1-(Benzo[d]thiazol-2-yl)-3-phenylureas as dual inhibitors of casein kinase 1 and ABAD enzymes for treatment of neurodegenerative disorders

Several neurodegenerative disorders including Alzheimer’s disease (AD) have been connected with deregulation of casein kinase 1 (CK1) activity. Inhibition of CK1 therefore presents a potential therapeutic strategy against such pathologies. Recently, novel class of CK1-specific inhibitors with N-(benzo[d]thiazol-2-yl)-2-phenylacetamide structural scaffold has been discovered. 1-(benzo[d]thiazol-2-yl)-3-phenylureas, on the other hand, are known inhibitors amyloid-beta binding alcohol dehydrogenase (ABAD), an enzyme also involved in pathophysiology of AD. Based on their tight structural similarity, we decided to evaluate series of previously published benzothiazolylphenylureas, originally designed as ABAD inhibitors, for their inhibitory activity towards CK1. Several compounds were found to be submicromolar CK1 inhibitors. Moreover, two compounds were found to inhibit both, ABAD and CK1. Such dual-activity could be of advantage for AD treatment, as it would simultaneously target two distinct pathological processes involved in disease’s progression. Based on PAMPA testing both compounds were suggested to permeate the blood-brain barrier, which makes them, together with their unique dual activity, interesting lead compounds for further development.     

Evaluation of an Allosteric BACE Inhibitor Peptide to Identify Mimetics that Can Interact with the Loop F Region of the Enzyme and Prevent APP Cleavage

The aspartyl protease BACE1 (BACE) has emerged as an appealing target for reduction of amyloid-β in Alzheimer's disease. The clinical fate of active-site BACE inhibitors may depend on potential side effects related to enzyme and substrate selectivity. One strategy to reduce this risk is through development of allosteric inhibitors that interact with and modulate the Loop F region unique to BACE1. Previously, a BACE-inhibiting antibody (Ab) was shown by co-crystallization to bind and induce conformational changes of Loop F, resulting in backbone perturbations at the distal S6 and S7 subsites, preventing proper binding of a long APP-like substrate to BACE and inhibiting its cleavage. In an effort to discover small Loop F-interacting molecules that mimic the Ab inhibition, we evaluated a peptide series with a YPYF(I/L)P(L/Y) motif that was reported to bind a BACE exosite. Our studies show that the most potent inhibitor from this series, peptide 65007, has a similar substrate cleavage profile to the Ab and reduces sAPPβ levels in cell models and primary neurons. As our modeling indicates, it interacts with the Loop F region causing a conformational shift of the BACE protein backbone near the distal subsites. The peptide-bound enzyme adopts a conformation that closely overlays with the crystal structure (PDB: 3R1G) from Ab binding. Importantly, peptide 65007 appears to be BACE substrate and enzyme selective, showing little inhibition of NRG1, PSGL1, CHL1, or Cat D. Thus, peptide 65007 is a promising lead for discovery of Loop F-interacting small-molecule mimetics as allosteric inhibitors of BACE.     

Focus: The Aging Brain: Amyloid-beta Alzheimer targets — protein processing,lipid rafts,and amyloid-beta pores

Amyloid beta (Aβ), the hallmark of Alzheimer’s Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aβ fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aβ aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aβ aggregates, and current therapeutics are summarized in this review.     

The amyloid-β1–42-oligomer interacting peptide D-AIP possesses favorable biostability,pharmacokinetics, and brain region distribution

We have previously developed a unique 8-amino acid Aβ42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer’s disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aβ42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid “seeding” in vitro (i.e., freshly monomerized versus preaggregated Aβ42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aβ42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer’s disease.     

