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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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High Throughput Screening for Small Molecule Inhibitors of Heparin-induced Tau Fibril Formation

, , and . , , , , and .

Author Information and Affiliations

Received: ; Last Update: September 2, 2010.

The microtubule-associated protein tau contributes directly or indirectly to key structural and regulatory cellular functions. Under pathological conditions, tau becomes sequestered into insoluble neurofibrillary tangles that promote axonal transport deficits, ultimately lead to synaptic dysfunction and neuronal loss. Beta-amyloid protein undergoes a similar type of aggregation as tau, and is implicated in the pathology of Alzheimer's disease. Recent reports describing screening of libraries of small molecules for their ability to inhibit tau fibrillization have shown that some classes of compounds, such as anthraquinones, phenothiazines, and porphyrins have the ability to inhibit tau fibril formation. While these hits may be unlikely to be developed into novel therapeutics for the treatment of tauopathies, the results suggest that in vitro assays used for these screenings are capable of detecting inhibitors of tau fibrillization. Thus, further qHTS for small molecule inhibitors of heparin-induced tau fibril formation promises to provide the identification novel and more tractable hits. HTS screening identified the probe ML103 (CID-9795907) as a member of a series of tau oligomerization/fibrillization inhibitors that can be used to study tau protein aggregation in vitro, as well as a starting point for drug development for the treatment of Alzheimer's Disease (AD) and other tauopathies.

Assigned Assay Grant #: X01 MH083262-01

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher Austin

Assay Submitter & Institution: Carlo Ballatore, University of Pennsylvania

PubChem Summary Bioassay Identifier (AID): AID-1475

Probe Structure & Characteristics

PubChem CIDCID-9795907
Chemical FormulaC13H9N3O3S
Molecular Weight287.29
Log P1.89
tPSA96
Number of hydrogen donors2
Number of hydrogen acceptors:5
Rotatable bond2
Image ml103fu1
CID/MLTarget NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (nM) [SID, AID]SelectivitySecondary Assay(s) % Inhibition Name: [SID, AID]
CID-9795907/ML103Human tau6,300 (100%)1 [SID-57288397, AID-1558]Aβ(1–42)35,500 (24%) [SID-57288397, AID-1712]5.6 (4.2)Tau Sedimentation Assay 84% [SID-57288397, AID-1720]
***

NCGC IDs and corresponding PubChem IDs are listed in Table 5.

Recommendations for the scientific use of this probe

This probe is a member of a series of tau oligomerization/fibrillization inhibitors. It can be used to study tau protein aggregation in vitro, as well as a starting point for drug development for the treatment of Alzheimer’s Disease (AD) and other tauopathies.

Specific Aim

SPECIFIC AIM 1. Screen the compound libraries of the MLSMR in an heparin-induced tau K18 protein fibrillization assay based on ThT fluorescence; generate dose-response curves for all compounds, select hits that exhibit IC50s of 5 μM or less; and confirm the results in a secondary sedimentation assay.

SPECIFIC AIM 2. Prioritize hits based on (a) inspection of the chemical structure of each hit (b) examination of dose-response curves (c) interpretation of structure-function relationships obtained by comparing hits with related compounds present in the chemical libraries.

SPECIFIC AIM 3. Based on the data generated in Specific Aim 1–2, validate the most representative hits in one or more assays designed to segregate false positives: (a) generate dose response curves in primary/secondary assay at different K18 concentration; (b) perform primary/secondary assay with and without reducing agents in the reaction mixture; (c) run primary/secondary assay using different anionic cofactor.

Significance

Six soluble tau isoforms are expressed in the normal adult human brain from a single gene by alternative splicing. Normally, tau binds to and stabilizes microtubules (MTs), thereby maintaining the network of MTs essential for axonal transport in neurons. While phosphorylation negatively regulates the binding of tau to MTs, tau becomes pathologically hyperphosphorylated in AD, resulting in reduced MT-binding, followed by aggregation and sequestration of tau as paired helical filaments (PHFs or PHFtau) into neurofibrillary tangles (NFTs). This loss of tau function leads to destabilization of the MT system, which is essential for axonal transport of proteins and other cargo to and from the cell body of neurons. By analogy with a railway, tau functions like the cross ties on railroad tracks (MTs), upon which trains (molecular motors) convey cargo (organelles, proteins) to and from nodes on the railway network (destinations in neuronal perikarya and their processes); the loss of a critical number of cross ties (the consequence of converting tau into PHFs) results in dissolution of the railroad tracks, leading to the failure to deliver cargo to assigned destinations (impaired axonal transport). The deleterious consequences of these events are the dysfunction and subsequent death of affected neurons. Importantly, recent advances in the development of in vitro tau fibrillization assays enable HTS to be used for interrogating compound libraries to identify fibrillization inhibitors. However, clinical drug candidates have not yet been identified. In addition, it is notable that the fibrillization assays reported to date differ in important ways, such as the tau isoforms or anionic cofactors employed, or concentrations of reducing agents in the reaction mixture. While the significance of such differences is not known, it is likely these differences will be reflected in the types and numbers of hits generated from HTS using different assays. Hence, it is timely to conduct further HTS for small-molecule inhibitors of tau fibrillization using more than one assay, and to compare results from different assays.

