Alzheimer’s disease (AD) is a chronic, progressive disorder associated with loss of memory or cognition. It is a leading cause of mortality worldwide There are no such drugs which can cure AD and are ineffective in the later stages. Such known drugs only ease the symptoms but do not prevent the onset or progression of the AD. Alzheimer’s is caused by the aggregation of the hyperphosphorylated tau which is one of the common characteristics of the neurodegenerative disorder. There are a number of kinases which hosts the excessive phosphorylation of tau protein. One of the kinases extensively targeted in the AD is GSK-3 ? ( Glycogen Synthase Kinase-3 ?). GSK-3 ? also designated as Tau Kinase 1, appears to phosphorylate over 30 distinct sites on protein tau. Alzheimer’s, structure-based drug designing was reported to be carried out with GSK-3 ?. As indicated by distinct studies, that by applying appropriate docking methods, a number of phyto compounds have shown enhanced target selectivity than the conventional Alzheimer’s drugs. This review summarizes the known drug targets in the AD, their conventional inhibitors and also the comparison between the current and future AD therapy based on their binding affinities. As a result, large libraries of compounds with inhibitory effect can be screened. It was also studied that Withanolide-A (extracted from roots of Withania somnifera ) has the potential to be the future drug for Alzheimer’s disease.
Introduction to AD
Alzheimer’s is a type of dementia associated with memory loss and other cognitive abilities, serious enough to interfere with daily life. Alzheimer’s disease accounts for 60 to 80 percent of dementia and the present Alzheimer’s disease therapies suffer from inefficient effects on its symptoms such as cognition especially in the later stages of the disease (http://www.alz.org). According to the report prepared by Alzheimer’s and related disorders society of India, In 2010, there are 3.7 million Indians with dementia and the total societal costs is about 14,700 crore. While the numbers are expected to double by 2030. The number of factors is thought to increase the progression of this disease, some of which are; increasing age, family history of the condition, previous severe head injuries etc. Over the past decade, much of the research on Alzheimer disease (AD) has focused on radical-induced oxidative stress and its importance in disease pathogenesis. Oxidative stress increases amyloid beta deposits in the brain which results in the synthesis of neurotoxic aggregates. The net effect of oxygen radicals is damaging as it may lead to neuronal cell death and contribute to AD (M.A.Smith., et al.,1998). Flavonoids also possess antioxidant activity and they regulate the redox status and prevent damage caused by oxidative stress. Protein Kinases are identified as promising target structures because of their involvement in AD progression pathways like pathophysiological tau protein phosphorylation and amyloid beta toxicity. It is believed that these pathophysiological factors all contribute to the AD progression.
Role of Flavonoids in the treatment of AD
Nature has endowed us with a lot of natural remedies in the form of fruits, leaves, bark, vegetables, and nuts, etc. The wide variety of bioactive nutrients present in these natural products play a vital role in prevention and cure of various neurodegenerative diseases. Flavonoids are a large group of non-nutrient polyphenolic compounds naturally obtained from plants. It was thought that the ability of flavonoids to improve neurological health was mediated by their antioxidant capacity. Flavonoids possess various biological activities like anti-amoebic activity, anti-inflammatory, anticoagulant, anti-cancer, anti-oxidants, and anti-spasmodic. There is an extensive role of flavonoids and even their metabolites in different signaling pathways by altering the phosphorylation state of target protein put forward their therapeutic potential and are beneficial in neurodegeneration (J.P.E. Spencer,2007). Increasing evidence shows their ability to improve brain function such as memory and learning by interacting with cellular as well as molecular components of the brain resulting in enhanced neuronal function and induce neurogenesis (J.P.E. Spencer,2010; Filipa I. Baptista et al., 2014). Phytocompounds such as myricetin and epicatechin-5-gallate have been involved in inhibiting heparin-induced assembly of tau into filaments (Taniguchi et al., 2004). In drug discovery, the major secondary metabolites (terpenoids, phenolics, and alkaloids) are of potential medicinal interest. Certain flavonoids such as indirubin and morin are capable of the inhibiting the activity of GSK-3 beta and thereby blocking tau hyperphosphorylation. Kinases are involved in tau phosphorylation and phosphatases reverse this action. Thus, flavonoids also play a crucial role in modulating the activity of phosphatases (Filipa I. Baptista et al., 2014).
