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Discovery of Inhibitors of Anti-Apoptotic Protein A1

, , , , , , , , , and .

Author Information

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

1 The Broad Institute Probe Development Center, Cambridge, MA
2 Cancer Program, Broad Institute, Cambridge, MA
3 Howard Hughes Medical Institute, Chemistry & Chemical Biology, Harvard University, Cambridge, MA
*Corresponding author email: gro.etutitsnidaorb@rekttibj

Received: ; Last Update: November 21, 2011.

Pro- and anti-apoptotic proteins maintain a regulated balance in cells that allow programmed cell death to occur in response to genetic damage and other stimuli. In cancer cells, this balance is often dysregulated by the overexpression of anti-apoptotic factors. A probe development project was carried out with the goal of identifying small molecules able to specifically inhibit the anti-apoptotic function of one of these proteins, the BCL-2-family member BCL2A1 (A1, Bfl1). A high-throughput screen (HTS) of 325,633 compounds was carried out by measuring caspase activation in a mouse fibroblast cell line primed for death by balanced overexpression of A1 and BIM, a pro-apoptotic BH3 domain-containing protein. A series of secondary assays was used to determine whether compounds identified in the primary screen were specific for caspase activation in an A1-dependent manner. A series of compounds was identified that contained reactive functionality but showed highly specific induction of apoptosis, including a probe (CID701939/ML214) that activates caspases in A1-overexpressing cells at low micromolar concentrations. Due to the reactive nature of the probe series and the necessity of this reactive functionality for biological activity, these compounds are not intended to be candidates for drug development. However, the surprisingly specific activation profile exhibited by these compounds suggests that they may be selectively modifying A1 or a related apoptotic protein. Therefore, the probe ML214 is a valuable research tool for further study of targets or interaction sites that can lead to overcoming the apoptotic blockage of A1 in cancer cells.

Assigned Assay Grant No.: 1 R03 DA028853-01

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

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

Assay Submitter and Institution: Todd Golub, Broad Institute

PubChem Summary Bioassay Identifier (AID): 2526

Probe Structure & Characteristics

Compound Summary in PubChem

IUPAC Chemical Name4-chloro-1-methyl-3-nitroquinolin-2-one
PubChem CID701939
Molecular Weight238.62718 g/mol
Molecular FormulaC10H7ClN2O3
XlogP1.9
H-Bond Donor0
H-Bond Acceptor3
Rotatable Bond Count0
Exact Mass238.01452
Topological Polar Surface Area66.1
CID/ML No.Target NameEC10 (nM) [SID, AID]Anti-target Name(s)EC10 (nM) [SID, AID]Fold SelectiveSecondary Assay(s) Name:
EC10 (nM)
[SID, AID]
CID 701939/ML214MEF-A1-2A-BIM1700 [SID 99432301, AID 504345]Bax/Bak −/−IA [SID 99432301, AID 504344]>10MEF-Flag-A1-Ires-BIM: 1880 [SID 99432301, AID 504342].
MEF-A1-2A-tBID: 1300 [SID 99432301, AID 504359].
MEF WT: 19950 [SID 99432301, AID 504392]
HMC-1-8: Inactive [SID 99432301, AID 504343.
MEWO: Inactive [SID 99432301, AID 504354]
HMC-1-8 viability: >35 μM [SID 99432301, AID 504415]
Cytochrome c release: <10000 [SID 99432301, AID 504412]

Recommendations for scientific use of the probe

Currently, there are no known compounds that specifically target the anti-apoptotic protein A1 relative to other BCL-2-family members. This probe (CID701939/ML214) shows specific activation of caspases in cells primed with various A1 constructs and does not activate caspases in human cancer cells that overexpress other BCL-2-family members (such as HMC-1-8 [MCL-1] or MeWo [BCL-2]). As this compound was discovered through a screening strategy solely involving cell-based assays, it is also more physiologically relevant than compounds found through in vitro screening of an isolated target.

The probe ML214 possesses an electrophilic center and could be viewed as a non-selective alkylating agent. However, it shows selective activity in A1-primed cell lines compared to control cell lines and is completely inactive in Bax/Bak −/− cell lines, suggesting that its mode of action involves a selective caspase activation pathway. Moreover, we have shown that the probe reacts very poorly with thiol nucleophiles such as glutathione (GSH, only 16% after 3h incubation time). Hence we recommend this probe for in vitro and cellular assays that can be completed in 3–5 hours.

The electrophilic nature of the probe is an advantage for identifying the target or modification site of proteins of interest (A1 or other closely related proteins). We have proposed additional studies using probe ML214, including adding an affinity tag to more readily identify cellular targets. Other researchers in the cancer biology field may also use ML214 for characterization of cancer cell vulnerabilities, possibly leading to additional target discovery. Overall, as a tool compound with novel specificity for a particular member of the anti-apoptotic protein family implicated in tumor maintenance, probe ML214 will be beneficial in advancing the field of oncology and related therapeutic development.

1. Introduction

Scientific Rationale

Dysregulated apoptotic mechanisms are central to the pathogenesis and maintenance of cancer, and are major barriers to effective treatment. The BCL-2-protein family comprises both pro- and anti-apoptotic members, and controls the activation of downstream caspases, which are the major effectors of apoptosis. Thus, developing small molecule probes that selectively target each anti-apoptotic protein family member may have relevance for both basic research and future clinical applications.

BCL-2-family and apoptosis. Impaired apoptosis is both critical to cancer development and a major barrier to effective treatment (1). It is now thought that one or more components of the apoptosis pathway are dysregulated in all cancers either by genomic mutation of the genes encoding these proteins (e.g., via point mutation, copy number abnormalities, or chromosomal translocation) or by other mechanisms (e.g., epigenetic mechanisms). The BCL-2-protein family (see Figure 1), which is highly conserved from worm to human, controls the activation of downstream caspases that are the major effectors of apoptosis (2).

Figure 1. The BCL-2-Family.

Figure 1

The BCL-2-Family.

The BCL-2-family, comprised of both pro- and antiapoptotic members, can be divided into three main subclasses. These subclasses are defined in part by the homology shared within four conserved regions termed “BCL2 homology (BH) 1–4 domains”, roughly corresponding to α helices, which dictate structure and function (1, 2). Anti-apoptotic family members BCL-2, BCL-XL, MCL-1, and A1 display conservation in all four BH domains. The BH1, BH2, and BH3 domains of those proteins are in close proximity and create a hydrophobic pocket that can accommodate the BH3 domain of a pro-apoptotic member (3–6). The “multidomain” pro-apoptotic members (Bax, Bak) possess BH1-3 domains. Cells doubly deficient in the pair of multidomain pro-apoptotic molecules Bax and Bak proved resistant to all tested intrinsic death pathway stimuli; thus, Bax and Bak constitute a requisite gateway to the intrinsic pathway operative (7, 8). In contrast, the pro-apoptotic molecules (such as BAD, BID, BIM, PUMA, and NOXA) share homology only within the minimal death domain, the BH3 amphipathic α-helix, prompting the title “BH3-only.” The BH3-only members serve as upstream sentinels that selectively respond to specific proximal death and survival signals. In response to various apoptotic stimuli, the “activator” BH3-only members, such as BIM or truncated BID (tBID), trigger the conformational activation of Bax or Bak leading to caspase activation through the apoptosome complex (9).

Working in opposition, the anti-apoptotic proteins BCL-2, BCL-XL, MCL-1, or A1 sequester BH3-only proteins, inhibiting Bax and Bak activation and apoptosis (2)]. The “sensitizer” BH3-only members (e.g., BAD, NOXA) neutralize the activity of specific anti-apoptotic proteins. Each anti-apoptotic protein specifically interacts with particular BH3-only proteins (10, 11, 12). For example, BAD binds strongly to BCL-2 and BCL-XL but not to MCL-1 or A1, while NOXA only binds to MCL-1 and A1 but not to BCL-2 or BCL-XL (10, 11, 12). Among anti-apoptotic proteins, A1 has the highest affinity to BID (12).

The anti-apoptotic protein A1. A1, also called Bcl2A1 or BFL-1, is preferentially expressed in hematopoietic and endothelial cells and can be induced in mast, smooth muscle, T, lung, and neuron cells in normal development (13–17). A1 has protective effects in a variety of settings including drug-induced and growth factor withdrawal–induced apoptosis (13, 18–23). The structure of A1 overall is similar to that of other anti-apoptotic BCL-2-family proteins; however, some features (such as an acidic patch in the binding groove and local plasticity of hydrophobic interactions) may explain the specificity of A1 binding to BH3 only proteins (4, 5).

