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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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Probe Reports from the NIH Molecular Libraries Program [Internet].

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Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays

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Author Information

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

1 University of New Mexico Center for Molecular Discovery
2 Carnegie Mellon University Technology Center for Networks and Pathway
3 Vanderbilt Specialized Chemistry Center, Vanderbilt University Medical Center

Received: ; Last Update: March 22, 2013.

A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. A highly potent compound (ML342, CID 2953239) was declared as a Molecular Libraries Probe Center Network (MLPCN) probe molecule.

Assigned Assay Grant #: R03 DA031668-01

Screening Center Name & PI: University of New Mexico Center for Molecular Discovery, Larry A. Sklar

Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center, Craig Lindsley

Assay Submitter & Institution: Jonathan Jarvik, Carnegie Mellon University

PubChem Summary Bioassay Identifier (AID): 651701

Probe Structure & Characteristics

ML342.

ML342

CID/ML#Target NameEC50 (nM) [SID, AID]Antitargets Name(s)Activity [SID, AIDs]Fold SelectiveCytotoxicityReversibilitySoluble Fluorogen Binding
CID 2953239/ML342FAP-tag2.24±0.51 nMMG13Inactive

AID 651698
AID 651873
>800non-cytotoxic at 100 μM

AID 651694
Reversible

AID 651936
Active

AID 651938
SID 125240931
SID 136348701

AID 588775, AID 651872

1. Recommendations for Scientific Use of the Probe

This probe (ML342, CID 2953239, SID 125240931) is a potent disruptor of the activation of thiazole-orange fluorogens TO1-2p by FAP AM2.2 and is selective as compared to the MG13 FAP-tag that activates fluorogen malachite green.

FAP tags can be genetically fused to proteins of interest. These fusion proteins are not fluorescent except when exposed to fluorogen, whereupon the fluorogen binds to the FAP and its fluorescence immediately increases by as much as 20,000-fold rendering the FAP fusion protein highly fluorescent. Fluorogens can be made membrane impermeant, thus allowing detection of only those receptors expressed on the cell surface. Researchers in both basic biology and in high throughput screening can benefit greatly from this new technology (5,6). There is currently no inhibitor of the single chain human antibody (scFv) AM2.2 FAP-tag that blocks the binding of its fluorogen, thiazole orange. Probes that specifically inhibit or reverse fluorogen activation by a FAP will have a number of immediate applications.

  1. Study of receptor proteins – By adding probe, one may selectively eliminate the fluorescence signal from FAP-tagged receptor molecules at the cell surface without affecting internalized signal. This enables more accurate quantitation of receptor (GPCR) internalization post-stimulation. We have demonstrated the utility of such an approach using trypsin treatment to remove the surface signal. Trypsin indiscriminately digests all surface proteins however, which can produce a variety of off-target effects. Use of the new probe in place of trypsin is expected to yield fewer false positives than the current trypsin-based protocols.
  2. Pulse-chase experiments – Enablement of multi-color pulse-chase experiments in which signal from one FAP but not another is specifically eliminated by the addition of the probe. By splitting the sample and adding probe over time, it would be possible to assess the level of extracellular and intracellular protein.
  3. High throughput screening - The proposed probe can be used as a baseline control for high throughput assays. This is particularly useful for assays where there is no known inhibitor of the target protein. In addition, the new probe would be used to make more precise measurements of the fraction of fluorescent signal that is due to background fluorescence. This will be done by measuring the fluorescence of cells that have been treated with fluorogen in the presence of excess eraser compound and subtracting this background value from the values collected from cells treated with fluorogen only. This more accurate determination of the net pre-stimulation and post-stimulation signal should lead to higher Z′ factors, which should permit the identification of weak (but genuine) agonists and antagonists in the library which would otherwise be missed.
  4. In vivo - The FAP-tags are likely to find utility in vivo. The total FAP signal would represent the amount of labeled protein. Through additional SAR and targeted design it may be possible to develop new impermeable probes to distinguish the amount of protein available to the extracellular from the intracellular environment.

2. Materials and Methods

2.1. Assays

HTS for Beta-2AR agonists (UNMCMD)

This is the primary assay to measure fluorescence signal decrease induced by sample compounds blocking the interaction between fluorogen TO1-2p and AM2.2.

