Bis(1,8-anilinonaphthalenesulfonate). A novel and potent inhibitor of microtubule assembly

Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving tubulin.     

Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.     

Detection of energy transfer between tryptophan residues in the tubulin molecule and bound bis(8-anilinonaphthalene-1-sulfonate), an inhibitor of microtubule assembly, that binds to a flexible region on tubulin

The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) is a potent inhibitor of microtubule assembly that binds to tubulin at a hitherto uncharacterized site distinct from those of the antimitotic drugs. We have found that energy transfer between tryptophan residues and bound Bis-ANS leads to quenching of the intrinsic tubulin fluorescence. The quenching is biphasic, implying two types of Bis-ANS binding sites. The estimated Kd values are 2.7 and 22.2 microM, consistent with reported values for the primary and secondary Bis-ANS binding sites. Preincubation of tubulin at 37 degrees C results in increased quenching of tryptophan fluorescence without any effect on the Kd values, suggesting localized structural change in the protein around the Bis-ANS binding sites. Concentration-dependent depolarization of Bis-ANS fluorescence was observed, suggesting energy transfer among bound Bis-ANS molecules. Such a concentration-dependent decrease in fluorescence polarization was not observed with 8-anilinonaphthalene-1-sulfonate (1,8-ANS), the monomeric form of Bis-ANS. Perrin-Weber plots were obtained for bound Bis-ANS and 1,8-ANS by varying the viscosity with sucrose. The rotational relaxation times calculated for Bis-ANS and 1,8-ANS are 18 and 96 ns, respectively. Comparison with the theoretical value (125 ns) suggests that Bis-ANS binds to a flexible region of tubulin. This, coupled with the fact that Bis-ANS, but not 1,8-ANS, inhibits microtubule assembly, suggests that the region in the tubulin molecule responsible for microtubule assembly is relatively flexible.     

The fluorescent characterization of the polymerized microtubule-associated protein Tau

A new fluorescence formed while microtubule-associated protein tau was incubated at 25 and 37C for hours, with its maximum excitation at 230 and 280 nm, respectively. The fluorescence completely formed after tau was incubated in phosphate buffer and Tris-HCl buffer for approximately 20 h, with a relaxation phase about 2-4 h. The light scattering of the sample solution improved during formation of the fluorescence when tau was incubated. Both the fluorescence and tau oligomers did not form when tau was incubated in the buffers containing DTT. On the other hand, heparin improved both tau aggregation and the fluorescence formation. It suggests that the fluorescence comes from tau polymerization, which may follow the mechanism of tyrosine-tyrosinate emission for a protein not containing any tryptophan residues. This new fluorescence could be used as a probe to tau polymers.     

Tetramerization of the S100B Chaperone Spawns a Ca2+ Independent Regulatory Surface that Enhances Anti-aggregation Activity and Client Specificity

Alzheimer’s disease (AD) hallmarks include the aggregation of amyloid-β (Aβ), tau and neuroinflammation promoted by several alarmins. Among these is S100B, a small astrocytic homodimeric protein, upregulated in AD, whose multiple biological activities depend on localization, concentration, and assembly state. S100B was reported to inhibit the aggregation and toxicity of Aβ42 and tau similarly to a holdase-type chaperone. This activity is dependent of Ca²⁺-binding, which triggers the exposure of a regulatory binding cleft at the S100B dimer interface with which amyloidogenic clients dynamically interact. Although the dimer prevails, a significant portion of secreted S100B in the human brain occurs as higher order multimers, whose protective functions remain uncharacterized and which we here investigate. Resorting to ThT-monitored aggregation kinetics, we determined that unlike the dimer, tetrameric S100B inhibits Aβ42 aggregation at sub/equimolar ratios, an effect that persists in the absence of Ca²⁺ binding. Structural analysis revealed that S100B tetramerization spawns a novel extended cleft accommodating an aggregation-prone surface that mediates interactions with monomeric Aβ client via hydrophobic interactions, as corroborated by Bis-ANS fluorescence and docking analysis. Correspondingly, at high ionic strength that reduces solvation and favours hydrophobic contacts, the inhibition of Aβ42 aggregation by tetrameric S100B is 3-fold increased. Interestingly, this extended Ca²⁺-independent surface favours Aβ42 as substrate, as tau K18 aggregation is not inhibited by the apo tetramer. Overall, results illustrate a mechanism through which oligomerization of the S100B chaperone fine-tunes anti-aggregation activity and client specificity, highlighting the potential functional relevance of S100B multimers in the regulation of AD proteotoxicity.     

