Structural basis for specificity of TGFβ family receptor small molecule inhibitors

Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC₅₀ (EC₅₀ = 1 μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway.     

Identification of small molecules that inhibit GSK-3β through virtual screening

Glycogen synthase kinase-3β (GSK-3β) is involved in glycogen metabolism, neuronal cell development, osteoblast differentiation. Small molecule inhibitors of GSK-3β have various therapeutic potential for the treatment of diabetes type II, bipolar disorders, stroke and chronic inflammatory disease.To identify GSK-3β inhibitors with novel scaffold from chemical library, we primarily screened out putative inhibitors through computer modeling and subsequently evaluated the inhibitory activity of selected compounds against GSK-3β by in vitro Z’-LYTE? assay. A series of compound KRMs strongly inhibited phosphorylation of its substrate with IC₅₀ value of approximately 0.5 μM. Also, we demonstrated that KRM-189 and KRM-191 competed with ATP for GSK-3β, leading to decreased V_max and constant K_m with increasing concentrations of ATP as determined from Lineweaver–Berk equation. Moreover, they showed the selectivity for GSK-3β over other kinases with IC₅₀ values of 2 to 10 μM or more Incubation of cells with KRM-191 with highly selective and potent inhibitory activity caused accumulation of β-catenin, downstream of GSK-3β signaling pathway, indicating that small molecule can prevent degradation of β-catenin via GSK-3β inhibition. Our results suggest that modeling in combination with in vitro assays can be used for the identification of selective and potent inhibitors.     

Identification of distinct physiochemical properties of toxic prefibrillar species formed by Aβ peptide variants

The formation of amyloid-β peptide (Aβ) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of Aβ. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of Aβ-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various Aβ aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those Aβ peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the Aβ peptide to form nontoxic versus toxic species.     

The identification of novel PLC-γ inhibitors using virtual high throughput screening

Phospholipase C-γ (PLC-γ) has been identified as a possible biological target for anticancer drug therapy but suitable inhibitors are lacking. Therefore, in order to identify active compounds (hits) virtual high throughput screening was performed. The crystal structure of the PLC-δ isoform was used as a model docking scaffold since no crystallographic data are available on its γ counterpart. A pilot screen was performed using ～9.2 × 10⁴ compounds, where the robustness of the methodology was tested. This was followed by the main screening effort where ～4.4 × 10⁵ compounds were used. In both cases, plausible compounds were identified (virtual hits) and a selection of these was experimentally tested. The most potent compounds were in the single digit micro-molar range as determined from the biochemical (Flashplate) assay. This translated into ～15 μM in a functional assay in cells. About 30% of the virtual hits showed activity against PLC-γ (IC₅₀ < 50 μM).     

Identification of novel scaffold of benzothiazepinones as non-ATP competitive glycogen synthase kinase-3β inhibitors through virtual screening

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that has been proved as a key target for neurodegenerative diseases and diabetes. Up to date, most of known inhibitors are bound to the ATP-binding pocket of GSK-3β, which might lead widespread effects due to the high homology between kinases. Recently, some of its non-ATP competitive inhibitors had been confirmed having therapeutical effects owing to their high selectivity. This finding opens a new pathway to study hopeful drugs for treatment of these diseases. However, it is still a challenge nowadays on how to efficiently find non-ATP competitors. Here, we successfully discovered a novel scaffold of benzothiazepinones (BTZs) as selective non-ATP competitive GSK-3β inhibitors through virtual screening approach. A 3D receptor model of substrate binding site of GSK-3β was constructed and applied to screen against drug-like Maybridge database through Autodock program. BTZ compounds were top ranked as efficient hits and were then synthesized for further screening. Among them, the representative compound 4j showed activity to GSK-3β (IC₅₀: 25 μM) in non-ATP competitive mechanism, and nearly no inhibitory effect on other 10 related protein kinases. Overall, the results point out that BTZ compounds might be useful in treatment of Alzheimer’s disease and diabetes mellitus as novel GSK-3β inhibitors. It also suggests, on the other hand, that virtual screening would provide a valuable tool in combination with in vitro assays for the identification of novel selective and potent inhibitors.     

