The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-luciferase fusion protein. To ensure ultra-high-throughput screening (uHTS) compatibility, the assay was scaled to a 1536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogeneous assay demonstrated robust performance, with a calculated Z′ factor of 0.65 +/− 0.05. The assay was screened against a publicly available library of approximately 218,000 compounds to identify Wee1 stabilizers. Nonselective, cytotoxic, and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of an N-cyclin B-luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of 4 unrelated cell-permeable small molecules that showed selective Wee1-luciferase stabilization with micromolar potency. One of these compounds, SID-4243143 (ML118), was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. This probe was found to be inactive in a whole-cell assay against its anti-target, cyclin B. In contrast, the current state-of-the-art probe, MG132, inhibits degradation of both Wee1 and cyclin B in the same assays. More importantly, flow-cytometry assays confirm that the probe is able to induce an increase in the G2/M population after cell treatment, without increasing the sub-G1 population, suggesting the probe is not toxic to cells. These results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.

Recommendations for scientific use of the probe (ML118)

Limitations in state of the art. Although selective inhibitors of Wee1 kinase activity have been reported, none have been shown to inhibit Wee1 turnover. For example, inhibition of the Wee1 kinase activity by compounds that abrogate the G2/M checkpoint improve the cytotoxic effects of DNA damaging agents on p53-negative cells [1–4]. However, these compounds are dual Src/Wee1 kinase inhibitors (i.e. they are not inhibitors of Wee1 degradation). In other efforts, 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.

The new probe ML118 acts as an inhibitor of cell cycle progression, but, unlike MG132, is not a proteasome inhibitor since it did not affect turnover of another proteasome substrate, N-cyclin B-luciferase. One other small molecule has been reported in the literature to stabilize Wee1 in HeLa cells: the dihydropteridinone BI 2536 [5]. 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.

Probe Applications. The probe can be used to elucidate the specific role of Wee1 in cell cycle-related tumorigenesis. 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 [6]. 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.

Expected end-users of the probe in the research community. The probe can be used by academic researchers studying cell cycle biology, cancer biology, cellular kinases, and development. Thus it is conceivable that scientists in diverse fields will be able to apply this chemical probe to elucidate the role of Wee1 in these cellular pathways.

Relevant biology of the probe. Wee1 is a kinase that phosphorylates the Cdk1/cyclin B complex and delays entry of cells into mitosis (M phase). The phosphatase Cdc25 counteracts Wee1 by dephosphorylating Cdk1/cyclin B. Wee1 is also a kinase substrate. As G2 progresses phosphorylated Wee1 accumulates and is recognized by E3 ubiquitin ligases and is degraded via the proteosome. Cdc25 is then able to remove the inhibitory phosphorylation on Cdk1 and mitotic entry proceeds. Thus, the goal of this probe development project was to identify small molecules that prevent mitotic entry, via a novel whole-cell HTS assay that measures Wee1-K328M-luciferase degradation.

1. Introduction

Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling. Wee1 is a highly conserved tyrosine kinase that inhibits mitotic entry by inactivating the mitosis-specific kinase Cdk1/cyclin B complex during the S and G2 phases through Cdk1 phosphorylation at tyrosine 15. [7] By contrast, the phosphatase Cdc25 abrogates the Wee1-mediated effect by removing Cdk1 phosphorylation.[8, 9] Therefore, there is a competition between Wee1 and Cdc25 in controlling Cdk1/cyclin B complex activity, which ultimately determines mitotic entry or division arrest.[10] Upon the onset of mitosis, Wee1 is inactivated both by protein phosphorylation on specific residues and subsequent degradation via the ubiquitin proteasome pathway.[11, 12] This mechanism tips the balance in favor of Cdc25, triggering a positive feedback loop driven by activated Cdc25 and Cdk1/cyclin B, thus conferring unidirectionality to mitosis.[13] Maintaining the right amount of Wee1 is essential for cell growth and proliferation, and hence Wee1 is likely to participate in tumor progression. Lung cancer biopsies have low levels of Wee1 protein.[14] By contrast, increasing Wee1 levels by reducing its degradation in a prostate cancer model was beneficial as it limited cell growth.[15] Moreover, an anticancer compound that increases the steady-state levels of Wee1 by inhibiting Plk-1 dependent Wee1 turnover entered phase I clinical trials.[5] In addition, many cancer cells are lacking Wee1-dependent checkpoint pathways needed to ensure proper correction of DNA defects prior to mitosis, causing the cells to divide with incompletely replicated DNA.[16] Tight regulation of Wee1 activity in these cells may prevent the genomic instability caused by premature mitosis entry. Taken together, these studies suggest Wee1 is a promising target in cancer and the regulation of its degradation to be point of choice for chemotherapeutic intervention. In addition to providing potential novel drug leads, small-molecule inhibitors of Wee1 degradation could yield valuable probes to decipher pathways controlling Wee1 turnover and cell cycle transit. However, no effort to identify such small molecule probes has been reported thus far.

