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Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry

, , , , , , , , , and .

Author Information and Affiliations

Received: ; Last Update: March 22, 2013.

Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling. The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclinB protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur. Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome. Studies showing that Wee1 expression is reduced in colon carcinoma cells and that Wee1 overexpression can block cell division, suggest that Wee1 may act as a tumor suppressor. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), utilized a chemical biology approach to uncover kinases controlling Wee1 proteolysis by identifying small molecule inhibitors stabilizing a reporter of Wee1 degradation, K328M-Wee1-luciferase. High-throughput screening followed by medicinal chemistry optimization driven by secondary assays yielded a selective stabilizing probe, ML177, which is 200 times more potent than our previously described probe, ML118. ML177 exhibits a Wee1 stabilization EC50 value of 40 nM. The probe has been tested against several cell-cycle related targets and anti-targets, including Wee1, cyclin B, p21, p27, and ability to induce HeLa (cancer) cell apoptosis. The identified probe exhibited significantly improved selectivity and potency in these assays compared to the previous probe. These results, combined with additional kinase profiling by the assay provider, confirm the value of this probe and its usefulness in biologically relevant, cell-based assays.

Assigned Assay Grant #: 1R21NS056991-01

Screening Center Name & PI: Scripps Research Institute Molecular Screening Center (SRIMSC), Hugh Rosen

Chemistry Center Name & PI: SRIMSC, Hugh Rosen

Assay Submitter & Institution: Nagi Ayad, The Scripps Research Institute (TSRI), University of Miami

Summary Bioassay Identifier (AID): 1807

Probe Structure & Characteristics

ML177.

ML177

CID/ML#Target NameEC50 (nM) [SID, AID]Anti-targetEC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 45100431/ML177Wee1 Degradation Inhibition40nM [SID 92092801, AID 463080] Active

23.3-fold activation, [SID 92092801, AID 463169] Active		Cyclin B2.6-fold activation [SID 92092801, AID 463186] Inactive23.3/2.6 = 8.9-foldCK1d IC50 Assay: 49 nM [SID 92092801, AID 463077] Active
FLT3 IC50 Assay: 0.305 nM [SID 92092801, AID 463076] Active
p21 Stabilization Assay: 3.41-fold change [SID 92092801, AID 463170] Inactive
p27 Stabilization Assay: 4.61-fold change [SID 92092801, AID 463171] Inactive
HeLa Apoptosis Assay: Active [SID 92092801, AID 463177]
Amyloid Assay: 60% signal; Active [SID 92092801, AID 504929]
GCP (Granule Cell Proliferation) Assay: IC50 = 0.46; Active [SID 92092801, AID 504930]

1. Recommendations for Scientific Use of the Probe

What limitations in current state of the art is the probe addressing?

Although selective inhibitors of Wee1 kinase activity have been reported, none have been shown to inhibit Wee1 turnover [1–2]. For example, inhibition of Wee1 kinase activity by compounds that abrogate the G2/M checkpoint improve the cytotoxic effects of DNA damaging agents on p53-negative cells [3–6]. However, these compounds are dual Src/Wee1 kinase inhibitors (i.e. they are not inhibitors of Wee1 degradation). In addition, the assay provider screened LOPAC and other libraries that contain Src inhibitors and identified no hits; thus, Src activity is unrelated to inhibition of Wee1 degradation. As a result, the current state of the art lacks potent inhibitors that selectively inhibit degradation of Wee1. Non-selective inhibitors of protein degradation exist but these compounds are not useful for elucidating the specific roles of Wee1 in cell cycle progression, cancer development, and other cellular events. For example, MG132 is not selective as it inhibits general protein degradation via proteasome inhibition, as measured by similar EC50 values (~5 μM) in Wee1 and cyclin B assays.

