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Identification of a small molecule that selectively inhibits alpha-synuclein translational expression

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Author Information

,1 ,1 ,1 ,1 ,2 ,1 ,1 ,1 ,1 ,2 and 1,3.

1 The Broad Institute Probe Development Center, Cambridge, MA
2 Massachussetts General Hospital, Charlestown, MA
3 Howard Hughes Medical Institute, Chemistry & Chemical Biology, Harvard University, Cambridge, MA

Received: ; Last Update: February 15, 2011.

Alpha-synuclein is a protein implicated in the pathogenesis of neurodegenerative alpha-synucleinopathies including Parkinson’s disease (PD), the most prevalent movement disorder in humans. Evidence suggests that misregulation of alpha-synuclein translation plays a clear role in the pathology of this disease. Consequently, our therapeutic strategy is to limit alpha-synuclein translation. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, and selective small-molecule inhibitors of alpha-synuclein expression, which will aid the development of therapies for PD and other neurodegenerative diseases. Of the 303,224-screened compounds, 36 hits were identified as inhibitors of alpha-synuclein expression and subsequently validated to confirm their inhibitory activity and selectivity. One of these compounds (ML150) displayed greater than 200-fold selective inhibition of alpha-synuclein expression compared with a related system and is inactive in a control system at the highest tested dose. Our results indicate that ML150 has a drug-like structure with no obvious chemical liabilities, excellent solubility, and stability in aqueous conditions. This new probe should be very useful in future cell-based investigations and in vivo studies of alpha-synuclein expression inhibition.

Assigned Assay Grant #: 1 R21 NS05943401

Screening Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber, PhD

Chemistry Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber, PhD

Assay Submitter & Institution: Jack Rogers, PhD. Massachusetts General Hospital

PubChem Summary Bioassay Identifier (AID): AID-1827

Probe Structure & Characteristics

Image ml150fu1
IUPAC Chemical NameN,N-dimethyl-6-({[1-(1-naphthyl)-1H-tetrazol-5-yl]thio}methyl)-1,3,5- triazine-2,4-diamine
PubChem CID/ML#CID-1517919/ML150
Molecular Weight379.44
Molecular FormulaC17H17N9S
XlogP3
H-Bond Donor1
H-Bond Acceptor8
Rotatable Bond Count5
Exact Mass379.13
Topological Polar Surface Area137
CID/ML#Target NameIC50/EC50 (μM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (μM) [SID, AID]
CID-1517919/ML150α-synuclein 5′-UTR (H4-2a)1.8 [SID-87218804, AID-2460]Prion 5′-UTR (H4-PRP)inactive [SID-87218804, AID-2468]1.8 μM vs inactiveH4-C inactive [SID-87218804, AID-2463]

Recommendations for scientific use of the probe

The goal of this project is to identify novel small molecule probes that inhibit alpha-synuclein translational expression in dopaminergic neurons by targeting the 5′-untranslated region (5′UTR) stem-loop of alpha-synuclein as a major new therapeutic target to retard the progression of Parkinson’s disease (PD). The 5′UTR stem-loop of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction causes an increase in alpha-synuclein mRNA translation. Compounds that can successfully reduce alpha-synuclein expression levels as measured in a luciferase reporter assay will be further tested for specificity in cells lacking the alpha-synuclein 5′UTR stem-loop (H4-C) to confirm that compounds are acting through the intended target. Probe selectivity will be tested in H4 cells containing the prion protein (H4-PRP) 5′UTR mRNA stem-loop, which is also fused to a luciferase reporter. The positive control for screening in these three cell lines will be strophanthidine (Figure 1), which is cytotoxic to H4 cells. Finally, probe candidates will be verified by an alpha-synuclein enzyme linked immunosorbent assay (ELISA) and using Western blot analysis.

Figure 1. Compound CID-6185 (strophanthidine) was used as a positive control in the alpha-synuclein H4-2a, H4-C, and H4-PRP assays (H4-2a data shown).

Figure 1

Compound CID-6185 (strophanthidine) was used as a positive control in the alpha-synuclein H4-2a, H4-C, and H4-PRP assays (H4-2a data shown).

The probe (CID-1517919/ML150) described in this report selectively inhibits alpha-synuclein expression in transfected H4 neuroglioblastoma cells and also reduces protein levels in native H4 cells without affecting actin levels. The probe has an IC50 of 1.8 μM in the H4 primary screening cell line (H4-2a) and was found to be over 100-fold selective versus the H4-C and H4-PRP counterscreen cell lines. In ELISA assays using SH-SY5Y neuroglioma cells, the IC50 of the probe was approximately 1 μM, and a dose-response knock-down of alpha-synuclein levels was observed in H4 cells by Western blotting.