Expression of A1 is elevated in several types of cancer, and data indicates that it may be required for tumor initiation, maintenance, and chemoresistance. For example, the overexpression of A1 is a potential anti-apoptotic mechanism in patients with diffuse large B-cell lymphoma (DLBCL) (24). In addition, A1 is necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive anaplastic large cell lymphoma cells (25), and A1 has been shown to contribute to tumor cell survival in B-cell chronic lymphocytic (26). Gene expression profiling of 732 cancer cell lines was also performed, spanning over 40 tumor types (see Figure 2A). The expression of A1 is limited to specific tumor types including acute myeloid leukemia (AML), lymphoma, and melanoma (see Figure 2B). Within those tumor types, A1 expression has wide distribution, leading to the hypothesis that A1 may be more crucial in cells with high A1 expression (see Figure 2C-2D). A1 is highly expressed in a subset of DLBCL cell lines and about 30% of DLBCL primary tumor samples. In contrast, A1 is highly expressed in the majority of melanoma cell lines and patient samples, and its expression increases with tumor progression (27, 28). It is interesting that the expression of pro-apoptotic BID and NOXA tightly correlates with that of A1 in DLBCL primary tumors as well as in cell lines, suggesting that A1 may be required to sequester BID and other pro-apoptotic proteins and to prevent them from activating Bax/Bak and the downstream apoptosis cascade. Taken together, these data all point to the important role of A1 as a regulator of cell survival in cancer.

Figure Icon

Figure 2

Evidence for a Key Role of A1 Expression in Cancer.

A1 as a therapeutic target. To test whether A1 is required for the survival of DLBCL or melanoma cells, RNA interference was used to knock down the expression of A1. The shRNA-mediated knockdown efficiency was over 70%. When tested in a panel of DLBCL and melanoma cell lines, knock-down of A1 led to significant induction of apoptosis in cell lines expressing high levels of A1 (see Figure 2). In accordance with this finding, it was reported that two other DLBCL cell lines highly expressing A1 required A1 for survival (29). In contrast, cells expressing low or undetectable levels of A1 are not sensitive to A1 knock down (see Figure 2), thus pointing to A1 as an “Achilles’ heel” in these tumor types, and suggesting A1 as a potential therapeutic target in such tumors. Therefore, specific small molecule inhibitors of A1 are of great need, and yet none have reported to date. We sought to identify small molecules capable of inhibiting A1 function using a novel cell-based screen.

Many chemotherapeutic drugs act through the BCL-2-family proteins to induce apoptosis in cancer cells (1). For example, glucocorticoids, cornerstone drugs for the treatment of acute lymphocytic leukemia (ALL), depend on BIM induction to effectively initiate apoptosis (30). The Golub laboratory recently reported that rapamycin overcomes glucocorticoid resistance in ALL cells by repressing MCL-1 expression (30). Similarly, imatinib elicits apoptosis in chronic myeloid leukemia (CML) cells mainly through induction of BIM and BAD (31). BIM is required for epidermal growth factor receptor (EGFR) inhibitors to induce apoptosis in non-small cell lung carcinoma cells bearing EGFR mutations, and failure to induce BIM correlates with drug resistance (32–35).

Recently, several approaches have been undertaken to directly target the BCL-2-family proteins, including antisense oligonucleotide (36, 37), stapled BID-BH3 peptide (38), and small molecules (39). Several small molecules have been reported as direct inhibitors of the BCL-2-family proteins, but these molecules typically show a wide range of potency and selectivity for the different anti-apoptotic BCL-2 proteins (BCL-2, BCL-XL, BCL-W, BCL-B, MCL-1, and A1) (40). Table 1 summarizes the affinity (Ki) of BCL-2 inhibitors for the different anti-apoptotic members of the Bcl2-family proteins.

Table 1. Affinity (Ki) of BCL-2 Inhibitors for Different Anti-apoptotic BCL-2-Family Protein Members.

Table 1

Affinity (Ki) of BCL-2 Inhibitors for Different Anti-apoptotic BCL-2-Family Protein Members.

ABT-737 (Figure 3A), a BAD-BH3 mimetic small molecule, exhibits single-agent, mechanism-based killing of primary follicular lymphoma, chronic lymphocytic leukemia (CLL) cells, and small-cell lung carcinoma lines where BCL-2 is commonly highly expressed (41, 42). ABT-737 binds strongly to BCL-2 and BCL- XL but not to MCL-1 or A1 (41, 42). Thus, its effectiveness is limited to cells where BCL-2 or BCL- XL is the major survival factor, but it is ineffective in cells where MCL-1 or A1 is the major force counteracting the pro-apoptotic proteins (43-47). Gossypol (Figure 3B), apogossypolone (Figure 3C), and apogossypol (Figure 3D) show binding to BCL-2, BCL-XL, BCL-W, and MCL-1, but do not bind to A1 (48, 49). More recently, Pellecchia and collaborators described apogossypol derivatives that were found to inhibit, BCL-XL, MCL-1, and A1 with submicromolar IC50 values (50). One of these apogossypol derivatives (compound 8k) inhibited the binding of BH3 peptide to A1 with an IC50 of 0.4 μM but showed similar potency against BCL-2, BCL-XL, and MCL-1. GX15-070 (Figure 3E) is a pan-BCL-2 inhibitor with Ki in the low micromolar range, and has been shown to inhibit the viability of 15 human myeloma cell lines (HMCL) with a mean IC50 value of 246 nM (51). BH3I-1 (Figure 3F) and EGCG (Figure 3G) are other pan-BCL-2 inhibitors with Ki values in the low micromolar range and have Ki values for A1 of 4.65 μM and 1.79 μM, respectively (52). Gambogic acid (Figure 3H), a natural product isolated from the tree Garcinia Hanburyi, has been reported as a BCL-B and MCL-1 selective inhibitor but also shows activity against BCL-2, BCL-XL, BCL-W, and A1 in the low micromolar range (53). More recently, Reed and collaborators reported N-aryl maleimides (see Figure 3I) as A1 inhibitors (54).

Figure 3. BCL-2-Family Protein Member Inhibitors.

Figure 3

BCL-2-Family Protein Member Inhibitors.

Overall, these previously reported A1 inhibitors are mainly pan-BCL-2-family inhibitors and lack selectivity for the A1 protein. The most potent A1 inhibitor is ABT-263 (Figure 3J), with a Ki of 0.34 μM; however, ABT-263 binds preferentially to BCL-2, BCL-XL, and BCL-W over A1. A1 expression actually confers resistance to ABT-263, so it is not suitable for studying A1-driven cancers. The N-aryl maleimide family, recently described by Reed and collaborators, also presents similar selectivity problems. In this context, the discovery of novel selective A1 inhibitors appears to be crucial and highly desired for studying A1-dependent cancer biology.

2. Materials and Methods

The screening strategy in this probe development project relied on a novel physiologically relevant cell-based approach to identify inhibitors of the anti-apoptotic protein A1 (see Figure 4). The fate of cell survival versus apoptosis is determined by the balance of anti- and pro-apoptotic proteins. Expression of activator BH3-only proteins (such as BIM or tBID; see Figure 1) leads to downstream caspase activation and apoptosis (7, 8). A1 can functionally bind to and sequester BIM or tBID (7, 8). In this assay, the parental control cells do not depend on A1 for survival; however, they can be primed to depend on A1 by co-expressing A1 and BIM. The primed cells still maintain a balance between anti- and pro-apoptotic proteins, but rely on A1 to sequester BIM. The cells contain a construct that allows A1 to be co-expressed with BIM. To make sure that A1 and BIM are expressed at comparable levels, A1 and BIM were cloned into the same retroviral construct, separating them with the 2A peptide (55). A1 and BIM are transcribed in the same transcript and translated together. The 2A peptide, which signals for “self-cleaving” (55), separates A1 and BIM after translation. An A1 inhibitor can release A1-bound BIM, which activates Bax/Bak, and leads to caspase activation that can be quantitatively measured (see Figure 4). Murine embryonic fibroblast (MEF) cells stably expressing the A1-2A-BIM construct via retroviral-mediated infection were generated. The expression of A1-2A-BIM does not affect cell proliferation, and is stable even after the cells were carried for months.

Figure 4. Screen Strategy.

Figure 4

Screen Strategy. MEF cells (A) stably co-expressing A1 and BIM (B) are sensitive to A1 inhibitors (D), while unprimed cells are not (C).

This cell-based assay has several advantages that allow the discovery of compounds that modulate A1 function through multiple possible mechanisms. In particular, the assay preserves A1 in its physiological state (as opposed to a biochemical assay), such that hit compounds are more likely to be physiologically relevant. In contrast, cell-free biochemical screens using recombinant proteins lacking the trans-membrane domain are not performed in the native membrane and cellular context. As a result, hits from those screens may not be specific, and are more likely to induce caspase activation through an A1-independent mechanism. For example, chelerythrine was identified as an inhibitor of BCL-XL function by a fluorescence polarization assay (56). Chelerythrine binds to BCL-XL in vitro (56, 57); however, it induces caspase activation in Bax/Bak−/− cells, arguing that its action in cells is independent of BCL-2-family proteins (58).

Materials and Reagents

All reagents and solvents were purchased from commercial vendors and used as received. Phosphate-buffered saline (PBS; catalog no. 08J07A0022) was acquired from the Broad Institute Supply and Quality Management (SQM; Cambridge, MA). CaspaseGlo 3/7 (catalog no. G8092, lot no. 292850) and CellTiter Glo (catalog no. G7573) were purchased from Promega (Fitchburg, WI), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES, catalog no. 15630, lot no. 697278) was purchased from Gibco/Invitrogen (Grand Island, NY). TC-treated, 384-well plates (catalog no.3570) were acquired from Corning (Corning, NY).

