2.1. Assays

qHTS assay for inhibitors of Hutingtin toxicity [AID: 1471, 2669, 2672]

A stable PC12 cell line containing a gene fusion of Exon 1 of the Huntingtin gene linked to GFP under the control of the inducible ecdysone promoter were used as the cell-based model of Huntington Disease for high throughput screening. Exon 1 of the Huntingtin gene contained an expansion of 103 polyglutamines (Q103HTT) which, when expressed, induced cell death and formed distinct, bright GFP aggregates. The amount of cell death and the size and intensity of GFP aggregates increased with time and induction level. Cell death was quantified by measurement of ATP content in the cells. A maximal 40–50% of cell death was observed when the Huntington gene was induced in this cell line.

Rat pheochromocytoma PC12 cells harboring HTT Q103 or Q25 fused to GFP under tebufenozide (Sigma Aldrich) induction were supplied by the Eric Schweitzer lab (UCLA)6. Cells were maintained in Phenol red free DMEM (Invitrogen) at 37°C under a humidified atmosphere containing 5% CO₂ and 95% air. The medium contained + 5% supplemented calf serum, + 5% horse serum (sera from HyClone), 250 μg/ml Geneticin and 1x pen/strep 2mM L-glutamine. Cells were passaged when they reached 85 to 90% confluency. Cells were plated at a density of 1000 to 1500 cells/well in black, clear bottom, tissue culture treated, microclear 1536 well plates (Aurora Biotechnologies) in 5 μl/well in phenol red free DMEM containing 2% serum (1% each) without geneticin using a Multidrop Combi dispenser (Thermo Scientific). Tebufenozide (Sigma Aldrich) inducer at a 200nM final concentration and test compounds were sequentially added to the cells using a Kalypsys pintool transfer station at a volume of 23nl/well after the cells were incubated for 24 hrs. Compound libraries and Tebufenozide were dissolved in DMSO and the final concentration of DMSO in the cell plates was 0.46% v/v. Plates were incubated at 37C in 5 % CO2 for 48 hours before being assayed. The assay protocol is listed in Table 1.

Table 1

PC-12 Q103 GFP Huntington qHTS.

For ATP quantitation, 3μl of ATPLite (PerkinElmer) was added to each well. The plates were spun for 1 minute at 1500 PRM to remove bubbles and luminescent signal was recorded on a Viewlux CCD Plate reader (30 second integration time). Compounds were normalized against induced (200nM tebufenozide considered maximum cell death) and uninduced (considered min. cell death). Controls were; Columns 1–3 DMSO only, Column 2 Tebufenozide 16 point dose response from 1μM to 0μM, Column 4 (and the rest of the plate) received 200nM tebufenozide. The multiplexed assay also measured aggregate status, but was not used for SAR studies on compounds which affected cytotoxicity (PubChem AID 1688).

Confirmatory assays

Protease release assay

Another measurement of cell health is membrane integrity. Cells with perturbed membrane integrity release proteins and cofactors from the cytosol into the media, a “dead cell protease” being one of them. Promega has developed a kit (Cytotox Glo) which contains a cell impermeable pro-luciferin substrate that becomes activated upon cleavage by such a protease7. Therefore, cells with altered membrane permeability will release this protease and generate an increase in luminescent signal. This assay was chosen as a follow-up assay because of its more reliable nature (average S/B of 9.6 and Z’ of 0.80). The protocol for the protease release assay is listed in Table 2.

Table 2

PC-12 Q103 GFP huntingtin-induced protease release qHTS protocol.

Striatal cell model

One hallmark of Huntington’s disease is a selective neurodegeneration of striatal cells in patients. To confirm the activity of lead compounds in an orthogonal assay, we tested the capacity of compounds to prevent cell death induced by serum deprivation in mouse wild type (wt) STHdh^Q7/7 cells and mutant STHdh^Q111/111 Huntington striatal cells8. The cells were plated in normal serum conditions or in serum deprivation conditions and treated at the same time with 30 μM solutions of our two initial hits. Cell health was quantitated by measuring ATP levels in cells. Figure 1 shows that the STHdh^Q111/111 Huntington striatal cells were more susceptible to the serum deprivation challenge than the wild type cells.