This probe will be used to examine the effect of reducing alpha-synuclein levels in neurons and mouse models of PD. The probe may also be further developed to optimize “drug-like” properties and may serve as a lead compound for therapeutic development.

1. Introduction

Scientific Rationale

Alpha-synuclein is an approximately 15 KDa protein implicated in the pathogenesis of neurodegenerative alpha-synucleinopathies (1), including Parkinson’s disease (PD), the most prevalent movement disorder in humans. In these disorders, alpha-synuclein undergoes a conformational change and subsequent oligomerization, which causes a toxic gain of function, neurodegeneration, and deposition of alpha-synuclein aggregates in the form of Lewy bodies. Increased alpha-synuclein levels caused by gene duplication causes familial PD (2) and GATA transcription factors (3) directly regulate the PD-linked alpha-synuclein gene. In this regard, regulation of alpha-synuclein translation is physiologically relevant to Lewy body dementia brains, which exhibit lowered alpha-synuclein mRNA but higher insoluble protein. This, suggests misregulation of alpha-synuclein translation in addition to protein clearance by chaperones (3, 4). Consequently, our therapeutic strategy is to limit alpha-synuclein translation.

Alpha-synuclein translation is governed, at least in part, by an iron-responsive element (IRE) in the 5′ untranslated region (5′-UTR) stem-loop of alpha-synuclein mRNA. Iron-responsive elements (IREs) are RNA stem loops in the UTRs of ferritin and transferring-receptor mRNAs that are critical to iron homeostasis (5, 6). However, the protein binding profile of the alpha-synuclein IRE is different from the canonical IREs of iron homeostasis. These findings reflect the sensitivity of this RNA sequence as a probe target. The IRE of alpha-synuclein is known to interact with iron regulatory protein-1 (IRP-1), which causes an increase in alpha-synuclein translation. There are currently no small molecules which inhibit the expression of alpha-synuclein by this mechanism (Section 4.1, Table 2).

Table 2. Search Strings and Databases Employed in Prior Art Search.

Table 2

Search Strings and Databases Employed in Prior Art Search.

Through this screening campaign we have discovered a probe that specifically reduces alpha-synuclein translation and likely acts by modulating this RNA-protein interaction. This probe, N,N-dimethyl-6-({[1-(1-naphthyl)-1H-tetrazol-5-yl]thio}methyl)-1,3,5-triazine-2,4-diamine, inhibits IRE-driven alpha-synuclein translation with an IC50 of 1.8 μM and is over 100-fold selective over cells lacking an IRE (H4-C) and cells with an IRE from Scrapies prion protein (H4-PRP).

2. Materials and Methods

Materials and Reagents

Dulbeccos modified essential medium (DMEM, catalog no. 12-614Q); FBS (catalog no.14-503E), L-glutamine (catalog no. 17-605E), penicillin/streptomycin (catalog no. 17-602E); phenol red free media (phenol red-free DMEM with 4.5 g/L D-glucose (catalog no. 12-917F) were purchased from Lonza (Portsmouth, NH). Geneticin (catalog no. 10131-027)) and alpha-synuclein ELISA kit KHB0061 were acquired from Invitrogen. Trypsin/EDTA (catalog no. 25-053-Cl) was purchased from Cellgro, and strophanthidine (catalog no. S6626-250MG, Lot 038K1036) was purchased from Sigma. Steady-Glo (catalog no. E2250) was purchased from Promega (Madison, WI). For the Elisa secondary screen, protease inhibitor (catalog no. 786-331) was acquired from G Biosciences. For the Western Blot secondary screen, penicillin/streptomycin was acquired from Bio-Whittaker (Walkerville, MD), mouse monoclonal anti-alpha-synuclein was purchased from BD Transduction Laboratories, and anti-beta-actin was acquired from Chemicon. 384-well plates (catalog no. 3570) were purchased from Corning and Falcon TC flasks (catalog no. 353112) were purchased from Becton Dickison (Waltham, MA).

2.1. Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided in Appendix A. Refer to Appendix B for the detailed assay protocols.