Discovery of thienoquinolone derivatives as selective and ATP non-competitive CDK5/p25 inhibitors by structure-based virtual screening

Calpain mediated cleavage of CDK5 natural precursor p35 causes a stable complex formation of CDK5/p25, which leads to hyperphosphorylation of tau. Thus inhibition of this complex is a viable target for numerous acute and chronic neurodegenerative diseases involving tau protein, including Alzheimer’s disease. Since CDK5 has the highest sequence homology with its mitotic counterpart CDK2, our primary goal was to design selective CDK5/p25 inhibitors targeting neurodegeneration. A novel structure-based virtual screening protocol comprised of e-pharmacophore models and virtual screening workflow was used to identify nine compounds from a commercial database containing 2.84 million compounds. An ATP non-competitive and selective thieno[3,2-c]quinolin-4(5H)-one inhibitor (10) with ligand efficiency (LE) of 0.3 was identified as the lead molecule. Further SAR optimization led to the discovery of several low micromolar inhibitors with good selectivity. The research represents a new class of potent ATP non-competitive CDK5/p25 inhibitors with good CDK2/E selectivity.     

Inhibition of the soluble form of testis adenylate cyclase by catechol estrogens and other catechols

The soluble form of rat germ cell adenylate cyclase was inhibited by compounds with a catechol moiety. Among the naturally occurring catechols tested, catechol estrogens were the most potent inhibitors. Catechol estrogens at 2-6 microM inhibited enzyme activity by 50% and almost completely at 30-100 microM concentration. The inhibitory activity of catechol estrogens depends on the catechol moiety of the molecule. Catechol per se also inhibited the activity of this enzyme, 50% inhibition being achieved at about 11 microM. The two hydroxyls of the catechol moiety are essential for the inhibitory interaction with the enzyme. Thus, aromatic compounds containing only one hydroxyl group in the benzene ring, such as tyrosine, phenylephrine, estradiol, and 6 alpha-hydroxyestradiol were either completely inactive or had marginal inhibitory activity at concentrations up to 0.3-1 mM. Moreover, methylation of the hydroxyl groups of the catechol moiety of the catechol estrogens as in 2-methoxyestradiol 3-methyl ether rendered the catechol estrogens inactive. The inhibitory potency of these compounds varied greatly depending on the structure associated with the catechol ring. Thus, compounds in which catechol is associated with an aliphatic side chain, such as dopamine, L-dopa, norepinephrine, and isoproterenol, were about 11- to 34-fold less potent than catechol. On the other hand, compounds in which catechol is associated either with a hydroaromatic ring system, as in apomorphine, or with an alicyclic ring system, as in catechol estrogens, were about 2- to 5-fold more potent than catechol. The inhibitory effect of dopamine, apomorphine, and catechol estrogens was not affected by specific D-1 or D-2 antagonist, indicating that they do not act via receptors for dopamine.     

Potent inhibition of tau fibrillization with a multivalent ligand

Small-molecule inhibitors of tau fibrillization are under investigation as tools for interrogating the tau aggregation pathway and as potential therapeutic agents for Alzheimer's disease. Established inhibitors include thiacarbocyanine dyes, which can inhibit recombinant tau fibrillization in the presence of anionic surfactant aggregation inducers. In an effort to increase inhibitory potency, a cyclic bis-thiacarbocyanine molecule containing two thiacarbocyanine moieties was synthesized and characterized with respect to tau fibrillization inhibitory activity by electron microscopy and ligand aggregation state by absorbance spectroscopy. Results showed that the inhibitory activity of the bis-thiacarbocyanine was qualitatively similar to a monomeric cyanine dye, but was more potent with 50% inhibition achieved at approximately 80nM concentration. At all concentrations tested in aqueous solution, the bis-thiacarbocyanine collapsed to form a closed clamshell structure. However, the presence of tau protein selectively stabilized the open conformation. These results suggest that the inhibitory activity of bis-thiacarbocyanine results from multivalency, and reveal a route to more potent tau aggregation inhibitors.     