In this probe report, we describe a novel homogeneous 1536-well plate assay to monitor Wee1 degradation using cryopreserved transiently transected cells. We also demonstrate the excellent performance of this assay in the context of an ultra-high-throughput screening (uHTS) campaign that led to the identification of potential selective cell-permeable Wee1-Luc stabilizers, and a novel probe, ML118 [17].

2. Materials and Methods

2.2. Probe Chemical Characterization

Synthetic route. Synthesis schemes for probe (Scaffold 1) and representative compounds from Scaffolds 2 and 3:

Figure

Scaffold 1 (PROBE). SID-87235992 Synthesis of SID-87235992 (liquid SID-4243143)

Figure

Scaffold 2. SID-87235990 Synthesis of SID-87235990 (liquid SID-3713089)

Figure

Scaffold 3. SID-87235991 Synthesis of SID-87235991 (liquid SID-4256064)

Probe chemical structure including stereochemistry. Separation of diastereomers (if necessary)

The structure of the probe ML118, is shown below. ML118 is an achiral molecule, and has no stereochemistry or stereoisomers.

Structure verification with 1H NMR and LCMS results

Probe ML118 was obtained as an oil with 99% purity (HPLC analysis): ¹H NMR (DMSO-d₆, 400MHz) δ 10.6 (br, s, 1H), 8.08-8.06 (m, 1H, 7.81-7.72 (m, 2H), 7.56-7.52 (m, 1H, 7.42-7.34 (m, 1H), 7.06-7.02 (m, 1H), 6.94-6.92 (m, 1H), 5.96 (s, 1H), 4.12 (t, 2H, J = 6), 3.48 (s, 3H), 2.59 (t, 2H, J = 7.2), 2.27 (s, 3H), 2.12-2.06 (m, 2H). ¹³C NMR (DMSO-d₆, 100MHz) δ 172.6, 162.3, 160.9, 152.4, 150.3, 144.0, 139.4, 131.3, 122.7, 121.2, 120.6, 115.5, 114.5, 114.3, 96.6, 67.9, 33.0, 28.5, 23.8, 21.2. IR (neat) 2945, 1720, 1636, 1579, 1505, 1368 cm⁻¹. LCMS 352.01 (M+H⁺). HRMS (ESI) 374.1484 m/z (calc M+Na⁺ C₂₀H₂₁N₃O₃Na 374.1475).

Solubility. The solubility of the probes was measured in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4) at room temperature (23°C). The solubility of probe ML118 was found to be 58 μM.

Stability. The stability of the probe was measured at room temperature (23ºC) in PBS (no antioxidants or other protectants; DMSO concentration below 0.1%). The stability, represented by the half-life, was found to be > 48 hours. Below is a graph showing loss of compound with time over a 48 hour period with a minimum of 6 time points. The table indicates the percent of compound remaining at the end of the 48 hours.

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Compound	SR Number	CID	SID	MLS ID	Solubility in PBS (μM)	Michael Acceptor 100 μM GSH trap (Yes/No)	Stability in PBS t1/2
Probe ML118	SR-01000621290	3237904	4243143	MLS000089581	58	No	> 48 hr (10 μM)

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Wee1 Probe ML118 (SR-01000621290)
Stability in PBS Buffer (pH 7.4)
Sample concentration: 10 μM
Storage condition: microcentrifuge tube on lab benchtop
Time (hr)	% remaining	In (%remaining)
0	100	4.61
1	101	4.62
2	90	4.50
4	94	4.55
8	99	4.60
24	110	4.70
48	109	4.69

The probe was measured for its ability to form glutathione adducts. At concentrations of 100 μM reduced GSH, 10 μM of the probe does not appear to be a Michael acceptor [18, 19].

