Novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamides as selective GSK-3 inhibitors

Synthesis, biological evaluation, and SAR dependencies for a series of novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamide inhibitors of GSK-3beta kinase are described. The inhibitory activity of the synthesized compounds is highly dependent on the character of substituents in the phenyl ring and the nature of terminal heterocyclic fragment of the core molecular scaffold. The most potent compounds from this series contain 3,4-di-methyl or 2-methoxy substituents within the phenyl ring and 3-pyridine fragment connected to the 1,2,4-oxadiazole heterocycle. These compounds selectively inhibit GSK-3beta kinase with IC(50) value of 0.35 and 0.41 microM, respectively.     

Rational design of 4-amino-5,6-diaryl-furo[2,3-d]pyrimidines as potent glycogen synthase kinase-3 inhibitors

4-Amino-5,6-diaryl-furo[2,3-d]pyrimidines have been identified as inhibitors of glycogen synthase kinase-3beta (GSK-3beta). One representative derivative, 4-amino-3-(4-(benzenesulfonylamino)-phenyl)-2-(3-pyridyl)-furo[2,3-d]pyrimidine (12) exhibited potent GSK-3beta inhibitory activity in low nanomolar level of IC(50). The binding mode was proposed from a docking study.     

Aryl[a]pyrrolo[3,4-c]carbazoles as selective cyclin D1-CDK4 inhibitors

The synthesis of new analogues of Arcyriaflavin A in which one indole ring is replaced by an aryl or heteroaryl ring is described. These new series of aryl[a]pyrrolo[3,4-c]carbazoles were evaluated as inhibitors of Cyclin D1-CDK4. A potent and selective D1-CDK4 inhibitor, 7a (D1-CDK4 IC(50)=45 nM), has been identified. The potency, selectivity profile against other kinases, and structure-activity relationship (SAR) trends of this class of compounds are discussed.     

4-Acylamino-6-arylfuro[2,3-d]pyrimidines: potent and selective glycogen synthase kinase-3 inhibitors

Modeling studies of a furo[2,3-d]pyrimidine GSK-3 hit compound 1 superimposed onto the X-ray crystal structure of a legacy pyrazolo[3,4-c]pyridazine GSK-3 inhibitor 2 led to the identification of 4-acylamino-6-arylfuro[2,3-d]pyrimidine template 3. Synthesis of analogues based on template 3 has resulted in a number of potent and selective GSK-3beta inhibitors. The most potent and selective compound was the m-pyridyl analogue 24.     

Pyrazolo[3,4-b]quinoxalines. A new class of cyclin-dependent kinases inhibitors

Protein kinases are involved in most physiological processes and in numerous diseases. Therefore, inhibitors of protein kinases have therefore a wide therapeutic potential. While screening for inhibitors of cyclin-dependent kinases (CDK's) and glycogen synthase kinase-3 (GSK-3), we identified pyrazolo[3,4-b]quinoxalines as sub-micromolar inhibitors of CDK1/cyclin B. A preliminary structure-activity relationship study suggests that this family of compounds can be optimized to inhibit CDK's and GSK-3. Compounds were tested for their anti-proliferative activity and the results show that several of them displayed a significant inhibitory effect on CDK1/cyclin B. The most active compound (1) was also tested against the brain kinases CDK5/p25 and GSK-3, and proved to be a good inhibitor of both of them. On the contrary, none of the compounds showed any activity in the CDC25 phosphatase assay. As an additional approach, affinity chromatography on immobilized pyrazolo[3,4-b]quinoxalines will be used to identify the intracellular targets of this family of compounds.     

Evidence for translational control of the binding of 7, 12-dimethylbenz[a]anthracene to DNA of murine epidermal cell in culture.