Interaction of silver nanoparticles with catechol O-methyltransferase: Spectroscopic and simulation analyses

Catechol O-methyltransferase, an enzyme involved in the metabolism of catechol containing compounds, catalyzes the transfer of a methyl group between S-adenosylmethionine and the hydroxyl groups of the catechol. Furthermore it is considered a potential drug target for Parkinson’s disease as it metabolizes the drug levodopa. Consequently inhibitors of the enzyme would increase levels of levodopa. In this study, absorption, fluorescence and infrared spectroscopy as well as computational simulation studies investigated human soluble catechol O-methyltransferase interaction with silver nanoparticles. The nanoparticles form a corona with the enzyme and quenches the fluorescence of Trp143. This amino acid maintains the correct structural orientation for the catechol ring during catalysis through a static mechanism supported by a non-fluorescent fluorophore–nanoparticle complex. The enzyme has one binding site for AgNPs in a thermodynamically spontaneous binding driven by electrostatic interactions as confirmed by negative ΔG and ΔH and positive ΔS values. Fourier transform infrared spectroscopy within the amide I region of the enzyme indicated that the interaction causes relaxation of its β?structures, while simulation studies indicated the involvement of six polar amino acids. These findings suggest AgNPs influence the catalytic activity of catechol O-methyltransferase, and therefore have potential in controlling the activity of the enzyme.     

An operator-induced conformational change in the C-terminal domain of the lambda repressor.

4,4'-bis(1-anilino-8-naphthalenesulfonic acid (Bis-ANS), an environment-sensitive fluorescent probe for hydrophobic region of proteins, binds specifically to the C-terminal domain of lambda repressor. The binding is characterized by positive cooperativity, the magnitude of which is dependent on protein concentration in the concentration range where dimeric repressor aggregates to a tetramer. In this range, positive cooperativity becomes more pronounced at higher protein concentrations. This suggests a preferential binding of Bis-ANS to the dimeric form of the repressor. Binding of single operator OR1 to the N-terminal domain of the repressor causes enhancement of fluorescence of the C-terminal domain bound Bis-ANS. The binding of single operator OR1 also leads to quenching of fluorescence of tryptophan residues, all of which are located in the hinge or the C-terminal domain. Thus two different fluorescent probes indicate an operator-induced conformational change which affects the C-terminal domain. The significance of this conformational change with respect to the function of lambda repressor has been discussed.     

Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP-SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488 nm was recorded on excitation at 385 nm. By combining HP-SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions. These structural changes of individual fractions, which were not detectable by online UV and multiangle laser light scattering (MALLS) or by stand-alone dynamic light scattering (DLS), intrinsic IgG fluorescence, and far-UV circular dichroism (CD), resulted in progressive aggregation on storage. The developed online fluorescent dye detection for HP-SEC or AF4 with Bis-ANS is a powerful method to detect both aggregation and structural changes of both monomeric and aggregated IgG in heat-stressed formulations.     

Novel multifunctional dopamine D2/D3 receptors agonists with potential neuroprotection and anti-alpha synuclein protein aggregation properties

Our ongoing drug development endeavor to design compounds for symptomatic and neuroprotective treatment of Parkinson’s disease (PD) led us to carry out a structure activity relationship study based on dopamine agonists pramipexole and 5-OHDPAT. Our goal was to incorporate structural elements in these agonists in a way to preserve their agonist activity while producing inhibitory activity against aggregation of α-synuclein protein. In our design we appended various catechol and related phenol derivatives to the parent agonists via different linker lengths. Structural optimization led to development of several potent agonists among which (−)-8a, (−)-14 and (−)-20 exhibited potent neuroprotective properties in a cellular PD model involving neurotoxin 6-OHDA. The lead compounds (−)-8a and (−)-14 were able to modulate aggregation of α-synuclein protein efficiently. Finally, in an in vivo PD animal model, compound (−)-8a exhibited efficacious anti-parkinsonian effect.     