Rationale

Specific Aim 1: Recent reports describing screening of libraries of small molecules for their ability to inhibit tau fibrillization have shown that some classes of compounds, such as anthraquinones (Pickhardt, Gazova et al. 2005), phenothiazines (Wischik, Edwards et al. 1996; Taniguchi, Suzuki et al. 2005) and porphyrins (Taniguchi, Suzuki et al. 2005), have the ability to inhibit tau fibril formation. While these molecules are unlikely to be developed into novel therapeutics for the treatment of tauopathies, they have importantly demonstrated that the in vitro assays used in the screenings were indeed capable of detecting inhibitors of tau fibrillization. Therefore, further HTS holds the promise of identifying novel and more tractable hits.

Specific Aim 2: In order to accurately assign a priority to the compounds, or classes of compounds populating Group III (see Table 4), an analysis of the data and the chemical structures will be essential at this point. Indeed, simple visual inspection of the chemical structure could rapidly highlight potential liabilities. For example, compounds containing reactive aldehydes or α,β-unsaturated systems that could easily react with the ɛ-amino group of lysines could be readily identified. Likewise, examination of the concentration-response curve will be helpful in identifying promiscuous inhibitors that act by forming large aggregates in solution. These compounds typically produce a steep concentration-response curve (Shoichet 2006) Last but not least, a first assessment of structure-activity relationship should be made at this point, if possible, by comparing hits with structurally related compounds in Group I or Group II (Table 4).

Table 4. Structure-activity relationships of the aminothienopyridazine series.

Table 4

Structure-activity relationships of the aminothienopyridazine series.

Specific Aim 3: The rationale for Specific Aim 3 is to screen the most representative hits emerging from Specific Aim 1–2 against additional assays that, based on our previous experiences (see Preliminary Studies; Section 4) and our current understanding of the assay, are designed to accurately test mechanisms of non-specific inhibition. For example, since non-specific binding of aggregates of small molecules onto proteins is often the source of false positives in HTS, concentration-response curves will be generated at different concentrations of K18 tau (McGovern, Caselli et al. 2002; McGovern, Helfand et al. 2003). It is expected that IC50s of compounds that exhibit inhibitory activity through the formation of aggregates will be independent of protein concentration. In addition, since it was previously demonstrated that compound-DTT interactions could lead to non-specific inhibition of tau filament formation via peroxide formation (see Table 1; Preliminary Studies), HTS will be conducted in the absence of DTT. However, secondary assays will be conducted in the presence of DTT to determine whether active compounds identified from HTS are also active in the presence of reducing agent.

Table 1. Final 1536-well assay protocol.

Table 1

Final 1536-well assay protocol.

Assay Implementation and Screening

PubChem Bioassay Name

Quantitative High-Throughput Screen for Inhibitors of Tau Fibril Formation

List of PubChem bioassay identifiers generated for this screening project (AIDs)

AIDTargetConcentrationBioassay type
1475TauSummary
1460Tau19 nM-57 µMPrimary qHTS (ThT)
1468Tau19 nM-57 µMPrimary qHTS (FP, mP)
1463Tau19 nM-57 µMPrimary Counterscreen qHTS (FP, Total Fluorescence)
1558Tau49 nM-100 µMSecondary (ThT)
1559Tau49 nM-100 µMSecondary (FP, mP)
1694Tau49 nM-100 µMSecondary Counterscreen (FP, Total Fluorescence)
1709Tubulin50 µMSecondary
1711Caspase-17 pM-41 µMSelectivity
1712Beta-amyloid16 nM-80 µMSelectivity
1719Tau100 µMSecondary
1720Tau100 µMSecondary