Antibodies and protein misfolding: From structural research tools to therapeutic strategies

Protein misfolding disorders, including the neurodegenerative conditions Alzheimer's disease (AD) and Parkinson's disease (PD) represent one of the major medical challenges or our time. The underlying molecular mechanisms that govern protein misfolding and its links with disease are very complex processes, involving the formation of transiently populated but highly toxic molecular species within the crowded environment of the cell and tissue. Nevertheless, much progress has been made in understanding these events in recent years through innovative experiments and therapeutic strategies, and in this review we present an overview of the key roles of antibodies and antibody fragments in these endeavors. We discuss in particular how these species are being used in combination with a variety of powerful biochemical and biophysical methodologies, including a range of spectroscopic and microscopic techniques applied not just in vitro but also in situ and in vivo, both to gain a better understanding of the mechanistic nature of protein misfolding and aggregation and also to design novel therapeutic strategies to combat the family of diseases with which they are associated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Isobavachalcone and bavachinin from Psoraleae Fructus modulate Aβ42 aggregation process through different mechanisms in vitro

Spontaneous aggregation of Aβ is a key factor in the development of Alzheimer’s disease. In searching for Aβ aggregation inhibitors from traditional Chinese herbal medicines, we identified two active compounds from Psoraleae Fructus, namely isobavachalcone and bavachinin. We further demonstrated that the two compounds modulate Aβ42 aggregation process through different mechanisms. Isobavachalcone significantly inhibits both oligomerization and fibrillization of Aβ42, whereas bavachinin inhibits fibrillization and leads to off-pathway aggregation. Both of the compounds attenuated Aβ42-induced toxicity in a SH-SY5Y cell model. These findings may provide valuable information for new drug development and Alzheimer’s therapy in the future.     

In silico and in vitro characterization of anti-amyloidogenic activity of vitamin K3 analogues for Alzheimer's disease

Background

Aggregation of amyloid-beta (Aβ) has been proposed as the main cause of Alzheimer's disease (AD). Vitamin K deficiency has been linked to the pathogenesis of AD. Therefore, 15 synthesized vitamin K3 (VK3) analogues were studied for their anti-amyloidogenic activity.

Methods

Biological and spectroscopic assays were used to characterize the effect of VK3 analogues on amyloidogenic properties of Aβ, such as aggregation, free radical formation, and cell viability. Molecular dynamics simulation was used to calculate the binding affinity and mode of VK3 analogue binding to Aβ.

Results

Both numerical and experimental results showed that several VK3 analogues, including VK3-6, VK3-8, VK3-9, VK3-10, and VK3-224 could effectively inhibit Aβ aggregation and conformational conversion. The calculated inhibition constants were in the μM range for VK3-10, VK3-6, and VK3-9 which was similar to the IC₅₀ of curcumin. Cell viability assays indicated that VK3-9 could effectively reduce free radicals and had a protective effect on cytotoxicity induced by Aβ.

Conclusions

The results clearly demonstrated that VK3 analogues could effectively inhibit Aβ aggregation and protect cells against Aβ induced toxicity. Modified VK3 analogues can possibly be developed as effective anti-amyloidogenic drugs for the treatment of AD.

General significance

VK3 analogues effectively inhibit Aβ aggregation and are highly potent as anti-amyloidogenic drugs for therapeutic treatment of AD.     

Identification of a Compound That Disrupts Binding of Amyloid-β to the Prion Protein Using a Novel Fluorescence-based Assay

The prion protein (PrP) has been implicated both in prion diseases such as Creutzfeldt-Jakob disease, where its monomeric cellular isoform (PrP^C) is recruited into pathogenic self-propagating polymers of misfolded protein, and in Alzheimer disease, where PrP^C may act as a receptor for synaptotoxic oligomeric forms of amyloid-β (Aβ). There has been considerable interest in identification of compounds that bind to PrP^C, stabilizing its native fold and thereby acting as pharmacological chaperones to block prion propagation and pathogenesis. However, compounds binding PrP^C could also inhibit the binding of toxic Aβ species and may have a role in treating Alzheimer disease, a highly prevalent dementia for which there are currently no disease-modifying treatments. However, the absence of a unitary, readily measurable, physiological function of PrP makes screening for ligands challenging, and the highly heterogeneous nature of Aβ oligomer preparations makes conventional competition binding assays difficult to interpret. We have therefore developed a high-throughput screen that utilizes site-specifically fluorescently labeled protein to identify compounds that bind to PrP and inhibit both Aβ binding and prion propagation. Following a screen of 1,200 approved drugs, we identified Chicago Sky Blue 6B as the first small molecule PrP ligand capable of inhibiting Aβ binding, demonstrating the feasibility of development of drugs to block this interaction. The interaction of Chicago Sky Blue 6B was characterized by isothermal titration calorimetry, and its ability to inhibit Aβ binding and reduce prion levels was established in cell-based assays.