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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Discovery and Development of Small Molecules That Reduce PNC Prevalence

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Author Information

The perinucleolar compartment (PNC) is a subnuclear body found at the periphery of the nucleolus in the nucleus. Its function is not completely known yet, but it is a highly dynamic body that is enriched with Ribonucleic acid (RNA) transcripts and RNA-binding proteins. This compartment is highly prevalent in metastatic tumors and metastatically transformed cancer cell lines, making it a potential pan marker for metastatic progression. A high throughput, high content assay was developed to identify novel small molecules that reduce the prevalence of this metastasis biomarker in cancer cells. We have identified and further optimized a potent pyrrolopyrimidine series that reduces PNC prevalence in PC3M cells at submicromolar concentrations, where the cell viability is not affected. These compounds also show dose-dependent inhibition of migration and anchorage-independent growth in invasion and soft-agar assays, respectively. The probe candidate, ML246, has drug-like physical properties and displays promising protein kinase, making it an ideal in vivo tool for establishing the link between the PNC and the metastatic transformation of tumor cells. ML246 will serve as a pivotal first step to further research the function of the PNC and its role in cancer metastasis.

Assigned Assay Grant #: R03MH082371

Screening Center Name & PI: NIH Chemical Genomics Center & Christopher P. Austin

Chemistry Center Name & PI: Kansas University & Jeffrey Aubé; NIH Chemical Genomics Center & Christopher P. Austin

Assay Submitter & Institution: Sui Huang, Northwestern University

PubChem Summary Bioassay Identifier (AID): 2431

Probe Structure & Characteristics

Image ml246fu1
CID/ML#Target NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 50985821/ML246PNCPNC Reduction (PC3M cells): 397 nM IC50 [SID 117695978, AID 540360]Cytotoxicity (ATP assay, PC3M cells)9960 nM IC50 [SID 117695978, AID 588740]25-foldMigration: 3.16 μM IC50 [SID 117695978, AID 588739]

1. Recommendations for Scientific Use of the Probe

The prevention and treatment of cancer metastasis is a prominent unmet medical need. Several molecular targets have been identified and explored for inhibition of the metastatic process, but these approaches have not yet yielded success in the clinic. The perinucleolar compartment (PNC) is a multicomponent subnuclear structure that is highly prevalent in metastatic tumors. [1, 2] Here, we present the discovery of a probe molecule that is able to disrupt the assembly of PNC in a metastatically transformed prostate cancer cell line without overt cytotoxicity. This structural class also impacts in vitro migration of tumor cells, making it useful for advanced cell-based studies. The probe displays promising in vitro ADME properties and in vivo mouse pharmacokinetics, making it an ideal candidate for understanding the therapeutic value of PNC disruption as a novel approach towards combating metastasis in cancer treatment.

2. Materials and Methods

The reagents and solvents used were commercial anhydrous grade and used without further purification. The column chromatography was carried out over silica gel (100–200 mesh). 1H NMR spectra were recorded with a Bruker 400 MHz spectrometer from solutions in CDCl3 and DMSO-d6. Chemical shifts in 1H NMR spectra are reported in parts per million (ppm, δ) downfield from the internal standard Me4Si (TMS, δ = 0 ppm). Chemical shifts in 13C NMR spectra are reported in parts per million (ppm, δ) calibrated from residual CHCl3 (δ = 77.0 ppm) signal and are reported using an APT pulse sequence displaying methyl and methine (CH3 and CH) signals as down, and quaternary and methylene (C and CH2) signals as up. Molecular weight confirmation was performed using an Agilent 6224 Time-Of-Flight Mass Spectrometer (TOF, Agilent Technologies, Santa Clara, CA). A 3 minute gradient from 5 to 100% Acetonitrile in water (0.03% formic acid) was used with a 5.1 minute run time at a flow rate of 0.4 mL/min. A Waters Atlantis T3 C18 column (1.8 micron, 2.1 × 50 mm) was used at a temperature of 25 °C. Confirmation of molecular formula was confirmed using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

2.1. Assays

High Content Assay for PNC Detection

The quantitative output for this assay is the reduction of PNC prevalence. The PNC can be detected in living cells by the expression of a green fluorescent protein (GFP) tagged to the PNC localized protein, PTB, as shown in Figure 1. We have created a PC3M cell line that stably expresses GFP-PTB to mark PNCs. This method eliminates the need for immunofluorescent staining. Previous studies demonstrate that the fusion proteins behave similarly to their endogenous counterparts: transient and stable over-expression of the fusion protein did not have detectable adverse effects on cell morphology or cell growth.[3] After treatment, cells are fixed and the nuclei are counterstained with Hoechst 33342 dye; the cells are then ready for analysis.