Growth Medium

Roswell Park Memorial Institute Medium (RPMI-1640, catalog no. 10-043-CV, lot no. 10043010) and Trypsin (catalog no. 25-053-Cl, lot no. 25053231) were purchased from MediaTech (Manassas VA). Fetal bovine serum (FBS; catalog no. SH30070.03, lot no. ATH32736) was purchased from Hyclone/Thermo-Fisher (Waltham, MA). Penn/Strep/Glutamine (catalog no. 10378, lot no. 622183) was purchased from Gibco.

Cell Lines

Throughout the project, different sets of cell lines were tested for activation of caspases to determine the specificity of small molecules for activity in A1-expressing cells. Caspase activity was detected using a commercially available reagent containing a caspase peptide substrate linked to a luciferase substrate; luminescence is generated in the presence of either active caspase 3 or caspase 7. Cell lines were either artificial, such as those containing the A1-2A-BIM construct described above, or were established cancer cell lines that have been characterized for A1 expression. The set of cell lines used is listed in Table 2.

Table 2. Cell Lines Used in Probe Development Assays.

Table 2

Cell Lines Used in Probe Development Assays.

In addition to the caspase activation assays, HMC-1-8 was also tested for viability using CellTiterGlo to determine if the compounds were generally toxic to cancer cells not expressing A1. CHL-1 cells were additionally used as the source of purified mitochondria for use in an enzyme-linked immunosorbent assay (ELISA) to measure the release of cytochrome c induced by compounds in the presence or absence of A1 expression, confirming activation of the apoptotic pathway.

2.1. Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided in Appendix A (Table A1). Refer to Appendix B for the detailed assay protocols.

2.1.1. Primary Screen for Caspase Activation in MEF-A1-2A-BIM

Cells were maintained in 35 ml of growth medium (per 1 liter: 890 ml RPMI-1640, 100 ml heat-inactivated FBS, 10 ml Penn/Strep/Glutamine, 0.5 to 1 mg blasticidin) in a T175 cell culture flask (Corning) and incubated in a TC incubator (Thermo-Fisher) at 95% humidity, 5% CO2, 37 °C. To passage, cells were harvested by first aspirating the media and rinsing the flask with 10 ml sterile PBS. Next, PBS was aspirated and 3 ml trypsin was added to the flask and incubated for 1 to 2 minutes at 22 °C. Then, 8 ml of growth medium was added to the trypsin to quench the reaction. The cells were resuspended, counted, and 4.5 million cells in approximately 2 ml were transferred to 33 ml fresh growth medium in a new T175 flask.

For screening, the cells were harvested, and the concentration was adjusted to 66,000 cells/ml in the above media without blasticidin. While gently stirring, the cells were dispensed with a Combi Multidrop (Thermo-Fisher) by adding 30 μl of suspension per well to white, sterile, TC-treated, 384-well plates (Corning) for a total of 2000 cells per well. The plates were incubated overnight in an automated TC incubator (Liconic) at 95% humidity, 5% CO2, 37 °C.

For compound screening, 50 nL of compound in DMSO was added using slotted steel pins (V&P Scientific) on a pin tool (HiRes Biosolutions). The plates were returned to the incubator for 3 hours. To read caspase activity, the cells were removed from the incubator and cooled to room temperature for 30 minutes. Lids were removed, and 10 μl of diluted CaspaseGlo (1:1 dilution with 50 mM HEPES buffer) was added to each well with a Combi Multidrop. The plates were shaken at 1250 rpm for 15 seconds (BigBear shaker), then incubated for 1 hour at room temperature. Luminescence was detected on an Envision (Perkin-Elmer) multimode reader (Ultrasensitive luminescence setting, 0.1 seconds per well).

2.1.2. Primary Retest for Caspase Activation in MEF-A1-2A-BIM

Repeat of primary screen at dose MEF-A1-2A-BIM using CaspaseGlo.

2.1.3. Counterscreen for Caspase Activation in MEF-Bak/Bak −/−

As described in the primary screen for MEF-A1-2A-BIM but using MEF Bax/Bak −/−.

2.1.4. Counterscreen for Caspase Activation in HMC-1-8

As described in the primary screen for MEF-A1-2A-BIM but using the HMC-1-8 cancer line.

2.1.4. Counterscreen for Caspase Activation in MEF Wild Type

As described in the primary screen for MEF-A1-2A-BIM but using MEF wild type.

2.1.5. Counterscreen for Caspase Activation in MEF Flag-A1-IRES-BIM

As described in the primary screen for MEF-A1-2A-BIM but using MEF Flag-A1-IRES-BIM.

2.1.6. Counterscreen for Caspase Activation in MeWo

As described in the primary screen for MEF-A1-2A-BIM but using MeWo.

2.1.7. Counterscreen for Caspase Activation in MeWo-A1 + TSA

As described in the primary screen for MEF-A1-2A-BIM but using MeWo-A1 (MeWo cells ectopically expressing A1) pre-activated with 1 μM trichostatin A following plating into 384-well plates.

2.1.8. Counterscreen for Caspase Activation in MEF-A1-2A-tBid

As described in the primary screen for MEF-A1-2A-BIM but using MEF-A1-2A-tBid.

2.1.9. Counterscreen for Caspase Activation in Mel501

As described in the primary screen for MEF-A1-2A-BIM but using the Mel501 cancer line.

2.1.10. Counterscreen for Caspase Activation in CHL-1 Cells

As described in the primary screen for MEF-A1-2A-BIM but using the CHL-1 cancer line.

2.1.11. Secondary Screen for Caspase Activation in CHL-1 Cells

As described in the primary screen for MEF-A1-2A-BIM but using the CHL-1 cancer line expressing the A1-2A-BIM construct.

2.1.12. Counterscreen for Cell Toxicity in HMC-1-8

As described in the primary screen for MEF-A1-2A-BIM but using HMC 1–8 cells and measuring viability after 24 hours of compound treatment. CellTiterGlo reagent (10 μl/well), which measures intracellular ATP levels as a proxy for viable cell numbers, was used to generate the luminescent signal.

2.1.13. Counterscreen for Cytochrome c Release from Mitochondria Isolated from CHL-1

CHL-1 cells were passed and harvested as for other cell lines. After the cells were dislodged with trypsin, 10 ml of cold media were added to collect the cells. The cells were centrifuged at 1000 rpm at 4 °C for 4 minutes. The cell pellets were washed with cold PBS once, resuspended in 1X AT buffer (300 mM trehalose, 10 mM HEPES-KOH pH 7.7, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, and 0.1% BSA), and homogenized with a Potter Elvehjem homogenizer for 30–40 strokes at 1,600 rpm on ice. After centrifuging the cell homogenate at 600Xg for 10 minutes at 4 °C, the supernatant containing mitochondria was collected and centrifuged at 7000g for 10 minutes at 4 °C. The mitochondria pellets were carefully dislodged and resuspended in AT buffer with 80 mM KCl. The mitochondria were then aliquoted in 1.5-ml centrifuge tubes, and incubated with appropriate compounds for 30 minutes at 37 °C, then centrifuged at 7000Xg for 10 minutes. Negative controls were treated with an equivalent amount of DMSO to compound treatment. Positive controls were treated with equivalent amounts of DMSO and 15 ng recombinant pro-apoptotic protein tBid. The supernatant was collected, diluted at appropriate fold into the 96-well assay kit, and detected with a human cytochrome c ELISA kit (R&D No. SCTC0). Absorbance was read at 450 nm in an M5e plate reader (Spectramax), normalized at 540 nm.

2.1.14. Secondary Screen for Cytochrome c Release from Mitochondria Isolated from CHL-1 expressing A1-2A-BIM

As described for the cytochrome c release assay for CHL-1 but using CHL-1 expressing A1-2A-BIM.

2.2. Probe Chemical Characterization

The probe compound (CID 701939, ML214, SID 99432301, MLS003370521) is commercially available and can be obtained from Sigma Aldrich (catalog no. R47119). To confirm its structure and activity, we resynthesized the probe in three steps as shown in Scheme 1.

Scheme 1. Synthesis of the Probe.

Scheme 1

Synthesis of the Probe.

Treatment of 4-hydroxyquinolin-2(1H)-one with nitric acid afforded the corresponding 4-hydroxy-3-nitroquinolin-2(1H)-one, which was chlorinated using phosphorus oxychloride and N-methylated using trimethyloxonium tetrafluoroborate to afford the probe. Full experimental details and characterization are in Section 2.3.

The solubility of the probe was measured at room temperature and was found to be 0.6 μM in water and 0.8 μM in PBS in a thermodynamic equilibrium assay. As compound addition and treatment occurs prior to equilibration, this is not inconsistent with higher observed AC50 values.

The stability of the probe in PBS (0.1% DMSO) was measured over 48 hours, and the data is shown in Figure 5. From these results, it can be concluded that the probe is stable in PBS since more than 90% of the probe is still present after 48 hours of incubation in PBS.

Figure 5. Stability Data for the Probe ML214 in PBS.

Figure 5

Stability Data for the Probe ML214 in PBS. Total ion count of the probe ML214 over 48 hours in PBS.

The chemical structure and purity of the probe was established using 1H and 13C NMR spectroscopy as well as UPLC analysis (see Appendix C).