The specificities of small molecule inhibitors of the TGFß and BMP pathways

Small molecule inhibitors of type 1 receptor serine threonine kinases (ALKs1-7), the mediators of TGFß and BMP signals, have been employed extensively to assess their physiological roles in cells and organisms. While all of these inhibitors have been reported as “selective” inhibitors of specific ALKs, extensive specificity tests against a wide array of protein kinases have not been performed. In this study, we examine the specificities and potencies of the most frequently used small molecule inhibitors of the TGFß pathway (SB-431542, SB-505124, LY-364947 and A-83-01) and the BMP pathway (Dorsomorphin and LDN-193189) against a panel of up to 123 protein kinases covering a broad spectrum of the human kinome. We demonstrate that the inhibitors of the TGFß pathway are relatively more selective than the inhibitors of the BMP pathway. Based on our specificity and potency profile and published data, we recommend SB-505124 as the most suitable molecule for use as an inhibitor of ALKs 4, 5 and 7 and the TGFß pathway. We do not recommend Dorsomorphin, also called Compound C, for use as an inhibitor of the BMP pathway. Although LDN-193189, a Dorsomorphin derivative, is a very potent inhibitor of ALK2/3 and the BMP-pathway, we found that it potently inhibited a number of other protein kinases at concentrations sufficient to inhibit ALK2/3 and its use as a selective BMP-pathway inhibitor has to be considered cautiously. Our observations have highlighted the need for caution when using these small molecule inhibitors to assess the physiological roles of BMP and TGFß pathways.     

Aβ peptide fibrillar architectures controlled by conformational constraints of the monomer

Anomalous self-assembly of the Aβ peptide into fibrillar amyloid deposits is strongly correlated with the development of Alzheimer's disease. Aβ fibril extension follows a template guided "dock and lock" mechanism where polymerisation is catalysed by the fibrillar ends. Using surface plasmon resonance (SPR) and quenched hydrogen-deuterium exchange NMR (H/D-exchange NMR), we have analysed the fibrillar structure and polymerisation properties of both the highly aggregation prone Aβ1-40 Glu22Gly (Aβ(40Arc)) and wild type Aβ1-40 (Aβ(40WT)). The solvent protection patterns from H/D exchange experiments suggest very similar structures of the fibrillar forms. However, through cross-seeding experiments monitored by SPR, we found that the monomeric form of Aβ(40WT) is significantly impaired to acquire the fibrillar architecture of Aβ(40Arc). A detailed characterisation demonstrated that Aβ(40WT) has a restricted ability to dock and isomerise with high binding affinity onto Aβ(40Arc) fibrils. These results have general implications for the process of fibril assembly, where the rate of polymerisation, and consequently the architecture of the formed fibrils, is restricted by conformational constraints of the monomers. Interestingly, we also found that the kinetic rate of fibril formation rather than the thermodynamically lowest energy state determines the overall fibrillar structure.     

Affinity-based screening of MDM2/MDMX–p53 interaction inhibitors by chemical array: Identification of novel peptidic inhibitors

MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein–protein interactions (PPIs) of MDM2–p53 and MDMX–p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2–p53 and MDMX–p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2–p53 and MDMX–p53 interactions.     

NF-κB inducing kinase (NIK) inhibitors: Identification of new scaffolds using virtual screening

As a wide variety of pro-inflammatory cytokines are involved in the development of rheumatoid arthritis (RA), there is an urgent need for the discovery of novel therapeutic strategies. Among these, the inhibition of the NF-κB inducing kinase (NIK), a key enzyme of the NF-κB alternative pathway activation, represents a potential interesting approach. In fact, NIK is involved downstream of many tumor necrosis factor receptors (TNFR) like CD40, RANK or LTβR, implicated in the pathogenesis of RA. But, up to now, the number of reported putative NIK inhibitors is extremely limited. In this work, we report a virtual screening (VS) study combining various filters including high-throughput docking using a 3D-homology model and ranking by using different scoring functions. This work led to the identification of two molecular fragments, 4H-isoquinoline-1,3-dione (5) and 2,7-naphthydrine-1,3,6,8-tetrone (6) which inhibit NIK with an IC₅₀ value of 51 and 90 μM, respectively. This study opens new perspectives in the field of the NF-κB alternative pathway inhibition.     