One other small molecule has been reported in the literature to stabilize Wee1 in HeLa cells: the dihydropteridinone BI 2536 [2]. The mechanism by which BI 2536 leads to Wee1 stabilization is unknown. BI 2536 is an inhibitor of Plk-1, which phosphorylates many proteins associated with cell cycle progression and mitotic entry. A sample of BI 2536 was obtained from commercial sources [Selleck Chemicals, catalog number S1109] and was determined by the assay provider to be inactive in the Wee1 degradation inhibition assay. Thus, dissecting the role of BI 2536 on Wee1 biology relative to its multiple other cellular roles is an exceedingly challenging process. A second commercially available Plk-1 inhibitor, [5-(5,6-dimethoxybenzimidazol-1-yl)-3-(4-methanesulfonyl-benzyloxy)-thiophene-2-carboxamide, purchased from EMD Chemicals, catalog number 528282-5MG] was determined by the assay provider to be inactive in the Wee1 degradation inhibition assay. Moreover, probe compound SID 4243143/CID 3237904/powder SID 87235992 does not inhibit Plk-1, so its mechanism of action for Wee1 stabilization is distinct from that of BI 2536.

Recent improvements have been made to the field, as demonstrated by a previous probe report submitted by the SRIMSC and assay provider Dr. Nagi Ayad. In this prior report, the authors describe the identification of a first generation selective inhibitor of Wee1 degradation [7]. This prior probe, (CID 3237904/SID 4243143/powder SID 87235992/ML118) exhibits a Wee1 EC50 value of ~8 μM and a Cyclin B EC50 of >50 μM, demonstrating a >6.3-fold selectivity towards Wee1 over cyclin B.

In an effort to improve upon this current art by improving the potency and selectivity for Wee1, the SRIMSC and Dr. Ayad extended their chemistry and biology efforts, resulting in a more potent and selective probe (CID 45100431/SID 92092801/ML177). This new probe exhibits a Wee1 stabilization EC50 of 40nM, and thus is clearly an improvement over the prior art compounds. It exhibits an 8-fold selectivity for Wee1 over cyclin B. In summary, this new probe ML177/CID 45100431 addresses the limitations of the current art by specifically inhibiting turnover of Wee1 with a greater potency and selectivity than other available chemical tools such as the prior probe and the prior art compound MG132, without acting as a general proteasome inhibitor. Thus, the new probe ML177/CID 45100431 represents a 200-fold improvement in potency over prior probe ML118.

What will the probe be used for?

The probe can be used to elucidate the specific role of Wee1 in cell cycle-related tumorigenesis and other cellular events. The importance of this probe is underscored by recent reports suggesting that Wee1 is shuttled from the nucleus to the cytoplasm, and thus there may be different pools of Wee1 that are located in different intracellular compartments and turned over via distinct mechanisms [8]. In this regard, the probe can also be used to elucidate Wee1 turnover in different intracellular compartments. This is not possible with siRNA mediated knockdown of intracellular components that generally inhibit turnover of all proteins since such treatments are often toxic and nonspecific.

Who in the research community will use the probe?

The probe can be used by academic researchers studying cell biology, molecular biology, and tumor biology. In addition, this probe was recently applied to studies of central nervous system neurogenesis. Thus it is conceivable that scientists in diverse fields will be able to apply this powerful chemical probe to elucidate the role of Wee1 in disease pathways.

What is the relevant biology to which the probe can be applied?

Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling [8–9]. The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclin B protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur [3, 8–9]. Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome [4, 6]. Studies showing that Wee1 expression is reduced in colon carcinoma cells suggest that Wee1 may act as a tumor suppressor [10]. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis. The assay provider hypothesizes that compounds that inhibit Wee1 degradation will provide information about the role of Wee1 in mitotic entry and cell cycle progression. As a result, the current probe can be applied to the characterization of Wee1 in cellular events, and may also be useful in identifying proteins that bind to, and/or modify the post-translational state of Wee1.

