Multitargeting the Action of 5-HT6 Serotonin Receptor Ligands by Additional Modulation of Kinases in the Search for a New Therapy for Alzheimer’s Disease: Can It Work from a Molecular Point of View?

In view of the unsatisfactory treatment of cognitive disorders, in particular Alzheimer’s disease (AD), the aim of this review was to perform a computer-aided analysis of the state of the art that will help in the search for innovative polypharmacology-based therapeutic approaches to fight against AD. Apart from 20-year unrenewed cholinesterase- or NMDA-based AD therapy, the hope of effectively treating Alzheimer’s disease has been placed on serotonin 5-HT6 receptor (5-HT6R), due to its proven, both for agonists and antagonists, beneficial procognitive effects in animal models; however, research into this treatment has so far not been successfully translated to human patients. Recent lines of evidence strongly emphasize the role of kinases, in particular microtubule affinity-regulating kinase 4 (MARK4), Rho-associated coiled-coil-containing protein kinase I/II (ROCKI/II) and cyclin-dependent kinase 5 (CDK5) in the etiology of AD, pointing to the therapeutic potential of their inhibitors not only against the symptoms, but also the causes of this disease. Thus, finding a drug that acts simultaneously on both 5-HT6R and one of those kinases will provide a potential breakthrough in AD treatment. The pharmacophore- and docking-based comprehensive literature analysis performed herein serves to answer the question of whether the design of these kind of dual agents is possible, and the conclusions turned out to be highly promising.

Both ligand-based (LBDD) and structure-based drug design (SBDD) approaches are useful in the rational design of 5-HT 6 R ligands. However, a limitation to SBDD was the lack of a crystallographic structure for this receptor in PDB until now, thus condemning designs based on homology models. Last-minute lines of evidence [46] point to obtaining the first 5-HT 6 R crystal, which gives hope for increasing SBDD efficiency as soon as the crystallographic structure becomes available. Nevertheless, the ligand-based design has dominated the exploration of the chemical space for new 5-HT 6 R ligands so far, in which the pharmacophore model for antagonists developed by the team of Lopez-Rodriguez in 2005 seems to be the number one for the computer-aided LBDD, until now.

Molecular Modeling Approaches to Evaluate the Potential 5-HT 6 R Compound Activity
We decided to combine both LBDD and SBDD to assess the potential of considered kinases inhibitors that also present 5-HT 6 R activity (detailed results are presented in the respective subchapters). At first, we used the pharmacophore-based method in order to examine the possibility of dual modulation of the 5-HT 6 R and each kinase selected from the considered ones (MARK4, ROCK I, ROCK II, CDK5) in a wider group of chemical compounds.
Taking into account the significant increase in the number of new highly active 5-HT 6 R antagonists for more than 15 years since the pharmacophore of Lopez-Rodriguez occurred, an update based on the extensive library of current antagonists (ligands) available in the CHEMBL database for the 5-HT 6 R pharmacophore model was made in the first step.
The developed pharmacophore was further used to analyze common structural features for the active kinase inhibitors and 5-HT 6 R.
For all 5-HT 6 R ligands with affinity to the receptor below 500 nM (expressed in K i , data fetched from the ChEMBL database) [47], the clustering procedure was carried out (with compounds represented by MOLPRINT2D fingerprint [48] and Tanimoto similarity metric used to measure distance between the clusters formed). The number of clusters was forced to be 50, and centroids together with compounds with the shortest distance to the centroid formed the set of compounds, which was used to construct the pharmacophore model (the total number of compounds was equal to 136). The clustering procedure was applied to ensure that the chemical space of ligands used for the pharmacophore model construction was representative to the whole set of 5-HT 6 R ligands to the highest possible extent.
The pharmacophore model was constructed using Phase [49] from the Schrödinger Suite 2022 (Figure 2).
Due to the relatively high fraction of 5-HT 6 R ligands with low basicity, the positive ionizable group, which was present in pharmacophore models developed in previous studies [22], is now not included in the model. The model is composed of three features: aromatic moiety (R7), hydrogen bond acceptor(A1) and hydrophobic moiety (H2). The features are arranged in the triangle-like shape with the distance between the aromatic ring and hydrogen bond acceptor equal to 2.77 Å, between hydrogen bond acceptor and hydrophobic feature: 2.43 Å, and 4.80 Å between the aromatic feature and hydrophobic moiety. The pharmacophore model was constructed using Phase [49] from the Schrödinger Suite 2022 (Figure 2). Due to the relatively high fraction of 5-HT6R ligands with low basicity, the positive ionizable group, which was present in pharmacophore models developed in previous studies [22], is now not included in the model. The model is composed of three features: aromatic moiety (R7), hydrogen bond acceptor(A1) and hydrophobic moiety (H2). The features are arranged in the triangle-like shape with the distance between the aromatic ring and hydrogen bond acceptor equal to 2.77 Å, between hydrogen bond acceptor and hydrophobic feature: 2.43 Å, and 4.80 Å between the aromatic feature and hydrophobic moiety.