Small molecule peptidomimetic inhibitors of importin α/β mediated nuclear transport

Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Translocation of proteins and many RNAs between the nucleus and cytoplasm is carried out by shuttling receptors of the β-karyopherin family, also called importins and exportins. Leptomycin B, a small molecule inhibitor of the exportin CRM1, has proved to be an invaluable tool for cell biologists, but up to now no small molecule inhibitors of nuclear import have been described. We devised a microtiter plate based permeabilized cell screen for small molecule inhibitors of the importin α/β pathway. By analyzing peptidomimetic libraries, we identified β-turn and α-helix peptidomimetic compounds that selectively inhibit nuclear import by importin α/β but not by transportin. Structure–activity relationship analysis showed that large aromatic residues and/or a histidine side chain are required for effective import inhibition by these compounds. Our validated inhibitors can be useful for in vitro studies of nuclear import, and can also provide a framework for synthesis of higher potency nuclear import inhibitors.     

Discovery of new GSK-3β inhibitors through structure-based virtual screening

Glycogen synthase kinase-3β (GSK-3β) is an attractive therapeutic target for human diseases, such as diabetes, cancer, neurodegenerative diseases, and inflammation. Thus, structure-based virtual screening was performed to identify novel scaffolds of GSK-3β inhibitors, and we observed that conserved water molecules of GSK-3β were suitable for virtual screening. We found 14 hits and D1 (IC₅₀ of 0.71?μM) were identified. Furthermore, the neuroprotection activity of D1–D3 was validated on a cellular level. 2D similarity searches were used to find derivatives of high inhibitory compounds and an enriched structure–activity relationship suggested that these skeletons were worthy of study as potent GSK-3β inhibitors.     

In silico screening and identification of potential GSK3β inhibitors

Abstract

Glycogen synthase kinase-3β (GSK3β) has been reported for its impact on multitude biological processes from cell proliferation to apoptosis. The increase in the ratio of active/inactive GSK3β is the major factor associated in the etiology of several psychiatric diseases, diabetes, muscle hypertrophy, neurodegenerative diseases, and some cancers. These findings made GSK3β a promising therapeutic target, and the interest in the discovery, synthesis of novel drugs to effectively attenuate its function with probably no side effects has been increasing in the chronology of GSK3β drug discovery. In the present study, we applied a combination of computational tools on a chemical library for the virtual discovery of their potency to inhibit GSK3β. The chemical library was screened against a set of filters at different levels. Finally, five compounds in the chemical library were found to potentially inhibit GSK3β with no toxic effects. Furthermore, binding mode analysis revealed that all the compounds bound to the ATP site and most of the hydrogen bonding interactions are conserved as in GSK3β structures deposited in PDB.     

Inhibition of choline acetyltransferase as a mechanism for cholinergic dysfunction induced by amyloid-β peptide oligomers

Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-β peptide (Aβ). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aβ oligomers (AβOs). AβOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AβOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AβOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AβOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AβO binding sites, fully prevented AβO-induced inhibition of ChAT. Interestingly, we found that AβOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AβO-targeted neurons. Reduction in ChAT activity instigated by AβOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.     

Small molecule inhibitors of IκB kinase β: A chip-based screening and molecular docking simulation

A chip-based screening system for IκB kinase β (IKKβ) has been developed by physically immobilizing the substrate IκBα on a glass matrix using a calixarene linker. Phosphorylation of IκBα by IKKβ and ATP was quantitated using a fluorescently labeled antibody. Using this efficient assay system a chemical library of 2000 bioactive compounds was screened against IKKβ and four were identified as good inhibitors, namely, aurintricarboxylic acid, diosmin, ellagic acid, and hematein. None of them have been reported to be an inhibitor of IKKβ although they were implicated in various NFκB-mediated biological processes. Our enzyme-based assay showed that IC50 of the four inhibitors is comparable with that of IKK-16, a previously known strong inhibitor. Molecular docking simulation shows that the hydrophobic moiety of an inhibitor interacts with the four hydrophobic residues (Leu21, Val29, Val152, and Ile165) of the active site. The MM-PBSA calculation suggests that these hydrophobic interactions appear to be the predominant contributor to the binding free energy. As IKKβ is ubiquitously expressed in various cell types and executes many biological functions, the enzyme and cell specificity of the four inhibitors need to be rigorously tested before accepted as a drug candidate.     