2.3. Probe Preparation

Chemistry Experimental Methods

General details. All reagents and solvents were purchased from commercial vendors and used as received. NMR spectra were recorded on a Bruker 300 MHz or Varian UNITY INOVA 500 MHz spectrometer as indicated. Proton and carbon chemical shifts are reported in ppm (δ) relative to tetramethylsilane or CDCl3 solvent (1H δ 7.26, 13C δ 77.0). NMR data are reported as follows: chemical shifts, multiplicity (obs. = obscured, br = broad, s = singlet, d = doublet, t = triplet, m = multiplet); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25 °C. Flash chromatography was performed using 40–60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash Rf system. Tandem Liquid Chromatography/Mass Spectrometry (LCMS) was performed on a Waters 2795 separations module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with ultraviolet (UV) light and aqueous potassium permanganate (KMnO4) stain followed by heating.

4-Hydroxy-3-nitroquinolin-2(1H)-one: 4-hydroxyquinolin-2(1H)-one (5.0 g, 31.0 mmol) was dissolved in nitric acid (30 ml) and stirred at room temperature for 10 minutes. The reaction was heated to 75 °C and stirred for 15 minutes. The reaction was cooled to room temperature and added to an ice-water mixture. The yellow precipitate was filtered and dried to obtain the product 5.5 g (26.7 mmol, 86% yield) as a yellow powder.

1H NMR (300 MHz, DMSO-d6): δ 12.01 (s, 1H), 8.03 (d, J = 8.1, 1H), 7.66 (dd, J = 8.2, J = 7.2, 1H), 7.38-7.24 (m, 2H); LRMS (ESI): calculated mass for C9H7N2O4 [M-H] 205.16, found 204.91.

4-Chloro-3-nitroquinolin-2(1H)-one: 4-hydroxy-3-nitroquinolin-2(1H)-one (3.36 g, 16.3 mmol) and N-benzyl-N,N-diethylethanaminium chloride (14.85 g, 65.2 mmol) were dissolved in acetonitrile (30 ml) and phosphorus oxychloride (6.69 ml, 71.7 mmol) was added. The reaction mixture was heated to 40 °C for 30 minutes and refluxed for 1 hour. Solvents were then evaporated, water was added, and the mixture was stirred for 3 hours at room temperature. The aqueous phase was extracted using ethyl acetate. The organic phase was dried on sodium sulfate, concentrated, and purified on silica gel using a gradient of ethyl acetate in hexanes to afford 3.35 g (14.92 mmol, 92% yield) of the desired product as a white solid.

1H NMR (300 MHz, DMSO-d6): δ 13.04 (s, 1H), 8.00 (d, J = 8.0, 1H), 7.80 (dd, J = 7.7, J = 7.3 Hz, 1H), 7.51-7.42 (m, 2H); LRMS (ESI): calculated mass for C9H6ClN2O3 [M+H]+ 225.60, found 224.90.

4-Chloro-1-methyl-3-nitroquinolin-2-one: 4-chloro-3-nitroquinolin-2(1H)-one (500 mg, 2.23 mmol) was dissolved in dichloromethane (20 ml) and cooled to 0 °C. Trimethyloxonium tetrafluoroborate (659 mg, 4.45 mmol) and di-isopropylethylamine (736 μl, 4.45 mmol) were added, and the reaction mixture was stirred at 0 °C until completion. The reaction mixture was concentrated and purified on silica gel using a gradient of ethyl acetate in hexanes to afford 210 mg (0.88 mmol, 41% yield) of the probe as an off-white powder.

1H NMR (500 MHz, CDCl3): δ 8.13 (d, J = 8.1, 1H), 7.78 (dd, J = 8.7, J = 7.2, 1H), 7.50-7.42 (m, 2H), 3.80 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 153.8, 138.7, 135.9, 134.0, 127.6, 124.1, 116.7, 114.9, 30.5. HRMS (ESI): calculated mass for C10H8ClN2O3 [M+H]+ 239.0223, found 239.0219.

The 1H NMR spectra,13C spectra, and UPLC chromatograms of the probe ML214 and analogs are provided in Appendix C.

3. Results

Probe Attributes

  • IC50 < 10 μM
  • < 3-fold caspase activation at IC50 in Bax/Bak−/− cell line.
  • Active in human cancer cells expressing A1-BIM cells (IC50 = 10 μM)
  • Preferentially active in A1-dependant cells compared to BCL-XL or MCL-1-dependant cells. (A1/BCL-XL = 2X)
  • Nontoxic in wild-type human cancer cells not expressing A1 (TC50/IC50 = 10X)

The project included a primary HTS of the entire MLSMR collection (greater than 320,000 substances) as well as a diversity-oriented synthesis (DOS) compound library of over 20,000 substances generated at the Broad Institute. Approximately 0.3% of the compounds tested were selected for dose retest and selectivity. Of these, three chemical classes showed varying levels of selectivity in the panel of cell-based specificity assays. One compound showed the desired profile but was of moderate potency (approximately 10 μM). This series was expanded through two rounds of SAR with both commercially available and internally synthesized analogs. A more potent compound, probe ML214, was identified that maintained the desired selectivity pattern.

3.1. Summary of Screening Results

Figure 9 displays the critical path for probe development. Some of the indicated assays were substituted with equivalent cell lines due to availability of reagents or robustness of assay parameters. For example, viability was measured in HMC1-8 rather than MeWo since both are A1-independent human cancer cell lines.

Figure 9. Critical Path for Probe Development.

Figure 9

Critical Path for Probe Development.

A pilot high-throughput screen (HTS) of 23,653 substances generated at the Broad Institute (AID 2465) was executed, followed by an HTS of the full Molecular Libraries Small Molecule Repository collection (325,633 substances; AID 2462.) Both HTS campaigns were performed in 30 μl reactions in 384-well plates, and all compounds were screened in duplicate. Caspase activation was measured using the CaspaseGlo 3/7 kit (Promega). Values were scaled to DMSO negative controls and a nonspecific positive control (clofoctol) that strongly induces caspase activation 10- to 20-fold depending on the cell line. A second positive control (ABT-263) that is more mechanistically relevant but less potent for A1, was also included to ensure that cell constructs were responding as expected. Because the clofoctol control was so potent, compounds with a score statistically significant from DSMO, corresponding to 5% or greater (approximately 1.5-fold caspase activation) were selected for retest at dose.

Next, 1065 compounds were retested in an 8-point, 2-fold dilution. Because of the cost of the caspase assay, these compounds were confirmed only in the primary screen (AID 2765). Based on this result, a second pick of 255 compounds was further tested in four additional cell lines:

In the course of determining potencies in these assays, it was found that the most potent compounds exhibited a bell-shaped curve. As the assay measures caspase activation, this may be due to cell death at higher activities, so curves were fit to the increasing part of the concentration response series. In addition, because a nonspecific control was needed for all assays, clofoctol was used, which very potently activates caspases in MEFs. It was determined that a biologically relevant and statistically significant level of activation corresponded to 10% of the clofoctol response. Therefore, potencies were measured based on the response curves crossing this threshold (AC10 relative to clofoctol).

Based on these results three classes of compounds were selected for purchase and further testing from dry powders, along with synthetic analogs to develop the SAR of the best candidate class. This SAR study included initial attempts to remove the undesired reactive functionality (see SAR Tables 58) These 39 compounds were tested in the above five assays (see Appendix A, Table A2 for SIDs) plus counterscreen MeWo (AID 488914), MeWo-A1 TSA (AID 488914), MEF-A1-2A-tBid (AID 488897), and Mel501 (AID 488934). The nitroquinolinone series was determined to have the best selectivity profile, and an additional round of SAR was performed to determine if potency or selectivity could be improved any further. These 21 compounds were tested in duplicate on two separate days in five of the assays and in duplicate on one day in the additional assays and repeated once more in the primary and MEF WT lines (see Appendix A, Table A1 for AIDs). They were also tested in the HMC-1-8 viability assay to determine toxicity to a non-A1 dependent cancer line. The probe ML214 was selected based on these results and was tested in the CHL-1 cytochrome c release assay to confirm specificity and mechanism of action. Additional cytochrome c experiments will also be performed to confirm consistent performance across different cell lines and growth conditions.

Table 5. Summary of SAR on the Quinolinone’s Nitrogen.

Table 5

Summary of SAR on the Quinolinone’s Nitrogen.

Table 6. Summary of SAR on Nitrocoumarin Analogs.

Table 6

Summary of SAR on Nitrocoumarin Analogs.

Table 7. Summary of SAR on Quinoline Analogs.

Table 7

Summary of SAR on Quinoline Analogs.

Table 8. Other Compounds Synthesized During the SAR Study.

Table 8

Other Compounds Synthesized During the SAR Study.

3.2. Dose Response Curves for Probe

Figure 10. displays the dose response curves for the probe ML214. For results of all replicates, refer to the Pubchem AIDs indicated in Appendix A (Table A1).

Figure Icon

Figure 10

Concentration-dependent Activities (%) of the Probe ML214 in A1-expressing and Non-expressing Cell Lines (Clofoctol = 100%).