Figure 1. PTB-GFP PC-3M cells.

Figure 1

PTB-GFP PC-3M cells. PNCs are detected as intense, punctuated fluorescence dots at the perinucleolar regions, as indicated with the arrows.

The IN Cell Analyzer 1000 automated fluorescent imaging system (GE Healthcare, Piscataway, NJ) was used for automated image acquisition. Images were acquired with a 20× objective using a 475/20nm excitation filter, a 535/50 nm HQ emission filter, a Q505LP dichroic filter, and an exposure time of 100 to 150 ms (adjusted to obtain a dynamic range of ~200 to 1750), with no camera binning. The instrument acquired images of each well in a 1536-well plate with a laser-based autofocus system. To score PNC prevalence in a high-content throughput, we used the Multi-Target Analysis (MTA) algorithm (GE Healthcare, Investigator v3.5) to identify individual cells and granules (PNCs) within these cells. The nucleus was segmented via a region growing method (50 μm2 minimum area) with light shading and noise removal to allow “touching” nuclei to be separated. Granules in the nucleus (PNCs) were segmented using a multiscale top hat method, which measures granules of 1 to 2 μm in size and used a smart masking method to identify the boundaries of each segmented granule. The algorithm was optimized and validated using positive and negative controls (50 μM camptothecin and DMSO, respectively). In particular, the MTA algorithm allows for the identification of multiple subcellular compartments and organelles (or granules) within those compartments. In this instance, we took advantage of the algorithm’s capability to identify objects within the same color channel that only differ in size or fluorescent intensity. Also, the algorithm allows for building complex hierarchical classification systems, using output measures within the algorithm to filter and define subpopulations. For this particular assay, PNC-positive cells were scored when 1 to 3 PNC granules were detected per nucleus. Cells that contained 0 granules were scored as PNC negative, and cells with >3 granules were assumed to be false positives (very few cells have more than 3 PNCs in one focus plane), and were also scored as PNC negative.

1Cells5 μL750–1000 cells/well
2Time4 hrsIncubate at 37 ºC and 5% CO2
3Reagent23 nLCompound library, camptothecin as control
4Time16 hrsIncubate at 37 ºC and 5% CO2
5Reagent4 μLFixation step with 6% EM grade paraformaldehyde and 0.1% Triton X-100
6Time20 minRT incubation
7Wash5 μLLiquid was aspirated and 5μL of PBS was added
8Wash5 μLLiquid was aspirated and 5μL of PBS was added
9Reagent5 μLStaining with PBS containing 1 μg/mL Hoechst 33342
10DetectorFluorescenceIN Cell 1000, 20 × objective

BellBrooks Labs® Tumor Cell Migration Assay

High-content tumor cell migration assays in 3-dimensional extracellular matrices are powerful tools for modeling and understanding the biology of this critical step in the process of metastasis. However, most of the available methods are not amenable to the throughput required by studies of comparative pharmacology or small scale screening. For this reason, we worked with BellBrook Labs® to test our compounds in their automated high-content tumor cell invasion assays (Figure 7).[4] A standard screening-sized plate with an array of embedded microchannels was designed and constructed from common thermoplastics.

Figure 7. A) Brief outline of BellBrook Labs® assay.

Figure 7

A) Brief outline of BellBrook Labs® assay. B) Invasion dose response curves for ML246 and an inactive analog with PC3M cells.

PC3M cells were tested for invasion through 3D fibrillar collagen in the Iuvo Single Microchannel Plate, in the presence of varying levels of test compounds. Channels were prefilled with 820 nL of 3-dimensional type I collagen at 1 mg/mL, through the input port. Following gelation, 2,000 PC3M cells were seeded into the output port using growth media (RPMI + 10% FBS with antibiotics) in a volume of 5 μL. Cells were incubated in a 37 ºC incubator inside a humidified container to control evaporation (Bioassay dish, Corning). Media, including test compounds, was changed daily for 5 days. At the end of the assay, cells were fixed and stained with Hoechst 33342, then imaged with 4× objective under epifluorescence. Under these conditions, we can reliably identify cells across the 140 μm height range of the microchannel. Test compounds were serially diluted by a factor of 3 to produce 10 concentrations ranging from 50 or 100 μM to 2.5 nM. All assays were conducted in the presence of 0.1% DMSO. Four replicates were performed for all test concentrations. Each plate had 4 dose response curves, as well as 16 channels with no compound and 16 channels with 50 μM blebbistatin (positive control). Analysis was done by automatically cropping each image at the right edge of the channel and counting cells via the ‘count nuclei’ function on Metamorph (Molecular Devices). Non-linear regression analysis was performed with GraphPad Prism.