2. Materials and Methods

Following a successful HTS campaign, which resulted in the identification of a Wee1 degradation inhibitor probe (CID 3237904/SID 4243143/powder SID 87235992/ML118), a Center-based initiative was implemented to further characterize the mechanisms of Wee1 stabilization and its role in cell cycle regulation. The approach taken was to uncover kinases controlling Wee1 proteolysis by identifying small molecule kinase inhibitors stabilizing a reporter of Wee1 degradation, K328M-Wee1-luciferase. We screened a proprietary 16,000 compound kinase inhibitor library at Scripps Florida with K328M-Wee1-luciferase expressing cells and identified several structurally related members of a purine scaffold that increased the steady-state levels of K328M-Wee1-luciferase. In order to further examine the specific kinases involved the assay provider tested several leads against fms-related tyrosine kinase 3 (FLT3) and casein kinase 1 delta (CK1d). Furthermore, since Wee1, p21, and p27 are targeted by the same class of ubiquitin ligases (the SCF ubiquitin ligases), the assay provider performed counter-screens against p21 and p27, which served to identify and eliminate non-selective inhibitors of SCF ligases (compounds active against p21 and p27 were not pursued). Counter-screening against cyclin B was also performed to eliminate non-selective inhibitors. Finally, to characterize the mechanism of action of probe candidates, studies were performed to determine the cytotoxicity of probe candidates against neuroblastoma and HeLa cancer cells.

2.1. Assays

The specific assays are listed in Table 1.

Table 1. Related PubChem Bioassays.

Table 1

Related PubChem Bioassays.

Descriptions and protocols for each assay

AID 434972, AID 463080, AID 463169 (Wee1 Degradation Inhibitor Assays)
Late stage results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to determine inhibition of Wee1 degradation by kinase inhibitors

The purpose of this assay is to identify whether powder samples of compounds identified as possible WEE1 probe candidates can act as inhibitors of WEE1 degradation. The assay uses HeLa cells transfected with a kinase negative mutant of WEE1 (WEE1K328M) fused to a luciferase reporter gene. As designed, compounds that increase WEE1K328M-luciferase stability and/or prevent its degradation will lead to increased well luminescence compared to untreated wells. Specifically, compounds that increase luminescence are considered WEE1 degradation inhibitors. Compounds were tested in quadruplicate at a final nominal concentration of 250 nM.

HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 μg of the WEE1K328M-luciferase plasmid complexed with 87 μL of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and resuspended at a concentration of 850,000 cells per mL in growth media. Then, 50 μL of cells were added in quadruplicate to wells of a 96 well plate. Compounds were made in DMSO at 2,000 times the required concentration and diluted in media 1:1000. 50 μL of each concentration was added to 50 μL of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37 degrees Celsius (10% CO2, 95% RH). The assay was stopped by adding 100 μL of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. K328M-WEE1-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for RJR159, analog, or DMSO. The ratio for each of the compounds or controls was then divided by the ratio for RJR159 to yield %RJR159 levels.

For dose response assays, a ratio of luminescence signals was then calculated and plotted against the concentration to generate the EC50s. Compounds with an EC50 greater than 1 μM were considered inactive. Compounds with an EC50 equal to or less than 1 μM were considered active.

List of Reagents
  • Dulbecco’s Modified Eagle’s Media (Invitrogen, part 11965-092)
  • Fetal Bovine Serum (Hyclone, part SH 30088.03)
  • Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)
  • SteadyLite HTS luciferase substrate (PerkinElmer, part 6016989)
  • Reference agonist MG132 (American Peptide, part 81-5-15)
  • TransIT-LT1 (Mirus, part MIR2306)
  • T75 flasks (Corning, part 430641)
  • 1536-well plates (Greiner, part 789173)
AID 463076
Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay for inhibitors of fms-related tyrosine kinase 3 (FLT3)

The purpose of this assay is to determine whether powder samples of probe candidates can inhibit the activity of FLT3. This in vitro kinase assay was performed by Reaction Biology Corporation (RBC). For FLT3, 20 μM final Abltide was used, sequence: [EAIYAAPFAKKK] in a standard kinase assay with 33P-ATP and purified kinase. Incorporation of 33P-ATP into the peptide was measured after a filter-binding assay. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 50 μM.