Molecular causes of AD
The key events the leading to the AD are:
a. Beta-amyloid toxicity
Brains of a patient with the AD are characterized by amyloid toxicity. Amyloid beta denotes peptides of 36-43 amino acids long processed from an amyloid precursor protein (APP) which is digested by beta secretase and gamma secretase to yield amyloid beta(A ? ) found in brains of patients suffering from Alzheimer’s (Haml ey IW,2012;M. Paul Murphy et al.,2010).Several mechanisms have been developed on the ability of flavonoids to delay the onset or prevent progression of the AD. Some processes include disruption of amyloid beta aggregates, alterations in the precursor of amyloid beta protein processing through the inhibition of beta-secretase.Thus, modulating the beta-secretase activity is the one suggested a therapeutic avenue to treat AD (Yin YI et al.,2007). Certain flavonoids may protect against the Alzheimer’s disease by interfering with the generation of beta-amyloid peptides into neurotoxic aggregates. It is a matter of contention that interfering with the activity of beta and gamma-secretase enzymes may disrupt their other functional roles besides playing an important part in amyloidogenic pathways. Thus such interference using ? secretase can result in skin cancers and cognitive dysfunction (Kikuchi et al.,2017). The recent failure of amyloid-?-targeted therapeutics in Phase III clinical trials suggests that it is timely to consider alternative drug discovery strategies for Alzheimer’s disease.
Therefore, Kurt.R.Brunden et al., (2009) focus on strategies directed at reducing misfolded tau ( due to hyperphosphorylation) which is one of the disease-causing agents.
b. Tau protein hyperphosphorylation
For more than a decade, researchers have found ‘tau’ protein as one of the causes other than the Beta-amyloid plaques (Emily Underwood, 2016). Tau hyperphosphorylation and accumulation of insoluble aggregates is strongly related to reduced cognitive performance. Hence, tau is a reliable marker of the neurodegenerative process. Incorporation of phosphate groups into tau depends on; tau’s confirmation and a balance between the activity of kinases and phosphatases (Anna Kremer et al.,2011). Changes in tau confirmation could lead to excessive phosphorylation resulting in the formation of neurotoxic aggregates and tau-mediated neurodegeneration (Dixit et al.,2008). Tau belongs to the family of proteins involved in stabilizing the microtubules. They are common in neurons of Central Nervous System and also present at low levels in CNS astrocytes and oligodendrocytes(Shin RW et al.,1991). The tau proteins have been formed as a result of alternative splicing from a single gene that in humans is termed as MAPT( microtubule-associated protein tau) and is located on chromosome 17 (Goedert M et al.,1989, Jesus Avila et al., 2016). The hydrophilic nature of the tau protein and its existence as intrinsically disordered protein was unfolded by many biophysical studies. (Anna Mietelska- Porowska et al., 2014).One of the critical function of tau protein is to prevent the depolymerization of microtubules by regulating its stability in two ways: isoforms, and phosphorylation. On the basis of the number of binding domains, six variants of tau protein exist in human brain tissue of 352-441 amino acids and with an apparent molecular weight between 60-74kDa (Ludovic Martin et al.,2011).Out of six variants, three isoforms have 3 tubulin binding domains and other three have 4 tubulin binding domains (Meaghan Morris et al.,2011). The domain structure of tau is such that its positively charged binding domain is located in carboxy-terminal which binds to the microtubule which is negatively charged. Tau is a phosphoprotein with 79 potential Serine (Ser) and Threonine (Thr) phosphorylation sites on the longest tau isoform. It has been reported in a study that phosphorylation is possible in about 30 sites in a normal tau protein. PKN, a serine/threonine kinase is one such enzyme among the plethora of kinases which regulates the phosphorylation of tau(Billingsley ML et al.,1997).When PKN is activated, it results in disorganization of microtubule assemble due to phosphorylation of tau. As revealed by primary sequence analysis, the tau molecule has three major domains: N-terminal(acidic), a proline-rich region, C-terminal domain(basic).These domains were characterized on the basis of their amino acid character and even on their microtubule interactions. Thus, tau protein act as a dipole with two domains having the opposite charge (Michala Kolarova et al., 2012; Anna Mietelska-Porowska et al.,2014).