In addition to the assays described in the Chemical Probe Development Plan (CPDP) and shown in Figure 11, we further tested the ability of different constructs to rescue the cytotoxic effects of the probe ML214 in a melanoma cell line expressing A1, Mel501 (see Figure 12). WT Mel501 cells displayed sensitivity to the probe ML214, while Mel501 cells transformed with constructs expressing anti-apoptotic BCL-2 proteins BCL-XL and MCL-1 were less sensitive. However, cells were partially but less significantly rescued by cells expressing additional A1, suggesting that the mode of action of the probe ML214 may involve specific inhibition of apoptotic blockage by A1 relative to other BCL-2-family members. While this supporting data illustrating viability rescue shows less selectivity than the caspase activation data used in probe development, it suggests that ML214 is targeting a cell survival process involving the BCL-2 family anti-apoptotic proteins.

Figure 11. Activity of the Probe ML214 in CHL-1 Human Cancer Cell Constructs.

Figure 11

Activity of the Probe ML214 in CHL-1 Human Cancer Cell Constructs. Treatment of CHL-1 cells with the probe ML214. Caspase activation in CHL-1 wild-type and CHL-1 expressing two different A1 constructs (A); Cytochrome c release (% of tBid poscon) measured (more...)

Figure 12. Viability of Mel501 Constructs Treated with the Probe ML214.

Figure 12

Viability of Mel501 Constructs Treated with the Probe ML214. Treatment of the Mel501 melanoma cell line overexpressing various BCL-2 constructs. More significant rescue of viability is observed with BCL-XL and MCL-1 than with A1, suggesting specific targeting (more...)

3.3. Scaffold/Moiety Chemical Liabilities

The probe ML214 contains an activated chloroquinolinone moiety and could potentially be a nonselective alkylating agent. As shown by the SAR study presented in Section 3.4, this reactive functionality was essential for activity and could not be removed despite generation of analogs attempting to do so. Thus, stability of the probe ML214 in the presence of glutathione (GSH) was investigated over 3 hours, the same time period for incubation in all cell assays used in this probe development project. After 3 hours of incubation of the probe ML214 at 10 μM nominal concentration in PBS (0.1% DMSO) at room temperature, in the presence of a physiologically relevant amount of glutathione (5 mM), no trace of the corresponding GSH adduct could be detected; unmodified probe was detected even after 3 hours, suggesting that the probe ML214 is stable to nucleophiles such as GSH present in cells. Since the solubility of the probe ML214 in PBS is not high, we wanted to further confirm GSH stability in the presence of organic solvents which can better dissolve the probe. Therefore, the experiment described above was repeated in a 6:1 mixture of DMSO:water (where the probe is completely soluble) and 1.5 equivalents of GSH. The concentration of the probe in this experiment was 2.5 mM, which is more than 1,000-fold above the concentration used in the biological screening experiments. We ran this assay under high concentration for two reasons: 1) We wanted to determine if GSH will form an adduct with the probe under these kinetically favorable conditions that are far more stringent than what the probe will typically encounter under biological settings., and 2) It is easier to detect the GSH adduct by LCMS under these higher concentrations. Formation of the GSH adduct was followed by LCMS over a 3-hour period (see Figure 13). Even under these forcing conditions, only 16% of the probe was converted to the corresponding GSH adduct and 84% of the probe remained intact after 3 hours of incubation at room temperature, further confirming the reasonable stability of the probe ML214 toward GSH. Since the caspase activation assays were also run over the same 3-hour period under which the probe showed relatively low thiol adduct formation, and under much milder conditions than those described above, it is unlikely that the probe is acting through non-specific alkylation to generate the observed selectivity profile. This suggests the notion of a selectivity window that is useful for biological study; over long time periods or at higher concentrations, the compound may be promiscuous, but when used at the appropriate concentration and time ML214 can be a useful tool for targeting a specific apoptotic pathway for study.

Figure 13. Stability of the Probe ML214 in the Presence of Glutathione.

Figure 13

Stability of the Probe ML214 in the Presence of Glutathione. Stability of the probe ML214 measured in DMSO:H2O (6:1) in the presence of 1.5 equivalent of GSH.

Interestingly, the coumarin analog of the probe (see Figure 14), which was found to be inactive in all A1-dependent cell lines, when subjected to the same experiments led to the complete formation of the corresponding GSH adduct in less than 2 hours. This shows that highly reactive compounds are unlikely to show desired selectivity profiles. The probe ML214, although slightly reactive toward nucleophiles like GSH, probably behaves as a mild and somewhat selective alkylating agent; thus, it could be useful to study cancer biology related to the A1 anti-apoptotic protein.

Figure Icon

Figure 14

Activity Versus GSH Stability for the Probe ML214 and its Coumarin Analog. Relative GSH stabilities of the probe ML214 and its coumarin analog. While the probe is slightly reactive, the coumarin analog is highly reactive and is correspondingly less selective (more...)

Furthermore, Hur et al. recently described a high-throughput cell-based screen to look for compounds that are capable of activating the antioxidant response element (ARE) in human neuroblastoma IMR-32 cells (59). They identified a small molecule (AI-1) that acts as an ARE activator by covalently modifying Keap1, the negative regulator of Nrf2 (see Figure 15). The probe ML214 has been tested as an analog of AI-1 in the same assay and was found to be inactive. This shows that reactive electrophilic compounds can be elaborated to display a desired selectivity profile and further utilized as a probe to study specific pathways.

Figure Icon

Figure 15

Structure of AI-1 Versus the Probe ML214. Chemically related compound AI-1 is active in an antioxidant response element (ARE) assay while the probe ML214, despite its apparently similar reactive functionality, is inactive in the ARE assay.

3.4. SAR Tables

The compounds synthesized during the course of the SAR study have been tested in a panel of four A1-dependent cell lines (MEF-A1-2A-BIM, MEF-Flag-A1-Ires-BIM, MEF-A1-2A-tBID, and MEWO-A1-TSA) and four A1-independent cell lines (Bax/Bak −/−, MEF-WT, HMC-1-8, and MEWO). For readability reasons, the SAR tables only present results obtained for the primary cell line (MEF-A1-2A-BIM) and the counter-screen cell line (Bax/Bak −/−); however, data for all the other cell lines are available in PubChem for every analog.

For the reasons described in Section 3.1, activity was measured by fitting a curve in the increasing section of concentration response plots, and the point at which the curve crosses the 10% of clofoctol threshold was used to determine relative activity of compounds (AC10). AC10 values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3). AC10 values were calculated up to the active concentration limit described for each sample. pAC was set to equal −1*log(AC10).

The hit compound (analog CID776319) (see Figure 16) has an AC10 of 7.52 ± 2.28 μM in the primary cell line and is inactive in the Bax/Bak −/− cell line; thus, the hit compound meets probe requirements. However, the hit compound (analog CID776319) possesses two main liabilities. The first liability is the presence of a nitro group, which is usually undesirable in drug discovery because it can be metabolized to toxic reactive intermediates such as nitroso or hydroxylamines. The second liability is the presence of the chloro-enone that could potentially lead to nonselective alkylations in cells.

Figure 16. Structure of the Hit Compound (Analog CID776319).

Figure 16

Structure of the Hit Compound (Analog CID776319).

First, we investigated the possibility of removing or replacing the nitro group. Replacing it with hydrogen (Table 3, entry 3) or an ethyl ester (Table 3, entry 2) led to inactive compounds. During the resynthesis of the hit compound (analog CID776319) from 4-chloro-3-nitroquinolinone and tolyl mercaptan, a by-product was identified where the nitro group has been replaced with a STolyl group (Table 3, entry 4). This compound was also found to be inactive. Together, these results suggest that the nitro group is necessary for activity.

Table 3. Summary of SAR to Replace the Nitro Group.

Table 3

Summary of SAR to Replace the Nitro Group.

To better understand the influence of the electrophilicity of the 4-position, we next investigated the substitution on the 4-position by using various replacements for the STolyl substituent (Table 4). Replacing the p-tolyl substituent with a 2-pyridyl substituent (Table 4, entry 2) to increase the leaving group capacity led to an increase in activity. Replacing the sulfide leaving group by sulfone analogs (Table 4, entries 3 and 4) led to a slight increase in activity indicating that increased electrophilicity at C4 (and other centers of the compounds) is beneficial for activity. Amine substituents were also used to replace the p-tolyl-sulfide (Table 4, entries 5–9). Although they led to an increase in solubility, amines were a very poor leaving group. All of these analogs were found to be inactive except the benzimidazole analog (Table 4, entry 7), which could have behaved as a weak heterocyclic leaving group. Replacing the p-tolyl-sulfide with an aliphatic ether (Table 4, entry 10), a poor leaving group, led to an inactive compound. An aromatic ether analog (Table 4, entry 11) was also found to be inactive. Finally, replacing the p-tolyl-sulfide with a good leaving group such as a chloride (Table 4, entry 12) led to a 4.5-fold increase in activity. This further confirms the necessity of a leaving group at the 4-position for activity.

Table 4. Summary of SAR at the 4-position.

Table 4

Summary of SAR at the 4-position.

Next, we studied the influence of substitution of the quinolinone ring system on the nitrogen atom (Table 5). Removing the methyl substituent on the nitrogen atom (Table 5, entry 1) led to a decrease in activity. Replacing the methyl group by an ethyl group (Table 5, entry 3) or a methoxyethyl group (Table 5, entry 4) led to a slight decrease in activity, but shows that small aliphatic groups are tolerated at that position. Replacing the methyl group by a benzyl group (Table 5, entry 5) led to an 8-fold decrease in activity. A similar trend was observed in the STolyl series (Table 5, entries 6–9), where replacement of the methyl group by small aliphatic groups (Table 5, entries 7–8) was tolerated, but bulkier substituents such as a benzyl groups (Table 5, entry 9) led to a 3-fold decrease in activity.