Kinase reactions were performed using the “Hotspot” kinase profiling service of Reaction Biology Corporation.

  1. Prepare indicated substrate in freshly prepared Base Reaction Buffer.
  2. Deliver any required cofactors to the substrate solution above.
  3. Deliver indicated kinase into the substrate solution and gently mix.
  4. Deliver compounds in DMSO into the kinase reaction mixture.
  5. Deliver 33P-ATP (specific activity 500 μCi/μl) into the reaction mixture to initiate the reaction.
  6. Incubate kinase reaction for 120 min. at room temperature.

A ratio of radioactivity signals was then calculated and plotted against the concentration to generate the IC50s. Compounds with an IC50 greater than 1 μM were considered inactive. Compounds with an IC50 equal to or less than 1 μM were considered active.

List of Reagents
  • Base Reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO)

Additional details can be found at http://www.reactionbiology.com/

AID 463077
Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay to identify CK1d inhibitors

The purpose of this assay is to determine whether powder samples of probe candidates can inhibit the activity of CK1d. This in vitro kinase assay was performed by Reaction Biology Corporation (RBC). For CK1, 20 μM final CK1tide was used, sequence: [KRRRAL[pS]VASLPGL] in a standard kinase assay with 33P-ATP and purified kinase. Incorporation of 33P-ATP into the peptide was measured after a filter-binding assay. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 50 μM.

Kinase reactions were performed using the “Hotspot” kinase profiling service of Reaction Biology Corporation.

  1. Prepare indicated substrate in freshly prepared Base Reaction Buffer.
  2. Deliver any required cofactors to the substrate solution above.
  3. Deliver indicated kinase into the substrate solution and gently mix.
  4. Deliver compounds in DMSO into the kinase reaction mixture.
  5. Deliver 33P-ATP (specific activity 500 μCi/μl) into the reaction mixture to initiate the reaction.
  6. Incubate kinase reaction for 120 min. at room temperature.

A ratio of radioactivity signals was then calculated and plotted against the concentration to generate the IC50s. Compounds with an IC50 greater than 1 μM were considered inactive. Compounds with an IC50 equal to or less than 1 μM were considered active.

List of Reagents
  • Base Reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO)

Additional details can be found at http://www.reactionbiology.com/

AID 434972
Late stage assay provider results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of Wee1 degradation

The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates can induce stabilization of a Wee1 luciferase fusion construct. This assay uses HeLa cells transfected with a plasmid that encodes a Wee1-luciferase fusion protein to monitor Wee1 levels. The Wee1-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit Wee1 degradation will increase Wee1-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nanomolar (nM).

Protocol Summary

HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 micrograms of the Wee1-luciferase plasmid complexed with 87 microliters of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and re-suspended at a concentration of 850,000 cells per mL in growth media. Then, 50 microliters of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty microliters of compound was added to 50 microliters of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37°C (10% CO2, 95% RH). The assay was stopped by adding 100 microliters of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. Wee1-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.

The fold-change inhibition for each compound was calculated as follows:

Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).

The average fold-change of each compound tested was calculated.

PubChem Activity Outcome and Score:

Compounds that induced a fold-change less than 10 were considered inactive. Compounds that induced a fold-change equal to or less than 10 were considered active.

Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.

AID 463170
Late stage assay provider counter-screen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of p21 (CDKN1A) degradation

The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates are non-selective inhibitors of protein turnover by the proteasome. Furthermore, since Wee1 and p21 are targeted by the same class of ubiquitin ligases (the SCF ubiquitin ligases), this p21 assay serves to identify non-selective inhibitors of SCF ligases. This assay uses HeLa cells transfected with a plasmid that encodes a p21-luciferase fusion protein to monitor p21 levels and its turnover. The p21-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit p21 degradation will increase p21-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nanomolar (nM).

HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 micrograms of the p21-luciferase plasmid complexed with 87 microliters of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and re-suspended at a concentration of 850,000 cells per mL in growth media. Then, 50 microliters of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty microliters of compound was added to 50 microliters of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37°C (10% CO2, 95% RH). The assay was stopped by adding 100 microliters of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. P21-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.

The fold-change inhibition for each compound was calculated as follows:

Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).

The average fold-change of each compound tested was calculated.

PubChem Activity Outcome and Score:

Compounds that induced a fold-change less than 10 were considered inactive. Compounds that induced a fold-change equal to or less than 10 were considered active.

Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.

AID 463171
Late stage assay provider counter-screen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify p27 (CDKN1B) degradation

The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates are non-selective inhibitors of protein turnover by the proteasome. Furthermore, since Wee1 and p27 are targeted by the same class of ubiquitin ligases (the SCF ubiquitin ligases), this p27 assay serves to identify non-selective inhibitors of SCF ligases. This assay uses HeLa cells transfected with a plasmid that encodes a p27-luciferase fusion protein to monitor p27 levels and its turnover. The p27-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit p27 degradation will increase p27-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nanomolar (nM).

Protocol Summary

HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 micrograms of the p27-luciferase plasmid complexed with 87 microliters of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and re-suspended at a concentration of 850,000 cells per mL in growth media. Then, 50 microliters of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty microliters of compound was added to 50 microliters of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37°C (10% CO2, 95% RH). The assay was stopped by adding 100 microliters of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. p27-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.

The fold-change inhibition for each compound was calculated as follows:

Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).

The percentage of cells in each stage of the cell cycle was determined directly from FACS.

PubChem Activity Outcome and Score: Compounds that induce >20% of the HeLa cell population to enter the “subG1” phase of the cell cycle at concentrations greater than100nM are considered Active. Compounds that induce <20% of the HeLa cell population to enter the “subG1” phase of the cell cycle at all concentrations are considered inactive. Both compounds were active in this assay.

Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.

AID 463177
Late stage assay provider counter-screen results from the probe development effort to identify inhibitors of Wee1 degradation: fluorescence activated cell sorting (FACS)-based cell-based assay to identify inducers of Hela cell apoptosis

The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates can induce apoptosis in parental HeLa cells, as measured by the percentage of cells in the subG1 phase of the cell cycle (9). The prediction is that a small-molecule inhibitor of Wee1 degradation would inhibit cell cycle progression because overexpression of non-degradable Wee1 induces a G2-M arrest. In this assay, compounds were tested using a 7-point dose response starting at a nominal high concentration of 5 micromolar, followed by determination of the percentage of cells in subG1, G1, S, and G2 phases of the cell cycle.

Protocol Summary

HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). HeLa cells were treated for 20 hours with different concentrations of test compounds. Next, compound-treated HeLa cells were harvested by collecting culture media, washing once in phosphate-buffered saline (PBS), and trypsinizing. Cells were centrifuged at 400 g for 5 min at 4°C. Cells were then washed with cold PBS, centrifuged at 400 g for 5 min at 4°C, and rapidly resuspended with 7.5 mL 70% ethanol and incubated at −20°C overnight. Cells were then centrifuged at 400 g for 10 min at 4°C and washed with 10 mL cold PBS. The supernatant was removed and the cell pellet re-suspended in 38 mM sodium citrate containing 69 mM of propidium iodide and 19 mg/mL of Rnase A. Fluorescence-activated cell sorting (FACS) analysis was performed on a BD Biosciences (San Jose, CA) LSR II system and analyzed using Flowjo 8.7.3 software.

The fold-change inhibition for each compound was calculated as follows:

Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).

The percentage of cells in each stage of the cell cycle was determined directly from FACS. Compounds that induce >20% of the HeLa cell population to enter the “subG1” phase of the cell cycle at concentrations greater than100nM are considered Active. Compounds that induce <20% of the HeLa cell population to enter the “subG1” phase of the cell cycle at all concentrations are considered inactive. Both compounds were active in this assay.