Primary Assay Description as Defined in PubChem

Overview

The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other essential cellular constituents to travel along the axons. Under pathological conditions, tau becomes sequestered into insoluble aggregates called neurofibrillary tangles. This phenomenon is believed to have pathological consequences by promoting axonal transport deficits that ultimately lead to synaptic dysfunction and neuronal loss. To identify inhibitors of tau aggregation, a heparin-induced tau fibril formation assay was used that employed a recombinantly expressed fragment of tau, K18 (Q242-E372), bearing a P301L mutation (Gustke, Trinczek et al. 1994; Hong, Zhukareva et al. 1998). This assay monitors tau fibrillization through the two complementary readouts of Thioflavin T (ThT) fluorescence and fluorescence polarization (FP) of Alexa 594-labeled K18 P301L which incorporates into growing multimers of unlabeled tau.

Protocol

Two K18 mutants were produced: P301L (K18PL), which fibrillizes faster than the wild-type form (von Bergen, Barghorn et al. 2001) and K311D (K19KD), which does not fibrillize (Khlistunova, Biernat et al. 2006) and was thus used as non-fibrillizing control in the assay. For screening, 2 μL/well human K18 P301L (15 μM unlabeled and 0.24 μM Alexa 594-labeled K18PL tau, final concentrations) in reagent buffer (100 mM sodium acetate pH 7) was dispensed into black solid 1536-well plates (Grenier) using a solenoid-based dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin tool, 2 μL/well heparin (20 μM final concentration) in reagent buffer was added and the plate centrifuged 15 s at 1000 RPM. Plates were incubated 6 hr at 37 °C and then 1 μL/well ThT (30 μM final concentration) was added. After a 1 hr ambient incubation, the plates were read twice by an Envision (Perkin Elmer) plate reader to sequentially monitor ThT fluorescence (450/8 nm excitation and 510/23 nm emission) and Alexa 594 FP (555 nm excitation and 632 nm emission).

Summary of the Primary Screen

Assay principle and protocol

The assay principle and protocol are indicated in Figure 1 and Table 1, respectively.

Figure 1. Schematic of Tau fibrillization assay.

Figure 1

Schematic of Tau fibrillization assay. Upon the addition of heparin, tau monomers oligomerize into fibrils. The fluorescent dye, Thioflavine T, binds to tau oligomers, which results in increased fluorescence intensity upon light excitation. Tau oligomerization (more...)

qHTS summary of assay results

The combined ThT/FP assay was optimized and validated using 1536-well test plates to ensure that automated procedures would provide adequate signal to background (S:B) ratios, and Z factor values exceeding 0.5 (Table 2). Subsequently, a ~292,000 compound library underwent qHTS at six concentrations, ranging from 56 μM to 18 nM for each compound. The combination of good signal-to-background and low CV values resulted in average plate Z-scores of 0.85 ± 0.12 and 0.79 ± 0.03 for the ThT and FP analyses, respectively (text adapted from (Crowe, Huang et al. Submitted)).

Table 2. Assay optimization, validation, and screen performance.

Table 2

Assay optimization, validation, and screen performance.

Identification of tau inhibitors

Following the screen, the concentration-response curve (CRC) data were classified to rank the curve quality, as described (Inglese, Auld et al. 2006). Briefly, CRCs were placed into four classes. Class 1 contains complete CRCs showing both upper and lower asymptotes and r2 values > 0.9. Class 2 contains incomplete CRCs lacking the lower asymptote and shows r2 values > 0.9. Class 3 curves are of the lowest confidence because they are poorly fit or based on activity at a single concentration point. Class 4 are inactives having either no curve-fit or an efficacy below threshold activity (3 SD of the mean activity). While both activators and inhibitors were recovered from the qHTS, activators were not considered further, as their activity likely arose from compound fluorescence. The qHTS resulted in 17,911 FP and 51,266 ThT actives (Class 1–3) of which 1,011 FP and 7,092 ThT actives were scored high quality (Class 1 and 2.1, Figures 2 and 3).

Figure 2. Titration-response plots of tau inhibitors.

Figure 2

Titration-response plots of tau inhibitors. Inhibitors from the ThT binding (top panel) and fluorescence polarization (bottom panel) measures are shown for class 1 (left), class 2 (middle), and class 3 (right). For classes 1 and-2, blue and orange curves (more...)