Mapping of the example 5-HT6R ligand (CHEMBL267615, Ki = 13 nM) on this model is presented in Figure 3. Mapping of the example 5-HT 6 R ligand (CHEMBL267615, K i = 13 nM) on this model is presented in Figure 3. Due to the relatively high fraction of 5-HT6R ligands with low basicity, the positive ionizable group, which was present in pharmacophore models developed in previous studies [22], is now not included in the model. The model is composed of three features: aromatic moiety (R7), hydrogen bond acceptor(A1) and hydrophobic moiety (H2). The features are arranged in the triangle-like shape with the distance between the aromatic ring and hydrogen bond acceptor equal to 2.77 Å, between hydrogen bond acceptor and hydrophobic feature: 2.43 Å, and 4.80 Å between the aromatic feature and hydrophobic moiety.
Mapping of the example 5-HT6R ligand (CHEMBL267615, Ki = 13 nM) on this model is presented in Figure 3. In order to strongly support the compound activity via molecular modeling approaches, docking studies were carried out in the next step (inactive-state homology model of 5-HT 6 R deposited in the GPCRdb [50] database was used, and the docking was carried out in Glide [51] from the Schrödinger Suite 2022). The models were created based on the GPCRdb homology modeling pipeline [52]. It uses a chimeric approach in which a single template is selected as a main template; however, the template is screened locally and when a better template for a particular protein region is found, it is used to model the respective protein fragment The example ligand-receptor complex obtained for CHEMBL267615 is presented in Figure 4.
In order to strongly support the compound activity via molecular modelin approaches, docking studies were carried out in the next step (inactive-state homolog model of 5-HT6R deposited in the GPCRdb [50] database was used, and the docking w carried out in Glide [51] from the Schrödinger Suite 2022). The models were created base on the GPCRdb homology modeling pipeline [52]. It uses a chimeric approach in which single template is selected as a main template; however, the template is screened local and when a better template for a particular protein region is found, it is used to model th respective protein fragment The example ligand-receptor complex obtained f CHEMBL267615 is presented in Figure 4. The compound presented in Figure 4 fits well in the 5-HT6R binding site, forming number of hydrophobic and polar interactions. Most importantly, the charge-assiste hydrogen bond with the aspartic acid from the third transmembrane helix (D3x according to the GPCRdb numbering) is formed, but other important residues indicate as important to 5-HT6R activity also make contact with the compound, such as C3x3 S5x43, F6x51, F6x52, etc.
Additionally, the kinase inhibitors were docked to the 5-HT6R homology model in a analogous manner. The distribution of the docking score values for particular compoun sets were examined ( Figure 5, examples of docking poses are provided in the subseque chapters).
The analysis of docking scores to 5-HT6R indicates that the distribution of their valu is similar to both 5-HT6R ligands and ligands of the examined kinases. Although, the lo docking score value does not guarantee the desired activity profile, its favorable valu increases the probability of biding to the considered protein. For all ligand sets, the highe fraction of docking score values falls in the range of −8 to −6, and the second mo populated group of values is between −6 and −4. CDK5, MARK4, ROCK I and ROCK ligands possess a slightly higher fraction of compounds with a docking score between and −2, but at the same time, for these targets, there is also a higher number of compound The compound presented in Figure 4 fits well in the 5-HT 6 R binding site, forming a number of hydrophobic and polar interactions. Most importantly, the charge-assisted hydrogen bond with the aspartic acid from the third transmembrane helix (D3x32 according to the GPCRdb numbering) is formed, but other important residues indicated as important to 5-HT 6 R activity also make contact with the compound, such as C3x36, S5x43, F6x51, F6x52, etc.
Additionally, the kinase inhibitors were docked to the 5-HT 6 R homology model in an analogous manner. The distribution of the docking score values for particular compound sets were examined ( Figure 5, examples of docking poses are provided in the subsequent chapters).
The analysis of docking scores to 5-HT 6 R indicates that the distribution of their values is similar to both 5-HT 6 R ligands and ligands of the examined kinases. Although, the low docking score value does not guarantee the desired activity profile, its favorable value increases the probability of biding to the considered protein. For all ligand sets, the highest fraction of docking score values falls in the range of −8 to −6, and the second most populated group of values is between −6 and −4. CDK5, MARK4, ROCK I and ROCK II ligands possess a slightly higher fraction of compounds with a docking score between −4 and −2, but at the same time, for these targets, there is also a higher number of compounds with docking score values between −10 and −8 (in comparison to compounds active towards 5-HT 6 R). with docking score values between −10 and −8 (in comparison to compounds active towards 5-HT6R). In addition, the compounds were evaluated in terms of their ability to penetrate the blood-brain barrier. This was carried out via the determination of logP (calculations were performed in InstantJChem, https://chemaxon.com/products/instant-jchem [53]) for analogous compound sets, as in the case of docking. It was previously reported that logP values for the majority of drugs fall in the range of −0.5-6 [54]; however, the optimal logP range was set to 1.5-2.5 [55]. All the examined ligands fall in the similar logP distribution with the majority of ligands adopting predicted logP values between 3 and 4 (Supplementary Materials Figure S1).