Hyperphosphorylation of tau leads to the formation of paired helical fragments (PHF’s) due to the loss of affinity with microtubules and they bind with one another which further aggregates in neurofibrillary tangles via. post-translational modifications (Avila,2006 ; Martin et al.,2011,2013).When misfolded, this otherwise very soluble protein forms extremely insoluble tangles or aggregates which contribute to the number of neurodegenerative disorders. The mutations in posttranslational modifications are the main cause of this failure i.e. they form nonfunctional aggregates. One of the studies demonstrated that dephosphorylation of the hyperphosphorylated tau converts abnormal tau protein into a normal like protein which then regulates microtubule assembly(Khalid Iqbal et al.,2010).Therefore abrogating the abnormal tau and recovery of the microtubule organization are the most promising therapeutic interventions to combat AD.
GSK-3 beta as a drug target
GSK-3 is encoded by two genes: GSK-3 ?, located on chromosome 19 and GSK-3 ?, located on chromosome 2. GSK-3 is ubiquitously expressed in mammals as well as in yeast ( Miguel Medina et al.,2011). GSK3 is a serine/threonine protein kinase that mediates the addition of phosphate molecules to serine and threonine amino acid residues. The kinase domain of these 2 isoforms are highly homologous (Stambolic V et al.,1994) but are differentiated in the N- and C-terminal regions. GSK3 ? has a molecular mass of 46-47 kDa consisting of 433 and 420 amino acids in human and mouse respectively. The protein contains an N-terminal domain, a kinase domain, and a C-terminal domain. Binding domain (BD) includes GSK3 ? specific binding sites for substrates and protein complexes (e.g., p53) (Atlas of Genetics and Cytogenetics in Oncology and Haematology).
A number of protein kinases are involved in tau phosphorylation such as CdK5(Cyclin-dependent Kinase 5), JNK( C-Jun amino-terminal Kinase), CK1(Casein Kinase1), Dyrk1A, AMPK(Adenosine-monophosphate activated protein kinase),MARK5( Microtubule affinity-regulating Kinases), PKA( Cyclic AMP-dependent protein Kinase), GSK-3 ?(Glycogen Synthase Kinase- 3 ?) (Leslie crews et al.,2010). But it has been reported that 31% of the pathological phosphorylation sites of tau protein are phosphorylated by GSK3? (Martin et al., 2013). The classical approach to treat misfolding of tau protein provides inhibition of protein kinases (Glycogen synthase kinase 3 ?) which hosts tau phosphorylation. According to the ‘GSK-3 hypothesis of AD’, tau hyperphosphorylation, memory impairment and enhanced ?- amyloid production is due to the overexpression of GSK-3, all of which are characteristic features of the haveIf this hypothesis is consolidated then inhibition of GSK-3 ? by novel inhibitors provides a better pathway against the effect of this destructing disorder (Claudie Hooper et al.,2008). There are two isoforms of GSK-3 gene; GSK-3 alpha and GSK-3 ?. GSK3? also exists as longer splice variants (Mukai et al.,2002; Schaffer et al.,2003). Moreover, GSK-3 ? results in a neuronal decline in the AD because of the fact that it is a causal mediator of apoptosis. Increased level of GSK-3 has been found in post-mortem analysis of brain of AD patients (Peri JJ et al.,1997). It is also demonstrated that a spatial and temporal pattern of increased GSK-3 expression correlates with the progression of NFT and neurodegeneration (Leroy K et al.,2002).