Figure 3. Classification of tau actives.

Figure 3

Classification of tau actives. A) Pie charts indicating overall FP and ThT activity of screened compounds. Inhibitors (grey) and activators (maroon) comprised 4.9% and 9.4%, respectively, of the collection while the remainders were inactive (yellow). (more...)

To derive nascent structure-activity relationships, an in-house auto scaffold detection program was used to cluster 2989 class 1 and 2 FP actives (including class 2.2 < −50% efficacy), yielding 514 structural series and 755 singletons. Each series contained at least three compounds, of which at least one of which was active. In addition, compounds could occupy multiple series. The series were then flagged for potential liabilities using activities from the tau and other assays (Table 3), resulting in 42 series with no liabilities.

Table 3. Criteria for identifying liabilities of tau active series.

Table 3

Criteria for identifying liabilities of tau active series.

Series were filtered and prioritized further using the following criteria:

  1. Active selection- Compounds with increasing total Alexa Red fluorescence in the FP assay were excluded, and only compounds active in both the FP and ThT assays (class 1 and 2; AC50 < 20 μM and efficacy <−40%) were included for further consideration.
  2. Known scaffolds- Compounds containing previously identified scaffolds (Crowe, Ballatore et al. 2007; Honson, Johnson et al. 2007), including pyrimidotriazines, depsidones, anthraquinones, phenothiazines, porphyrins, sulfonated dyes, and cyanine dyes, were excluded.
  3. Undesirable functional groups- Compounds containing undesirable functional groups with known chemical liabilities, such as quinones, metal complexes, reactive halides, imines (except hydrazones), aldehydes, aliphatic thiols, and potential Michael acceptors (except rhodanines), were excluded.
  4. Lipinski’s rule of five (Ro5)- Compounds having more than two Ro5 violations were excluded. In general, only compounds with molecular weight <600 and AlogP <6 were included.

Twenty three series passed this filtering process and were chosen for retest. An additional six series were selected, where the most potent compound in each series showed low efficacy in the FP readout, but showed 50% or greater efficacy in the ThT readout. For retest, 98 compounds (actives, inactive, and analogs) were chosen and of these, 85 were available for testing. Of the 85 compounds, 70 were identified from the screen (70 actives, 2 inconclusives, and 6 inactives) and 7 were analogs. When checked by LCMS, 75% of the compounds passed (correct mass and 90% or greater purity). The compounds were tested at both the NCGC and CNDR, and 94% and 73% of the qHTS actives retested positive in the ThT and FP assays, respectively. The compounds were tested in a sedimentation assay that measures the percentage of soluble and insoluble tau (Crowe, Ballatore et al. 2007), where 46% of the 76 qHTS actives and analogs were scored as active (40% or greater inhibition) and 11% as inconclusive (20–40% inhibition). All of the 47 samples scored active and inconclusive in the sedimentation assay were examined by transmission electron microscopy; 66% were scored active, showing a lack of fibrillar clumps, with only single filaments or pieces evident (data not shown). Following the retest studies, an aminothienopyridazine series that showed good performance in the follow-up assays and suitable chemical tractability was chosen for probe optimization.

Probe Characterization

The ability of aminothienopyridazines (ATPZ’s) to inhibit tau fibril formation was evaluated in the K18PL fluorescence and sedimentation assays to determine their AC50 values and percent maximal inhibition (AID-1558;AID-1720). As shown in Table 4, N-acylations (R1; entries 3–5) as well as substitution in the C-5 position (R2; entries 5 and 6) result in a dramatic loss of activity. Structural modifications in the fragment linked at C-4 (R3; entries 7–12) appear to be generally well tolerated. Indeed, several compounds bearing R3 modifications (entries 7–8, and 11–12) appear to cause greater maximal inhibition than the original hits identified during qHTS (entries 1 and 2). Furthermore, although the correlations between biological activity and the stereo-electronic properties of phenyl moieties (R4) are not entirely clear, the nature, number and position of the substituents in the aromatic ring appeared to modulate ATPZ activity (entries 1–2 and 13–18). Although previous studies have demonstrated that tau fragments such as K18PL form fibrils which resemble those observed in tauopathies (Crowe, Ballatore et al. 2007), it was important to demonstrate that the compounds could disrupt assembly of full-length tau40. Two ATPZ’s (NCGC-00053250 and NCGC-00031883) were tested at 50 µM concentration and caused at least a 60% inhibition of ThT fluorescence in the tau40 assay. This result thus confirms that ATPZ’s affect full-length tau in addition to the truncated tau fragment. Since an important function of tau protein is to stabilize microtubules (MTs), we evaluated whether these inhibitors interfered with the ability of tau to promote tubulin polymerization into MTs. Unlike methylene blue, a promiscuous compound active in a high percentage of PubChem screens (data not shown), the ATPZ’s had no effect on tau-mediated tubulin polymerization (Figure 4, AID-1709; text adapted from (Crowe, Huang et al. Submitted)).