Figure 1

Cell growth of wild type and Huntington mutant STHdh^Q111/111 striatal cells upon serum deprivation. ATP levels quantitated using ATPlite, a luciferase-based ATP quantitation assay, after 24 hour incubation. Data represented in RLU counts. Serum deprivation (more...)

2.2. Probe Chemical Characterization

Molecular weight confirmation was performed using an Agilent Time-Of-Flight Mass Spectrometer (TOF, Agilent Technologies, Santa Clara, CA). A 3 minute gradient from 4 to 100% Acetonitrile (0.1% formic acid) in water (0.1% formic acid) was used with a 4 minute run time at a flow rate of 1 ml/min. A Zorbax SB-C18 column (3.5 micron, 2.1x 30 mm) was used at a temperature of 50°C. Confirmation of molecular formula was obtained using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

1H NMR (400 MHz, DMSO-d6) δ ppm: 3.94 (s, 3H), 7.57 (m, 3H), 7.61 (m, 2H), 7.99 (s, 1H), 8.74 (s, 1H)

LC/MS (Agilent system) Retention time t1 (short) = 3.47 min

Purity: UV220 > 99%, UV254 > 99%; MS m/z 327.1 (M+H);

Column: 3x 75 mm Luna C18, 3 micron

Run time: 4.5 min (short)

Gradient: 4% to 100%

Mobile phase: Acetonitrile (0.025% TFA), water (0.05% TFA).

Flow rate: 0.8 to 1.0ml

Temperature: 50°C

UV wavelength: 220 nm, 254 nm

ML168/MLS00089318/CID 2432214 is commercially available from Enamine (Cat# T0516-1746). In addition, several analogs were submitted to the SMR collection:

Compound is soluble at 10mM in DMSO. The compound is not fluorescent with blue excitation wavelengths (~340nm). Solubility in buffer has not been determined.

2.3. Probe Preparation

We decided to evaluate the SAR of the thienopyrimidine series identified through the primary screen. In initial studies, we focused on exploring analogs in the 5 position of the thienopyrimidine ring. Scheme 1 shows the synthetic methodology used for the synthesis of these analogs. The commercially available thieno[2,3-d]pyrimidin-4(3H)-one 3 was regioselective halogenated in the 6 position with bromo in acetic acid to obtain compound 4. Transformation of the pyrimidinone into 6-bromo-4-chlorothieno[2,3-d]pyrimidine 3 was done with phosphorus oxychloride. LDA induced bromo rearrangement and bromo-iodo replacement promoted by ethyl Grignard yield intermediate 7. Regioselective Suzuki coupling and halogen displacement with several nucleophiles yielded final compound 9. Note that carrying out the displacement first followed by cross coupling yields no final product. We speculate that intermediate 10 complexed with the palladium catalyst, preventing the progression of the reaction.

Scheme 1

General synthetic methodology for the synthesis of analogues.

3.1. Summary of Screening Results

A total of 1052 plates were screened; the Z score averaged 0.22 and the SB was 1.9 fold on average in the primary screen. The PC12 cells require rather stringent growth conditions. Cells in wells that become overly confluent perish readily, so plating at a consistent 1000–1500 cells/well is crucial for optimal responsiveness.

A qHTS screen in the Q103HTT cell line against 227,407 compound samples revealed several series with the capacity to selectively impact Huntingtin aggregation, impact expression-related-toxicity, or both. Those compounds that affected the formation of protein aggregates were not investigated in detail, as another series with this activity was recently reported on in detail9. There were very few compounds that were solely cytoprotective, and we initially focused on these. Two of the lead candidates from the screen, MLS000089318 (CID 2432214) 1 and MLS000058474 (CID 2526122) 2, both thienopyrimidines, were quite cytoprotective, and we ordered them as powder samples from Enamine for further confirmation. Based on this activity, the chemotype was chosen for SAR studies of its anti-apoptotic activity10. 1 also had an appealing molecular weight (326), total polar surface area (65), and lipophilicity (ClogP 3.4) for a probe (Figure 2). These compounds were then confirmed in the protease release and the striatal cell assays.