5-HT6R/MARK4 as Dual Target Approach in Search for Therapeutic Solution against AD
Concerning the kinases, the mitogen-activated protein kinases (MAPKs) govern meaningful cellular programs and are crucial intermediate pathways in signaling, while microtubule affinity-regulating kinase 4 (MARK4) is a part of the kinases family recognized for actively phosphorylating neural microtubule-associated proteins (MAPs) i.e., MAP2, MAP4, and especially important for AD, tau protein. The kinase MARK4 is a member of the Ser/Thr kinase family and has been confirmed as a significant contributor in phosphorylating specific residues of tau, followed by its accumulation, and contributing in tauopathy. Phosphorylated tau also leads to neurofibrillary deposits and the formation of APP [56]. Tau phosphorylation effects are, therefore, correlated with neurodegeneration. Consequently, an overexpression of MARK4 is associated with numerous neurodegenerative disorders and neuropathy [57,58]. Thus, inhibiting MARK4 can be considered essential to cure some neurodegenerative diseases, including AD [59,60].
On the other hand, the highly important role of serotonin and 5-HT receptors in AD particularly accented in the case of the 5-HT6R due to its unique function and CNS distributions, seems to be indisputable in light of the results of research conducted for over 20 years. Furthermore, recent lines of evidence, based on the fluorescence binding study, isothermal calorimetry, molecular docking and MD simulation studies for In addition, the compounds were evaluated in terms of their ability to penetrate the blood-brain barrier. This was carried out via the determination of logP (calculations were performed in InstantJChem, https://chemaxon.com/products/instant-jchem [53]) for analogous compound sets, as in the case of docking. It was previously reported that logP values for the majority of drugs fall in the range of −0.5-6 [54]; however, the optimal logP range was set to 1.5-2.5 [55]. All the examined ligands fall in the similar logP distribution, with the majority of ligands adopting predicted logP values between 3 and 4 (Supplementary Materials Figure S1).

5-HT 6 R/MARK4 as Dual Target Approach in Search for Therapeutic Solution against AD
Concerning the kinases, the mitogen-activated protein kinases (MAPKs) govern meaningful cellular programs and are crucial intermediate pathways in signaling, while microtubule affinity-regulating kinase 4 (MARK4) is a part of the kinases family recognized for actively phosphorylating neural microtubule-associated proteins (MAPs), i.e., MAP2, MAP4, and especially important for AD, tau protein. The kinase MARK4 is a member of the Ser/Thr kinase family and has been confirmed as a significant contributor in phosphorylating specific residues of tau, followed by its accumulation, and contributing in tauopathy. Phosphorylated tau also leads to neurofibrillary deposits and the formation of APP [56]. Tau phosphorylation effects are, therefore, correlated with neurodegeneration. Consequently, an overexpression of MARK4 is associated with numerous neurodegenerative disorders and neuropathy [57,58]. Thus, inhibiting MARK4 can be considered essential to cure some neurodegenerative diseases, including AD [59,60].
On the other hand, the highly important role of serotonin and 5-HT receptors in AD, particularly accented in the case of the 5-HT 6 R due to its unique function and CNS distributions, seems to be indisputable in light of the results of research conducted for over 20 years. Furthermore, recent lines of evidence, based on the fluorescence binding study, isothermal calorimetry, molecular docking and MD simulation studies for estimating the binding affinity and inhibiting potential of serotonin with MARK4, have demonstrated serotonin as an inhibitor of this important kinase. Hence, targeting MARK4 by serotonin opens a "new gate" in managing the clinical manifestations of neurodegenerative diseases such as AD and dementia [60].
In contrast to the 5-HTR-nonselective serotonin, selective 5-HT 6 R agents that also inhibit MARK4 would offer the possibility of a specific and better controlled pharmacological profile, thus guaranteeing more favorable therapeutic effects. In this context, the search for new structures of dual modulators of MARK4 and 5-HT 6 R gives new hope for a breakthrough in AD treatment, which is highly justified taking into account the signal transduction pathways at the cellular level. The question then arises as to whether it is possible to find these suitable double-agent structures from a chemical point of view.
Although lines of evidence indicate hundreds of 5-HT 6 R ligands with nanomolar affinities and different intrinsic profiles, the number of identified families of MARK4 inhibitors is much lower, and it is difficult to find any report on compounds simultaneously showing both MARK4 and 5-HT 6 R action in a high enough activity range. Among more than 100 dual MARK4/5-HT 6 R records in the CHEMBL database, none have been found to exhibit submicromolar effects for both purposes simultaneously. However, this state of the art does not seem to be associated with a distinct structural limitation but, more probably, with a lack of attention to this direction of research, until now.