Figure 4. Effects of ATPZ tau fibrilization inhibitors on tau-mediated MT assembly.

Figure 4

Effects of ATPZ tau fibrilization inhibitors on tau-mediated MT assembly. Representative traces of tau-induced microtubule assembly in the presence of 50 μM test compounds or DMSO, as described in the assay methods below.

The ATPZ’s were also tested for their ability to block the fibrillization of Aβ(1–42). We found that these compounds were less effective in blocking Aβ(1–42) fibril formation, as judged by ThT fluorescence, than they were in inhibiting tau fibrillization (Figure 5). Among the tested compounds, none caused >45% inhibition of Aβ(1–42) fibril formation when tested at 80 µM (AID: 1712).

Figure 5. Comparison of K18PL tau and Aβ(1–42) fibrillization in the presence of CID-9795907 (NCGC-00183199).

Figure 5

Comparison of K18PL tau and Aβ(1–42) fibrillization in the presence of CID-9795907 (NCGC-00183199). CID-9795907 (NCGC-00183199) was tested at the indicated concentrations in ThT binding assays for K18PL tau and Aβ(1–42) (more...)

Synthesis of analogs

CID-647821 (NCGC-00031883) (8a), CID-719449 (NCGC-00053250) (8b), CID-798725 (NCGC-00183206) (8c), CID-25181381 (NCGC-00183204) (8d), CID-25181382 (NCGC-00183205) (8e), CID-25181375 (NCGC-00183195) (8f), and CID-9795907 (NCGC-00183199) (9) were synthesized (Scheme 1) following a modified literature procedure (Ferguson, Valant et al. 2008). Commercially available anilines 4a–e were converted to aryl diazonium salts 5, which reacted with β-ketoesters to form hydrazones 6 as a mixture of E/Z isomers. Condensation reactions of the hydrazones with ethyl cyanoacetate gave pyridazines 7. Subsequently reactions of pyridazines 7 with sulfur under Gewald conditions generated 8a–f. Saponification of 8a gave the corresponding 9.

Scheme 1. Reagents and conditions: (i) X = BF4; HBF4, NaNO2; X = Cl; isoamyl nitrile; (ii) NaOAc, EtOH, R2CH2COCH2CO2Et; (iii) 4-aminobutyric acid, ethyl cyanoacetate; (iv) S8, morpholine, MW, 150 °C, 10 min; (v) NaOH.

Scheme 1

Reagents and conditions: (i) X = BF4; HBF4, NaNO2; X = Cl; isoamyl nitrile; (ii) NaOAc, EtOH, R2CH2COCH2CO2Et; (iii) 4-aminobutyric acid, ethyl cyanoacetate; (iv) S8, morpholine, MW, 150 °C, 10 min; (v) NaOH.

CID-25181378 (NCGC-00183201) (11a), CID-25181379 (NCGC-00183202) (10b), and CID-25181383 (NCGC-00183207) (10c) were prepared by a reaction sequence starting from 7a as depicted in Scheme 2. Saponification of 7a under acidic condition yielded 10a. Amide 10b was formed by reacting 7a with ammonium. Coupling reactions of acid 10a with appropriate amines followed by Gewald reaction yielded the desired amides 11a–c.

Scheme 2. Reagents and conditions: (i) X = OH: HCl/HOAc, MW, 150 °C, 15 min; X = NH2: NH3.H2O, DMF, MW, 150 °C, 10 min; (ii) DMC, iPr2NEt, DCM, r.t. o/n; (iii) S8, morpholine, MW, 150 °C, 10 min;.

Scheme 2

Reagents and conditions: (i) X = OH: HCl/HOAc, MW, 150 °C, 15 min; X = NH2: NH3.H2O, DMF, MW, 150 °C, 10 min; (ii) DMC, iPr2NEt, DCM, r.t. o/n; (iii) S8, morpholine, MW, 150 °C, 10 min;.

