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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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Selective small molecule activator of the apoptotic arm of the UPR

, , , , , , , , , , , , , , , , , , , , , , , , , and .

Author Information

,1 ,1 ,2 ,4 ,4 ,4 ,4 ,5 ,5 ,5 ,5 ,4 ,4 ,4 ,5 ,5 ,5 ,4 ,4,* ,4 ,3 ,6 ,6 ,6 ,6 and 7.

1 University of Kansas Specialized Chemistry Center, Delbert M. Shankel Structural Biology Center, 2034 Becker Drive, Lawrence, KS, USA
2 University of Kansas COBRE Center for Cancer Experimental Therapeutics, Delbert M. Shankel Structural Biology Center, 2034 Becker Drive, Lawrence, KS, USA
3 Department of Medicinal Chemistry, University of Kansas, Malott Hall, Lawrence, KS, USA
4 Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
5 Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL 32827, USA; John C. Reed, PI
6 Wayne State University, Carmen and Ann Adams Department of Pediatrics Division of Hematology/Oncology, 2228 Elliman Building 421 E. Canfield Detroit, MI 48201, USA
7 Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Rd, La Jolla, CA 92037, USA
* Corresponding author: Thomas D.Y. Chung, Ph.D. gro.mahnrubdrofnas@gnuhct

Received: ; Last Update: February 28, 2013.

Many diseases, such as diabetes, Alzheimer’s, Parkinson’s, hemophilia, and lysosomal storage diseases and cancer involve folding defects or impaired transport of proteins from the endoplasmic reticulum (ER). Cells activate the Unfolded Protein Response (UPR) to restore homeostatic protein processing by clearing out the stress of accumulated misfolded proteins (adaptive arm). However, with prolonged stress an apoptotic arm of the UPR leads to selective cell death. We herein report the successful development of a first-in-class, potent (762 nM EC50), not generally cytotoxic, chemical probe, ML291, that selectively activates the apoptotic but not the adaptive arm of the UPR, that demonstrates efficacy in inducing cell death through activation of the apoptotic arm in relevant cells, and moreover activate genes associated with the apoptotic arm of the UPR (by qRT-PCR). The progenitor of this probe was identified through a high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) of >350,000 compounds through complementary cell-based reporter assays using stably transfected CHO-K1 cells that specifically identify activators of the PERK/eIF2a/CHOP (apoptotic), but not the IRE1/XBP1 (adaptive) UPR subpathways. The identification, validation, structure activity relationship (SAR) elucidation and development of this probe are described. The probe may serve as the basis for eventual proof-of-concept tool compound for activation of the apoptotic arm of UPR as a therapeutic modality in certain diseases.

Assigned Assay Grant #: 1 R03 MH089782-01

Screening Center Name & PI: Sanford-Burnham Center for Chemical Genomics, John C. Reed, PI

Chemistry Center Name & PI: University of Kansas Specialized Chemistry Center, Jeffrey Aubé, PI

Assay Submitter & Institution: Randal J Kaufman, Sanford-Burnham Medical Research Institute (formerly at U. Michigan) & Collaborating PI: Andrew M. Fribley, Wayne State University

PubChem Summary Bioassay Identifier (AID): 449771

Probe Structure & Characteristics

Image ml291fu1
CID/ML#Target NameEC50 (nM) [SID, AID]*Anti-target NameEC50 (nM) [SID, AID] **Fold Selectivity (XBP1/CHOP)
CID 52940465/ML291UPR-CHOP762 nM*[SID123083137 & SID134228465, AID 602434]*UPR-XBP1> 80 μM**[SID123083137 & SID134228465, AID 602416]> 105
*

Reported value is an average of values from two separate batches of probe compound, SID123083137, EC50 = 740 nM and for SID 134228465, EC50 = 780 nM, respectively.

**

Reported value is an average of values from two separate batches of probe compound, SID123083137, EC50 > 80 μM and for SID 134228465, EC50 > 80 μM, respectively.

1. Recommendations for Scientific Use of the Probe

What limitations in current state of the art is the probe addressing? Prior to this report, there were no published reports of any pharmacologically and chemically attractive, selective small molecule activator probes to dissect the molecular mechanisms the apoptotic arm of the UPR activation, even at the phenotypic cell death level.