  1. Spin down AM2.2-beta2AR cells, discard supernatant, and resuspend in fresh RPMI1640 full medium. Final cell density will be 5×106 cells/mL.
  2. Add 5 μL serum free RPMI to the assay plate except for columns 11 and 23 by Microflo.
  3. Add 5 μL of freshly prepared 32 μM ISO in RPMI full media to Column 11 and 23 of all the plates as PCntrls by Microflo.
  4. Add 100 nL of library compounds between 3 nM and 100 μM (final concentration) to assay plates by FX.
  5. Add 3 μL of cells to Columns 1 – 11, 13–23 of the assay plates by Microflo.
  6. Shake the plates and put them in 37°C incubator for 90 mins.
  7. Add 3 μL 650nM TO1-2p to assay plates by Microflo or Nanoquot to assay plates and read by high-throughput flow cytometers immediately.
Calculation

If EC50 is reported and EC50 < 10 then PUBCHEM_ACTIVITY_SCORE = 10

Otherwise PUBCHEM_ACTIVITY_SCORE = 100

IF PUBCHEM_ACTIVITY_SCORE <= 10 then PUBCHEM_ACTIVITY_OUTCOME = 2 (or active)

IF PUBCHEM_ACTIVITY_SCORE >10 then PUBCHEM_ACTIVITY_OUTCOME = 1 (or inactive)

High throughput counter screen assay with AM2.2-β2AR/MG13-CCR5 cells (UNMCMD)

This is a counter screen for the beta-2AR screens. This assay measures binding of fluorogen MG-2p to FAP MG13-tagged mouse CCR5 in addition to the binding of TO1-2p to FAP AM2.2-tagged β2AR in the presence of test compounds to assess if the compound interferes the binding between fluorogen MG-2p and FAP MG13.

  1. Spin down AM2.2-β2AR/MG13-mCCR5 dual expressing cells, discard supernatant, and resuspend in fresh RPMI1640 full medium. Final cell density will be 5×106 cells/mL.
  2. Add 5 μL serum free RPMI to Columns 2–24 of the assay plate by Nanoquot.
  3. Add 5 μL of freshly prepared Rantes in RPMI full media to Column 1 of all the plates as PCntrls by Microflow.
  4. Add 100 nL of library compounds to assay plates by FX or NX.
  5. Add 3 μL of cells to Columns 1 – 22 of the assay plates by Nanoquot.
  6. Shake the plates and put them in 37°C incubator for 90 mins.
  7. Add 3 μL 650nM TO1-2p/210 nM MG-2p mixture to assay plates by Microflow or Nanoquot to assay plates and read by high-throughput flow cytometers immediately.
Calculations

Median Channel fluorescence is calculated from flow cytometric data by HyperView (IntelliCyt, Albuquerque, NM). These values for the entire concentration range of a test compound are fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics are used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.

Compounds with percent viability at the highest concentration is less than 50% are labeled active and the PubChem_Score is calculated based on EC50 of cytotoxicity by the following equation:

PubChem Score = 100 * (1 − EC50/30 μM)

Fluorogen binding competition assay using AM2.2-β2AR cells and AM2.2-GPR32 cells (UNMCMD)

This is a counter screen for the beta-2AR screens. This assay measures binding of fluorogen to FAP-tagged GPR32 in the presence of test compounds to assess whether the compound interferes with FAP binding.

  1. Spin down AM2.2-GPR32 cells, discard supernatant, and resuspend in fresh RPMI1640 full medium. Final cell density will be 5×10^6 cells/mL.
  2. Add 5 μL serum free RPMI to Columns 2–24 of the assay plate by Nanoquot.
  3. Add 5 μL of RPMI full media to Column 1 of all the plates, i.e., there is no Positive control.
  4. Add 100 nL of library compounds to assay plates by FX or NX.
  5. Add 3 μL of cells to Columns 1 – 22 of the assay plates by Nanoquot.
  6. Shake the plates and put them in 37 °C incubator for 90 mins.
  7. Add 3 μL 650 nM TO1-2pto assay plates by Microflo or Nanoquot to assay plates and read by high-throughput flow cytometers immediately.
Calculations

Median Channel fluorescence is calculated from flow cytometric data by HyperView (IntelliCyt, Albuquerque, NM). These values for the entire concentration range of a test compound are fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics are used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.

Compounds with EC50 less than 10 μM are labeled active and the PubChem_Score is calculated based on EC50 by the following equation:

PubChem Score = 100 * (1 − EC50/10 μM)

Fluorogen/soluble FAP binding competition assay (CMU)

Competition binding assays are performed by adding differing amounts of compounds to mixtures of soluble AM2.2 (FAP-tag) and TO1-2p (fluorogen), and the fluorescent signal measured by spectrofluorometry.

Compound cytotoxicity in U937 cells (UNMCMD)

This assay is used to determine whether a compound is causing a decrease in signal by killing the cell instead of actually inhibiting beta2AR internalization.