CID-4185185 (NCGC-00182502) (12a) and CID-2814251 (NCGC-00183197) (12b) were prepared by reactions of 8a with appropriate acid chlorides, Scheme 3.

The preparation of CID-25181383 (NCGC-00183207) (18) is illustrated in Scheme 4. EDC-mediated coupling reaction of acid 10a with tert-butylcarbazate followed by Boc-deprotection gave hydrazide 14. The hydrazide reacted with nitrous acid in acetic acid produced carboazide 15. Curtius rearrangement of 15 in ethanol gave ethyl carbamate 17. Under basic condition, the carbamate was converted to the desired amine 18.

Scheme 4. Reagents and conditions: (i) EDC, NH2NHBoc; (ii) MeOH, HCl/dioxane; (iii) HOAc, NaNO2; (iv) EtOH, MW, 150 °C, 10 min; (v) S8, morpholine, MW, 150 °C, 10 min; (vi) Et3N, EtOH/H2O, MW, 170 °C, 30 min.

Scheme 4

Reagents and conditions: (i) EDC, NH2NHBoc; (ii) MeOH, HCl/dioxane; (iii) HOAc, NaNO2; (iv) EtOH, MW, 150 °C, 10 min; (v) S8, morpholine, MW, 150 °C, 10 min; (vi) Et3N, EtOH/H2O, MW, 170 °C, 30 min.

CID-25181376 (NCGC-00183196) (19) was prepared by LiAlH4 reduction of ester 8a, Scheme 5.

Probe

a. Chemical name of probe compound

5-amino-4-oxo-3-phenylthieno[3,4-d]pyridazine-1-carboxylic acid (ML103)

b. Probe chemical structure including stereochemistry

Image ml103fu15

c. Structural Verification Information of probe (SID-57288397)

Structural verification and purity quantification were performed by 1H NMR analysis using a Varian spectrometer and by LC-MS analysis using a Waters Acquity UPLC in the following conditions:

  • Column: Phenomenex 2.5um Luna C18(2)-HST 100x2mm
  • Column Temperature: 45 degrees C
  • Flow: 0.5mL/min
  • Solvent A: 0.05% TFA in Water
  • Solvent B: 0.025% TFA in Acetonitrile
  • Gradient: 2% to 100% Solvent B over 2.2 minutes.
  • Run Time: 3.0 min
  • Detectors: PDA, ELSD, and MS (ESI+).

Both NMR and LC-MS analyses showed purity greater than 95% for CID-9795907 (NCGC-000183199-01; SID-57288397). 1H NMR (400 MHz, DMSO-d6) δ 7.12 (s, 1H), 7.35–7.39 (m, 1H), 7.44–7.54 (m, 4H), 7.60 (bs, 2H), 13.31 (bs, 1H); HPLC: tR = 1.89 min, ELSD = 100%, UV220 = 100%; HRMS (ESI): m/z calcd for C13H9N3O3S [M+1]+ 288.0437, found 288.0441.

d. PubChem CID (corresponding to the SID)

CID-9795907

e. Availability from a vendor

N/A.

f. Mode of action for biological activity of probe

The probe is a member of a series of Tau Oligomerization/Fibrillization inhibitors.

g. Detailed synthetic pathway for making probe

CID-9795907 (NCGC00183199) (9) was synthesized (Scheme 3) following a modified literature procedure (Ferguson, Valant et al. 2008).

Scheme 3. Reagents and conditions: (i) HBF4, NaNO2; (ii) NaOAc, EtOH, CH3COCH2CO2Et; (iii) 4-aminobutyric acid, ethyl cyanoacetate; (iv) S8, morpholine, MW, 150 °C, 10 min; (v) NaOH.

Scheme 3

Reagents and conditions: (i) HBF4, NaNO2; (ii) NaOAc, EtOH, CH3COCH2CO2Et; (iii) 4-aminobutyric acid, ethyl cyanoacetate; (iv) S8, morpholine, MW, 150 °C, 10 min; (v) NaOH.

This compound has been made available through the MLSMR (MLS-002472423).