2.3. Probe Preparation

Detailed experimental procedures for the synthesis of Probe ML118 (SR-01000621290; CID-3237904; SID-4243143) follow.

tert-Butyl 4-bromobutanoate (2): Trifluoroacetic anhydride (6 mL) was added slowly to a −40 °C solution of 4-bromobutyric acid (1.7 g, 10.2 mmol) in dry THF (20 mL). The resulting solution was stirred at −40 °C for approximately 30 min. tert-Butanol (25 mL) was added and the solution allowed to warm up to ambient temperature and was stirred for an additional 16 h. The reaction mixture was poured slowly into a mixture of crushed ice and saturated sodium bicarbonate solution (50 mL). The product was extracted into EtOAc (2×100 mL) and the organic phase was washed with water (2×100 mL) and brine (2×100 mL). The organic extracts were dried over Na₂SO₄, then the solution was concentrated under reduced pressure to obtain an oil. The oil was dissolved in MTBE (100 mL) and filtered through a short pad of silica gel. The silica pad was washed with MTBE (100 mL). The combined filtrate was concentrated under reduced pressure to obtain 4.1 g (72%) of the knownⁱ t-butyl ester 2 as a liquid: ¹H NMR (CDCl₃, 400MHz) δ 3.45 (t, 2H, J = 6.4 Hz), 2.40 (t, 2H, J = 7.2 Hz), 2.16-2.10 (m, 2H), 1.45 (s, 9H).

tert-Butyl 4-(1-methyl-2-oxo-1,2-dihydroquinolin-4-yloxy) butanoate (3). To a stirred solution of 4-hydroxy-1-methylquinolin-2(1H)-one (1.0 g, 5.71 mmol) in anhydrous DMF (10 mL) was added tert-butyl 4-bromobutanoate (1.25 g, 5.71 mmol) and cesium carbonate (2.23 g, 6.85 mmol). The reaction mixture was stirred at room temperature for 14 h. After completion of the reaction (monitored by LCMS), the solid was filtered off and DMF removed under vacuum. The residue was dissolved in ethyl acetate and washed successively with water and brine. The organic phase was dried over sodium sulfate and filtered. Removal of solvent by rotary evaporation gave crude 3 (1.59 g, 88% yield), which was directly used in the next step without further purification. LCMS 317.98 (M+H⁺).

4-(1-Methyl-2-oxo-1,2-dihydroquinolin-4-yloxy) butanoic acid (4). Crude 3 from the previous experiment (500 mg, 1.58 mmol) was dissolved in anhydrous dichloromethane (5 mL). Triethylsilane (0.629 mL, 3.94 mmol) and trifluoroacetic acid (1.57 mL, 20.5 mmol) were then added to reaction mixture. The mixture was stirred for 5 h under nitrogen until the reaction was complete (monitored by LCMS). The solvent was removed under vacuum and the residue was dissolved in diethyl ether, causing the product to separate as a white solid which was filtered and dried under vacuum to give the carboxylic acid 4 (400 mg, quantitative yield): ¹H NMR (CDCl₃, 400MHz) δ 8.02 (dd, J = 8), 1H), 7.644-7.600 (m, 1H), 7.40-7.26 (m, 2H), 6.30 (s, 1H), 4.23 (t, 2H, J = 6.4), 3.72 (s, 3H), 2.67 (t, 2H, J = 6.8), 2.30-2.27 (m, 2H); LCMS 262.01 (M+H⁺).