CellTiter-Glo, a luminescent cell viability assay kit from Promega (Madison, WI), will be used according to the manufacturer’s instruction. Briefly, the cell cultures of AM2.2-beta2AR cells are seeded in complete medium at twelve different cell densities in 96-well white polypropylene opaque plates (50 μL/well per 384 well) (Corning, Corning, NY). At the time of passage, 90 μL of cell suspension (10^5 cells/mL) are added into the plates. After stabilization for 2h, test compounds are added to the wells at 10 μL per well to a final concentration range of 380 nM to 100 μM. Vehicle control wells contain 0.01% DMSO alone. Following treatment, cells are incubated at 37°C and 5% CO2 for 18 hours. At the end of the respective time point, 1x CellTiter-Glo is added to each well (10 μL/well per 384 well). Plates are read after 30 minutes. Luminescence intensity (LI) is collected using a Wallac 1420 plate reader (PerkinElmer, Norwalk, CT).

Calculations

Background luminescence were subtracted from all readings and then luminescence values for the entire concentration range of a test compound were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the following parameters of the model: EC50, μM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.

Compounds with percent viability at the highest concentration is less than 50% are labeled active and the PubChem_Score is calculated based on EC50 of cytotoxicity by the following equation:

PubChem Score = 100 * (1 − EC50/50 μM)

Compound reversibility (UNMCMD)

This is a secondary assay to determine whether the binding between sample compound and the FAP is covalent. Binding between non-covalent compounds and the FAP is reversible.

  1. Spin down AM2.2-GPR32 cells and resuspend in serum free RPMI media and yielded a final cell density of 5×105/mL.
  2. Add 99 μL of cells to two sets of assay tubes followed by 1 μL of 100 × compound in DMSO or 1 μL of DMSO only for NCntrls to both sets of tubes.
  3. Mix well by mild vortexing and keep it at 37°C incubator for 90 mins.
  4. Spin down cells from one set of the assay samples, discard supernatant, wash the cells in 500 μL of serum free RPMI, then spin and discard supernatant. Resuspend the cells in 100 μL RPMI.
  5. Add 150 nM TO1-2p to the cells and read by Accuri flow cytometer immediately.

2.2. Probe Chemical Characterization

Synthetic procedure and spectral data for ML342 (CID 2953239, SID 125240931, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide:

Probe compound ML342 (CID 2953239) was prepared according to Scheme 1 and provided the following characterization data: LCMS (>99% 215 nm, 254 nm), Rt = 0.65 min; m/z (M+H)+ = 389. 1H NMR (400 MHz, CDCl3) δ 8.1–8.2 (m, 1H), 7.9–8.0 (m, 1H), 7.7 (d, J=8.2 Hz, 2H), 7.4 (d, J=8 Hz, 2H), 7.1 (d, J=9.2 Hz, 1H), 7.0 (t, J=6.5 Hz, 1H), 3.8–4.0 (m, 10H), 2.8 (s, 3H), 2.5 (s, 3H); 13C NMR 166.2, 152.3, 144.1, 144.1, 138.1, 133.0, 129.9, 128.9, 127.5, 113.3, 111.8, 52.2, 45.9, 45.7, 43.8, 40.8, 35.7, 21.4; HRMS (ESI) m/z 389.1646 ([M+H]+, 100%) calcd for C19H25N4O3S, 389.1647.

Scheme 1. Synthesis of ML342.

Scheme 1

Synthesis of ML342.

Solubility. In-house solubility assay in PBS at pH 7.4 was determined for ML342 to be 95.1±2.7 μM or 38 μg/mL based upon triplicate testing. ML342 shows excellent solubility up to 10 mM DMSO. Overall ML342 is in the moderate to highly soluble range for an in vitro tool compound at neutral pH.

Stability. Stability was determined for ML342 in PBS buffer at room temperature with time course evaluation over 48h. After 1.5 hour, the percent of parent compound remaining was >95%. After 24 and 48h, there is <10% apparent loss of compound. Thus ML342 appears to have excellent stability after prolonged exposure to PBS buffer.

Compounds added to the SMR collection (MLS#s): MLS004645659 (ML342, CID 2953239, 24.5 mg); MLS004645660 (CID 16323094, 8.3 mg); MLS004645661 (CID 3164412, 9.7 mg); MLS004645662 (CID 16365258, 7.7 mg); MLS004645663 (CID 17477879; 9.1 mg); MLS004645664 (CID 1001608, 5.4 mg)

2.3. Probe Preparation

Image ml342fu2

Step 1. Preparation of 2-bromo-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (1)

To a round bottom flask equipped with a magnetic stir bar was added bromoacetyl bromide (201 mg, 1 mmol), followed by dichloromethane (10 mL). This mixture was cooled in an ice bath, and 1-(pyridin-2-yl)piperazine (326 mg, 2 mmol) was added dropwise as a solution in 5 mL dichloromethane. The mixture was allowed to stir at 0 °C for 30 min., then allowed to warm to ambient temperature and stirred for an additional 60 min. The mixture was diluted with a saturated aqueous ammonium chloride solution and extracted with diethyl ether. The combined organic layers were dried and evaporated under reduced pressure to afford 2-bromo-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (1) as a clear oil, which solidified on standing. Bromide 1 was used in the next step without further purification.