AID 463186
Late stage assay provider results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based dose response cell-based assay to identify inhibitors of cyclin B degradation

The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates are non-selective due to stabilization of a cyclin B luciferase fusion construct. This assay uses HeLa cells transfected with a plasmid that encodes a cyclin B-luciferase fusion protein to monitor cyclin B levels. The cyclin B-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit cyclin B degradation will increase cyclin B-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nanomolar (nM).

Protocol Summary

HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 micrograms of the Cyclin B-luciferase plasmid complexed with 87 microliters of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and re-suspended at a concentration of 850,000 cells per mL in growth media. Then, 50 microliters of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty microliters of compound was added to 50 microliters of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37°C (10% CO2, 95% RH). The assay was stopped by adding 100 microliters of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. Cyclin B-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.

The fold-change inhibition for each compound was calculated as follows:

Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).

The average fold-change of each compound tested was calculated.

PubChem Activity Outcome and Score: Compounds that induced a fold-change less than 10 were considered inactive. Compounds that induced a fold-change equal to or less than 10 were considered active. Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.

List of Reagents
  • Dulbecco’s Modified Eagle’s Media (Invitrogen, part 11965-092)
  • Fetal Bovine Serum (Hyclone, part SH 30088.03)
  • Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)
  • Brite-lite reagent (PerkinElmer, part 6016769)
  • TransIT-LT1 (Mirus, part MIR2306)
  • T75 flasks (BD Falcon 353136)
  • 96-well plates (Corning part 3917)

2.2. Probe Chemical Characterization

Graphical representations of the stability of the new and prior probe compounds are shown in Figure 1. The new probe (ML177; CID 45100431) has been tested in numerous cell-based assays, demonstrating the solubility and stability of the probe.

Figure 1. Stability of Probes ML177 and ML118.

Figure 1

Stability of Probes ML177 and ML118.

The results summarized in Table 2, indicating that ML177 is unstable in PBS solution is very surprising and highly suspect, given that the SRIMSC medicinal chemistry team have observed absolutely no issues of chemical instability of this compound. Indeed, in one test, a 5 mg sample of ML177 was dissolved in 0.5 mL of d6-DMSO and monitored by 1H NMR spectroscopy. After one week at ambient temperature, the 1H NMR spectrum of this sample was identical to that of the freshly prepared sample at the beginning of the experiment. This experiment verifies the chemical stability of the probe. However, ML177 has poor solubility in PBS buffer and aqueous solution. Indeed, when a 10μM solution of ML177 in DMSO was titrated with water, ML177 began to precipitate from solution after 5% water had been added. Therefore, the “apparent” instability of ML177 suggested by the graph in Figure 1, is attributed to solubility problems of the probe.

Table 2. Summary of Results.

Table 2

Summary of Results.

Preliminary efforts have been made to improve the solubility of ML177 by generation of salts. However, the several salts that we have attempted to generate (by concentrating a solution of ML177 in the presence of the indicated acids) are even less soluble than the parent ML177. Therefore, solubility of the probe is an issue that will be addressed in further optimization of ML177 for in vivo applications.

ML177 SaltsSolubility (μM)
ML177·AcOH0.6
ML177·HCl1.3
ML177·TFA0.7
ML177·Sulfuric Acid5.6

2.3. Probe Preparation

Figure 2. Synthesis of Wee1 Degradation Inhibitor Probe (ML177/CID 45100431).

Figure 2Synthesis of Wee1 Degradation Inhibitor Probe (ML177/CID 45100431)

Methods: All reactions were magnetically stirred, and monitored by thin-layer chromatography using 0.25-mm precoated silica gel Kieselgel 60 F254 plates, unless otherwise noted. Visualization of all TLC plates was performed by UV. Product purifications were performed by silica gel flash chromatography (Kieselgel 60; 230–400 mesh) packed in glass columns and eluted with CH2Cl2/MeOH, or with EtOAc/MeOH buffered with 1% NH4OH.