4-(1-Methyl-2-oxo-1,2-dihydroquinolin-4-yloxy)-N-(4-methylpyridin-2-yl)butanamide (ML118, SID-4243143, 5a). A mixture of carboxylic acid 4 (100 mg, 0.38 mmol), HATU (218mg, 0.57 mmol), and triethylamine (0.08 mL, 0.57 mmol) in anhydrous DMF (2 mL) was stirred under argon atmosphere for 15 min. 2-Amino-4-methylpyridine (49 mg, 0.46mmol) was then added. The reaction mixture stirred for 3 h. After completion of the reaction, DMF was removed by rotary evaporation, and the residue was dissolved in ethyl acetate. The organic phase was washed with saturated solution of NaHCO₃ (50mL). Removal of solvent and purification of the residue by preparative HPLC afforded ML118 (69 mg, 51% yield) as an oil with 99% purity (HPLC analysis): ¹H NMR (DMSO-d₆, 400MHz) δ 10.6 (br, s, 1H), 8.08-8.06 (m, 1H, 7.81-7.72 (m, 2H), 7.56-7.52 (m, 1H, 7.42-7.34 (m, 1H), 7.06-7.02 (m, 1H), 6.94-6.92 (m, 1H), 5.96 (s, 1H), 4.12 (t, 2H, J = 6), 3.48 (s, 3H), 2.59 (t, 2H, J = 7.2), 2.27 (s, 3H), 2.12-2.06 (m, 2H). ¹³C NMR (DMSO-d₆, 100MHz) δ 172.6, 162.3, 160.9, 152.4, 150.3, 144.0, 139.4, 131.3, 122.7, 121.2, 120.6, 115.5, 114.5, 114.3, 96.6, 67.9, 33.0, 28.5, 23.8, 21.2. IR (neat) 2945, 1720, 1636, 1579, 1505, 1368 cm⁻¹. LCMS 352.01 (M+H⁺). HRMS (ESI) 374.1484 m/z (calc M+Na⁺ C₂₀H₂₁N₃O₃Na 374.1475).

3. Results

3.1. Summary of Screening Results

Following primary HTS in singlicate to identify Wee1 degradation inhibitors (AID-1321), confirmation of hit activity in triplicate (AID-1410), titration assays to determine compound potency (AID-1412), cytotoxicity (AID-1413), and selectivity against cyclin B (AID-1414), compounds were identified as possible candidates for probe development. Powder assay data is available in late stage AID-2088.

3.2. Dose Response Curves for Probes

Probe ML118 (SR-01000621290; CID-3237904; SID-4243143).

Dose-response curves for Wee1 Degradation inhibitor probe ML118 in three cell-based assays.

Ten-point, 1:3 serial dilution of probe compound ML118 (SID-4243143) were tested in triplicate in both the Wee1 (circles), N-Cyclin B (squares), and viability (diamonds) assays. Note the limited cytotoxicity and activity in the N-cyclin B assay at higher test concentrations. Data points represent mean ± SD (n = 3).

3.4. SAR Tables

WEE1 Degradation Inhibitor SAR Table (Scaffold 1: 4-alkoxy quinolone scaffold)

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Compound Information							HTS Information					Probe Development Assays (AID-2088)
Compound	Structure	CID	SID	MLS	Vendor	Synthesized at the SRIMSC	Wee1 Primary (AID-1321)	Wee1 Confirmation (AID-1410)	Wee1 Dose Response (AID-1412)	Cytotoxicity Assay (CC50) (AID-1413)	Cyclin B EC50 (AID-1414)	SID	Wee1 MAX % ACT	Wee1 EC50	Cyclin B MAX % ACT	Cyclin B EC50 (uM)	G2/M Arrest Assay
WEE1 Probe		3237904	4243143	MLS002473427	Chem Div	No	Active	Active	Active (7.87 μM)	Inactive (49.75 μM)	Inactive (49.75 μM)	87235992	79.19	6.19	6.78	Inactive (>55.7)	Active (30.5% of cells in the G2/M phase, compared to 16.5% for the DMSO group.
Analog 1		20862056	56432192	MLS 002473429	Chem Div	Yes	These compounds were not in the MLSMR collection.					56432192	11.34	Inactive	2.78	Inactive	Not tested due to lower % activation of Wee1 compared to the probe.
Analog 2		20862034	56431992	MLS002473428	ChemNavigator	Yes						56431992	11.1	Inactive	8.47	Inactive
Analog 3		20862013	56432194	MLS002473425	Chem Div	Yes						56432194	6.32	Inactive	6.13	Inactive
Analog 4		20862086	56432196	MLS002473426	Chem Div	Yes						56432196	7.82	Inactive	10.59	Inactive
Analog 5		20862025	56431991	MLS002473424	ChemNavigator	Yes						56431991	3.53	Inactive	7.67	Inactive
Analog 6		20862012	56432190	None	TSRI	Yes						56432190	8.29	Inactive	9.82	Inactive
Analog 7		20862057	56432174	None	TSRI	Yes						56432174	7.64	Inactive	15.78	Inactive
Analog 8		6622317	56432193	None	TSRI	Yes						56432193	5.44	Inactive	13.49	Inactive
Analog 9		20862014	56431995	None	TSRI	Yes						56431995	16.07	Inactive	4.55	Inactive
Analog 10		20862079	56431993	None	TSRI	Yes						56431993	8.98	Inactive	11.31	Inactive
Analog 11		20862084	56432195	None	TSRI	Yes						56432195	5.12	Inactive	9.46	Inactive
Analog 12		20862053	56432191	None	TSRI	Yes						56432191	8.15	Inactive	13.99	Inactive
Analog 13		20862019	56431996	None	TSRI	Yes						56431996	6.00	Inactive	10.35	Inactive
Analog 14		20862080	56431994	None	TSRI	Yes						56431994	6.97	Inactive	15.3	Inactive
Analog 15		20861758	56432173	None	TSRI	Yes						56432173	5.8	Inactive	3.33	Inactive
Analog 16		20861840	56432172	None	TSRI	Yes						56432172	12.2	Inactive	11.31	Inactive
Prior Art (MG132)		462382	24892475	None	Sigma- Aldrich	C2211	This compound is not in the MLSMR collection		4.5 μM (control wells)	This compound is not in the MLSMR collection	4.7 μM (control wells)	85261504	Not tested due to lack of Wee1 selectivity.