Soft Agar Assay

In vitro cellular transformation detection assays are semi-quantitative and measure the morphological transformation of cell colonies modulated by chemicals. This transformation is associated with certain phenotypic changes, such as loss of contact inhibition (cells can grow over one another) and anchorage independence (cells form colonies in soft agar). Anchorage independence can be described in the light of primary fibroblasts and many other cell lines (e.g. BALB/c3T3, NIH-3T3, etc.) that must attach to a solid surface before they can divide. They fail to grow when suspended in a viscous fluid or gel (e.g. agar or agarose); however, when these cell lines are transformed, they are able to grow in a viscous fluid or gel and become anchorage-independent. This process, by which these phenotypic changes occur, is assumed to be closely related; hence, it is a good predictor of in vivo carcinogenesis.

PC3M cells were gently suspended in 0.35% agar in serum/antibiotic free RPMI supplemented with 10% FBS, 1% penicillin-streptomycin, and compounds whose concentrations was based on the EC10 and EC20. Then, 5000 cells/mL per well were seeded onto 1:1 mixture of 1% agar RPMI containing supplements into 6-well plates. After the top layer had dried, the agarose was overlaid with 3 mL RPMI +10% FBS and drug concentration at EC10 or EC20. The overlaying media was changed twice per week. After 14 and 21 days, the number and size of colonies were visualized and measured, using 40× and 20× objectives. Triplicate wells were used for every treatment condition, and the same field was used for analysis and then averaged. After counting and measuring the colonies, the soft agar cell plates were incubated for another 21 days to observe a phenotype.

ATP Quantitation Assay (viability assay)

We conducted this follow-up assay to measure the effect of compounds on cell health by measuring ATP levels (ATPLite™). ATPLite™ is an Adenosine TriPhosphate (ATP) monitoring system based on firefly (Photinus pyralis) luciferase. The level of ATP in a metabolically active cell is a general marker for its viability. ATP levels are often reduced during necrosis or apoptosis. In this assay, the luciferase enzyme catalyzes the conversion of the added substrate D-luciferin to oxyluciferin and light with ATP. Thus, the emitted light is proportional to the ATP concentration. For this assay, the highly metastatic PC3M reporter cell line stably expressing the PTB-GFP was provided by Professor Sui Huang of Northwestern University. The media and cell culture reagents were purchased from Invitrogen (Carlsbad, CA), ATPLite™ came from PerkinElmer.

1Cells5 μL2000 cells/well
2Time4 hrsIncubate at 37 ºC and 5% CO2
3Reagent23 nLCompound library, camptothecin as control
4Time24 hrsIncubate at 37 ºC and 5% CO2
5Reagent3 μLATPLite
6Time20 minRT incubation
7Centrifuge1 min1500 RPM

PC3M Caspase 3/7 Activation Assay

This assay was used as an orthogonal measurement of cytotoxicity of the compounds that reduced PNC prevalence. The activation of caspases is a pivotal step during apoptosis, because these cysteine-dependent, aspartate-directed proteases enable the systematic structural disassembly of dying cells. Their activation is typically triggered by a variety of stimuli, including growth factor withdrawal, exposure to radiation or chemotherapeutic agents, or initiation of the Fas/Apo-1 receptor-mediated cell death process. The caspase-3/7 substrate rhodamine 110, bis-(N-CBZL- aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide; Z-DEVD-R110) is a pro-fluorescent substrate. To perform the Apo-ONE® Homogeneous caspase-3/7 Assay, the buffer and substrate are mixed and added to the sample. Upon sequential cleavage and removal of the DEVD peptides by caspase-3/7 activity and excitation at 499 nm, the rhodamine 110 leaving group becomes intensely fluorescent. The emission maximum is 521 nm.

The media and cell culture reagents were purchased from Invitrogen (Carlsbad, CA), and Caspase Glo 3/7 came from Promega. The highly metastatic PC3M-GFP reporter cell line was provided by Professor Sui Huang of Northwestern University.