2.1.1. Primary Screen in H4 Cells

Stably transfected H4 neuroglioblastoma cells were grown to confluency in 35 mL complete DMEM with 4.5 g/L D-glucose supplemented with 10% FBS, 200 μM L-glutamine, 100 μM penicillin/streptomycin, and 200 μg/mL geneticin in a T175 TC flask in a TC incubator (37ºC, 95% humidity, 5% CO2) (doubling time = 24 h). Cells were harvested by washing the monolayer quickly with 5 mL trypsin/EDTA (1X), aspirating, then adding 5 mL trypsin/EDTA and incubating for 5 min at 37ºC, 95% humidity, 5% CO2. Then, 5 mL of complete media was added and mixed by pipetting up and down. Then, 10 mL of cell solution was aspirated and added to a conical tube; cells were pelleted for 3 min at 1000 rpm in a swinging bucket centrifuge. The media was aspirated, and the cell pellet was resuspended in 10 mL fresh complete media. Cells were then counted and expanded by plating 1×106 cells in 35 mL complete media per T175 flask (20xT175 flasks per 200 assay plate run) and allowing 72 h for cells to grow to confluency. Cells were harvested as above, but were resuspended in phenol-red free complete media (phenol red-free DMEM with 4.5 g/L D-glucose supplemented with 10% FBS, 200 μM L-glutamine, 100 μM penstrep, and 200 μg/mL geneticin, and then were diluted to 60,000 cells/mL in phenol-red free complete media prewarmed to 37ºC in 1-liter sterile plastic bottles. Cells were plated into white TC treated 384-well plates (batch size = 200–220 assay plates) at 3,000 cells/well in 50 μL phenol-red free complete media using a Thermo MultiDrop Combi liquid dispenser and a sterilized dispensing cassette and stir bar, in a TC hood. Assay plates were loaded into 22 slot holders which were then placed into an online Liconic (STX 2201C) incubator set to 37ºC, 95 % humidity, 5% CO2, and were incubated overnight.

Screening was performed using an open system (HiRes Biosolutions). Each run was initiated in CBIP (Broad Chemical Biology Informatics Platform) and scheduled with Cellario software (HiRes Biosolutions). Staubli arms moved plates from different instruments on the robotic system. Replicate assay plates were delidded and then pinned with 100 nL of 3.75 mM compound (final concentration = 7.5 μM) each with a 100 nL pin head using a MicroPin pin tool (HiRes Biosolutions). The positive control used in this assay was strophanthidine (Sigma, Cat. S6626-250MG, Lot 038K1036), which was used in dose (12-point, 2-fold dilution, from 20 μM final [compound]), and was incorporated at the beginning and end of each run. The median raw value of the 20-μM wells on the dose plates was used for normalizing the compound plates throughout the run. Plates with compound were then relidded and returned to the Liconic incubator (STX 2201C) and incubated for 48 h at 37ºC, 95% humidity, 5% CO2. Plates were then individually moved to a room temperature Liconic Carousel (LPX 220) and were allowed to temperature equilibrate for 30 min. Plates were then delidded and moved to a MultiDrop Combi liquid dispenser where 30 μL of 0.5X Steady-Glo was added (Steady-Glo was maintained at 4ºC and warmed to room temperature using a Combi cassette with a 6-foot input line submerged in a room temperature water bath). Plates were then relidded, returned to the Liconic Carousel, and incubated at room temperature for 30 min. Plates were then delidded and moved to an Envision plate reader (Perkin Elmer); luminescence values were collected using with 100 ms read time per well. Plates were then relidded and discarded.

2.1.2. Primary Retest in H4 Cells

As described in primary screen in H4 cells, but the retest samples were cherry picked from the central library by Discovery Partners Inc.

2.1.3. Secondary Assay in H4-C Cells

As described in primary retest in H4 cells but using the H4-C cell line.

2.1.4. Secondary Assay in H4-PRP Cells

As described in primary retest in H4 cells but using the H4-PRP cell line.