Drug research is an important tool in the field of medicine. Use of computers to predict the efficiency of binding of a set of small molecules or ligands with the target is an important component of drug discovery process. There is a wide range of software packages used to conduct molecular docking such as Dock, Autodock, GOLD, ICM, Glide, AutoDock Vina, FlexX etc (Nataraj S et al., 2017).Automated docking is widely used for prediction of biomolecular complexes, in structure and function analysis and in computer-aided molecular designing. A dozen of methods is available, incorporating different energy evaluation methods. But in several structural based drug designing approaches docking is performed by the Autodock4 which is a molecular modeling simulation software. Due to the enhanced docking speed, AutoDock 4.2 is the most recent version which has been widely used for virtual screening (Collignon et al., 2011). Its default search function is based on a hybrid genetic algorithm(Lamarckian Genetic Algorithm) with local optimization that uses a parameterized free-energy scoring function to estimate the fast prediction of binding energy (Madeswaran et al., 2011).Two main programs are involved in AutodockTools: Autodock for docking of the ligand within the set of grids( within the binding site) in the target protein and Autogrid for selection of grid parameters, size of the box, its location etc (http://autodock.scripps.edu/). It is especially effective for protein-ligand docking in which we predict the position and orientation of a ligand (a small molecule) when it is bound to a protein receptor. It is used to select likely drug candidates. Typically, ligands are drug candidates and the macromolecule is the protein or receptor of the known three-dimensional structure. In this docking simulations, the ligand being docked were kept as flexible while target protein was kept as rigid. The graphical user interface i.e. Autodock Tools was used to prepare, run, analyze the docking simulations.
Current and future AD therapy
There are no such drugs/treatments available that can cure AD completely. However, there are several medications developed for Alzheimer’s disease that can temporarily attenuate the symptoms. The U.S. Food and Drug Administration (FDA) has approved two medications- acetylcholinesterase inhibitors and Memantine. Drugs such as tacrine, rivastigmine, galantamine, and donepezil are the widely used conventional drugs to treat AD ( M.R. Islam et al.,2013). Memantine is a dissociative hallucinogenic and anesthetic drug of the adamantane class of chemicals that are currently used as an FDA approved drug in the treatment of AD (www.alz.org). Therefore, traditional drugs like memantine and donepezil are being extendedly used as the reference in molecular docking studies. Hence, the goal of eventual AD therapy is to develop potential compounds that could inhibit the tau protein and thereby it can be used for the treatment of neurodegenerative diseases( Anja Schneide et al.,2008).The study related to the AD is focused more towards the traditional medicinal plants and its components such as Withania somnifera (Ashwagandha), Celastrus paniculatus(Jyotismati), Convolvulus pluricaulis(Shankhpushpi), Bacopa monnieri(Brahmi). By analyzing the binding energies of various ligands such as acacatechin, catechin, galangin, scopoletin, silibinin, memantine ( as standard), it was observed that flavonoids showed binding energy ranging between 7.07 kcal/mol to -4.85 kcal/ mol. Silibinin showed better binding energy -7.07 kcal/mol than the standard memantine (-5.89 kcal/mol) (Arumugam Madeswaran et al.,2013). Quercetin ( with binding energy -8.8 kcal/mol) also showed the same drug-likeness than conventional drugs such as donepezil( with binding energy -7.9 kcal/mol) (M.R Islam et al., 2013).
Withania somnifera, a potential inhibitor of GSK-3 ?
Withania somnifera commonly called Ashwagandha, Indian ginseng and wind cherry have been an important herb in Indigenous and ayurvedic medical system. Historically, the plant has been used therapeutically for boosting the brain function including memory retrieval. Thus has a cognition promoting effect in adults and children (N Singh et al.,2011). It consists of two components: withanolides, withanamides. Withanolide A is extracted from the roots of the plant and promotes antioxidant properties that protect nerve cells from harmful free radicals. Many clinical trials and excessive research on animals support the use of Ashwagandha for anxiety, cognitive and neurological disorders (S. Rajasekar et al.,2011).Withanolides have also been used for the treatment of AD (Khan SA et al.,2016). Withanolide A is used as an inhibitor of acetylcholinesterase activity and reduces beta-amyloid protein formation. Also, it has been involved in the regeneration of pre and postsynaptic neurons. Instead of the root extract, a study also suggested fruits and leaves of Egyptian plant have strong antioxidant activity ( Rahma SR Mahrous et al., 2017).
The field of molecular docking has emerged during the last three decades for structural based drug designing. It has become an integral part of drug discovery and development which is utilized for the accurate prediction of protein-ligand complexes. To explore potent and effective drugs for the treatment of AD, different phytocompounds were compared against the standard using Autodock4. Appropriate ligands were docked into the active site of the receptor GSK-3 ? and analyzed for the effective protein-ligand interactions.Therefore molecular docking identified many more promising, efficacious, selective new drugs against Alzheimer’s reducing the time span of complex drug discovery process.
The authors are thankful to the Department of Biotechnology, University Insti