Interestingly, the studies of Shamsi et al. [57] demonstrated the moderate micromolar MARK4 inhibiting action for known AD drugs acting as AChE inhibitors, i.e., donepezil and rivastigmine; slightly more potent in the case of donepezil (Figure 6c).
For the 4,5-cyclic pyrimidines, the pyrimidine is condensed with 3,3-dimethylpyrrolidin-2one, with another moiety attached by nitrogen in the pyrrolidine ring. Two methyl groups function as a small substituent at position 5 and the amide group with attached moiety (2,3dihydroindene or ethylbenzene derivatives) serves as the bigger substituent at position 4. Additionally, an electron rich substituent (amine, methylsulfonamide or hydroxyl group) at the phenyl ring in the big substituent positively influenced the MARK4 inhibitory action.
However, lines of evidence indicate one unique compound 28 (Figure 7), which, unlike other discussed structures, possesses a very big substituent at position 6 instead of the small substituent at position 5; however, it still presents very good activity (IC50 = 21 nM).  Other crucial features in these structures are substituents at positions 4 and 5 of the pyrimidine ring, depending on these substituents, structures can be divided into 4,5disubstituted (A) and 4,5-cyclic (B). The substituent at position 5 is small and lipophilic, while substituents occurring at position 4 are rather bigger and contain an amide group and/or a heteroaromatic ring. In the case of 4,5-disubstituted pyrimidines, trifluoromethyl, iodine, bromine and cyclopropane seem to be the most favorable small substituents at position 5, while in position 4, the best results were obtained for the amine group linked by different alkyl chains with pyrazole, thiophene-2-carboxamide or cyclobutene carboxamide. For the 4,5-cyclic pyrimidines, the pyrimidine is condensed with 3,3-dimethylpyrrolidin-2one, with another moiety attached by nitrogen in the pyrrolidine ring. Two methyl groups function as a small substituent at position 5 and the amide group with attached moiety (2,3dihydroindene or ethylbenzene derivatives) serves as the bigger substituent at position 4. Additionally, an electron rich substituent (amine, methylsulfonamide or hydroxyl group) at the phenyl ring in the big substituent positively influenced the MARK4 inhibitory action.
However, lines of evidence indicate one unique compound 28 (Figure 7), which, unlike other discussed structures, possesses a very big substituent at position 6 instead of the small substituent at position 5; however, it still presents very good activity (IC 50 = 21 nM).
In order to examine possible dual MARK4/5-HT 6 R action, all potent MARK4 inhibitors 22-28 ( Figure 7) were fitted to the pharmacophore model of 5-HT 6 R, and a docking study to the 5-HT 6 R homology model was carried out. The results for the most active inhibitors 23, 24 and 25 are shown in Figure 9.

5-HT 6 R/ROCKI/ROCKII as Multitarget Approach to AD Therapy
In 1995, Rho-associated coiled-coil-containing protein kinase, otherwise known as ROCK, was first identified and described as a major effector of RhoA [75]. This protein with a molecular mass of~160 kDa belongs to the RhoA subfamily and the Ras GTPase superfamily with 25% homology to Ras [76]. Their structure comprises a N-terminally located catalytic Ser/Thr kinase domain, followed by a coiled-coil-forming region (~600 amino acids) with a Rho-binding domain (RBD) and a pleckstrin-homology (PH) domain with a cysteinerich repeat at the C terminus [77]. Two mammalian isoforms of ROCK, ROCK I (ROCK-β, Rho-kinase β, or p160) encoded by a gene located on chromosome 18 and ROCK II (ROCKα, p164) encoded by a gene located on chromosome 2, can be distinguished [78,79]. Despite a high structural similarity at approximately 65% overall amino acid identity and approximately 92% identity within the N-terminal kinase domain, these homologs have different locations in the body and, thus, different physiological functions have been identified for each [80].
In general, by phosphorylation of various molecular substrates, kinases ROCK are involved in many processes including cell contraction, adhesion, migration, growth, proliferation, inflammation, apoptosis and other various cellular functions [81]. Moreover, studies indicate that the activation of the RhoA/ROCK signaling pathway seems to induce Aβ aggregation [80], phosphorylated tau formation [82], neuroinflammation [83], synaptic damage [84], and other mechanisms, ultimately leading to AD [85].
Over the past decade, a whole host of structures have emerged as ROCK inhibitors for use in certain pathological conditions [86][87][88][89][90][91], including central nervous system diseases such as AD, Parkinson's disease (PD) and Huntington's disease (HD) [85,92,93]. So far, none have been sufficient for use in the treatment of neurodegenerative diseases. Furthermore, in the current literature, there are a lack of compounds with multitarget effects on ROCK and another dementia-related targets, such as 5-HT 6 R.