Canonical SMILES: C1=CC=C(C=C1)N2C(=O)C3=C(SC=C3C(=N2)C(=O)O)N

InChI: InChI=1S/C13H9N3O3S/c14-11-9-8(6-20-11)10(13(18)19)15–16(12(9)17)7-4-2-1-3-5-7/h1-6H,14H2,(H,18,19)

Description of secondary assays used to optimize/characterize probe structure

Assay: Tau Sedimentation

Overview: The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other essential cellular constituents to travel along the axons. Under pathological conditions, tau becomes sequestered into insoluble aggregates called neurofibrillary tangles. This phenomenon is believed to have pathological consequences by promoting axonal transport deficits that ultimately lead to synaptic dysfunction and neuronal loss. The ability of compounds to block tau fibrillization was assessed using a sedimentation assay (Crowe, Ballatore et al. 2007). Unlike tau monomers, tau fibrils readily sediment upon centrifugation. Samples from the tau ThT confirmation assay treated with 100 µM compound (AID-1558) were centrifuged and the amount of tau present in the supernatant and pellet was determined.

Protocol: Human K18PL (15 µM) in assay buffer (20 µM heparin, 12.5 µM Thioflavine T, 100 mM sodium acetate pH 7) was centrifuged at 186,000 x g for 30 min and the supernatant removed from the pellet. Pellets were resuspended in a volume equal to the supernatant and equal amounts of supernatants and pellet were analyzed by SDS–PAGE using a 12.5% acrylamide gel. The gel was stained with Coomassie Blue and the percentage of tau in each fraction was determined by densitometry.

Assay: Transmission Electron Microscopy

Overview: The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other essential cellular constituents to travel along the axons. Under pathological conditions, tau becomes sequestered into insoluble aggregates called neurofibrillary tangles. This phenomenon is believed to have pathological consequences by promoting axonal transport deficits that ultimately lead to synaptic dysfunction and neuronal loss. The ability of compounds to block tau fibrillization was visualized using transmission electron microscopy (TEM). Samples from the tau ThT confirmation assay (AID-1558) were centrifuged and imaged using TEM. Representative fields from electron micrographs of objects were imaged to judge fibril content using grading scale from 0–4: 0, all fibrils in clumps, no fragments; 1, small fibril clumps, free filaments and some pieces; 2, filaments and pieces but no clumps; 3, short filaments and tiny pieces; 4, only tiny pieces.

Protocol: Human K18PL tau (15 µM) in assay buffer (20 µM heparin, 30 µM Thioflavine T, 100 mM sodium acetate pH 7) was centrifuged at 186,000 g for 30 min through a 25% sucrose cushion and the supernatant removed from the pellet. Pellets were resuspended, stained with uranyl acetate, and imaged by electron microscopy

Assay: Aβ1-42 Fibrillization

Overview: The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other essential cellular constituents to travel along the axons. Under pathological conditions, tau becomes sequestered into insoluble aggregates called neurofibrillary tangles. This phenomenon is believed to have pathological consequences by promoting axonal transport deficits that ultimately lead to synaptic dysfunction and neuronal loss. Beta-amyloid protein undergoes a similar type of aggregation as tau and is implicated in the pathology of Alzheimer’s disease. To examine the selectivity of tau fibrillization inhibitors, compounds were tested in an in vitro assay for fibrillization of the Beta-amyloid fragment, Aβ1–42.

Protocol: Synthetic Aβ1–42 aliquots were reconstituted to 2 mg/ml in DMSO and then diluted to 15 μM in 25 mM Tris, pH 7.0 buffer to which test compound was added at 50 μM final concentration, or at several concentrations ranging from 0.16–40 μM. The reaction mixtures were dispensed at 25 μl/well into a 384-well plate and then incubated at 37°C for 4 hrs. Upon completion of the reaction, 25 μl of 25 μM ThT was added to each well followed by ThT fluorescence readings as described above for K18PL.

Known probe properties

CID9795907
IDNCGC00183199
Chemical FormulaC13HgN3O3S
Molecular Weight287.29
Log P1.89
tPSA96
Number of hydrogen donors2
Number of hydrogen acceptors:5
Rotatable bond2

Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.) and recommendations for the scientific use of probe as research tool

CID-9795907 (NCGC-000183199) is a yellowish solid and dissolves well in DMSO. It is stable at room temperature.

Compound preparation

Compound is prepared in DMSO at 10 mM stock concentration. Assays described above have 0.6% DMSO final concentration in buffer.

Probe availability

Aliquots of 10 mM DMSO solution are available from the NCGC upon request. In addition, compound has been made available through the MLSMR (MLS-002472423).