2.1.5. ELISA Secondary Screen in SH-SY5Y Neuroglioma Cells

A human ASYN ELISA kit was utilized for all ELISA experiments, and the kit protocol was followed. Briefly, SH-SY5Y neuroglioblastoma cells were plated at 10,000 cells/well in 50 μL phenol red free media (phenol red-free DMEM with 4.5 g/L D-glucose supplemented with 10 % FBS, 200 μM L-glutamine, 100 μM penicillin/streptomycin, and 200 μg/mL geneticin. Plates were lidded, and cells were then incubated for 24 hours at 37ºC, 5% CO2, 95% humidity in a tissue culture incubator. Next, 100 nL of each compound was then pinned into the assay plates spanning a final concentration range from 100 – 0.006 μM. Plates were then incubated for 48 h at 37ºC, 5% CO2, 95% humidity. Following incubation, 5 μL of Alamar blue solution was added per well and allowed to incubate for 1 h at 37ºC, 5% CO2, 95% humidity. Alamar blue fluorescence was then read using a Thermo Varioskan (570 nm excitation, 585 nm emission). Cell media was removed and 30 μL cell lysis solution with protease inhibitor (10 μL/mL) was added. Plates were then placed on a plate shaker for 1 h, before removing 20 μL lysate. Cell lysates were transferred to 500 μL Eppendorf tubes and centrifuged (Eppendorf Centrifuge 5417C) for 10 min at 13,000 rpm. Then, 12 μL of the cell supernatant was added to 48 μL of standard diluent buffer (1:5), and these samples were then added to ELISA plate wells. ASYN standard curve wells were prepared as described in the kit manual. Next, 50 μL of anti-α-Synuclein (Detection Antibody) solution was added to each well, tapping the plate to mix. The plate was then covered with a tape seal and incubated for 3 h at RT. Wells were washed four times with wash buffer using a multichannel pipette, and 100 μL of anti-rabbit IgG-HRP antibody solution was added to each well. The plate was covered and incubated for 30 m at RT. Wells were washed four times with wash buffer and 100 μL of Stabilized Chromogen was added to each well. The plate was then sealed and incubated in the dark at RT for 30 m. Finally, 100 μL of Stop Solution was added to each well, tapping plate to mix, and absorbance was read at 450 nm using an Envision plate reader (Perkin Elmer).

2.1.6. Western Blot Secondary Screen in H4 Cells

Human H4 neuroglioblastoma cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Cells were exposed to increasing concentrations of compound (0, 0.1, 1, 10, 100 μM) for 48 hours. Cytoplasmic protein lysates were prepared by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor and 10 mM DTT). Western blotting for alpha-synuclein was performed using mouse monoclonal anti-alpha-synuclein, and anti-beta-actin. The blots were developed using chemiluminescence (Pierce), visualized with a PhosphoImager (BioRad, Hercules, CA), and the bands were quantified using QuantityOne software (BioRad).

2.2. Probe Chemical Characterization

Image ml150fu2

The probe 1 (CID-1517919/ML150) was purchased from Chembridge (ID 7814700); however it could be synthesized starting from 1-isothiocyanatonapthalene 2 in two steps as shown in Scheme 1, based on literature precedent (7, 8, 9). Treatment of 2 with sodium azide would provide 1-(naphthalene-1-yl)-1H-tetrazole-5-thiol 3. Treatment of 3 with sodium hydride and 6-(bromomethyl)-N2,N2-dimethyl-1,3,5-triazine-2,4-diamine 4 would provide the probe compound (CID-1517919, CID-87556809, ML150). The probe was determined to have a solubility of <1 μM in PBS at room temperature. The probe and five analogs were submitted to the SMR collection (MLS002703075, MLS002703073, MLS002703074, MLS002703076, MLS002703077).

The 1H and 13C NMRspectra and LC/MS chromatograms of the probe (CID-1517919/ML150) and analogs are provided in Appendix C.

2.3. Probe Preparation

Not applicable. The probe (CID-1517919/ML150) was purchased from Chembridge (ID 7814700).

3. Results

Probe Attributes

  • IC50 ≤ 10 μM in the alpha-synuclein IRE containing H4 neuroglioblastoma primary screening cell line (H4-2a).
  • Greater than 10-fold selectivity as defined by: IC50 ratios of the primary screening cell line (H4-2a) and both the non-IRE containing H4 cell line (H4-C) and the prion protein IRE containing H4 cell line (H4-PRP).
  • Compound has a dose-response inhibitory effect in native non-transfected SH-SY5Y or H4 cells as observed by ELISA and Western blot.

3.1. Summary of Screening Results

A high-throughput screen of 303,811 compounds (AID-1813) was performed in duplicate in the alpha-synuclein IRE containing H4 neuroglioblastoma primary screening cell line (H4-2a). Using a screening “hit” cutoff of ≥62% inhibition at a screening concentration of 7.5 μM, 2498 compounds were identified as inhibitors of IRE-driven luciferase expression, among which 2193 compounds were available as cherry picks (Figure 2). These 2193 compounds were retested in 8-point dose (20 - 0.16 μM) in the primary screening cell line (AID-1988). Compounds that had IC50 values ≤ 200 μM were considered to be successful primary retests (1381/2193, 63% retest rate).

Figure 2. Critical Path for Probe Development.

Figure 2

Critical Path for Probe Development.