Integrating in vitro and in silico approaches to evaluate the “dual functionality” of palmatine chloride in inhibiting and disassembling Tau-derived VQIVYK peptide fibrils

Background

Alzheimer's disease (AD) is the most common neurodegenerative disorder which is characterized by the deposits of intra-cellular tau protein and extra-cellular amyloid-β (Aβ) peptides in the human brain. Understanding the mechanism of protein aggregation and finding compounds that are capable of inhibiting its aggregation is considered to be highly important for disease therapy.

Discovery of the catechol structural moiety as a Stat3 SH2 domain inhibitor by virtual screening

The Stat3 SH2 domain is essential for its activation, and development of a potent SH2 inhibitor will be therapeutically valuable in treating cancers with constant Stat3 activation. We report here the identification of the catechol (1,2-dihydroxybenzene) structural moiety by virtual screening as a Stat3 SH2 inhibitor. The catechol compound docked to the Stat3 SH2 domain in computer modeling forms hydrogen bonds with the conserved pTyr-interacting amino acids. In the biochemical assay, a catechol-containing compound, but not the hydroxyl group-acetalized analogue, was able to inhibit Stat3 DNA-binding activity. Furthermore, the catechol compound was demonstrated to compete with pTyr peptides in binding to the Stat3 SH2 domain, suggesting that the catechol moiety is a pTyr bioisostere and may potentially be used for designing cell-permeable SH2 inhibitors. In our preliminary effort, we also demonstrated that the potency of catechol compound as Stat3 SH2 inhibitors could be improved by modifying the non-catechol part of the compound structure.     

Phenylthiazolyl-hydrazide and its derivatives are potent inhibitors of tau aggregation and toxicity in vitro and in cells

One of the key pathological features of Alzheimer's disease is the aggregation of tau protein. We are therefore searching for compounds capable of inhibiting this reaction. On the basis of an initial screen of 200000 compounds [Pickhardt, M., Gazova, Z., von Bergen, M., Khlistunova, I., Wang, Y., Hascher, A., Mandelkow, E. M., Biernat, J., and Mandelkow, E. (2005) Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells, J. Biol. Chem. 280, 3628-3635], we performed an in silico screen and predicted a new phenylthiazolyl-hydrazide (PTH) compound as a possible hit [Larbig, G., Pickhardt, M., Lloyd, D. G., Schmidt, B., and Mandelkow, E. (2007) Screening for inhibitors of tau protein aggregation into Alzheimer paired helical filaments: A ligand based approach results in successful scaffold hopping. Curr. Alzheimer Res. 4 (3), 315-323.]. Synthesis of this compound showed that it was indeed active in terms of inhibiting de novo tau aggregation and disassembling preformed aggregates (IC50 = 7.7 microM and DC50 = 10.8 microM). We have now synthesized 49 similar structures and identified the core of the PTHs to be crucial for activity, thus representing a lead structure. Analysis of the binding epitope by saturation transfer difference NMR shows strong interactions between the tau protein and the ligand in the aromatic regions of the inhibitor. By chemical variation of the core, we improved the inhibitory potency five-fold. The compounds showed a low toxicity as judged by an N2A cell model of tau aggregation and lend themselves for further development.     

Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls

Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease‐specific ‘seed’ proteins; however, roles of other co‐aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control‐subject pools using magnetic‐bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high‐resolution proteomics. Although total detergent‐insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or Aβ_1–42 pulldown, the protein constituents of Alzheimer‐derived aggregates were more abundant, diverse, and post‐translationally modified than those from controls. Tau‐ and Aβ‐containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer‐specific protein enrichment in tau‐containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co‐aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co‐localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer‐specific enrichment in tau‐containing aggregates, were assessed for aggregation‐promoting roles in C. elegans by RNA‐interference ‘knockdown’. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ_1–42. These observations imply shared mechanisms driving both types of aggregation, and/or aggregate‐mediated cross‐talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.     