Appendices

Comparative data on probe, similar compound structures and prior probes

Prior State-of-the-Art for Probe Development

StructureNameClassStageCIDSourceReference
Image ml103fu16.jpg
BF-170Quinolineex vivo16219029Sigma-Aldrich cat. no. B4311Okamura et al 2005
Image ml103fu17.jpg
Methylene bluePhenothiazinephase II6099Sigma-Aldrich cat. no. 03978 Wischik et al 1996
Image ml103fu18.jpg
EmodinAnthraquinonein vitro3220Sigma-Aldrich cat. no. 45170 Pickhardt et al 2005
Image ml103fu19.jpg
NCGC00065256Quinoxalinein vitro663281Ambinter 4S-27438 Honson et al 2007
Image ml103fu20.jpg
N744Cyanine Dyein vitro1103407Commercially unavailableCongdon et al 2007
Image ml103fu21.jpg
113F08Quinoxalinein vitro2301472Chembridge cat. No. Crowe et al 2007
Image ml103fu22.jpg
19Rodalinein vitro24756229Commercially unavailableBulic et al 2009
Image ml103fu23.jpg
GossypetinPolyphenolin vitro5280647Microsource cat. No. MS-1505143 Taniguchi et al 2005
Image ml103fu24.jpg
BSc3094phenylthiazolyl-hydrazidein vitro25096749Commercially unavailablePickhardt et al 2007
Image ml103fu25.jpg
B4A1Pyrimidinein vitro7908145Asinex BAS 00234164 Khlistunova et al 2006
Table 5. MLS, NCGC and PubChem IDs of ATPZ series compounds.

Table 5

MLS, NCGC and PubChem IDs of ATPZ series compounds.

Bibliography

  1. Crowe A, Ballatore C, et al. High throughput screening for small molecule inhibitors of heparin-induced tau fibril formation. Biochem Biophys Res Commun. 2007;358(1):1–6. [PMC free article: PMC2646256] [PubMed: 17482143]
  2. Crowe A, Huang W, et al. The Identification of Aminothienopyridazine Inhibitors of Tau Assembly by Quantitative High Throughput Screening. Submitted. [PMC free article: PMC2773749] [PubMed: 19580328]
  3. Ferguson GN, Valant C, et al. 2-aminothienopyridazines as novel adenosine A1 receptor allosteric modulators and antagonists. J Med Chem. 2008;51(19):6165–72. [PMC free article: PMC2605720] [PubMed: 18771255]
  4. Gustke N, Trinczek B, et al. Domains of tau protein and interactions with microtubules. Biochemistry. 1994;33(32):9511–22. [PubMed: 8068626]
  5. Hong M, Zhukareva V, et al. Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science. 1998;282(5395):1914–7. [PubMed: 9836646]
  6. Honson NS, Johnson RL, et al. Differentiating Alzheimer disease-associated aggregates with small molecules. Neurobiol Dis. 2007;28(3):251–60. [PMC free article: PMC2194600] [PubMed: 17761424]
  7. Inglese J, Auld DS, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103(31):11473–8. [PMC free article: PMC1518803] [PubMed: 16864780]
  8. Khlistunova I, Biernat J, et al. Inducible expression of Tau repeat domain in cell models of tauopathy: aggregation is toxic to cells but can be reversed by inhibitor drugs. J Biol Chem. 2006;281(2):1205–14. [PubMed: 16246844]
  9. McGovern SL, Caselli E, et al. A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem. 2002;45(8):1712–22. [PubMed: 11931626]
  10. McGovern SL, Helfand BT, et al. A specific mechanism of nonspecific inhibition. J Med Chem. 2003;46(20):4265–72. [PubMed: 13678405]
  11. Pickhardt M, Gazova Z, et al. Anthraquinones inhibit tau aggregation and dissolve Alzheimer’s paired helical filaments in vitro and in cells. J Biol Chem. 2005;280(5):3628–35. [PubMed: 15525637]
  12. Shoichet BK. Interpreting steep dose-response curves in early inhibitor discovery. J Med Chem. 2006;49(25):7274–7. [PubMed: 17149857]
  13. Taniguchi S, Suzuki N, et al. Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J Biol Chem. 2005;280(9):7614–23. [PubMed: 15611092]
  14. von Bergen M, Barghorn S, et al. Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure. J Biol Chem. 2001;276(51):48165–74. [PubMed: 11606569]
  15. Wischik CM, Edwards PC, et al. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci U S A. 1996;93(20):11213–8. [PMC free article: PMC38310] [PubMed: 8855335]

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