Reactivities against Multiple Pathogenic Elements of Alzheimer’s Disease

The influence of orobola on the formation of metal-free and metal-induced A42 aggregates. a) Schematic of the inhibition experiment. Effect of flavonoids on the aggregation of metal-free and metal-treated A42. a) Schematic of the inhibition experiment.

Introduction

Alzheimer’s Disease (AD)

Cholinergic deficit
Proteopathy

Amyloid- (A)
Tau

Metal ion dyshomeostasis

Cu(I/II)
Zn(II)

Oxidative stress
Metal-associated amyloid- (Metal–A)

With this understanding of the cholinergic nervous system, its role in memory, and the observation of decreased ACh levels in the brain of AD patients, researchers have explored the potential pathological role of AChE. Challenges in selectively monitoring the levels of metal ions in the brain of living patients make it difficult to directly evaluate the region-specific effects of metal ion dyshomeostasis in a.

Figure 1.1. The production, aggregation, and amino acid sequence of A. (a) Non-amyloidogenic and amyloidogenic processing of the amyloid precursor protein (APP)

Conclusions

The above-mentioned relationship between Aβ and metal ions in AD suggests the pathogenic relevance and synaptic presence of metal Aβ. However, further research is needed to discover the neuropathologically relevant metal-Aβ complexes and conformations, evaluate the toxicity of the relevant metal-Aβ complexes to identify the species responsible for critical neurotoxicity, compare their cerebral localization with the regions of neurodegeneration in the AD-affected brain, ultimately resolving its toxic mechanisms to disrupt neuronal homeostasis.

Acknowledgments

Zemek, F.; Drtinova, L.; Nepovimova, E.; Sepsova, V.; Korabecny, J.; Klimes, J.; Kuca, K., Results of the treatment of Alzheimer's disease with acetylcholinesterase inhibitors and memantine. Andreasen, N.; Hesse, C.; Davidson, P.; Minthon, L.; Wallin, A.; Winblad, B.; Vanderstichele, H.; Vanmechelen, E.; Blennow, K., Cerebrospinal fluid β-Amyloid (1-42) in Alzheimer's disease: differences between early- and late-onset Alzheimer's disease and stability over the course of the disease. Xiao, Y.; Ma, B.; McElheny, D.; Parthasarathy, S.; Long, F.; Hoshi, M.; Nussinov, R.; Ishii, Y., Aβ(1–42) Fibril structure illuminates amyloid self-recognition and replication in Alzheimer's disease.

Serra-Batiste, M.; Tolchard, J.; Giusti, F.; Zoonens, M.; Carulla, N., Stabilization of a membrane-associated amyloid-β oligomer for its validation in Alzheimer's disease. House, E.; Collingwood, J.; Khan, A.; Korchazkina, O.; Berthon, G.; Exley, C. Aluminum, iron, zinc and copper affect the in vitro formation of amyloid fibrils from Aβ 42 in a manner that may have implications for metal chelation therapy in Alzheimer's disease.

Orobol: An Isoflavone Exhibiting Regulatory Multifunctionality against Four

Results and discussion

Rational selection of orobol (Oro)
Synthesis of orobol (Oro)
Modulative reactivity towards metal-free A 42 and metal–A 42 aggregation

Inhibitory activities against organic free radicals and acetylcholinesterase
Conclusions
Experimental section

Materials and methods
Synthesis of orobol (Oro)
A 42 aggregation experiments
Gel electrophoresis with Western blotting (Gel/Western Blot)
Transmission electron microscopy (TEM)
Cu(II) binding studies
Mass spectrometric analyses
Trolox equivalent antioxidant capacity (TEAC) assay
Acetylcholinesterase (AChE) activity assay
Docking studies

Acknowledgments
References

In the inhibition experiments, Oro noticeably changed the molecular weight (MW) distribution of metal-free A42 species by increasing the intensity of smear bands greater than ca. Influence of orobol on the formation of metal-free and metal-induced A42 aggregates. a) Schematic of the inhibition experiment. To visualize the larger A42 aggregates, transmission electron microscopy (TEM) was used to examine the morphology of the resulting metal-free A42 and metal A42 aggregates from. Influence of orobol on disassembly and/or aggregation of preformed metal-free and metal-associated A42 aggregates. a) Schedule for the disaggregation experiment.

The complexation of Oro with A42 dimer was observed to change the size distribution of the peptide dimer indicating a structural compaction, according to the ion mobility-mass spectrometry (IM-MS) data (Figure 2.4b; purple). Size distributions of the incubated A42 samples were analyzed by gel/Western blot using an anti-A.