1Cells5 μL2000 cells/well
2Time4 hrsIncubate at 37 ºC and 5% CO2
3Reagent23 nLCompound library, camptothecin as control
4Time24 hrsIncubate at 37 ºC and 5% CO2
5Reagent3 μLCaspase Glo 3/7
6Time5 minRT incubation
7Centrifuge1 min1500 RPM

PicoGreen Assay

The pilot screen for PNC prevalence reduction revealed several small molecules that acted as PNC reducers by intercalating DNA. Therefore, we conducted a DNA intercalation displacement assay on the lead candidates in order to exclude compounds with this mechanism of action. PicoGreen is a DNA intercalator that becomes excitable at 488 nm and emits at 520 nm when bound to DNA. Compounds that displaced the dye decreased fluorescence intensity and were considered DNA intercalators/displacers. In order to rule out potential fluorescence artifacts, a measurement of compound plus DNA only was also calculated.

1Reagent2 μLSupercoiled plasmid (pBR322) at 200 ng/mL in TE
3Reagent23 nLCompound library, camptothecin as control
4Time30 minRT incubation
5DetectorFluorescenceBaseline signal on Envision Ex488/Em520
6Reagent2 μLPicoGreen in TE
7Time10 minIncubate at 37 ºC
8DetectorFluorescenceEnvision Ex488/Em520

2.2. Probe Chemical Characterization

Probe Characterization of ML246.

Probe Characterization of ML246

*Purity 100% as determined by LC/MS and 1H NMR analyses

Trans-4-(7-benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl) cyclohexanol: LC/MS (Agilent system) Retention time t1 (long) = 2.73 min; 1H NMR (400MHz, DMSO-d6) δ1.66 (m, 4 H), 2.13 (d, J = 8.8 Hz, 4 H), 3.70 (m, 1 H), 5.14 (m, 1 H), 5.28 (s, 2 H), 6.45 (br s, 1 H),6.95 (m, 2 H), 7.04 (d, J = 6.8 Hz, 2 H), 7.18–7.26 (complex, 11 H), 7.80 (s, 1 H); HRMS (ESI) m/z calculated for C31H31N4O [M + H]+ 475.2498 found 475.2492.

Figure 2. LC/MS chromatogram of probe.

Figure 2LC/MS chromatogram of probe

Figure 3. 1H NMR and 13C APT NMR spectra of probe.

Figure 31H NMR and 13C APT NMR spectra of probe

Figure 4. A Stability of ML246 in D-PBS pH 7.

Figure 4

A Stability of ML246 in D-PBS pH 7.4 PBS at room temperature evaluated by LC/MS/MS. B. Stability of ML246 in mouse plasma at room temperature evaluated by LC/MS/MS.

Table 1Summary of Key Properties for ML246

CompoundAqueous Kinetic Solubility (μM)Stability in PBS buffer (% remaining at 48 hours)Stability in mouse plasma (% remaining at 2 hours)

Table 2Probe and analog submissions to the MLSMR (Evotec) for Identification of Compounds that Reduce PNC Prevalence

Internal IDMLS IDSIDCIDML#TypeSource
NCGC00247785MLS003678589SID 117695978CID 50985821ML246ProbeKansas
NCGC00244845MLS003678590SID 116933572CID 50985817AnalogKansas
NCGC00247790MLS003678591SID 117695983CID 51035471AnalogKansas
MLS000556915MLS003678592SID 113635282CID 5152963AnalogKansas
NCGC00247775MLS003678593SID 117695968CID 51035453AnalogKansas
NCGC00244849MLS003678594SID 116933577CID 50985820AnalogKansas
Figure 5. Structures of compounds submitted to the MLSMR compound repository.

Figure 5Structures of compounds submitted to the MLSMR compound repository

2.3. Probe Preparation

Scheme 1. Reagents and conditions.

Scheme 1Reagents and conditions

(a) benzyl amine, zinc chloride (cat), neat; (b) malononitrile, DMF (46%, two steps); (c) triethylorthoformate, neat (64%); (d) trans-4-aminocyclohexanol hydrochloride, MeOH (34%).

Preparation of trans-4-(7-benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl) cyclohexanol (ML246).