Inhibition of ROCK I/II kinases has become a dynamically developing trend in recent years, as evidenced by the huge number of compounds from various chemical classes. Among them, it is possible to distinguish compounds belonging to the following chemical groups: benzimidazole 29 [ One of the first and most important ROCK inhibitors was the isoquinoline derivative fasudil 30 [109], approved by the FDA for human use in 1995 in Japan for the treatment of cerebral vasospasm [110]. The compound is moderately potent with a K i of 330 nM and its structure consists of an isoquinoline ring, linked via a sulfonic group to the homopiperazine ring. Based on the current literature, it is worth noting that the sulfone moiety is also found in a great amount of potent 5-HT 6 ligands.
To date, fasudil, as well as its analogues, are the most investigated ROCK inhibitors. Many studies have shown that the compound improves memory deficits, significantly reduces Aβ and p-tau protein levels, restores cognitive function, reduces oxidative stress, and decreases neuronal apoptosis in the hippocampus [111][112][113][114].
The compound Y-27632 34 ( Figure 10) and its analogues that have the aminopyridine core were synthesized by Yoshitomi Pharmaceuticals [115]. Moreover, compounds consisting of an aromatic ring directly attached at position 4 of the pyridine, azaindole, or pyrimidine already showed activity at the nanomolar level [97]. SAR analysis indicated that a large aromatic surface area hiding in the kinase active site and the additional presence of the NH moiety as hydrogen bond donors/acceptors significantly increases the inhibitory potency [116,117].
The indazole scaffold reported mainly by GlaxoSmithKline Pharmaceutical and Lee's team has provided a number of compounds that can be considered potent inhibitors of ROCK I/II [104,105]. It should be noted that several essential 5-HT 6 ligands, such as Cerlapirdine (2, Figure 1), developed by Pfizer, also contain a central core of the indazole [30]. In addition, many compounds in this chemical class have piperazine, 1,3,5-triazine [106] or 1,3-diazine (pyrimidine) moieties in their structure, which may also be a required pharmacophore feature of ROCK kinases. Importantly, these elements are a crucial structural feature of many 5-HT 6 ligands, fitting into the current 5-HT 6 R pharmacophore.
The extensive literature used for this review also identified indole and 7-azaindole fused rings as structurally important moieties for both 5-HT 6 R ligands and ROCK I/II kinase inhibitors ( Figure 11). All the structural similarities indicated above point to a real opportunity to create compounds with multitarget action on 5-HT 6 /ROCK I/II. One of the first and most important ROCK inhibitors was the isoquinoline derivative fasudil 30 [109], approved by the FDA for human use in 1995 in Japan for the treatment of cerebral vasospasm [110]. The compound is moderately potent with a Ki of 330 nM and its structure consists of an isoquinoline ring, linked via a sulfonic group to the homopiperazine ring. Based on the current literature, it is worth noting that the sulfone moiety is also found in a great amount of potent 5-HT6 ligands.
To date, fasudil, as well as its analogues, are the most investigated ROCK inhibitors. Many studies have shown that the compound improves memory deficits, significantly reduces Aβ and p-tau protein levels, restores cognitive function, reduces oxidative stress, and decreases neuronal apoptosis in the hippocampus [111][112][113][114].
The compound Y-27632 34 ( Figure 10) and its analogues that have the aminopyridine core were synthesized by Yoshitomi Pharmaceuticals [115]. Moreover, compounds consisting of an aromatic ring directly attached at position 4 of the pyridine, azaindole, or pyrimidine already showed activity at the nanomolar level [97]. SAR analysis indicated that a large aromatic surface area hiding in the kinase active site and the additional presence of the NH moiety as hydrogen bond donors/acceptors significantly increases the inhibitory potency [116,117].
The indazole scaffold reported mainly by GlaxoSmithKline Pharmaceutical and Lee's team has provided a number of compounds that can be considered potent inhibitors of ROCK I/II [104,105]. It should be noted that several essential 5-HT6 ligands, such as Cerlapirdine (2, Figure 1), developed by Pfizer, also contain a central core of the indazole [30]. In addition, many compounds in this chemical class have piperazine, 1,3,5-triazine [106] or 1,3-diazine (pyrimidine) moieties in their structure, which may also be a required pharmacophore feature of ROCK kinases. Importantly, these elements are a crucial structural feature of many 5-HT6 ligands, fitting into the current 5-HT6R pharmacophore.
The extensive literature used for this review also identified indole and 7-azaindole fused rings as structurally important moieties for both 5-HT6R ligands and ROCK I/II kinase inhibitors ( Figure 11). All the structural similarities indicated above point to a real opportunity to create compounds with multitarget action on 5-HT6/ROCK I/II.  Figure 11. Common structural elements found in 5-HT6R ligands and ROCK I/II inhibitors. Figure 11. Common structural elements found in 5-HT 6 R ligands and ROCK I/II inhibitors.