Materials: Commercial grade reagents and solvents were used without further purification unless otherwise specified. DMF was dried by passing through a column of activated molecular sieves [11]. N,N-Diisopropylethylamine was distilled under argon from calcium hydride.

Instrumentation: 1H and 13C NMR spectra were recorded on a commercial NMR spectrometer (400 MHz for 1H, respectively, and 100 MHz for 13C, respectively), with chemical shifts reported relative to the residue peaks of solvent DMSO (δ 2.50 for 1H and 39.5 for 13C). IR spectra were obtained on a FT-IR spectrophotometer and are given in cm−1. Low resolution mass spectra (LRMS) and high resolution mass spectra were obtained on commercial instruments.

Synthesis of RJR-159 (probe ML177)[15]: A 2–5 mL Biotage microwave vial was charged with 2,6-dichloropurine-9-(3-thiophene)-purine (2) (0.136 g, 0.5 mmol), 4-nitro-2-(aminomethyl)-benzimidazole (5) (0.105 g, 0.55 mmol), freshly distilled N,N-diisopropylethylamine (0.435 mL, 2.5 mmol), and isopropanol (2 mL). The vial was sealed with a microwave cap, and the reaction mixture was heated to 90 °C for 30 minutes in a Biotage microwave unit. The cooled vial was then placed on a rotary evaporator and the reaction mixture was concentrated. Morpholine (2.5 mL) was added to the vial containing the concentrated crude mixture. The vial was resealed and the reaction was heated to 130 °C for 30 minutes in the microwave reactor. The cooled reaction mixture was concentrated in vacuo, and the crude product was subjected to flash chromatographic purification (98/2 (500mL) then 95/5 (500mL) [CH2Cl2/MeOH]) to give 0.156 g (65%) of RJR-159 as a tan-orange powder; m.p. = decomposed at 290 °C; 1H NMR (400 MHz, d6-DMSO) δ 13.1 (s, 1H), 8.39 (s, 1H), 8.15 (bs, 1H), 8.09 (d, J = 8.05 Hz, 1H), 8.07 (dd, J = 1.51 & 3.03 Hz, 1H), 8.03 (d, J = 7.32 Hz, 1H), 7.78 (dd, J = 1.51 & 5.30 Hz, 1H), 7.73 (dd, J = 3.28 & 5.30 Hz, 1H), 7.36 (t, J = 8.08 Hz, 1H), 4.95 (bs, 2H), 3.60 (bs, 4H), 3.53 (bs, 4H); 13C NMR (100 MHz, d6-DMSO) δ 158.6, 156.8, 154.0, 153.9, 150.0, 136.4, 133.6, 126.7, 121.0, 120.8, 118.1, 113.6, 112.8, 65.8, 48.5, 44.4; IR (neat) 1605, 1484, 1411, 1325, 1268, 1238, 1115, 1014, 901, 864, 783, 733 cm-1; LRMS-ESI (M+1) m/z: 478, 398, 279, 201, 171, 139.

3. Results

3.1. Dose Response Curves for Probe

The new probe RJR-159 (ML177/CID 45100431) is a more potent inhibitor of GCP (Granule cell progenitor) cell proliferation than FKL135. 3H-thymidine incorporation of GCPs treated with DMSO, FKL-135 (analog), or probe RJR-159 was measured after 24 hours. Well-established methods for purifying GCPs were developed by the Hatten laboratory [16]. In brief, to purify immature granule cells, cerebella from postnatal mice are isolated and triturated, and a mixed cell suspension containing GCPs is applied to a Percoll gradient (35%/65%), followed by three sequential panning steps to remove glia and fibroblasts from meninges and enrich GCPs. By electron microscopy (EM) and immunocytochemistry, the final suspension is 99% pure GCPs. 40 million GCPs or more are purified from a single litter of C57Bl6J mice. 3H-thymidine uptake has been extensively utilized to measure GCP proliferation [16]. The extent of 3H-thymidine incorporation was normalized to DMSO control and plotted. Moreover, these cell-based studies support the solubility and stability of the probe. These assays are available in PubChem as AID 504930.