Compounds were also tested in two counterscreens, the first of which was an H4 neuroglioblastoma cell line transfected with the same plasmid as the primary screening line, except the alpha-synuclein IRE stem-loop was removed from the 5′UTR (H4-C). The purpose of this counterscreen was to determine if active compounds were either cytotoxic, inhibiting luciferase activity, or causing global reductions in transcription/translation. Compounds were tested in dose, and the hit criteria were the same as the primary retest. Of these, 1216 compounds were active (AID-1990). The second counterscreen was with H4 neuroglioblastoma cells transfected with a construct containing the prion protein IRE in the 5′UTR upstream of luciferase (H4-PRP). The purpose of this counterscreen was to determine if compounds that were active in the primary retest were specific for the alpha-synuclein IRE stem-loop or if they were capable of binding a closely related IRE stem-loop. Compounds were tested in the same means as in the previous confirmatory screens using the same activity criteria. Of these, 1193 compounds were active (AID-1994).

Compounds with at least 10-fold selectivity for the primary H4-2a screening line over the H4-C cell line and 10-fold selectivity for H4-2a over the H4-PRP cell line were prioritized for further development. Of these, 36 compounds were obtained as dry powders and were then retested in the primary and counterscreen assays (AIDs: H4-2a = 2460, H4-C = 2463, H4-PRP = 2468).

One compound (1517919/ML150) met the selectivity criteria using dry powder samples and this became probe candidate 1. The inhibition curves of 1517919/ML150 in the aforementioned cell lines are depicted in Figure 3. The probe candidate was then tested in by ELISA in SH-SY5Y cells (Figure 4, AID = 2473), and a clear dose-response reduction in alpha-synuclein levels was observed.

Figure 3. Concentration-dependent Activities of the Probe (CID-1517919/ML150) in the Target and Counterscreen Assays.

Figure 3

Concentration-dependent Activities of the Probe (CID-1517919/ML150) in the Target and Counterscreen Assays. Activity curves for probe CID-1517919/ML150: the alpha-synuclein IRE containing H4 neuroglioblastoma primary screening cell line (A), the H4 neuroglioblastoma (more...)

Figure 4. ELISA Experiments with Probe CID-1517919/ML150 in SH-SY5Y Cells (AID = 2473).

Figure 4

ELISA Experiments with Probe CID-1517919/ML150 in SH-SY5Y Cells (AID = 2473).

Finally, the compound was also examined by Western blot, where it showed a dose-dependent reduction in alpha-synuclein levels without altering actin levels (Figure 5, AID = 2484). This compound became probe series 1.

Figure 5. Western Blot Results From Probe CID-1517919/ML150 in H4 Cells (AID = 2484).

Figure 5

Western Blot Results From Probe CID-1517919/ML150 in H4 Cells (AID = 2484).

3.2. Dose Response Curves for Probe

The probe (CID-1517919/ML150) met the defined probe criteria and has acceptable solubility and stability in aqueous conditions. The activity curves of the probe in the aforementioned cell lines are depicted in Figure 3.

3.3. Scaffold/Moiety Chemical Liabilities

A search of PubChem for the probe compound (CID-1517919) revealed that the probe has been tested in 552 BioAssays and was confirmed as active in only three other assays. Therefore, the compound is not promiscuous. The compound has a drug-like structure with no obvious chemical liabilities that would be a concern.

3.4. SAR Tables

The structures and activities of the probe and analogs in the primary assay and the suite of secondary assays are summarized in Table 1.

Table 1. SAR Analysis and Properties of the Alpha-synuclein Probe and Analogs.

Table 1

SAR Analysis and Properties of the Alpha-synuclein Probe and Analogs.

Analogs to the probe were generated and tested in the H4-2a primary screening cell line in dose format. The analogs had a range of activity from 8 μM to weaker than 200 μM (Figure 6, Table 1). Table 1 reveals some very interesting SAR involving structural perturbations to the probe scaffold. For example, the large naphthalene ring (entry 1) appears to be required for good potency, as the simple unsubstituted phenyl derivative (entry 5) is inactive. However, potency is regained by adding back substituents to the meta and ortho positions of the phenyl group (entries 2 and 3); substitution at the para position is likely not tolerated (entry 4), although this could be due to the lack of methyl groups on the triazine ring as well.

Figure 6. Concentration-dependent Activity of Alpha-synuclein Analogs.

Figure 6

Concentration-dependent Activity of Alpha-synuclein Analogs. Dose-response curves of five probe analogs: CID-1517919/ML150 (A), 17452347(B), CID-17425317(C), CID-7562110(D), 1248117(E)

In general, the probe compound (CID-1517919/ML150) should serve as a useful tool for cell biology research because of its’ low molecular weight and good physical properties. Furthermore, both the naphthalene and triazine rings could be further substituted to optimize drug metabolism and pharmacokinetic (DMPK) properties.