Figure 2.1. Rational identification of Oro as a multifunctional small molecule for regulating four pathological factors implicated in AD (i.e., metal-free A, metal–A, free radicals, and AChE)

Multiple Reactivities of Flavonoids towards Pathological Elements in Alzheimer’s

Results and discussion

Rational selection
Interaction with Cu(II)
Modulation of metal-free A 42 and metal–A 42 aggregation
Scavenging free organic radicals
Redox potentials
Inhibition against AChE
Computational studies for interactions with AChE

As depicted in Figure 3.3c, TEM images confirmed the effect of the flavonoids on the Cu(II)-induced aggregation of A42. The relevance of the 3-OH group in the modulatory reactivity of the flavonoids towards A42 was indicated by the distinction between (i) Kae and Api and (ii) Que and Lut. As shown in Figures 3.6b and 3.6c, a closer inspection of the bond configurations between the flavonoids and AChE revealed the hydrogen bond between the catechol moiety of the B ring and the carboxylate group of E202.

These observations highlight the important role of the catechol moiety on the B ring in controlling AChE activity. Computational parameters of interactions between selected flavonoids and AChE (PDB 1C2O51). a) The closest distance between the hydroxyl groups in flavonoids and the heteroatom (X.

Figure 3.2. Interaction of the flavonoids with Cu(II) monitored by UV–Vis spectroscopy

Conclusions

In addition, the relatively low level of fluctuations in the calculated SASAs indicated the increased stability of the binding pocket with Lut and Oro. This implied the unstable organization of the hydrophobic residues upon binding of HF to the active site. The number of water molecules in holo states decreased by more than 5 in the presence of the flavonoids, while 22 water molecules were present in the apo state.

Our structure-activity relationship study on the inhibition of AChE highlights the importance of (i) the catechol group on the B-ring to facilitate hydrogen bonding with E202, (ii) the 7-OH group for hydrogen bonding, and (iii) the chromone framework for – stacking with the hydrophobic residues along the active site cleft. The chromone framework of the flavonoids also demonstrated additional interactions with the active site AChE cleft via – stacking.

Experimental Section

Synthesis of 5-hydroxyisoflavone (HIF)
Ultraviolet–Visible (UV–Vis) measurements
A aggregation experiments
Transmission electron microscopy (TEM)
Calculation of redox potentials
Docking studies
Molecular dynamics (MD) simulation
Accelerated molecular dynamics (aMD) simulation
Analysis of the conformations sampled from the aMD simulations

The resultant A42 species from the inhibition and breakdown experiments were analyzed via gel/Western blot using an anti-A antibody (6E10).1 Samples (10 L) were split into 10-20%. The Amber FF99SB75 force field was used for the eeAChE dimer and the Generalized Amber Force Field (GAFF)76 was used to parameterize the covalent binding parameters of the ligands. During these two steps, all carbons in the ligand as well as Ca of the hydrophobic pocket residues present within 5.0 Å of the anchored ligand were inhibited with an inhibition force constant of 5.0 kcal/mol/Å2.

For each aligned trajectory, the coordinates were binned based on the ligand RMSD metric with a 1.5 Å cutoff. The SASA values of selected hydrophobic residues (Ala, Ile, Leu, Phe, Val, Pro, Gly, Met, and Trp) located within 6.0 Å of S203 in the AChE dimer model were calculated.

Acknowledgments

To determine the presence of water molecules within the binding site, for each frame we counted the number of water molecules within 8.0 Å of the Cα atom of S203 in both apo and holo cases.

Cao, S.; Jiang, X.; Chen, J., Effect of zinc(II) on the interactions of bovine serum albumin with flavonols with different numbers of hydroxyl substituents on the B ring. Bendary, E.; Francis, R.; Ali, H.; Sarwat, M.; El Hady, S., Antioxidant and structure-activity relationships (SARs) of some phenolic and aniline compounds. Labajos, M.; González-Correa, J., Role of the Catechol Group in the antioxidant and neuroprotective effects of virgin olive oil components in the brain of rats.

Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H., A consistent and accurate ab initio density functional dispersion correction (DFT-D) parameterization for 94 H-Pu elements. Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C., Comparison of multiple amber force fields and development of improved protein backbone parameters.