Biological evaluation of synthetic α,β-unsaturated carbonyl based cyclohexanone derivatives as neuroprotective novel inhibitors of acetylcholinesterase,butyrylcholinesterase and amyloid-β aggregation

A series of new α,β-unsaturated carbonyl-based cyclohexanone derivatives was synthesized by simple condensation method and all compounds were characterized by using various spectroscopic techniques. New compounds were evaluated for their effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds were also screened for in vitro cytotoxicity and for inhibitory activity for self-induced Aβ_1–42 aggregation. The effect of these compounds against amyloid β-induced cytotoxicity was also investigated. The findings of in vitro experiment revealed that most of these compounds exhibited potent inhibitory activity against AChE and self-induced Aβ_1–42 aggregation. The compound 3o exhibited best AChE (IC₅₀ = 0.037 μM) inhibitory potential. Furthermore, compound 3o disassembled the Aβ fibrils produced by self-induced Aβ aggregation by 76.6%. Compounds containing N-methyl-4-piperidone linker, showed high acetylcholinesterase and self-induced Aβ aggregation inhibitory activities as compared to reference drug donepezil. The pre-treatment of cells with synthetic compounds protected them against Aβ-induced cell death by up to 92%. Collectively, these findings suggest that some compounds from this series have potential to be promising multifunctional agents for AD treatment and our study suggest the cyclohexanone derivatives as promising new inhibitors for AChE and BuChE, potentially useful to treat neurodegenerative diseases.     

Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual inhibitors for amyloid-beta and tau aggregation

Amyloid-β (Aβ) and tau protein are two crucial hallmarks in Alzheimer’s disease (AD). Their aggregation forms are thought to be toxic to the neurons in the brain. A series of new 1,2,3,4-tetrahydro-1-acridone analogues were designed, synthesized, and evaluated as potential dual inhibitors for Aβ and tau aggregation. In vitro studies showed that compounds 25–30 (20?μM) with N-methylation of the quinolone ring effectively inhibited Aβ_1-42 aggregation by 84.7%–99.5% and tau aggregation by 71.2%–101.8%. Their structure-activity relationships are discussed. In particular, 30 could permeate the blood-brain barrier, bind to Aβ_1-42 and tau, inhibit Aβ_1-42 β-sheets formation, and prevent tau aggregation in living cells.     

Structure-activity relationship of clovamide and its related compounds for the inhibition of amyloid β aggregation

Alzheimer’s disease (AD), a neurodegenerative disorder, is characterized by aggregation of amyloid β-protein (Aβ). Aβ aggregates through β-sheet formation and induces cytotoxicity against neuronal cells. Inhibition of Aβ aggregation by naturally occurring compounds is thus a promising strategy for the treatment of AD. We have already reported that caffeoylquinic acids and phenylethanoid glycosides, which possess two or more catechol moieties, strongly inhibited Aβ aggregation. Clovamide (1) containing two catechol moieties, isolated from cacao beans (Theobroma cacao L.), is believed to exhibit preventive effects on Aβ aggregation. To investigate the structure-activity relationship of clovamide (1) for the inhibition of Aβ aggregation, we synthesized 1 and related compounds 2–11 through reaction between l-DOPA, d-DOPA, l-tyrosine, or l-phenylalanine and caffeic acid, p-coumaric acid, or cinnamic acid, and compounds 12 and 13 were derived from 1. Among tested compounds 1–13, those containing one or two catechol moieties exhibited potent anti-aggregation activity, whereas the non-catechol-type related compounds showed little or no activity. This suggests that at least one catechol moiety is essential for inhibition of Aβ42 aggregation, and this activity increases depending on the number of catechol moieties. Consequently, clovamide (1) and its related compounds may be a promising therapeutic option for inhibiting Aβ-mediated pathology in AD.