Coumarins are good isosteres of quinolinones and are usually more reactive toward nucleophilic attack at the 4-position. Thus, we compared the activity of the quinolinones with their coumarin analogs. Quite surprisingly, both sulfides (Table 6, entries 1–3) and chloride (Table 6, entry 4) substitutions at the 4-position led to completely inactive compounds. This shows that the reactivity at the 4-position is critical for activity. Coumarin seems to be too reactive and probably reacts nonselectively with cysteine or lysine residues. The quinolinone probe, being less reactive, probably reacts with cysteine or lysine residues in a more selective fashion to result in the desired phenotype; however, further testing of this hypothesis by identifying the target(s) for the chloroquinolinone probe is needed. Our current efforts and planned future studies for target identification are described in Section 4.2. To reduce the reactivity of the coumarin series, we considered amines at the 4-position (Table 6, entries 5–7); however, these analogs were all completely inactive.

To reduce the electrophilicity of the 4-position, we also synthesized quinoline and methoxyquinoline analogs of the probe (Table 7). All of these analogs were found to be inactive except the quinoline with a STolyl substituent at the 4-position, which showed an AC10 of 8.65 μM.

During the synthesis of analogs of the hit compound from 4-chloro-3-nitroquinolinone and different alklyl mercaptans, a by-product was identified where the nitro group has also been replaced with the thiol nucleophile. Several of these by-products are presented in Table 8 (entries 1–4). They were all found to be inactive, probably because they do not possess the 3-nitro substituent. An intermediate in the synthesis of the probe, 4-hydroxy-3-nitroquinolin-2(1H)-one (Table 8, entry 5), was also tested and found to be inactive. A quinoxaline analog, where the electrophilic carbon atom at the 4-position was replaced with a nitrogen atom (Table 8, entry 6) was found to be inactive as well, further confirming the necessity of an electrophilic position, ready for selective alkylation.

The probe ML214 shows an AC10 of 1.70 ± 0.82 μM (n = 4) in the MEF-A1-2A-BIM cell line and is completely inactive in the Bax/Bak −/− cell line (n = 3). It was also tested in six other cell lines: three A1-dependent cell lines (MEF-A1-Ires-BIM-Flag, MEF-A1-2A-tBID, MEWO-TSA) and three A1-independent cell lines (HMC 1–8, MEWO, MEF-WT). The probe ML214 showed good potency in MEF-Flag-A1-Ires-BIM (1.88 ± 0.77 μM, n = 2) and MEF-A1-2A-tBID (1.30 ± 0.58 μM, n = 2), but was inactive in MEWO-A1-TSA. Along with the Bax/Bak −/− cell line, the probe ML214 was also inactive in the HMC-1-8 cell line (n = 1), which is an MCL-1-dependent cancer cell line, and in the MeWo cell line (n = 2). Its activity in MEF-WT was found to be 20 ± 15 μM (n = 4; 2 replicates inactive, two levels of caspase activation at single digit micromolar concentration).

Figure 17 presents a visual summary of the SAR performed on the quinolinone scaffold. Four points of diversity were investigated. The nitro group was found to be necessary for activity. A leaving group at the 4-position was also found to be required to obtain the desired selectivity profile in the different cell lines used in the project. Substitution on the quinolinone nitrogen atom was necessary for activity, a methyl group being preferred and small aliphatic chains being tolerated. All synthesized coumarin and quinoline analogs were found to be inactive.

Figure 17. Summary of SAR Performed on the Quinolinone Scaffold (39 analogs, 28S/11P).

Figure 17

Summary of SAR Performed on the Quinolinone Scaffold (39 analogs, 28S/11P). SAR analysis of the quinolinone scaffold. A leaving group at the 4-position, a nitro group, and N-substitution with small aliphatic groups were essential for activity and selectivity (more...)

3.5. Cellular Activity

All assays used in this project were cellular cytotoxicity assays, with the probe selected on the basis of selective cellular caspase activation. Therefore, no additional testing of cell activity or cell permeability was necessary.

3.6. Profiling Assays

A search of PubChem shows that the probe ML214 has been screened in 314 different assays and was identified as active below 1 μM in only one assay (AID 485364): qHTS assay for the inhibitors of Schistosoma Mansoni Peroxiredoxins, and below 10 μM in 17 assays (AIDs 485364, 2044, 504322, 485313, 2685, 2010, 2753, 435010, 2382, 2326, 449749, 504313, 493014, 485297, 2551, and 1461). This represents a hit rate of 5.7% below 10 μM, and shows that the probe molecule could be moderately promiscuous. The apparent raw hit rate in Pubchem (42 marked “active” out of 314, 13.3%) could be misleadingly high, however. A naïve search of number of assays marked “active” frequently double counts the same biological assay (primary screen and dose retest) and does not take into account the relatively high concentration used in primary screens. In fact, due to the potentially reactive core structure, it is likely that the compound will be toxic at these higher concentrations. However, we have observed an activity window where selective A1-dependent activation of caspases occurs without gross cytotoxic effects. This window is where the probe will be a useful tool for investigating the specific role of Bcl2-A1.

The probe ML214 compound has also been tested for inhibition of a panel of cancer cell lines, including non-engineered lines and engineered lines overexpressing antiapoptotic protein BCL-XL (see Figure 18 & Appendix E). 45 indicated cell lines were treated with varying concentrations of ML214 for 24 hours and viability was measured using the Cell Titer Glo ATP viability assay (Promega); at least a 30-fold window in IC50 has been observed between sensitive and insensitive cancer lines. Indeed, the most sensitive cell lines appear to be lymphomas expressing a combination of the antiapoptotic proteins A1 and MCL-1, suggesting that ML214 could be a useful tool for further understanding the roles of different proteins in cancer survival.

Figure 18. Differential Inhibition of Cancer Cell Line Viability by the Probe ML214.

Figure 18

Differential Inhibition of Cancer Cell Line Viability by the Probe ML214. Viability of cell lines when treated with 0.8–25 μM ML214 for 24 hours (data in Appendix E.) Additional profiling and correlation with A1 expression and dependence (more...)

We have also added the probe ML214 to a small set of approximately 300 compounds that are being profiled against a larger (n≈1000) panel of cell lines as part of another initiative underway at the Broad Institute (60). This profiling analysis will correlate effects of the probe ML214 on cell viability and other cellular markers with gene expression data. Additional hypotheses can then be generated as to the molecular pathway or target of probe ML214.

4. Discussion

A novel, selective, potent probe ML214 was identified by high-throughput screening of the NIH molecular libraries screening collection, followed by optimization through SAR studies. While the probe ML214 itself and many related compounds in the collection are superficially unattractive for probe development due to the presence of an apparently nonselective reactive group, the activity profile of probe ML214 suggests that, in fact, many of these unattractive molecules may be developed into useful biological research tools. Further SAR confirmed that the reactive group is the key substituent for activity, but that a range of potency and selectivity can be engineered by adjusting other molecular functionality.

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

Investigation into relevant prior art entailed searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO) PatFT and AppFT, and World Intellectual Property Organization (WIPO) databases. The search terms applied and hit statistics are provided below in Table 9. Abstracts were obtained for all references returned and were analyzed for relevance to the current project. The searches were performed on, and are current as of, February 11, 2011.

Table 9. Search Strings and Databases Employed in the Prior Art Search.

Table 9

Search Strings and Databases Employed in the Prior Art Search.

Probe ML214 is an improvement over the prior art in that it displays cell-based specificity for A1 not previously demonstrated for any existing small molecule. Other known compounds (such as ABT-737 and ABT-263) have some activity against A1 but are much more potent for other BCL-2-family members. Probe ML214 has been demonstrated to activate caspases in A1 primed cells in three different constructs (A1-2A-BIM, A1-2A-tBid, and Flag-A1-IRES-BIM) and to not activate caspases in cancer cell lines weakly or not expressing A1 (MeWo, HMC 1–8, and CHL-1). Furthermore, Mel501 cancer cells are rescued by overexpression of anti-apototic proteins Bcl-XL and MCL-1 but less so by overexpression of A1. Due to its specific effects in the context of A1, probe ML214 has a unique mode of action not reported in the prior art.

4.2. Mechanism of Action Studies

Since this project has employed phenotypic screens to look for A1-selective inhibitors, we have no information on the target of the probe we have developed, which may be A1 or a closely related component of the apoptotic pathway. Although the probe ML214 showed stability in the presence of glutathione (see Section 3.3), it is an electrophile and, thus, a potential alkylating agent. Its mode of action probably involves a somewhat selective protein alkylation on cysteine or lysine residues. This mode of action can present a strong liability in the context of a drug development program but can be turned to our advantage in the context of a probe development program for target identification. If the mechanism of action involves alkylation, the covalent modification of the target will greatly facilitate target identification. In this context, an electrophilic probe, which is potentiallly an alkylating agent, would facilitate target identification using pull-down experiments.