Wee1 SAR Table (Scaffold 2: pyrazolyl pyrimidone; there are no probes in this scaffold)

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Compound Information							HTS Information					Probe Development Assays (AID-2088)
Compound	Structure	CID	SID	MLS	Vendor	Synthesized at the SRIMSC	Wee1 Primary (AID-1321)	Wee1 Confirmation (AID-1410)	Wee1 Dose Response (AID-1412)	Cytotoxicity Assay (CC50) (AID-1413)	Cyclin B EC50 (AID-1414)	SID	Wee1 MAX % ACT	Wee1 EC50	Cyclin B MAX % ACT	Cyclin B EC50 (uM)	G2/M Arrest Assay
Compound 1		2408467	3713089	MLS000036000	Enamine	No	Active	Active	Active (1.84 μM)	Inactive (49.75 μM)	Inactive (49.75 μM)	3713089	38.05	5.57	40.18	Inactive (>55.7)	Not tested due to lower % activation of Wee1 compared to the probe.
Compound 2		25110118	56432176	None	TSRI	Yes	These compounds were not in the MLSMR collection.					56432176	143	> 1.66	49.7	Inactive	Not tested due to lower Wee1 selectivity compared to the probe
Compound 3		8514124	56432177	None	TSRI	Yes						56432177	4.94	Inactive	4.76	Inactive	Not tested due to lower % activation of Wee1, compared to the probe.
Compound 4		25110119	56432178	None	TSRI	Yes						56432178	101.08	> 1.66	28.88	Inactive	Not tested due to lower Wee1 selectivity compared to the probe.
Compound 5		8514206	56432199	None	TSRI	Yes						56432199	17.64	Inactive	45.94	Inactive	Not tested due to lower % activation of Wee1, compared to the probe.
Compound 6		25110124	56432183	None	TSRI	Yes						56432183	6.37	Inactive	5.22	Inactive
Compound 7		25110123	56432182	None	TSRI	Yes						56432182	4.78	Inactive	4.46	Inactive	Not tested due to lower % activation of Wee1, compared to the probe.
Compound 8		25110122	56432181	None	TSRI	Yes						56432181	6.04	Inactive	11.92	Inactive
Compound 9		25110121	56432180	None	TSRI	Yes						56432180	4.99	Inactive	6.39	Inactive
Compound 10		25110120	56432179	None	TSRI	Yes						56432179	128.71	> 1.66	9.86	Inactive	Not tested due to lower Wee1 selectivity compared to the probe.
Compound 11		25110125	56432185	None	TSRI	Yes						56432185	6.39	Inactive	11.01	Inactive	Not tested due to lower % activation of Wee1, compared to the probe.
Compound 12		8514497	56432201	None	TSRI	Yes						56432201	22.6	Inactive	12.94	Inactive	Not tested due to lower % activation of Wee1, compared to the probe.
Compound 13		8514474	56432186	None	TSRI	Yes						56432186	4.37	Inactive	11.64	Inactive
Compound 14		8514425	56432198	None	TSRI	Yes						56432198	130.77	> 1.66	27.61	Inactive	Not tested due to lower Wee1 selectivity compared to the probe.
Compound 15		8514429	56432200	None	TSRI	Yes						56432200	184.08	> 1.66	8.26	Inactive
Compound 16		8514632	56432187	None	TSRI	Yes						56432187	71.9	> 1.66	4.07	Inactive	Not tested due to lower % activation of Wee1, compared to the probe.
Compound 17		8514620	56432203	None	TSRI	Yes						56432203	7.02	Inactive	3.41	Inactive
Compound 18		8514324	56432184	None	TSRI	Yes						56432184	134.5	> 1.66	7.34	Inactive	Not tested due to lower Wee1 selectivity compared to the probe.
Compound 19		18568950	56432202	None	TSRI	Yes						56432202	73.44	> 1.66	47.95	Inactive