3.5. Cellular Activity

The cell permeability and toxicity were not quantitatively measured. The probe and analogs were tested in cell-based assays. Since the analogs CID-17425317 and CID-1248117 do not display activity in the cell-based reporter assay while the other compounds (CID-1517919, CID-17452347, and CID-174254317) do display activity, it is highly unlikely that the triazine thio tetrazol scaffold itself is exhibiting cellular toxicity at the μM concentrations tested over the 48 h incubation time of the various cell-based assays. Furthermore, the specificity of effect observed between the H4-2a screening cell line dose response and H4-C cell line also indicates a lack of cellular toxicity from the probe molecule at the conditions tested (AID-2460, AID-2463, and AID-2468).

3.6. Profiling Assays

No profiling assays were conducted.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

The literature searches described in Table 2 were unable to find any small molecules that inhibited the 5′UTR of alpha synuclein mRNA. The only report of alpha synuclein expression inhibition was a patent utilizing RNAi.

4.2. Mechanism of Action Studies

No mechanism of action studies were conducted.

4.3. Planned Future Studies

The probe and analogs will be tested in primary neuronal cells to confirm their activity. Furthermore, they will be used in mouse models of Parkinson’s disease

5. References

1.
Uéda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero DA, Kondo J, Ihara Y, Saitoh T. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc Natl Acad Sci USA. 1993 Dec 1;90:12282–12286. [PMC free article: PMC47966] [PubMed: 8248242]
2.
Singleton A, Gwinn-Hardy K. Parkinson’s disease and dementia with Lewy bodies: a difference in dose. Lancet. 2004 Sep 25–Oct 1;364(9440):1105–1107. [PubMed: 15451205]
3.
Scherzer CR, Grass JA, Liao Z, Pepivani I, Zheng B, Eklund AC, Ney PA, Ng J, McGoldrick M, Mollenhauer B, et al. GATA transcription factors directly regulate the Parkinson’s disease-linked gene alpha-synuclein. Proc Natl Acad Sci USA. 2008 Aug 5;105(31):10907–10912. Epub 2008 Jul 31. [PMC free article: PMC2504800] [PubMed: 18669654]
4.
Cantuti-Castelvetri I, Klucken J, Ingelsson M, Ramasamy K, McLean PJ, Frosch MP, Hyman BT, Standaert DG. Alpha-synuclein and chaperones in dementia with Lewy bodies. J Neuropathol Exp Neurol. 2005 Dec;64(12):1058–1066. [PubMed: 16319716]
5.
Thomson AM, Rogers JT, Leedman PJ. Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation. Int J Biochem Cell Biol. 1999 Oct;31(10):1139–1152. [PubMed: 10582343]
6.
Thomson AM, Cahill CM, Cho HH, Kassachau KD, Epis MR, Bridges KR, Leedman PJ, Rogers JT. The acute box cis-element in human heavy ferritin mRNA 5′-untranslated region is a unique translation enhancer that binds poly(C)-binding proteins. J Biol Chem. 2005 Aug 26;280(34):30032–30045. Epub 2005 Jun 20. [PubMed: 15967798]
7.
Neidlein R, Tauber J. Tetrazolylmercaptomethyl-isocyanate und ihre Derivate. Chemische Berichte. 1967;100:736–740. [Cross Ref]
8.
Lebrun ME, Le Marquand P, Berthelette C. Stereoselective synthesis of Z alkenyl halides via Julia olefination. J Org Chem. 2006 Mar 3;71(5):2009–13. [PubMed: 16496987]
9.
Klenke B, Stewart M, Barrett MP, Brun R, Gilbert IH. Synthesis and biological evaluation of s-Triazine substituted polyamines as potential new anti-trypanosomal drugs. J Med Chem. 2001;44(21):3440–3452. Publication Date (Web): 10.1021/jm010854+September 18, 2001. [PubMed: 11585449]

6. Appendices

Appendix A. Compound Characterization

Table A1Summary of Completed Assays and AIDs

PubChem AID No.TypeTargetConcentration Range (μM)Samples Tested
1813PrimaryASYN 5′UTR7.5303811
1988ConfirmatoryASYN 5′UTR20 - 0.162913
1990CounterscreenH4-C20 - 0.162193
1994CounterscreenH4-PRP20 - 0.162193
2460Confirmatory-powderASYN 5′UTR20 - 0.1636
2463Counterscreen-powderH4-C20 - 0.1636
2468Counterscreen-powderH4-PRP20 - 0.1636
2473ELISAASYN100 – 0.0061
2484Western BlotASYN100 – 0.11
2471ConfirmatoryASYN 5′UTR20 - 0.164
2454ConfirmatoryASYN 5′UTR1.2-0.0006184
2452CounterscreenH4-PRP1.2-0.0006184
2458CounterscreenH4-C1.2-0.0006184
2442ConfirmatoryASYN 5′UTR20-0.1641
2627Western BlotASYN10-0.11
2670ConfirmatoryASYN 5′UTR20-0.000025