The effects of various inhibitors of aryl hydrocarbon hydroxylase (AHH), antioxidants, inhibitors of DNA, RNA, and protein synthesis, and protease inhibitors on the binding of [7,12-3H]dimethylbenz[a]anthracene ([3H] DMBA) to DNA of murine epidermal cells in culture have been investigated. 7,8-Benzoflavone, 5,6-benzoflavone and methyrapone (inhibitors of AAH) and antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), efficiently reduced the binding of [3H] DMBA to cellular DNA. Inhibitors of DNA and RNA synthesis did not affect this process whereas inhibitors of protein synthesis suppressed the binding of [3H] DMBA to cellular DNA. Protease inhibitors p-tosylamide-2-phenylchloromethyl ketone (TPCK) and p-tosyl-L-lysine chloromethyl ketone (TLCK) also reduced the interaction between DMBA and DNA. Thus, it appears that binding of DMBA to cellular DNA is regulated at the level of translation or/and post translation.     

Docking,molecular dynamics,binding energy-MM-PBSA studies of naphthofuran derivatives to identify potential dual inhibitors against BACE-1 and GSK-3β

BACE-1 and GSK-3β both are potential therapeutic drug targets for Alzheimer’s disease. Recently, both these targets received attention for designing dual inhibitors. Till now only two scaffolds (triazinone and curcumin) derivatives have been reported as BACE-1 and GSK-3β dual inhibitors. In our previous work, we have reported first in class dual inhibitor for BACE-1 and GSK-3β. In this study, we have explored other naphthofuran derivatives for their potential to inhibit BACE-1 and GSK-3β through docking, molecular dynamics, binding energy (MM-PBSA). These computational methods were performed to estimate the binding affinity of naphthofuran derivatives towards the BACE-1 and GSK-3β. In the docking results, two derivatives (NS7 and NS9) showed better binding affinity as compared to previously reported inhibitors. Hydrogen bond occupancy of NS7 and NS9 generated from MD trajectories showed good interaction with the flap residues Gln73, Thr72 of BACE-1 and Arg141, Thr138 residues of GSK-3β. MM-PBSA and energy decomposition per residue revealed different components of binding energy and relative importance of amino acid involved in binding. The results showed that the binding of inhibitors was majorly governed by the hydrophobic interactions and suggesting that hydrophobic interactions might be the key to design dual inhibitors for BACE1-1 and GSK-3β. Distance between important pair of amino acid residues indicated that BACE-1 and GSK-3β adopt closed conformation and become inactive after ligand binding. The results suggested that naphthofuran derivatives might act as dual inhibitor against BACE-1 and GSK-3β.     

Novel GSK-3 inhibitors with improved cellular activity

A novel series of [1-(1H-benzimidazol-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl] arylhydrazones was synthesized and shown to potently inhibit glycogen synthase kinase-3 (GSK-3). In light of detailed structure-activity relationships and structural knowledge of the GSK-3 binding pocket, a benzimidazole substituent was incorporated onto the pyrazolopyrimidine core resulting in improved potency over previous analogs. More importantly, these derivatives show low nanomolar efficacy for stimulating glycogen synthesis in vitro and therefore may be useful in the treatment of type 2 diabetes mellitus.     

Computer-aided molecular design of pyrazolotriazines targeting glycogen synthase kinase 3

Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several processes associated with Alzheimer’s disease (AD). Therefore, GSK-3 has become a crucial therapeutic target for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep synthesis of ethyl 4-oxo-pyrazolo[4,3-d][1–3]triazine-7-carboxylates and their biological evaluation as GSK-3 inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical structure. Potential binding mode determination in the enzyme and the analysis of the key features in the catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood–brain barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and permeation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphorylation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo studies and may be considered as new leads for further optimisation for the treatment of AD.     

Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.     

