystallized leucettine L41 with DYRK1A, DYRK2, CLK3, and PIM1 22

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26 To confirm the effects of leucettine L41 on endogenous cellular kinases in vivo, we used HT22 cells. Cells were exposed for 4 h to increasing concentrations of leucettine L41 and collected for analysis of their DYRK1A and GSK-3 expression and activity levels. Results showed that although endogenous GSK-3 activity remained constant, DYRK1A activity was dose-dependently inhibited. 26

27 Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3β. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimer s disease. 27

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