Preparation of trans-4-(7-benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl) cyclohexanol (ML246)

2-Amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile (C). A modified Voigt reaction/Knoevenagel condensation sequence was carried out using the procedure of Roth and Eger.[5] Thus, benzoin A (2.19 g, 10.3 mmol), benzylamine (1.66 g, 15.5 mmol, 1.5 equiv.), and zinc chloride (0.10 g, 0.73 mmol, 0.07 equiv.) were heated at reflux for 3 hours and the mixture was removed from the oil bath. Malononitrile (1.35 g, 20.6 mmol, 2.0 equiv.) in DMF (3 mL) was added to the mixture, which was still warm. The reaction mixture was allowed to cool to room temperature and stirred for 16 hrs, affording the crude pyrrole as a dark brown solid. The solid was partitioned between water and CH2Cl2, and the aqueous layer was extracted with additional CH2Cl2 (2×50 mL). The combined organics were dried with Na2SO4, and the solvent removed in vacuo to afford the previously reported pyrrole product C[6] as a light brown solid (1.67 g, 4.78 mmol, 46% yield), which was used without further purification.

Image ml246fu4

(E)-Ethyl N-(1-benzyl-3-cyano-4,5-diphenyl-1H-pyrrol-2-yl)formimidate (D). 2-Amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile C (1.07 g, 3.06 mmol) and triethylorthoformate (4.54 g, 30.6 mmol, 10 equiv.) were heated at 75 °C for 14 hrs, and the excess triethylorthoformate was removed in vacuo. The residue was dissolved in a minimum of CH2Cl2, adsorbed onto celite, and chromatographed on silica to afford the previously reported formimidate product[6]D as a tan solid (0.80 g, 1.97 mmol, 64% yield).

Image ml246fu5

trans-4-(7-Benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)cyclohexanol (CID 5098582, ML246). A solution of the formimidate D (40 mg, 0.099 mmol) and trans-4-aminocyclohexanol hydrochloride (23 mg, 0.15 mmol, 1.5 equiv) in MeOH (1.5 mL) was heated in a reaction vial at 60 ºC for 17 hrs, and then cooled to room temperature. Evaporation of the solvent and purification of the residue by mass-directed preparative reverse-phase HPLC afforded the pyrolopyrimidine product, ML246, as a tan solid (16 mg, 0.034 mmol, 34% yield).

3. Results

3.1. Dose Response Curves for Probe

Figure 6. PNC reduction dose response curve for ML246.

Figure 6PNC reduction dose response curve for ML246

3.2. Cellular Activity

After activity optimization in the PNC assay, it was necessary to examine if these compounds had an effect on migration. Table 3 summarizes preliminary results obtained in an invasion assay with ML246. An examination of the number of cells that have invaded through the 3D collagen channel reflects their migratory aptitude. Gratifyingly, the novel PNC-reducer probe ML246 showed dose dependent inhibition of migration with IC50s 3–4 μM. The numbers of cells in the output port after the assay duration were also counted as a measurement of proliferation. Here too, the probe showed dose-dependent inhibition activity, suggesting that the anti-invasive effect of the compound is concomitant with an anti-proliferative effect. With this data in hand, we measured the cytotoxicity of the probe (via the ATP viability assay, AID 588740) to be 9,960 nM. The ATP data and the pattern of activity demonstrated in the NCI-60 panel (vide infra, Figure 9) lead us to conclude that the cell proliferation modulation, especially at the concentrations causing PNC reduction, is mostly likely due to growth inhibition and not cytotoxicity.

Table 3. BellBrook Labs Proliferation and Migration Data for Key Compounds.

Table 3

BellBrook Labs Proliferation and Migration Data for Key Compounds.

Figure 9. NCI-60 panel: Dose response growth inhibition curves for ML246 in 60 cancer cell lines.

Figure 9

NCI-60 panel: Dose response growth inhibition curves for ML246 in 60 cancer cell lines. The curves are grouped according to the type of cancer. Concentrations able to produce a percentage of growth below zero indicate loss of cell density and can be correlated (more...)

We also wanted to test the ability of the chemical series to affect anchorage independent growth in a soft agar assay, a stringent method to detect malignant transformation of cells in vitro. To that end, Figure 8 depicts our initial assessment of ML246 in such an assay with PC3M cells. As shown in panel 8A, ML246 demonstrates a dose dependent reduction in the number of colonies after 14 days at very low concentrations (3.8, 18.6 nM), with no impact on cell viability. Thus, the probe exhibits potent inhibition of anchorage independent growth in PC3M cells. Figure 8B shows two images of representative colonies after treatment at the relevant concentrations; a clear reduction if the number of PC-3M cell colonies can be seen especially at 18.6 nM.

Figure 8. A: Histogram representing number and size (μm) of PC3M soft agar colonies after 14 days treatment with ML246 at 3.