Structure based de novo design of novel glycogen synthase kinase 3 inhibitors

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase that has captured great attention in drug discovery projects. Structure based design has been successfully carried out to find a novel class of GSK-3 inhibitors using the Ludi de novo ligand design program. A total of 15 potential leads are suggested from the study. The structures have been validated through detailed analysis of the Ludi score values and by molecular docking experiment using FlexX. The hits have been further verified through: (1) visual examination of how well the hits dock into the GSK-3beta binding site; (2) comparative analysis of their FlexX, G_Score, PMF_Score, ChemScore, and D_scores values; (3) a comparative investigation of the docking scores of the hits with those of the reported inhibitors after calibration of the docking procedure with 17 previously reported inhibitors; (4) determination of the binding mode of the hits and comparison with that of the so far known inhibitors. Hits retaining interactions with the common amino acids of GSK-3beta binding site were taken to represent potential leads. Structurally the hits designed are mainly flat nitrogen heterocycles. These hits are expected to be important additions to the search of GSK-3 inhibitors and may provide invaluable insights to further understand the structural basis of catalysis and inhibition of this kinase.     

Thiazolo[5,4-f]quinazolin-9-ones, inhibitors of glycogen synthase kinase-3

In an effort to identify new protein kinase inhibitors with increased potency and selectivity, we have developed the microwave-assisted synthesis of thiazolo[5,4-f]quinazolin-9-ones. The effects of eighteen derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3 were investigated. Several turned out to inhibit GSK-3 in the micromolar range. Molecular modeling studies suggest that the most selective GSK-3 inhibitors 7a-d bind into the ATP-binding site through a key hydrogen bond interaction with Val135 and target the specific hydrophobic backpocket of the enzyme.     

Elucidating substrate and inhibitor binding sites on the surface of GSK-3β and the refinement of a competitive inhibitor

A molecular understanding of substrate recognition of protein kinases provides an important basis for the development of substrate competitive inhibitors. Here, we explored substrate recognition and competitive inhibition of glycogen synthase kinase (GSK)-3β using molecular and computational tools. In previous work, we described Gln89 and Asn95 within GSK-3β as important substrates binding sites. Here, we show that the cavity bordered by loop 89-QDKRFKN-95, located in the vicinity of the GSK-3β catalytic core, is a promiscuous substrate binding subsite. Mutations within this segment highlighted Phe93 as an additional essential contact residue for substrates' recognition. However, unlike Gln89 and Asn95, Phe93 was also important for the binding of our previously described substrate competitive inhibitor, L803 [KEAPPAPPQS(p)P], and its cell-permeable variant L803-mts. The effects of the substitution of charged or polar residues within L803 further suggested that binding to GSK-3β is governed by hydrophobic interactions. Our computational model of GSK-3β bound to L803 was in agreement with the experimental data. It revealed L803 binding with a hydrophobic surface patch and identified interactions between Pro8 (L803) and Phe93 (GSK-3β). Computational modeling of new L803 variants predicted that inhibition would be strengthened by adding contacts with Phe93 or by increasing the hydrophobic content of the peptide. Indeed, the newly designed L803 variants showed improved inhibition. Our study identified different and overlapping elements in GSK-3β substrate and inhibitor recognition and provides a novel example for model-based rational design of substrate competitive inhibitors for GSK-3.     

Synthesis and evaluation of novel heterocyclic inhibitors of GSK-3

A set of novel heterocyclic pyrimidyl hydrazones has been synthesized as inhibitors of glycogen synthase kinase-3 (GSK-3) with the most active exhibiting low nanomolar activity. Quantum mechanical calculations indicate that of the conformational factors that could determine binding affinity, the planarity of the phenyl ring in relation to the central core and the conformation of the hydrazone chain may be the most influential.     

Binding characteristics of 4-S proteins from rat and mouse liver. High affinity of ellipticines