5-HT 6 & ROCK I/II common structural components
The potency of ROCK I and ROCK II inhibitors to also constitute good 5-HT 6 R ligands was tested in the following manner: all ROCK I and ROCK II data present in the ChEMBL database were filtered according to the activity values (K i or IC 50 ), which were supposed to be lower than 500 nM to consider the compound as active. There were 983 such inhibitors of ROCK I, and 1841 compounds inhibiting ROCK II. The majority of those ligands (80% and 76%, respectively) were successfully mapped on the 5-HT 6 R pharmacophore model, with example mappings presented for 31, 32 and 44 in Figure 12A.
The potency of ROCK I and ROCK II inhibitors to also constitute good 5-HT6R ligands was tested in the following manner: all ROCK I and ROCK II data present in the ChEMBL database were filtered according to the activity values (Ki or IC50), which were supposed to be lower than 500 nM to consider the compound as active. There were 983 such inhibitors of ROCK I, and 1841 compounds inhibiting ROCK II. The majority of those ligands (80% and 76%, respectively) were successfully mapped on the 5-HT6R pharmacophore model, with example mappings presented for 31, 32 and 44 in Figure 12A. Analogously to the MARK4 inhibitors, the ROCK I and ROCK II ligands were also docked to the 5-HT6R homology model ( Figure 12B). Despite the correct fitting of the ligands to the pharmacophore model, they also form energy-preferable complexes with the 5-HT6R. All the compounds presented in Figure 12B form a hydrogen bond with D3x32 (31 and 32 via piperidine moiety, 44 via the amine part). 31-5HT6R complex possesses an additional hydrogen bond interaction between the primary amine group and A5x43. All the compounds also interact with 5-HT6R via pi-pi stacking with F6x52 (31 forms also pi-pi contact with F6x51).

5-HT6R/CDK5 as Possible Dual Target Approach in Search for Innovative Therapy
First purified from bovine brain in 1992 [118], cyclin-dependent kinase 5 (CDK5) belongs to the family of proline-directed serine/threonine kinases and its gene is located on chromosome 7q36. The amino acids sequence of CDK5 is highly homologous to the sequence of other members of the CDKs family. In cells, it is responsible for various mechanisms including metabolic pathways, cell division and activation of transcriptional factors. To maintain its action, this kinase binds with unique activators such as p35 and p39 (expressed only in the CNS), the structure of which is more distinctive than typical CDKs. Structurally, the CDK5 protein consists of the N-terminus, C-terminus, ATP binding domain, activator binding domain, hinge region, PSSALRE helix and Tloop. Functions of the PSSALRE helix, Tloop, and ATP binding domain, which are essential for activation of CDK5, can be changed by different post-translational modifications (PTMs). Lines of evidence show that PTMs extend the functionality of the protein [119][120][121].
CDK5 in the human body can be found mainly in the central nervous system (CNS), where it participates in neuron migration, neurite overgrowth and synaptogenesis. Apart from CNS, CDK5 is also present in pancreatic β cells, corneal epithelial cells, and Analogously to the MARK4 inhibitors, the ROCK I and ROCK II ligands were also docked to the 5-HT 6 R homology model ( Figure 12B).
Despite the correct fitting of the ligands to the pharmacophore model, they also form energy-preferable complexes with the 5-HT 6 R. All the compounds presented in Figure 12B form a hydrogen bond with D3x32 (31 and 32 via piperidine moiety, 44 via the amine part). 31-5HT 6 R complex possesses an additional hydrogen bond interaction between the primary amine group and A5x43. All the compounds also interact with 5-HT 6 R via pi-pi stacking with F6x52 (31 forms also pi-pi contact with F6x51).

5-HT 6 R/CDK5 as Possible Dual Target Approach in Search for Innovative Therapy
First purified from bovine brain in 1992 [118], cyclin-dependent kinase 5 (CDK5) belongs to the family of proline-directed serine/threonine kinases and its gene is located on chromosome 7q36. The amino acids sequence of CDK5 is highly homologous to the sequence of other members of the CDKs family. In cells, it is responsible for various mechanisms including metabolic pathways, cell division and activation of transcriptional factors. To maintain its action, this kinase binds with unique activators such as p35 and p39 (expressed only in the CNS), the structure of which is more distinctive than typical CDKs. Structurally, the CDK5 protein consists of the N-terminus, C-terminus, ATP binding domain, activator binding domain, hinge region, PSSALRE helix and Tloop. Functions of the PSSALRE helix, Tloop, and ATP binding domain, which are essential for activation of CDK5, can be changed by different post-translational modifications (PTMs). Lines of evidence show that PTMs extend the functionality of the protein [119][120][121].