Single-cell screening of cytosolic [Ca2+] reveals cell-selective action by the Alzheimer’s Aβ peptide ion channel

Interaction of the Alzheimer’s Aβ peptides with the plasma membrane of cells in culture results in chronic increases in cytosolic [Ca²⁺]. Such increases can cause a variety of secondary effects leading to impaired cell growth or cell degeneration. In this investigation, we made a comprehensive study of the changes in cytosolic [Ca²⁺] in single PC12 cells and human neurons stressed by continuous exposure to a medium containing Aβ42 for several days. The differential timing and magnitude of the Aβ42-induced increase in [Ca²⁺] reveal subpopulations of cells with differential sensitivity to Aβ42. These results suggest that the effect produced by Aβ on the level of cytosolic [Ca²⁺] depends on the type of cell being monitored. Moreover, the results obtained of using potent inhibitors of Aβ cation channels such as Zn²⁺ and the small peptide NA7 add further proof to the suggestion that the long-term increases in cytosolic [Ca²⁺] in cells stressed by continuous exposure to Aβ is the result of Aβ ion channel activity.     

Combinatorial peptide library screening for discovery of diverse α-glucosidase inhibitors using molecular dynamics simulations and binary QSAR models

Human α-glucosidase is an enzyme involved in the catalytic cleavage of the glucoside bond and involved in numerous functionalities of the organism, as well as in the insurgence of diabetes mellitus 2 and obesity. Thus, developing chemicals that inhibit this enzyme is a promising approach for the treatment of several pathologies. Small peptides such as di- and tri-peptides may be in natural organism as well as in the GI tract in high concentration, coming from the digestive process of meat, wheat and milk proteins. In this work, we reported the first tentative hierarchical structure-based virtual screening of peptides for human α-glucosidase. The goal of this work is to discover novel and diverse lead compounds that my act as inhibitors of α-glucosidase such as small peptides by performing a computer aided virtual screening and to find novel scaffolds for further development. Thus, in order to select novel candidates with original structure we performed molecular dynamics (MD) simulations among the 12 top-ranked peptides taking as comparison the MD simulations performed on crystallographic inhibitor acarbose. The compounds with the lower RMSD variability during the MD, were reserved for in vitro biological assay. The selected 4 promising structures were prepared on solid phase peptide synthesis and used for the inhibitory assay, among them compound 2 showed good inhibitory activity, which validated our method as an original strategy to discover novel peptide inhibitors. Moreover, pharmacokinetic profile predictions of these 4 peptides were also carried out with binary QSAR models using MetaCore/MetaDrug applications.     

Identification of a small molecule β-secretase inhibitor that binds without catalytic aspartate engagement

A small molecule inhibitor of β-secretase with a unique binding mode has been developed. Crystallographic determination of the enzyme–inhibitor complex shows the catalytic aspartate residues in the active site are not engaged in inhibitor binding. This unprecedented binding mode in the field of aspartyl protease inhibition is described.     

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

Background

Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.

Methods

To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.

Results

LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.

Conclusion

Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.     

Identification of chemically diverse, novel inhibitors of 17β-hydroxysteroid dehydrogenase type 3 and 5 by pharmacophore-based virtual screening

17β-Hydroxysteroid dehydrogenase type 3 and 5 (17β-HSD3 and 17β-HSD5) catalyze testosterone biosynthesis and thereby constitute therapeutic targets for androgen-related diseases or endocrine-disrupting chemicals. As a fast and efficient tool to identify potential ligands for 17βHSD3/5, ligand- and structure-based pharmacophore models for both enzymes were developed. The models were evaluated first by in silico screening of commercial compound databases and further experimentally validated by enzymatic efficacy tests of selected virtual hits. Among the 35 tested compounds, 11 novel inhibitors with distinct chemical scaffolds, e.g. sulfonamides and triazoles, and with different selectivity properties were discovered. Thereby, we provide several potential starting points for further 17β-HSD3 and 17β-HSD5 inhibitor development. Article from the Special issue on Targeted Inhibitors.