Figure 3. Dose Response Curve.

Figure 3Dose Response Curve

3.2. Cellular Activity

The results of a Cell Titer Glo-based cytotoxicity assay of the probe RJR-159 (ML177/CID 45100431) performed using neuroblastoma cell lines are provided in Figure 4. The cell toxicity observed in this assay is presumed to be related to the ability of probe RJR-159 (ML177/CID 45100431) to inhibit kinases associated with cell-cycle progression.

Figure 4. Results of a Cell Titer Glo-based cytotoxicity assay.

Figure 4

Results of a Cell Titer Glo-based cytotoxicity assay.

3.3. Profiling Assays

The probe has been tested against several cell-cycle related targets and anti-targets, including Wee1, cyclin B, p21, p27, and ability to induce HeLa (cancer) cell apoptosis. The objective is to identify a compound that could stabilize (inhibit the degradation of) Wee1, without acting as a general protein stabilizer. For this reason, the assay provider selected cyclin B stabilization as the counter-screen target. These studies employed a luciferase-fusion protein to monitor protein degradation. Further studies were performed using p21 (CDKN1A) and p27 (CDKN1B) fusion constructs. These latter assays were performed by the assay provider. The identified probe exhibited significantly improved selectivity and potency in these AIDs, compared to the previous probe. These results, combined with additional kinase profiling by the assay provider, confirm the value of this probe and its usefulness in biologically-relevant, cell-based assays. These results are available in PubChem as AID 504930 and AID 504930 (GCP). The data for kinase profiling of probes against the casein kinases (Ck1delta, epsilon) and FLT3 have been reported in PubChem AID 463077 and AID 463076, respectively.

The probe has not been tested in other PubChem assays, as it is a newly synthesized molecule. The probe and its analog FKL-135 have been profiled against a set of 296 kinases (performed by Reaction Corporation Biology). These data, summarized in Figure 5 as an activity heat map for assays performed in a 1 μM inhibitor, demonstrate that the probe, CID 45100431/ML177 (e.g., RJR-159), is a highly selective kinase inhibitor—much more so than the initial hit FLK-135.

Figure 5. Summary of profile between FKL-135 and a set of 296 kinases.

Figure 5

Summary of profile between FKL-135 and a set of 296 kinases.

4. Discussion

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

As summarized in Table 3, the current probe ML177/CID 45100431 is significantly more potent and selective than the previous probe and the prior art compound MG132.

Table 3. Wee1 Degradation Inhibitor Probes and Prior Art.

Table 3

Wee1 Degradation Inhibitor Probes and Prior Art.

5. References

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Pangborn ABG, MA, Grubbs RH, Rosen RK, Timmers FJ. Safe and Convenient Procedure for Solvent Purification. Organometallics. 1996;15(5):1518–1520.
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Behrend L, et al. IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects. Oncogene. 2000;19(47):5303–13. [PubMed: 11103931]
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Rena G, et al. D4476, a cell-permeant inhibitor of CK1, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a. EMBO Rep. 2004;5(1):60–5. [PMC free article: PMC1298959] [PubMed: 14710188]
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Walton KM, et al. Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period. J Pharmacol Exp Ther. 2009;330(2):430–9. [PubMed: 19458106]
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Ding S, Gray NS, Ding Q, Schultz PG. A concise and traceless linker strategy toward combinatorial libraries of 2,6,9-substituted purines. J Org Chem. 2001;66(24):8273–6. [PubMed: 11722241]
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Hatten ME. Neuronal regulation of astroglial morphology and proliferation in vitro. J Cell Biol. 1985;100(2):384–96. [PMC free article: PMC2113456] [PubMed: 3881455]
6

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