CDK5 in the human body can be found mainly in the central nervous system (CNS), where it participates in neuron migration, neurite overgrowth and synaptogenesis. Apart from CNS, CDK5 is also present in pancreatic β cells, corneal epithelial cells, and monocytes, where it is responsible for apoptosis, cell motility and cell cycle progression. Moreover, in previous years, CDK5 action was also proved to be associated with dopaminergic signaling, neurotransmitter release, and membrane cycling [122]. Concerning its mechanism of action, the aforementioned protein was suggested as a new therapeutic target for cancer [123][124][125], along with CNS diseases including AD, HD, stroke, and PD [126][127][128][129]. Increased activity of CDK5 is suggested as one of the causes of AD development. Dysregulation of this protein induces apoptosis of neuronal cells through various mechanisms, including Bcl-2, JNK3 and MEF2 [128].
The first main group represents 5-cyclobutylthiazol-2-yl derivatives, from which SAR showed that the heteroaromatic ring connected in the 2-amino position and a small hydrophobic substituent in the thiazole 5-position increased the selectivity and potency towards the CDK5. Thiazole moiety can also be found in a few antagonists of 5-HT 6 receptor, e.g., 12 with K i = 119 nM (Figure 1) [44].
Compounds containing 9-isopropyl-9H-purine scaffold (Figure 13b) having various hydroxyalkylamine substituents at the 5-position were shown to be the most potent within this group. As examples, highly potent reference kinase inhibitors: roscovitine, olomoucine and purvalanol A, may be mentioned. In a diverse compilation of 5-HT 6 receptor antagonists, some of the structures (with nanomolar affinities towards 5-HT 6 R) can resemble the purine scaffold present in 48 and 49 (Figure 13b).
Importantly, several studies including X-ray and molecular modeling have indicated the pivotal role of cysteine (Cys83) in ligands binding to CDK5 in the ATP binding pocket [139,140]. This amino acid may be S-nitrosylated and, fascinatingly, the perturbation of such a process leads to the enhancement of dendrite development in cultured hippocampal neurons, which, of course, influences overall neuronal development [120]. Cys83, thanks to its characteristic structure, acts simultaneously as a hydrogen bond acceptor and donor. Hence, potent CDK5 inhibitors very often possess structural fragments that also consist of pairs, such as hydrogen bond acceptor and donor, placed closed to each other ( Figure 14). Thus, it is possible to form two hydrogen bonds with the protein via interaction with Cys83 ( Figure 14a) [141]. Interestingly, such chemical groups also occur in the structures of many 5-HT 6 R ligands, increasing the probability of their strong binding to CDK5 (Figure 14b). Along other inhibitors (Figure 13d), chemical structures vary genuinely, including structures of pyrazolo[1,5-a]pyrimidine (52), 2-aminopyrimidine (53), indoline-2-on (54), macrocycles (55) pyridopyrimidinone (44) and many others.
The first main group represents 5-cyclobutylthiazol-2-yl derivatives, from which SAR showed that the heteroaromatic ring connected in the 2-amino position and a small hydrophobic substituent in the thiazole 5-position increased the selectivity and potency towards the CDK5. Thiazole moiety can also be found in a few antagonists of 5-HT6 receptor, e.g., 12 with Ki = 119 nM (Figure 1) [44].
Compounds containing 9-isopropyl-9H-purine scaffold (Figure 13b) having various hydroxyalkylamine substituents at the 5-position were shown to be the most potent within this group. As examples, highly potent reference kinase inhibitors: roscovitine, olomoucine and purvalanol A, may be mentioned. In a diverse compilation of 5-HT6 receptor antagonists, some of the structures (with nanomolar affinities towards 5-HT6R) can resemble the purine scaffold present in 48 and 49 (Figure 13b).
Importantly, several studies including X-ray and molecular modeling have indicated the pivotal role of cysteine (Cys83) in ligands binding to CDK5 in the ATP binding pocket [139,140]. This amino acid may be S-nitrosylated and, fascinatingly, the perturbation of such a process leads to the enhancement of dendrite development in cultured hippocampal neurons, which, of course, influences overall neuronal development [120]. Cys83, thanks to its characteristic structure, acts simultaneously as a hydrogen bond acceptor and donor. Hence, potent CDK5 inhibitors very often possess structural fragments that also consist of pairs, such as hydrogen bond acceptor and donor, placed closed to each other ( Figure 14). Thus, it is possible to form two hydrogen bonds with the protein via interaction with Cys83 ( Figure 14a) [141]. Interestingly, such chemical groups also occur in the structures of many 5-HT6R ligands, increasing the probability of their strong binding to CDK5 (Figure 14b).  Dozens of CDK5 inhibitors have reached clinical trials, mainly as therapeutics for various cancers. Dinaciclib (52, Figure 13d), for example, is currently undergoing phase 1 of the clinical trials for the treatment of breast cancer [142]. The most potent inhibitors reach the 1 nM affinity. Despite the huge number of highly active CDK5 agents, dual 5-HT 6 R/CDK5 continue to be an underexplored area of scientific research.