Drug Repurposing: Multifunctional Molecules against Cu(II)–Amyloid-, Reactive

Results and discussion

Rational selection of repurposing candidates
Modulating activity of the repurposing candidates against the aggregation of metal-free
Cu(II) interaction studies
Mechanistic details behind the modulative reactivity against the aggregation of Cu(II)–A
Free radical scavenging capacity and inhibitory activity against acetylcholinesterase

Effects of repurposing candidates on A40 and A42 aggregate formation in the absence and presence of metal ions [i.e. Zn(II) and Cu(II)]. a) Scheme of inhibition experiments. Interestingly, the reuse candidates showed only appreciable modulatory reactivity against A aggregation in the presence of Cu(II) in both inhibition and breakdown experiments. QTP did not show modulatory reactivity against the A40 and A42 aggregation pathways even in the presence of Cu(II).

Treatment of reuse candidates (i.e., ISNZ, IPNZ, and BMX) with modulating reactivity toward Cu(II)–A led to the detection of singly oxidized A in samples of both Cu(II)–A40 and Cu(II)– A42 (Figure 4.5). Our MS studies indicate that oxidation of A in the presence of Cu(II) is responsible for the ability of ISNZ, IPNZ and BMX to specifically alter Cu(II)– aggregation.

Figure 4.2. Effects of the repurposing candidates on the formation of A 40 and A 42 aggregates in the absence and presence of metal ions [i.e., Zn(II) and Cu(II)]

Conclusions

Furthermore, the inhibitory capacity of the repurposing candidates against AChE was determined using a fluorometric AChE activity assay. As a reference, tacrine, a potent AChE inhibitor, was first tested and its inhibitory activity against AChE was determined under our conditions (IC nM). In contrast, QTP showed no notable inhibitory activity against free organic radicals and AChE under our experimental conditions.

Inhibitory activity against free organic radicals and AChE is determined by means of TEAC and fluorometric assays, respectively. The inhibitory activity of QTP against free organic radicals and AChE was not significant enough to be detected under our experimental conditions.

Experimental section

Synthesis of benmoxin (BMX)
A aggregation experiments
Cu(II) interaction studies
Electrospray Ionization Mass Spectrometry (ESI-MS)

The interaction of the recycling candidates with Cu(II) was monitored by UV-Vis spectroscopy. The free radical scavenging capacity of the selected compounds was determined by the TEAC assay based on decolorization of the ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] cation radical compared to the vitamin E analogue , Trolox, known for its antioxidant properties.50 The TEAC assay was performed in EtOH following previously reported methods.40 Blue ABTS+• cation radicals were formed by dissolving ABTS (7.0 mM) with potassium persulfate (2.5 mM) in ddH2O (5 mL) of water and incubating the solution for 16 hours at room temperature in the dark. The TEAC values for each time point were calculated as the ratio of the slope of the compounds to the slope of Trolox.

The AChE inhibitory activities of the selected compounds were determined using a fluorometric AChE assay kit (Abcam, Cambridge, MA, USA) following the manufacturer's protocol with slight modifications. Calculations of AChE inhibitory activity were made based on the fluorescence intensity (ex/em = 540/590 nm) of the wells detected after 15 min of incubation.

Acknowledgments

Various concentrations of compounds or Trolox were added to the 96 well plate and incubated at 25 ºC for various periods (1, 3, 6 and 10 min). Percentage inhibition was calculated based on the measured absorbance at 734 nm [% inhibition = 100 X (A0 – A)/A0; A0 = absorbance of control well without compound; A = absorbance of wells treated with compound] and plotted as a function of compound concentration. A reaction mixture solution containing ACh, AChE probe and AbRedTM was added to the 96-well plate to initiate the reaction.

Geewoo Nam,† Mannkyu Hong,† Hyuck Jin Lee, Yonghwan Ji, Juri Lee, Juhye Kang, Mu-Hyun Baik* and Mi Hee Lim* "Multiple Reactions of Flavonoids to Pathological Elements in Alzheimer's Disease: Structure-Activity Relationship" 2020 , Sent. Geewoo Nam and Mi Hee Lim*, “Amyloid-β and metal ion intertwining pathologies in Alzheimer's disease: metal–amyloid-β” Chem. Orobol: an isoflavone with regulatory versatility against four pathological factors of Alzheimer's disease” ACS Chem.

Orobol: An Isoflavone Exhibiting Regulatory Multifunctionality against Four Pathological Features of Alzheimer's Disease” Korea Dementia Association Fall Simposium, 2019. Orobol: An Isoflaone Exhibiting Regulatory Multifunctionality against Four Patological Features of Alzheimer’s Disease” Die Internasionale Konferensie oor Biogeïnspireerde Klein Molekule2019.