Figure 2

Structure and activity of MLS000089318 (CID 2432214) and MLS000058474 (CID 2526122). AC50 values from confirmation protease release assay.

3.2. Dose Response Curves for Probe

Figure 3Activity of ML168 (MLS000089318, CID 2432214) in preventing Q103HTT induced cytotoxicity as measured by the protease release assay

3.3. Scaffold/Moiety Chemical Liabilities

At the present time, liabilities of the present scaffold are not known.

3.4. SAR Tables

In the SAR table, Table 5, it can be seen that substituents at the ortho position of the phenyl ring diminish or abolish the activity of the molecule, indicating the need of a co-planarity between the substituent at the 5 position and the thienopyrimidine core. In addition, many different functional groups are well tolerated at the meta and para positions, although some strong electro withdrawing groups tend to reduce AC₅₀s. Last, the introduction of a functional group bearing a carbonyl or a sulfonyl group in the meta-position yields slightly more potent compounds, although the activity is similar to the initial leads.

Table 5

SAR of analogs with a modification at position 5; AC₅₀ values from protease release assay.

3.5. Cellular Activity

The primary screen was a cell-based assay. However, the most important results obtained in this project are from the striatal cell assay, the results of which are given in Table 4, below. As discussed earlier, striatal cells are of particular relevance to the pathology of Huntington’s disease. The phenotype observed with expression of the mutant Huntingtin protein is an enhanced sensitivity to cellular stress, in this case being serum deprived culture. For this experiment, striatal cells with wildtype (Q7/Q7) and mutant full-length protein (Q111/Q111) were grown without FBS and treated with the 2 probes of interest. Both compounds reversed the stress-induced apoptosis specifically in the cells expressing mutant Huntingtin protein, indicating a selective reversal of the Huntington phenotype with these cells.

Table 4

ATP quantitation of wild type and mutant Huntington striatal cells in normal growth media (DMEM + 10% FBS) and serum deprived media after compound treatment. Data are expressed as the AVG ± STDV of the ratio of the treatment to DMSO (vehicle) (more...)

3.6. Profiling Assays

No profiling assays were carried out.

4.1. Comparison to existing art and how the new probe is an improvement

A recent publication has reported on compounds that prevent cellular toxicity of Huntingtin protein expression by inhibiting protein aggregation9. The present series appears to protect cells by inhibiting activation of the intrinsic apoptotic pathway, which is a novel mode of action (see next section).

4.2. Mechanism of Action Studies

Because this series of compounds was identified from a phenotypic screen, we attempted to understand their mechanism of action by examining their effects on the apoptosis pathway. Figure 4 shows that ML168 (MLS000089318, CID 2432214) is able to block the activation of caspase 9 and its downstream target, caspase 3. Surprisingly, it prevents activation of apoptotic caspases in both wild type and mutant Huntington striatal cells under serum deprivation conditions. Caspases 9 and 3 are involved in the initiation of the intrinsic apoptotic pathway. It has been extensively reported that the apoptotic intrinsic pathway is activated in Huntington’s disease, ALS, Niemann-Pick disease type C, Lysosomal Cell Death, Ischemia, viral and bacterial infection, and ceramide induced neuronal death. Thus, the inhibition of this intrinsic apoptotic pathway looks to be the mechanism of action of this probe in the cell-based Huntington model.

Figure 4

Western blot of caspase protein following compound treatment. ML168 (MLS000089318) protects the cells from serum deprived apoptotic cell death by inhibiting the activation of caspase 9 and its downstream target, caspase 3. A) Caspase 9 and 3 activation (more...)

4.3. Planned Future Studies

An advanced characterization proposal has been submitted to the Molecular Libraries Network and has been accepted. Further mechanisms of action studies are ongoing.