We are currently working on the synthesis of a suitable biotinylated analog of the probe ML214 that could be used for pull-down experiments (see Scheme 2). Since aliphatic substituents are tolerated on the quinolinone’s nitrogen atom, we will use 4-chloro-3-nitroquinolin-2(1H)-one, an intermediate in the synthesis of the probe ML214, to alkylate the nitrogen with a functionalized PEG linker. First, we intend to prepare a N-acetyl version of it (Scheme 2A), and test it in the eight cell lines. If the compound is still active and selective for the A1-dependent cell lines, we would then prepare a biotin version (Scheme 2B) that could be used for target identification.

Scheme 2. Synthesis of a Probe Analog Possessing a Functionalized Linker on the Quinolinone’s Nitrogen Atom for Target Identification.

Scheme 2

Synthesis of a Probe Analog Possessing a Functionalized Linker on the Quinolinone’s Nitrogen Atom for Target Identification.

In the event that longer aliphatic chains are not tolerated on the quinolinone’s nitrogen atom, we envision positioning a functionalized linker on the eastern phenyl ring of the probe ML214 (see Scheme 3). Starting from 3-nitro-4-hydroxyquinolin-2(1H)-one, bromination will be performed using N-bromosuccinimide in sulfuric acid and afforded 61% of the desired 6-bromo product. This step will be followed by acetylation of the 4-hydroxyl group using acetic anhydride in pyridine. Methylation of the quinolinone’s nitrogen will then be performed using trimethyloxonium tetrafluoroborate. The 6-bromo substituent will be used to introduce a functionalized linker via a Suzuki coupling reaction. Once the functionalized linker is introduced, deprotection of the acetyl group will be performed with sodium methoxide in methanol. The obtained hydroxyl group will then be converted to the corresponding chloride using oxalyl chloride.

Scheme 3. Synthesis of a Probe Analog Possessing a Functionalized Linker on the Eastern Phenyl Ring.

Scheme 3

Synthesis of a Probe Analog Possessing a Functionalized Linker on the Eastern Phenyl Ring.

We aim to identify the target of the probe ML214 to understand its selectivity profile. This would greatly improve the utility of the probe ML214 and could allow the identification of new target proteins critical in A1-dependent cell lines.

4.3. Planned Future Studies

As described in Section 4.2, there are several routes for furthering the understanding of cancer cell survival and apoptosis using the novel research tool probe ML214. The outlined target identification method will determine whether the molecular target of the probe ML214 is A1 itself or a related protein. If A1 is the target, the binding site can also be determined, which may reveal a novel interaction site that confers specificity over other BCL-2-family members. More generally, viability profiling in additional cell lines, which is already underway, will further define the specificity and the scope of the activity of the probe ML214.

As a designated probe, the compound ML214 will, by default, be included in several planned studies at the Broad Institute that will characterize the MLSMR probe set. The Broad Institute Center Driven Research Project (CDRP) will profile probes, analogs, and a subset of less characterized compounds from the MLSMR using both multiparametric, image-based analysis and 1000-plex Luminex gene expression arrays in a well-defined cancer cell line (U2OS). In addition, probe compounds will be further characterized in 1000-plex gene expression arrays in up to 20 additional cell lines. These and other profiling approaches will continue to enhance the understanding of the mechanism of action and applicability of probes such as this probe ML214.

We envision that this probe ML214 will be a tool for the cancer biology research community for defining the role of apoptosis in oncology. Due to its chemical liabilities, we recognize that probe ML214 is intended to be a research tool and is not appropriate for therapeutic development; however, it is possible that probe ML214 may open new avenues for therapeutic development by elucidating a novel binding site or related target that allows specific targeting of A1 dependence in cancer.

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Appendix A. Compound Characterization

Table A1Summary of Completed Assays and AIDs

PubChem AID No.TypeTargetConcentration Range (μM)Samples Tested
AID 2465PrimaryMEF-A1-2A-BIM1023653
AID 2462PrimaryMEF-A1-2A-BIM10325633
AID 2765DR in primaryMEF-A1-2A-BIM10–501065
AID 449754CounterscreenMEF Bax/Bak−/−10–50255
AID 449755CounterscreenHMC-1-810–50255
AID 449761CounterscreenMEF WT10–50255
AID 449757SecondaryMEF-Flag-A1-IRES-BIM10–50255
AID 488858DR in primaryMEF-A1-2A-BIM0.068 – 15025
AID 488898CounterscreenMEF Bax/Bak−/−0.068 – 15025
AID 488885CounterscreenHMC-1-80.068 – 15025
AID 488902CounterscreenMEF WT0.068 – 15025
AID 488891SecondaryMEF-Flag-A1-IRES-BIM0.068 – 15025
AID 488948CounterscreenMEWO0.068 – 15025
AID 488914SecondaryMEWO-A1-TSA0.068 – 15025
AID 488897SecondaryMEF-A1-2A-tBid0.068 – 15025
AID 488934SecondaryMel 5010.068 – 15025
AID 504345DR in primaryMEF-A1-2A-BIM0.12 – 3021
AID 504344CounterscreenMEF Bax/Bak−/−0.12 – 3021
AID 504343CounterscreenHMC 1-80.12 – 3021
AID 504392CounterscreenMEF WT0.12 – 3021
AID 504342SecondaryMEF-Flag-A1-IRES-BIM0.12 – 3021
AID 504354CounterscreenMEWO0.12 – 3021
AID 504359SecondaryMEF-A1-2A-tBid0.12 – 3021
AID 504413DR in primaryMEF-A1-2A-BIM0.12 – 3021
AID 504403CounterscreenMEF Bax/Bak−/−0.12 – 3021
AID 504348CounterscreenHMC-1-80.12 – 3021
AID 504405CounterscreenMEF WT0.12 – 3021
AID 504409SecondaryMEF-Flag-A1-IRES-BIM0.12 – 3021
AID 504346CounterscreenMEWO0.12 – 3021
AID 504356SecondaryMEWO-A1-TSA0.12 – 3021
AID 504360SecondaryMEF-A1-2A-tBid0.12 – 3021
AID 504365SecondaryMel 5010.12 – 3021
AID 504347DR in primaryMEF-A1-2A-BIM0.015 – 3513
AID 504353CounterscreenMEF WT0.015 – 3513
AID 504415CounterscreenHMC-1-8 viability0.12 – 3021
AID 504407CounterscreenCHL-1 WT mitochondria10–501
AID 504412SecondaryCHL-1 A1-2A-BIM mitochondria10–501
AID 2526SummaryNANANA

NA=Not applicable

Appendix B. Detailed Assay Protocols

Protocol for Caspase Activation Measurement

  1. Maintain cells in 35 ml of growth medium (per 1 Liter: 890 ml RPMI-1640, 100 ml heat-inactivated fetal bovine serum (FBS) 10 ml Penn/Strep/Glutamine, 0.5–1 mg blasticidin) in a T175 cell culture flask.
  2. Incubate in a TC incubator at 95% humidity, 5% CO2, 37 °C.
  3. To passage, harvest cells by first aspirating the media and rinsing the flask with 10 ml sterile PBS.
  4. Aspirate PBS, add 5 ml trypsin to the flask, and incubate for 5 minutes at 22 °C.
  5. Add 8 ml of growth medium to the trypsin to quench the reaction.
  6. Resuspend the cells, count, and transfer approximately 4.5 million cells in approximately 2 ml to 33 ml fresh growth medium in a new T175 flask.
  7. For screening, harvest the cells and adjust concentration to 66,000 cells/ml in the above media without blasticidin. While gently stirring, dispense cells with a Combi Multidrop by adding 30 μl of suspension per well to white, sterile, TC-treated, 384-well plates for a total of 2000 cells per well. Incubate the plates overnight in an automated TC incubator at 95% humidity, 5% CO2, 37 °C.
  8. Add 50 or 100 nL of compound, depending on the desired final compound concentration, using slotted steel pins on a pin tool.
  9. Return the plates to the incubator for 3 hours.
  10. To read viability, remove the cells from the incubator and cool to room temperature for 30 minutes.
  11. Remove the lids and add 10 μl of diluted Caspase Glo (1:1 dilution with 50 mM HEPES buffer) to each well with a Combi Multidrop.
  12. Shake the plates at 1250 RPM for 15 seconds.
  13. Incubate for 1 hour at room temperature.
  14. Read luminescence on an Envision multimode reader (Ultrasensitive luminescence setting, 0.1 seconds per well).

Protocol for Cell Viability Measurement

  1. Maintain cells in 35 ml of growth medium (per 1 Liter: 890 ml RPMI-1640, 100 ml heat-inactivated fetal bovine serum (FBS) 10 ml Penn/Strep/Glutamine, 0.5–1 mg blasticidin) in a T175 cell culture flask.
  2. Incubate in a TC incubator at 95% humidity, 5% CO2, 37 °C.
  3. To passage, harvest cells by first aspirating the media and rinsing the flask with 10 ml sterile PBS.
  4. Aspirate PB, add 5 ml trypsin to the flask, and incubate for 5 minutes at 22 °C.
  5. Add 8 ml of growth medium to the trypsin to quench the reaction.
  6. Resuspend the cells, count, and transfer approximately 4.5 million cells in approximately 2 ml to 33 ml fresh growth medium in a new T175 flask.
  7. For screening, harvest the cells and adjust concentration to 66,000/ml in the above media without blasticidin. While gently stirring, dispense cells with a Combi Multidrop by adding 30 μl of suspension per well to white, sterile, TC-treated, 384-well plates for a total of 2000 cells per well. Incubate the plates overnight in an automated TC incubator at 95% humidity, 5% CO2, 37 °C.
  8. Add 50 or 100 nL of compound, depending on the desired final compound concentration, using slotted steel pins on a pin tool.
  9. Return the plates to the incubator for 24 hours.
  10. To read viability, remove the cells from the incubator and cool to room temperature for 30 minutes.
  11. Remove the lids and add 30 μl of diluted CellTiterGlo (1:3 dilution with 1X PBS) to each well with a Combi Multidrop.
  12. Incubate for 10 minutes at room temperature.
  13. Read luminescence on an Envision multimode reader (Standard luminescence setting, 0.1 seconds per well).