Image ml342fu3

Step 2. Preparation of 2-(methylamino)-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (2)

A solution of 2-bromo-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (300 mg, 1 mmol) in dichloromethane (10 mL) was added slowly to a solution of methylamine (1M in THF, large excess). This mixture was allowed to stir at ambient temperature for 10 minutes, then diluted with water and extracted with dichloromethane. The combined organic layers were dried and evaporated to give 205 mg of title compound 2 as an oil which was used without further purification (89%): LCMS (>99% 215 nm, 254 nm) m/z (M+H)+ = 235.

Step 3. Amide coupling to prepare ML342

2-(methylamino)-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (246 mg, 1 mmol) was added to a round bottom flask and dissolved in dichloromethane (20 mL). N,N-Diisopropylethylamine (0.35 mL, 2 mmol) was added, followed by 4-methylbenzene-1-sulfonyl chloride (380 mg, 2 mmol). Mixture allowed to stir at room temperature for 30 minutes, then washed with water. Organic layers collected, dried, evaporated and purified by HPLC to afford N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide as a colorless oil (see final full characterization data above).

3. Results

3.1. Dose Response Curves for Probe

Figure 1. Concentration Response Curve for Probe ML342 (CID 2953239) in AM2.2-β2AR/TO1-2p (fluorogen) assay.

Figure 1Concentration Response Curve for Probe ML342 (CID 2953239) in AM2.2-β2AR/TO1-2p (fluorogen) assay

Figure 2. Concentration Response Curve for Probe ML342 (CID 2953239) in MG13-CCR5 counterscreen binding assay.

Figure 2Concentration Response Curve for Probe ML342 (CID 2953239) in MG13-CCR5 counterscreen binding assay

3.2. Cellular Activity

This probe is directed at an extracellular target. We plan to pursue cellular permeability in future studies.

3.3. Profiling Assays

Due to the goal and unique nature of this probe development project, which is currently focused on in vitro studies, ancillary pharmacology was not assessed at this time for ML342. Should future efforts lead to the identification of more advanced and highly selective small molecule inhibitors of FAP-tagged cell surface proteins, applications could be envisioned which might require such an assessment of biological activity.

4. Discussion

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

In the course of identifying candidate β2AR agonists, we identified a number of compounds that act at the level of the FAP reporter and not at the level of the receptor to which it is fused. In the context of screen development, as described above, these compounds have provided a new and unpredicted opportunity. To date, no non-fluorescent analogs of fluorogen thiazole orange and its derivatives have been discovered or reported in literature. Thus ML342 represents the first probe molecule of this type.

5. References

1.
Szent-Gyorgyi C, Schmidt BF, Creeger Y, Fisher GW, Zakel KL, Adler S, Fitzpatrick JA, Woolford CA, Yan Q, Vasilev KV, Berget PB, Bruchez MP, Jarvik JW, Waggoner A. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nature biotechnology. 2008;26:235–240. [PubMed: 18157118]
2.
Holleran J, Brown D, Fuhrman MH, Adler SA, Fisher GW, Jarvik JW. Fluorogen-activating proteins as biosensors of cell-surface proteins in living cells. Cytometry. Part A : the journal of the International Society for Analytical Cytology. 2010;77:776–782. [PMC free article: PMC2945705] [PubMed: 20653017]
3.
Saunders MJ, Szent-Gyorgyi C, Fisher GW, Jarvik JW, Bruchez MP, Waggoner AS. Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors. Methods. 2012;57:308–317. [PMC free article: PMC3432715] [PubMed: 22366230]
4.
Keseru GM, Makara GM. The influence of lead discovery strategies on the properties of drug candidates. Nat Rev Drug Discov. 2009;8:203–212. [PubMed: 19247303]
5.
Wu Y, Tapia PH, Fisher GW, Simons PC, Strouse JJ, Foutz T, Waggoner AS, Jarvik J, Sklar LA. Discovery of regulators of receptor internalization with high-throughput flow cytometry. Mol Pharm. 2012;82:645–657. [PMC free article: PMC3463215] [PubMed: 22767611]
6.
Wu Y, Tapia P, Fisher GW, Waggoner AS, Jarvik J, Sklar LA. High-Throughput Flow Cytometry Compatible Biosensor Based on Fluorogen Activating Protein Technology. Cytometry. in Press. [PMC free article: PMC3621705] [PubMed: 23303704]

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