MARK4 Inhibited by AChE Inhibitors, Donepezil and Rivastigmine Tartrate: Insights into Alzheimer's Disease Therapy
Authors: Gulam Mustafa Hasan
Taj Mohammad
Md Tabish Rehman
Md Imtaiyaz Hassan
Saleha Anwar
Anas Shamsi
Asimul Islam
Afzal Hussain
Mohamed F Alajmi
Journal: Biomolecules (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Alzheimer’s disease; MARK4; acetylcholinesterase inhibitors; drug design and discovery; isothermal titration calorimetry
Affiliations: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India., Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia., Department of Biochemistry, College of Medicine, Prince Sattam Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Saudi Arabia.

Abstract:  Microtubule affinity-regulating kinase (MARK4) plays a key role in Alzheimer's disease (AD) development as its overexpression is directly linked to increased tau phosphorylation. MARK4 is a potential drug target of AD and is thus its structural features are employed in the development of new therapeutic molecules. Donepezil (DP) and rivastigmine tartrate (RT) are acetylcholinesterase (AChE) inhibitors and are used to treat symptomatic patients of mild to moderate AD. In keeping with the therapeutic implications of DP and RT in AD, we performed binding studies of these drugs with the MARK4. Both DP and RT bound to MARK4 with a binding constant (K) of 107 M-1. The temperature dependency of binding parameters revealed MARK-DP complex to be guided by static mode while MARK-RT complex to be guided by both static and dynamic quenching. Both drugs inhibited MARK4 with IC50 values of 5.3 μM (DP) and 6.74 μM (RT). The evaluation of associated enthalpy change (ΔH) and entropy change (ΔS) implied the complex formation to be driven by hydrogen bonding making it seemingly strong and specific. Isothermal titration calorimetry further advocated a spontaneous binding. In vitro observations were further complemented by the calculation of binding free energy by molecular docking and interactions with the functionally-important residues of the active site pocket of MARK4. This study signifies the implications of AChE inhibitors, RT, and DP in Alzheimer's therapy targeting MARK4. 

Retracted: No

PubPeer comments:
 This paper cites one of my articles which critically discusses the very popular methodology of using fluorescence quenching to determine ligand binding characteristics. I wrote this article with a colleague, as we were quite upset about the numerous basic mistakes made in this type of analysis in the scientific literature (with currently well over 1,000 such flawed papers). One would hope that authors who cite this paper are aware of its contents, and either rebut it, or accept the criticism and apply it in their own work. However, this is not the case here, as is evidenced in e.g. the discussion on distinguishing dynamic and static quenching, the lack of any attempt to correct for the inner filter effect, and the improper use of the double log equation 4 - as a result the authors do analyses and draw conclusions that are at odds with my article. Can the authors please explain why they cite my article, but largely ignored its contents? , 
 #1 We read your paper and took all the content while analysis. Its a beautifully written article. , 
 Moreover if u do find any discrepancy, I will go through your beautifully written article again , 
 #3 Allow me to point out a few things to consider to check up: 1. Did you correct for the inner-filter effect? It is always difficult to determine whether it plays any role if UV-VIS spectra of the ligands at relevant concentrations are provided, but upward curvature in Stern-Volmer curves can be due to a) the inner-filter effect; b) multiple binding sites with slightly different binding constants but the same similar quenching efficiency; and c) combination of dynamic + static quenching. The latter is, however, only relevant for situations where *collisional* quenching plays an important role, and this is very unlikely at the ligand concentrations chosen. If you ligand has a combined absorbance at excitation and emission wavelength of 0.05 at the highest used concentration, you already should consider correcting for the inner-filter effect 2. The distinction of dynamic and static quenching is not as simple as discussed in your paper. You are essentially trying to distinguish collisional quenching from binding-related quenching. Even then temperature effects are not always very useful (binding may increase with higher temperatures - and thus quenching efficiency, too - in the case of entropy-driven binding). This is explained in our article in sections 2.3+2.4 3. The double log equation is problematic for various reasons: a) contrary to the authors suggestion, it does *not* give the number of binding sites, but the Hill coefficient, which is always the same or lower than the number of binding sites; b) it implicitly assumes the complex is completely non-fluorescent. This is very unlikely when dealing with a protein, and even more so if that protein has multiple fluorophores - it appears MARK4 has five tryptophan residues, and it is very unlikely that binding of a ligand fully quenches all of these residues; c) the equation assumes the added ligand concentration is the same as the free ligand concentration. This is very unlikely to be a valid assumption if the protein and ligand concentrations are similar. In this case for DP the concentration of the ligand is even much lower than that of the protein. This is discussed in Section 2.8 in my article. Now, it appears to me the authors also made at least one, but possibly several mistakes in Table 2 for the binding constants. For example, the 298K data for MARK4-DP make no sense when looking at the graphs (n most definitely is not 0.1, but more likely 1), and the deltaG value does not fit the reported binding constant (factor 100 too small). A concern mainly based on experience of the temperature effect on binding for many ligand-protein pairs is the reported enormous temperature sensitivity for these two ligands based on the binding constants noted in Table 2. Here, a mere 5 degrees increase supposedly decreases the binding constant by several orders of magnitude - almost 4 orders for RT. I have never seen such enormous temperature sensitivity. , 
 Thanks for all these concerns and making us aware of all these. All the fluorescence intensities were corrected for inner filter effect. Temperature dependency was used to distinguish quenching as per reviewer s suggestion and also based on recent literature reporting drug protein interactions. The article written by you is a beautifully written piece of work and that can help us in overcoming all the errors that are usually deployed. I value it and and try to follow each and every point and before carrying out my mext project i will revert back to you if any query arises. Thanks for this opprotunity and making us aware , 
 Moreover, the reference was cited in terms of types of quenching active for protein ligand interaction. If you still feel that citing your literature is irrelevant , we can request the production team to have it altered. Thanks for understanding, let us know. , 
 #6 I do not quite see any benefit in just removing a citation. I d rather you correct the paper, but it looks like that is not going to happen. We ll just leave it at that. , 
 Thanks for making us aware of all these. In earlier studies, I have also not found such variation in binding with temp but this was the case for MARK4. Another study of MARK$ also reported high variations in binding with temp. In this case also, we repeated experiments tine and gain but similar results were obtained. Thank you. ,