Figure 8

A: Histogram representing number and size (μm) of PC3M soft agar colonies after 14 days treatment with ML246 at 3.8 and 18.6 nM. B: Representative images of colonies at these concentrations.

3.3. Profiling Assays

ML246 is a novel chemical entity and has no previously disclosed interactions with biological targets. The probe has been screened against 44 GPCR and CNS relevant targets in the PDSP comprehensive binding panel (Table 4). With the exception of the sigma 2 receptor, ML246 was mostly inactive against all these targets. Notably, this includes dopaminergic activity, as the original hit (8) was found to be an allosteric D1 agonist in PubChem AID 504660.

Table 4. Profiling of the probe, ML246, in the PDSP comprehensive panel.

Table 4

Profiling of the probe, ML246, in the PDSP comprehensive panel.

ML246 has good stability in PBS buffer. It also has good stability in mouse plasma and is not rapidly metabolized by mouse liver microsomes in the presence of NADPH (Table 5). At a 50 mpk high dose, the probe provided significant in vivo exposure in mice, with a Cmax of 20.3 μM @ 30 min and a Clast of 1.3 μM @ 48 hrs. Some mortality was observed (3/18), but this is expected, as the exposure is well beyond the expected therapeutic levels. Subsequent experiments with 25 mpk intraperitoneal (IP) dosing provided very good exposure without any sign of toxicity (Table 6).

Table 5. In vitro ADME data for ML246.

Table 5

In vitro ADME data for ML246.

Table 6. Comparison of Mouse Pharmacokinetics Parameters of Probe after IP Dosing of 50 or 25 mpk in Male C57BL/6 Mice.

Table 6

Comparison of Mouse Pharmacokinetics Parameters of Probe after IP Dosing of 50 or 25 mpk in Male C57BL/6 Mice.

The NCI-60 panel is a collection of human cancerous cell lines used by the National Cancer Institute as a profiling tool for the development and screening of novel anticancer drugs.[5] This panel allows the evaluation of compounds in 60 cell lines of various origins and compares the pattern of activity with other known anticancer agents to impute a possible mechanism of action. Compounds are tested at a single concentration (10 μM) first, and if a growth was below 40% in at least eight cell lines, they are sent for dose-response testing. The dose response curves for ML246 are displayed in Figure 9.

In general, we observed potent dose dependent growth inhibition ≥1 μM concentration across all cell lines. At the highest concentration (100 μM), we observed cell death (as represented by curves below 0% percentage growth) in all cell lines. Thus, the chemical series appears to exhibit growth inhibitory effects at concentrations that cause reduction of the PNC. Fortunately, cytotoxicity is observed only at higher concentrations, and it should be possible to decouple this effect from PNC disruption with proper dosage.

4. Discussion

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

Existing PNC reducers are chemotherapy drugs that induce PNC disruption by known cytotoxic mechanisms, like topoisomerase or polymerase III inhibition; or DNA intercalation. Drugs used against metastatic cancers also operate through such cytotoxic mechanisms. Hence, we chose to develop PNC reducers that did not show activity in assays which measured cytotoxicity via these mechanisms. Extensive SAR explorations yielded a probe molecule that is able to reduce the PNC population in PC3M cells at submicromolar concentrations with cytotoxicity observed at ~10μM (25-fold selectivity). The probe shows ~10-fold selectivity between growth inhibition and cytotoxicity in the NCI-60 panel. Thus the chemical series represents first-in-class molecules that reduce the prevalence of the PNC and do not operate via known cytotoxic mechanisms.

5. References

Kamath RV, Thor AD, Wang C, Edgerton SM, Slusarczyk A, Leary DJ, Wang J, Wiley EL, Jovanovic B, Wu Q, Nayar R, Kovarik P, Shi F, Huang S. Cancer Res. 2005;65(1):246–53. [PubMed: 15665301]
Slusarczyk A, Kamath R, Wang C, Anchel D, Pollock C, Lewandowska MA, Fitzpatrick T, Bazett-Jones DP, Huang S. Cold Spring Harb Symp Quant Biol. 2010;75:599–605. [PMC free article: PMC4374480] [PubMed: 21289045]
Kamath RV, Leary DJ, Huang S. Mol Biol Cell. 2001;12:3808–20. [PMC free article: PMC60757] [PubMed: 11739782]
Roth HJ, Eger K. Arch. Pharmaz. 1975;308:179–185. [PubMed: 1130955]
Mohamed MS, Rashad AE, Zaki MEA, Fatahala SS. Acta Pharm. 2005;55:237–249. [PubMed: 16375835]


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