In terms of exploring the structural possibility for the desirable CDK5/5-HT 6 R dual action, the CDK5 ligands, which were filtered according to the same criteria as ROCK I and ROCK II compounds, formed the set of 263 compounds. Among them, 219 compounds were properly mapped to the 5-HT 6 R pharmacophore model, indicating their high potency for possessing a 5-HT 6 R activity component. Examples of three (56-58) out of 44 compounds, which were not mapped to the model, are presented in Figure 15.  Figure 13d), for example, is currently undergoing phase 1 of the clinical trials for the treatment of breast cancer [142]. The most potent inhibitors reach the 1 nM affinity. Despite the huge number of highly active CDK5 agents, dual 5-HT6R/CDK5 continue to be an underexplored area of scientific research.
In terms of exploring the structural possibility for the desirable CDK5/5-HT6R dual action, the CDK5 ligands, which were filtered according to the same criteria as ROCK I and ROCK II compounds, formed the set of 263 compounds. Among them, 219 compounds were properly mapped to the 5-HT6R pharmacophore model, indicating their high potency for possessing a 5-HT6R activity component. Examples of three (56-58) out of 44 compounds, which were not mapped to the model, are presented in Figure 15. In addition, the compounds were docked to the 5-HT6R homology model. Results for representatives (44 and 47) of both pharmacophore mapping and docking are presented in Figure 16.  In addition, the compounds were docked to the 5-HT 6 R homology model. Results for representatives (44 and 47) of both pharmacophore mapping and docking are presented in Figure 16.  Figure 13d), for example, is currently undergoing phase 1 of the clinical trials for the treatment of breast cancer [142]. The most potent inhibitors reach the 1 nM affinity. Despite the huge number of highly active CDK5 agents, dual 5-HT6R/CDK5 continue to be an underexplored area of scientific research.
In terms of exploring the structural possibility for the desirable CDK5/5-HT6R dual action, the CDK5 ligands, which were filtered according to the same criteria as ROCK I and ROCK II compounds, formed the set of 263 compounds. Among them, 219 compounds were properly mapped to the 5-HT6R pharmacophore model, indicating their high potency for possessing a 5-HT6R activity component. Examples of three (56-58) out of 44 compounds, which were not mapped to the model, are presented in Figure 15. In addition, the compounds were docked to the 5-HT6R homology model. Results for representatives (44 and 47) of both pharmacophore mapping and docking are presented in Figure 16.  Both ligands (44 and 47) are well aligned to the pharmacophore features of 5-HT 6 R ligands ( Figure 16A). Compound 44, which has a smaller structure, is almost fully covered by the 5-HT 6 R pharmacophore model, in contrast to 47, for which quite a significant part of the molecule is outside of the model. Despite this extending part, 47 is very well fitted to the three considered features. Compound 47 also did not enter very deeply into the 5-HT 6 R binding site, rather it occupies its upper part ( Figure 16B). Nevertheless, both compounds are strongly fitted in their ligand-protein complexes through the extended network of polar and hydrophobic contacts. Additionally, both 44 and 47 form hydrogen a bond with D3 × 32 and pi-pi interaction with a phenylalanine cluster from the 6 th transmembrane helix of 5-HT 6 R.

Conclusions
As polypharmacology approaches may result in long-awaited breakthroughs in AD treatment, a computer-aided and visual analysis of the possibility of designing molecules with as yet unreported action via both 5-HT 6 R and the AD pathology-related kinase (MARK4, ROCKI/II or CDK5) within this paper has been performed. The deep insight into recent lines of evidence allowed us to identify the structural fragments that occur simultaneously in 5-HT 6 R agents and inhibitors of the considered above-mentioned kinases, as well as their appropriate bioisosteres. Interestingly, mapping the known kinase inhibitors to the 5-HT 6 R pharmacophore model showed the high potency of the majority of them to interact with the 5-HT 6 R. Hence, all potent pyrimidine-derived MARK4 inhibitors meet the 5-HT 6 R pharmacophore features criteria, as well as 80% of ROCKI, 76% of ROCKII and 83% of CDK5 ligands. Additionally, the results of docking to the 5-HT 6 R homology model confirmed the high probability of the investigated structures to form key interactions with this protein target. More than 60% of all four groups of the tested agents were docked with very good docking scores (values in the range between −10 and −6).
Summarizing, the overall analysis of the results from the pharmacophore-based and the docking-based (docking score values distribution) approaches indicated the high potency of inhibitors for all four investigated kinases to be also 5-HT 6 R antagonists. Furthermore, this very initial prediction of ADMET properties [143] for the most active kinase inhibitors that also fit in the 5-HT 6 R ligand pharmacophore features (23, 24, 25, 31, 32, 44 and 47) demonstrated rather a satisfactory profile for most of them, comparable to that of the reference drug, donepezil (see Table S2, Figure S1 in Supplementary Materials).
Such results give real hope for the design of structurally novel anti-AD agents with pioneering multifunctional (dual) action. Simultaneously, it seems to be strongly justified to test already reported 5-HT 6 R ligands in terms of potency to inhibit the investigated herein kinases, as well as to examine ADMET properties for the most promising dual-target agents found following this process.