Wee1 SAR Table (Scaffold 3: pyrrolo-benzoxadiazole N-oxide; there are no probes in this scaffold)

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Compound Information							Screening Assays					Probe Development Assays (AID-2088)
Compound	Structure	CID	SID	MLS	Vendor	Synthesized at the SRIMSC	Wee1 Primary (AID-1321)	Wee1 Confirmation (AID-1410)	Wee1 Dose Response (AID-1412)	Cytotoxicity Assay (CC50) (AID-1413)	Cyclin B EC50 (AID-1414)	SID	Wee1 MAX % ACT	Wee1 EC50	Cyclin B MAX % ACT	Cyclin B EC50 (uM)	G2/M Arrest Assay
Compound 1		690911	4256064	MLS 000061388	Chem Bridge	No	Active	Active	Active (1.842 μM)	Inactive (49.75 μM)	Inactive (49.75 μM)	87235991	186.39	6.19	47.27	Inactive (>55.7)	Not tested due to lower Wee1 selectivity compared to the probe.
Compound 2		755456	56432189	None	TSRI	Yes	These compounds were not in the MLSMR collection.					56432189	3.75	Inactive	14.7	Inactive	Not tested due to lower % activation of Wee1, compared to the probe.
Compound 3		2837745	56431998	None	TSRI	Yes						56431998	6.78	Inactive	8.09	Inactive	Not tested due to lower Wee1 selectivity compared to the probe.

Following the initial SAR effort, the SRIMSC synthesized an additional 20 analogs in a second round. The results are summarized in the tables below. All analogs are acylated 2-amino pyridine derivatives. The top 4 of these analogs (SR-03000001762, SR-03000001772, SR-03000001774, and SR-03000001775) appear to be more potent than the first generation Wee1 degradation inhibitor probe ML118. Three of these new analogs were submitted for solubility and stability testing; data are provided following the new SAR table. In addition, the assay provider repeated dose response assays on the top 3–4 inhibitors to confirm the potency of the individual compounds. Analysis of these results reveals that one of these new analogs (SR-03000001762-1) exhibited an EC50 of 346nM and is 10-fold more potent than the original probe ML118, and thus is an improvement over ML118. Moreover, data presented in Section 3.5 indicates that SR-03000001762 is more effective in arresting cells at the G2/M checkpoint.

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S. No.	SR#	Structure	SID	CID	Wee1-Luc/Luc	%621290-1 at 1.11 μM	EC50 (M)
1	01000621290-2 (ML118)		87235992	44552613	0.278	100	2.01e-05 (Original Hit)
2.	03000001762-1		99367847	46907627	0.766	275.82	3.46e-07
3.	03000001775-1		99367857	46907636	0.693	249.507	6.00e-07
4.	03000001772-1		99367855	46907622	0.348	125.275	Unable to determine; problems fitting the data
5.	03000001774-1		99367856	46907630	0.476	171.319	5.89e-07
6.	01000621258-2		99367844	46907633	0.065	23.52	-
7.	03000001760-1		99367845	46907616	0.053	19.22	-
8.	03000001761-1		99367846	46907626	0.132	47.62	-
9.	03000001763-1		99367848	46907638	0.150	54.11	-
10.	03000001764-1		99367849	46907619	0.062	22.30	-
11.	03000001765-1		99367850	46907614	0.070	25.08	-
12.	03000001766-1		99367851	46907635	0.061	22.11	-
13.	03000001767-1		99367852	46907615	0.077	27.792	-
14.	03000001769-1		99367854	46907624	0.064	22.891	-
15.	01000130705-3		4242355	3237217	0.073	26.321	-
16.	03000001768-1		99367853	46907628	0.053	19.193	-
17.	01000130703-2		99367879	46907653	0.206	74.249	-
18.	03000001771-1		99367881	46907654	0.103	37.221	-
19.	03000001773-1		99367882	46907655	0.133	47.784	-
20.	03000001776-1		99367858	46907629	0.086	30.782	-
21.	03000001770-1		99367880	46907652	0.071	25.720	-