Appendix B. Detailed Assay Protocols

Primary Screen in H4 Cells

  1. Grow stably transfected H4 neuroglioblastoma cells to confluency in 35 mL complete media (DMEM with 4.5 g/L D-glucose [Lonza, 12-614Q] supplemented with 10% FBS [Lonza, 14-503E], 200 μM L-glutamine [Lonza, 17-605E], 100 μM penstrep [Lonza, 17-602E] and 200 μg/mL geneticin [Invitrogen, 10131-027]) in a T175 TC flask (BD Falcon, 353112).
  2. Incubate in a TC incubator (37ºC, 95% humidity, 5 % CO2) (doubling time = 24 h).
  3. Harvest cells by washing the monolayer quickly with 5 mL trypsin/EDTA (1X, Cellgro, 25-053-Cl). Aspirate, then add 5 mL trypsin/EDTA.
  4. Incubate for 5 min (37ºC, 95% humidity, 5% CO2).
  5. Add 5 mL of complete media and mix by pipetting up and down. Aspirate 10 mL of cell solution and add to a conical tube.
  6. Pellet the cells for 3 min at 1000 rpm in a swinging bucket centrifuge.
  7. Aspirate the media and resuspend the cell pellet in 10 mL fresh complete media.
  8. Count the cells and expand by plating 1×106 cells in 35 mL complete media per T175 flask (20xT175 flasks per 200 assay plate run). Allowing 72 h for cells to grow to confluency.
  9. Harvest the cells as above, but resuspend cells in phenol-red free complete media (phenol red-free DMEM with 4.5 g/L D-glucose [Lonza, 12-917F] supplemented with 10% FBS [Lonza, 14-503E], 200 μM L-glutamine [Lonza, 17-605E], 100 μM penstrep [Lonza, 17-602E], and 200 μg/mL geneticin [Invitrogen, 10131-027]).
  10. Dilute resuspended cells to 60,000 cells/mL in phenol-red free complete media prewarmed to 37ºC in 1-L sterile plastic bottles.
  11. Plate cells into white TC-treated 384-well plates (Corning, 3570; batch size = 200–220 assay plates) at 3,000 cells/well in 50-μL phenol-red free complete media using a Thermo MultiDrop Combi liquid dispenser, a sterilized dispensing cassette, stir bar in a TC hood.
  12. Load assay plates into 22 slot holder and place into an online Liconic (STX 2201C) incubator set to 37ºC, 95% humidity, 5% CO2. Incubate plates overnight.
  13. Perform screening using an open system (HiRes Biosolutions). Initiate each run in Broad Chemical Biology Informatics Platform (CBIP) and schedule with Cellario software (HiRes Biosolutions). Staubli arms moved plates from different instruments on the robotic system.
  14. Delid replicate assay plates and pin with 100 nL of 3.75 μM compound (final concentration = 7.5 μM) each with a 100 nL pin head using a MicroPin pin tool (HiRes Biosolutions).
  15. Use strophanthidine (Sigma, Cat.# S6626-250MG, Lot#038K1036) as the positive control in dose (12-point, 2-fold dilution, from 20 μM final [compound]) at the beginning and end of each run. Relid plates with compound and return to the Liconic incubator (STX 2201C).
  16. Incubate plates for 48h at 37ºC, 95% humidity, 5% CO2. Move plates individually to a room temperature Liconic Carousel (LPX 220) and allow to temperature equilibrate for 30 min.
  17. Delid plates and move to a MultiDrop Combi liquid dispenser and add 30 μL of 0.5X Steady-Glo (Promega, E2250). Maintain Steady-Glo at 4ºC and warm to room temperature using a Combi cassette with a 6-foot input line submerged in a room temperature water bath.
  18. Relid plates and return to the Liconic Carousel. Incubate at room temperature for 30 min.
  19. Delid plates and move to an Envision plate reader (Perkin Elmer). Collect luminescence values using with 100 ms read time per well. Relid the plates and discard.

ELISA Secondary Screen in SH-SY5Y Neuroglioma Cells

A human ASYN ELISA kit was utilized for all ELISA experiments (Invitrogen alpha-synuclein ELISA kit KHB0061) and the kit protocol was followed.