The binding characteristics of 4-S components (carcinogen-binding protein) from livers of Sprague-Dawley rats, C57BL/6 and DBA/2 mice have been examined before and after separation from other binding components presents in the cytosol. Competitive potency of 3-methylcholanthrene, benzo[alpha]pyrene, beta-naphthoflavone and 20 ellipticines, a series of compounds differently substituted on the dimethyl-pyrido-carbazole nucleus and deprived of carcinogenic activity, has been determined with [3H]3-methylcholanthrene and/or [3H]benzo[a]pyrene as radioligands. The inducing ability of the same compounds for aryl hydrocarbon hydroxylase and for ethoxyresorufin-O-deethylase has been compared to their affinity for the 4-S protein and the Ah receptor respectively. The main results of this study are as follows. 1. The intrinsic binding characteristics of 4-S proteins were dependent on both the nature of the radioligand used and the presence or absence of other cytosolic binding components. 2. The heterocyclic ellipticines were revealed as strong ligands for the carcinogen-binding protein (stronger than benzo[alpha]pyrene for five derivatives substituted in the A ring of ellipticine), with IC50 values ranging from 0.047 microM (8-hydroxyellipticine) to 5.8 microM (N2-ethyl-9-hydroxyellipticinium). 3. When the affinity of ellipticines was plotted versus their inducing ability of aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase, it appears that a good correlation exists for the Ah receptor but not for the 4-S protein. It is concluded that these data, as well as the lack of enzymatic induction after benzo[alpha]pyrene treatment of DBA/2 mice, which display a high level of 4-S protein, do not support the implication of this binding component in the positive control of cytochrome P-450 induction.     

Structure-based design of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3β inhibitors to activate Wnt/β-catenin pathway

Deregulation of Wnt/β-catenin pathway is closely related to the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD), and glycogen synthase kinase 3β (GSK-3β), the central negative regulator of Wnt pathway, is regarded as an important target for these diseases. Here, we report a series of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3β inhibitors by rational-design and synthesis, which show high selectivity against GSK-3β over Cyclin-dependent kinase 2 (CDK2), and significantly activate the cellular Wnt/β-catenin pathway. The structure–activity relationship of these GSK-3β inhibitors was also explored by in silico molecular docking.     

1-[3-Aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one (BMS-740808) a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa

Attempts to further optimize the pyrazole factor Xa inhibitors centered on masking the aryl aniline P4 moiety. Scaffold optimization resulted in the identification of a novel bicyclic pyrazolo-pyridinone scaffold which retained fXa potency. The novel bicyclic scaffold preserved all binding interactions observed with the monocyclic counterpart and importantly the carboxamido moiety was integrated within the scaffold making it less susceptible to hydrolysis. These efforts led to the identification of 1-[3-aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one 6f (BMS-740808), a highly potent (fXa Ki=30 pM) with a rapid onset of inhibition (2.7x10(7) M-1 s-1) in vitro, selective (>1000-fold over other proteases), efficacious in the AVShunt thrombosis model, and orally bioavailable inhibitor of blood coagulation factor Xa.     

4-amino-5-aryl-6-arylethynylpyrimidines: structure-activity relationships of non-nucleoside adenosine kinase inhibitors

A series of non-nucleoside adenosine kinase (AK) inhibitors is reported. These inhibitors originated from the modification of 5-(3-bromophenyl)-7-(6-morpholin-4-ylpyridin-3-yl)pyrido[2,3-d]pyrimidin-4-ylamine (ABT-702). The identification of a linker that would approximate the spatial arrangement found between the pyrimidine ring and the aryl group at C(7) in ABT-702 was a key element in this modification. A search of potential linkers led to the discovery of an acetylene moiety as a suitable scaffold. It was hypothesized that the aryl acetylenes, ABT-702, and adenosine bound to the active site of AK (closed form) in a similar manner with respect to the orientation of the heterocyclic base. Although potent acetylene analogs were discovered based on this assumption, an X-ray crystal structure of 5-(4-dimethylaminophenyl)-6-(6-morpholin-4-ylpyridin-3-ylethynyl)pyrimidin-4-ylamine (16a) revealed a binding orientation contrary to adenosine. In addition, this compound bound tightly to a unique open conformation of AK. The structure-activity relationships and unique ligand orientation and protein conformation are discussed.     

5-aryl-pyrazolo[3,4-b]pyridines: potent inhibitors of glycogen synthase kinase-3 (GSK-3)

A novel series of pyrazolo[3,4-b]pyridines has been identified that are potent inhibitors of glycogen synthase kinase-3 (GSK-3).