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Compound	SR Number	CID	SID	MLS ID	Solubility in PBS (μM)	Michael Acceptor 100 μM GSH trap (Yes/No)	Stability in PBS t1/2
Probe ML118	SR-01000621290	3237904	4243143	MLS000089581	58	No	> 48 hr (10 μM)
Analog	SR-03000001762 (synthesis)	46907627	99367847	MLS003329219	7.3	No	> 48 hr (7 μM)
Analog	SR-03000001772 (synthesis)	46907622	99367855	MLS003329220	6.2	No	> 48 hr (6 μM)
Analog	SR-03000001775 (synthesis)	46907636	99367857	MLS003329222	45.9	No	> 48 hr (10 μM)

Solubility and Stability data for the new analogs

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Wee1 Analog (SR-03000001772)
SR-03000001772 Stability in PBS Buffer (pH 7.4)
Sample concentration: 6 μM
Storage condition: microcentrifuge tube on lab benchtop
Time (hr)	% remaining	In (%remaining)
0	100	4.61
1	106	4.66
2	101	4.62
4	104	4.65
8	97	4.57
24	99	4.60
48	98	4.59

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Wee1 Analog (SR-03000001775)
Stability in PBS Buffer (pH 7.4)
Sample concentration: 10 μM
Storage condition: microcentrifuge tube on lab benchtop
Time (hr)	% remaining	In (%remaining)
0	100	4.61
1	104	4.65
2	103	4.64
4	99	4.59
8	106	4.66
24	96	4.56
48	96	4.56

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Wee1 Analog (SR-03000001762)
Sr-03000001762 Stability in PBS Buffer (pH 7.4)
Sample concentration: 7 μM
Storage condition: microcentrifuge tube on lab benchtop
Time (hr)	% remaining	In (%remaining)
0	100	4.61
1	102	4.63
2	99	4.59
4	103	4.63
8	94	4.55
24	100	4.60
48	99	4.60

3.5. Cellular Activity

Probe ML118 was tested in a variety of cell-based assays performed by the SRIMSC and assay provider. These assays were performed to determine the probe’s selectivity and mechanism of action. The results of these studies demonstrated that probe ML118 is a stabilizer of Wee1 that inhibits Wee1 degradation.

FACS analysis/G2/M Arrest Assays

In order to determine whether the identified inhibitors of Wee1 degradation inhibit cell cycle progression, the effect of probe compound ML118 (SID-4243143; powder SID-87235992/CID-44552613) on cell cycle was tested by performing FACS analysis. Compound-treated HeLa cells were resuspended in 70% ethanol, incubated at −20°C overnight, and washed with 10ml cold PBS. The supernatant was removed and the cell pellet was resuspended in 38 mM sodium citrate containing 69 mM of propidium iodide and 19 mg/mL of RNase A. FACS analysis was performed on a BD Bioscience LSR II system and analyzed using Flowjo 8.7.3 software.

As shown in the figure below, probe compound ML118/SID-4243143/CID-3237904 (powder SID-87235992) induces G2/M arrest and does not increase sub G1. A) Cell sorting images of ML118/SID-4243143/CID-3237904 (powder SID-87235992) at 1:10 increasing concentrations of 50nM, 500nM, and 5000nM for 20 hours. B) Histogram showing the percentage of cells in each stage of the cell cycle. Whereas DMSO-treated cells contained 16.7% of cells in the G2/M phase, SID-4243143/CID-3237904 (powder SID-87235992)-treated cells contained 30.5%. Furthermore, no increase in the sub-G1 population after SID-4243143 treatment was observed, suggesting that compound ML118/SID-4243143/CID-3237904 (powder SID-87235992) is not toxic. While this compound acts as an inhibitor of cell cycle progression, it is not a proteasome inhibitor since it did not affect turnover of another proteasome substrate, N-cyclin B-luciferase.

FACS analysis/G2/M arrest assay data for the three most potent new analogs, SR-1762, DR-1772 and SR-1775, are summarized below. These experiments were performed using the test compounds at 1.11 μM. These data demonstrate that the new analogs, and especially SR-03000001772, are more potent than the original probe, ML118, in these experiments.