Protocol for Cytochrome c Release

  1. Maintain cells in 35 ml of growth medium (per 1 Liter: 890 ml RPMI-1640, 100 ml heat-inactivated fetal bovine serum (FBS) 10 ml Penn/Strep/Glutamine, 0.5–1 mg blasticidin) in a T175 cell culture flask.
  2. Incubate in a TC incubator at 95% humidity, 5% CO2, 37 °C
  3. To passage, harvest cells by first aspirating the media and rinsing the flask with 10 ml sterile PBS.
  4. Aspirate PBS, add 5 ml trypsin to the flask, and incubate for 5 minutes at 22 °C.
  5. Add 8 ml of growth medium to the trypsin to quench the reaction.
  6. Resuspend the cells, count, and transfer approximately 4.5 million cells in approximately 2 ml to 33 ml fresh growth medium in a new T175 flask.
  7. Centrifuge the cells at 1000 rpm at 4 °C for 4 minutes.
  8. Wash cell pellets with cold PBS once, and resuspend in 1X AT buffer (300 mM trehalose, 10 mM HEPES-KOH pH 7.7, 10 mM KCl, 1 mM EGTA, 1 mM EDTA and 0.1% BSA)
  9. Homogenize cells with a Potter Elvehjem homogenizer for 30–40 strokes at 1,600 rpm on ice.
  10. Centrifuge at 600g for 10 minutes at 4 °C.
  11. Collect supernatant and centrifuge at 7000g for 10 minutes at 4 °C.
  12. Carefully dislodge mitochondria pellets and resuspend in AT buffer with 80 mM KCl.
  13. Aliquot resuspended mitochondria.
  14. Incubate with appropriate compounds or controls for 30 minutes at 37 °C.
  15. Centrifuge at 7000 g for 10 minutes at 4 °C.
  16. Collect supernatant and dilute appropriate fold into the 96-well ELISA kit (R&D No. SCTC0) and detect cytochrome c according to kit protocol.
  17. Read absorbance at 450 nm in an M5e plate reader (Spectramax), normalized at 540 nm.

Appendix C. NMR and LC Data of Probe ML214 and Analogs

1H NMR (500 MHz, CDCl3) Spectrum of the Probe ML214

13 C NMR (125 MHz, CDCl3) Spectrum of the Probe ML214

UPLC chromatogram of the Probe ML214 Showing 99% Purity

Spectroscopic Data for SAR Analogs

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 776319 (Hit compound)

UPLC Chromatogram of Analog CID 776319 (Hit compound)

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49835867

UPLC Chromatogram of Analog CID 49835867

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 46926579

LCMS Chromatogram of Analog CID 46926579

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789901

UPLC Chromatogram of Analog CID 49789901

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789893

UPLC Chromatogram of Analog CID 49789893

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789892

UPLC Chromatogram of Analog CID 49789892

1HNMR(300 MHz, CDCl3) Spectrum of Analog CID 49789900

UPLC Chromatogram of Analog CID 49789900

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 3128936

UPLC Chromatogram of Analog CID 3128936

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 3125011

UPLC Chromatogram of Analog CID 3125011

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 769909

UPLC Chromatogram of Analog CID 769909

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 3408272

UPLC Chromatogram of Analog CID 3408272

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 3636391

UPLC Chromatogram of Analog CID 3636391

1HNMR 300 MHz, CDCl3) Spectrum of Analog CID 779355

UPLC Chromatogram of Analog CID 779355

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 2786843

UPLC Chromatogram of Analog CID 2786843

1HNMR (300 MHz, DMSO-d6) Spectrum of Analog CID 2785467

UPLC Chromatogram of Analog CID 2785467

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 800048

UPLC Chromatogram of Analog CID 800048

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789899

UPLC Chromatogram of Analog CID 49789899

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 800654

UPLC Chromatogram of Analog CID 800654

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789897

UPLC Chromatogram of Analog CID 49789897

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789891

UPLC Chromatogram of Analog CID 49789891

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789903

UPLC Chromatogram of Analog CID 49789903

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 46926574

LCMS Chromatogram of Analog CID 46926574

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 823866

LCMS Chromatogram of Analog CID 823866

1HNMR 300 MHz, CDCl3) Spectrum of Analog CID 14614200

UPLC Chromatogram of Analog CID 14614200

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 688979

UPLC Chromatogram of Analog CID 688979

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 1417123

UPLC Chromatogram of Analog CID 1417123

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 2888950

UPLC Chromatogram of Analog CID 2888950

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 38045

UPLC Chromatogram of Analog CID 38045

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 46926573

UPLC Chromatogram of Analog CID 46926573

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 46926575

UPLC Chromatogram of Analog CID 46926575

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789896

UPLC Chromatogram of Analog CID 49789896

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789902

UPLC Chromatogram of Analog CID 49789902

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789898

UPLC Chromatogram of Analog CID 49789898

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789895

UPLC Chromatogram of Analog CID 49789895

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789904

LCMS Chromatogram of Analog CID 49789904

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 49789894

UPLC Chromatogram of Analog CID 49789894

1HNMR (300 MHz, DMSO-d6) Spectrum of Analog CID 2785039

UPLC Chromatogram of Analog CID 2785039

1HNMR (300 MHz, CDCl3) Spectrum of Analog CID 763757

UPLC Chromatogram of Analog CID 763757

Appendix D. Compounds Provided to BioFocus

Table A2Probe and Analog Information

BRDSIDCIDP/AMLSIDML
BRD-K78867378-001-08-1SID 110167736CID 701939PMLS003370521ML214
BRD-K03857568-001-10-8SID 110167733CID 776319AMLS003370520NA
BRD-K39580048-001-03-9SID 110167735CID 2785467AMLS003370522NA
BRD-K08463963-001-10-1SID 110167734CID 800048AMLS003370524NA
BRD-K91349888-001-03-7SID 110167737CID 800654AMLS003370523NA
BRD-K32010074-001-01-5SID 99432298CID 46926575AMLS003370519NA

A=analog; NA=not applicable; P=probe

Appendix E. Cell line cytotoxicity data

Table A3Fractional viability of cell lines at various concentrations of ML214 (plotted in Figure 18).

Cells indicated by BCLX are engineered to overexpress antiapoptotic protein BCL-XL

[ML214] μ0.81.63.16.312.525.0
AU5650.90.80.70.70.50.0
AU565 BCLX0.80.90.80.91.00.2
CAL85-11.00.90.90.70.91.4
CAL85-1 BCLX0.80.90.80.61.01.3
EVSA-T0.90.90.80.70.60.4
EVSA-T BCLX0.80.91.01.01.11.3
HMC-1-80.80.80.80.70.50.6
HMC-1-8 BCLX0.80.80.90.90.80.8
MDA-MB-1570.90.90.70.40.10.0
MDA-MB-157 BCLX0.90.90.90.70.50.1
MDA-MB-2310.80.80.70.60.60.6
MDA-MB-231 BCLX0.80.90.90.80.70.7
SKBR30.80.80.70.80.40.2
ZR-75-10.80.90.90.90.80.7
G3610.80.80.70.30.30.4
G361 BCLX0.80.90.80.70.70.7
M140.80.80.80.60.60.5
M14 BCLX0.80.80.80.80.80.8
MEL5010.80.90.80.70.60.6
MEL501 BCLX0.80.80.80.70.70.7
UACC620.91.00.90.90.60.6
UACC62 BCLX0.90.90.90.80.90.9
DB0.50.60.70.70.70.5
DHL-40.60.70.50.20.20.1
DHL-50.30.00.00.00.00.0
DHL-60.70.60.20.00.00.0
DHL-70.70.80.20.10.00.0
DHL-80.60.70.80.70.30.3
DHL-100.60.50.30.10.00.0
FARAGE0.60.50.10.00.10.1
KARPOS 1106P0.40.20.00.00.00.0
KARPOS 4220.60.70.60.40.10.1
KMH20.60.70.80.70.40.4
L-12360.90.80.40.10.00.0
L-4280.80.80.80.80.90.9
LY10.70.40.40.40.30.4
LY100.60.40.00.00.00.0
LY180.60.70.50.20.10.1
LY190.70.70.40.10.10.1
LY30.70.80.90.80.70.7
LY40.70.60.10.00.00.0
PFIEFFER0.70.80.70.70.20.2
RCK80.70.70.60.40.20.2
TOLEDO0.60.40.00.00.00.0
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