  1. Plate SH-SY5Y neuroglioblastoma cells (Corning, 3570) at 10,000 cells/well in 50 μL phenol red free media (phenol red-free DMEM with 4.5 g/L D-glucose [Lonza, 12-917F] supplemented with 10 % FBS [Lonza, 14-503E], 200 μM L-glutamine [Lonza, 17-605E], 100 μM penstrep [Lonza, 17-602E] and 200 μg/mL geneticin [Invitrogen, 10131-027]).
  2. Lid plates, then incubate cells for 24 hours at 37ºC, 5% CO2, 95% humidity in a tissue culture incubator.
  3. Pin 100 nL of each compound into the assay plates spanning a final concentration range from 100 – 0.006 μM.
  4. Incubate plates for 48h at 37ºC, 5% CO2, 95% humidity. Following incubation, add 5 μL of Alamar blue solution per well and allow to incubate for 1h at 37ºC, 5% CO2, 95% humidity.
  5. Read Alamar blue fluorescence using a Thermo Varioskan (570 nm excitation, 585 nm emission). Remove cell media and add 30 μL cell lysis solution with protease inhibitor (G Biosciences, 786-331; 10 μL/mL).
  6. Place plates on a plate shaker for 1h, before removing 20 μL lysate. Transfer cell lysates to 500 μL Eppendorf tubes and centrifuge (Eppendorf Centrifuge 5417C) for 10 min at 13,000 rpm.
  7. Add 12 μL of the cell supernatant to 48 μL of standard diluent buffer (1:5). Add these samples to ELISA plate wells. Prepare ASYN standard curve wells as described in the kit manual.
  8. Add 50 μL of anti-α-Synuclein (Detection Antibody) solution to each well, tapping the plate to mix. Cover the plate with a tape seal and incubate for 3 h at RT.
  9. Wash wells four times with wash buffer using a multichannel pipette and add100 μL of anti-rabbit IgG-HRP antibody solution to each well. Cover the plate and incubate for 30 min at RT. Wash wells four times with wash buffer and add 100 μL of Stabilized Chromogen to each well. Seal the plate and incubate in the dark at RT for 30 min.
  10. Finally, add 100 μL of Stop Solution to each well, tapping the plate to mix, and read absorbance at 450 nm using an Envision plate reader (Perkin Elmer).

Western Blot Secondary Screen in H4 Cells

  1. Culture human H4 neuroglioblastoma cells in Dulbeccos modified essential medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (Bio-Whittaker, Walkerville, MD).
  2. Expose cells to increasing concentrations of compound (0, 0.1, 1, 10, 100 μM) for 48 hours.
  3. Prepare cytoplasmic protein lysates by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor and 10 mM DTT).
  4. Perform Western blotting for alpha-synuclein using mouse monoclonal anti-alpha-synuclein (BD Transduction Laboratories), and anti-beta-actin (Chemicon). Develop the blots using chemiluminescence (PIERCE) and visualize with a PhosphoImager (BioRad, Hercules, CA). Quantify the bands using QuantityOne software (BioRad).

Appendix C. NMR and LC Data of Probe and Analogs

1H NMR Spectra of Probe (CID-1517919/ML150).

1H NMR Spectra of Probe (CID-1517919/ML150)

LC/MS Chromatogram of Probe (CID-1517919/ML150).

LC/MS Chromatogram of Probe (CID-1517919/ML150)

1H NMR Spectra of analog CID-17425317.

1H NMR Spectra of analog CID-17425317

LC/MS Chromatogram of analog CID-17425317.

LC/MS Chromatogram of analog CID-17425317

1H NMR Spectra of analog CID-17425347.

1H NMR Spectra of analog CID-17425347

LC/MS Chromatogram of analog CID-17425347.

LC/MS Chromatogram of analog CID-17425347

1H NMR Spectra of of analog CID-17562110.

1H NMR Spectra of of analog CID-17562110

LC/MS Chromatogram of analog CID-17562110.

LC/MS Chromatogram of analog CID-17562110

1H NMR Spectra of analog CID-1248117.

1H NMR Spectra of analog CID-1248117

LC/MS Chromatogram of analog CID-1248117.

LC/MS Chromatogram of analog CID-1248117

Appendix D. Compounds submitted to BioFocus

Table A2Probe and Analog Information

BRDSIDCIDP/AMLSIDML
BRD-K51608872875568091517919PMLS002703075ML150
BRD-K257741478755680417425317AMLS002703073NA
BRD-K315539178755680717562110AMLS002703074NA
BRD-K400941918755680617452347AMLS002703076NA
BRD-K61627029874571491248117AMLS002703077NA

P = probe, A = analog

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