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Compound	%G1	%S	%G2/M
ML118	39.7	34.7	19.5
SR-03000001762-1	50.8	29.6	12.3
SR-03000001772-1	33.7	40.4	18.5
SR-03000001775-1	35.6	34.3	18.5
DMSO	48.7	28.3	16.8

3.6. Profiling Assays

In addition to the above cellular assays, we have also obtained profiling results for probe ML118 at 10 μM using the NCI-60 DTP Human Tumor Cell Line Screen (Background and Methods can be found at http://dtp.nci.nih.gov/branches/btb/ivclsp.html). This compound advanced to dose response testing and has been assigned NIH code 754211. The profiling screen examined the effect of a single high concentration (10 μM) of the probe on cellular growth of 60 well-characterized cell lines during 48 hours of exposure http://dtp.nci.nih.gov/branches/btb/onedose_interp.html

The One-dose data is reported as a mean graph of the percent growth of treated cells. The number reported for the One-dose assay is growth relative to the no-drug control, and relative to the time zero number of cells. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). This is the same as for the 5-dose assay, described on http://dtp.nci.nih.gov/branches/btb/ivclsp.html. For example, a value of 100 means no growth inhibition. A value of 40 would mean 60% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of −40 would mean 40% lethality. A value of −100 means all cells are dead. Importantly, probe ML118 was able to inhibit the growth of the majority of cancer cell lines tested (mean growth value between 0 and 100). Thus, it is clear that this probe represents a useful tool for cell-based studies involving cell cycle regulation and cancer-related events.

Results of the NCI-60 profiling assays preformed with ML118 are on the next page.

Kinase profiling

Wee1 degradation has been shown to be regulated by complex pathways involving kinases and phosphatases.[20] In an attempt to identify potential new proteins involved in Wee1 phosphorylation, we profiled probe ML118/SID-4243143 against a panel of kinases involved in cell cycle progression, including Wee1 itself. The mitotic kinase profiling assay was performed by Reaction Biology Corporation (Malvern, PA) using a radiometric-based filtration binding assay as previously described.[21] Briefly, compound SID-4243143 was tested in duplicate at 10 μM on a panel of purified kinases in the presence of ³²P-γ-ATP. Incorporation of ³²P-γ-ATP into peptide substrates was measured and compared to DMSO controls. The kinase pan-inhibitor staurosporine was used as a positive control for these experiments. As shown below, the hit compound we identified significantly inhibited CDK9, either coupled to cyclin K or cyclin T1. As shown below, black bars indicate activities lower than the average of activities measured for all kinases minus 3 times their standard deviation. Error bars represent the standard deviation (n = 2).

IC50’s for the inhibition by ML118 of kinases reported in the literature to act on Wee1 are summarized below [5, 11]. The data for inhibition of CDK9/cyclin K and CDK9/cyclinT1 track well with the IC50 value for inhibition of Wee1 degradation by ML118. Because the pyridine carboxamide is essential for activity, a plausible hypothesis is that ML118 inhibits Wee1 degradation by inhibiting a kinase (or kinases) responsible for phosphorylation of Wee1 and thereby targeting Wee1 for degradation via the proteosome [5, 11].

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Target	Activity (μM)
CDK9/cyclin K	3.4
CDK9/cyclinT1	3
CDK3/cyclin E	inactive
CDK4/cyclin D3	inactive
CK1d	30
FLT3	6

4. Discussion

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

The new probe ML118 is a significant improvement over prior art compound MG132 because it is a selective inhibitor of Wee1 degradation, and unlike MG132, is not a generic proteosome inhibitor. Furthermore, given that it also inhibits CDK9/cyclin K and CDK9/cyclin T1 selectively relative to a panel of kinases tested, it is possible that stabilization of Wee1 mediated by ML118 is due to inhibition of CDK9/cyclin K and/or CDK9/cyclin T1. In this model, CDK9 would phosphorylate either Wee1 directly or a protein required for Wee1 degradation. Thus, ML118 would act upstream of the proteasome.

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Compound	Structure	CID	Wee1 EC50	Cyclin B EC50	PubChem Activity Profile
Probe ML118 (SR-01000621290)		3237904	7.87 μM	>49.75 μM	26/523 assays (5.0%)
Prior art MG132		462382	4.5 μM	4.7μM	11/456 assays (2.4%)

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