What would the probe be used for? A probe of the type described would be used to characterize the molecular mechanism of UPR activation through PERK and ATF6. We would use such probes to test the hypothesis that small molecules that induce ER stress and overwhelm the hyperactive secretory pathway of head and neck squamous cell carcinoma cells will be an effective therapeutic approach to kill tumor cells.

Who in the research community will use the probe? The ability to chemically activate the apoptotic arm of the UPR (in the absence of the adaptive arm) would broadly appeal to the interests of the UPR-focused scientific community. Currently we are without selective non-contrived methods or models to activate the PERK-eIF2α-CHOP (apoptotic) or ATF6-CHOP pathways that do not involve genetic manipulation or viral infection. Studies by our group and others have reported that CHOP-null cells are resistant to death induced by a variety of chemical and pathophysiological stresses, however, the mechanism by which CHOP actives cell death remains incompletely elucidated. Interestingly, it has been proposed that non-UPR related insults, such as DNA damage, might involve or rely on CHOP activation for the efficient induction of cell death. CHOP-specific probes identified from this screen will provide a unique panel of tools to identify novel pathways by which CHOP is activated and glean a clearer picture of the mechanism by which CHOP orchestrates UPR-mediated cell death, a subject of tremendous interest in the UPR field.

What is the relevant biology to which the probe can be applied? CHOP-specific probes will permit the more careful dissection of the apoptotic UPR pathway in a fashion that will shed new insight into its potential as a therapeutic target. Current therapies for patients suffering from UPR related diseases are limited to minimally effective gene therapy or extensive costly enzyme replacement therapies. We would also assess the potential of the apoptosis-inducing compounds to kill head and neck cancer cells using a variety of in vitro and xenograft animal models. Many different human cancer cells (cell lines and surgical specimens) have been shown to have increased levels of UPR activation. We hypothesize that CHOP-specific probes will overwhelm the adaptive UPR capacity of malignant cells, while healthy cells, with low or no basal UPR activation, would be able to mount an effective UPR and overcome the chemotherapeutic challenge and directly induce apoptosis. The advantage of such as strategy is apparent in that the treatment could be delivered systemically, inducing UPR-mediated cell death specifically in malignant cells. Our laboratory’s multi-disciplinary approach will help us to maximize the potential of the most promising probe leads to improve diverse disease states ranging from diabetes and Alzheimer’s disease to a wide range of cancers including breast, lung, and head and neck squamous cell carcinoma.

2. Materials and Methods

The details of the primary HTS and additional assays can be found in the “Assay Description” section in the PubChem BioAssay view under the AIDs as listed in Table 1. Additionally, the details for the primary HTS are provided in the Appendix A at the end of this probe report.

Table 1. Summary of Assays and AIDs.

Table 1

Summary of Assays and AIDs.

2.1. Assays

The parental cell line for both reporter cell lines is CHO-K1. The construction of the XBP1 reporter was described by Back et al.48 and the stable clone used for screening was identified by Andrew Fribley (unpublished). The CHOP luciferase construct originally contained a GFP reporter and was described by Novoa et al.49 The re-construction of the luciferase reporter and a subsequent screen for activators of CHOP were described by Harding et al.50

2.2. Probe Chemical Characterization

Chemical name of probe compound & structure including stereochemistry

The IUPAC name of the probe ML291 is N-(4-((4-chloropiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide. The actual batch prepared, tested and submitted to the MLSMR is archived as SID 134228465 corresponding to CID 52940465.

Image ml291fu2

For detailed characterization see Section 2.3. Images of spectral data (1HNMR, 13CNMR, and LC/MS) used to support the structural assignment of ML291 can be found in Supplementary Information at end.

Availability from a vendor

This probe is not commercially available. A 25 mg sample of ML291 synthesized at the KUSCC has been deposited in the MLSMR (see Probe Submission Table 2).

Table 2. Probe and Analog Submissions to the MLSMR for the UPR CHOP Activator Probe.

Table 2

Probe and Analog Submissions to the MLSMR for the UPR CHOP Activator Probe.

Solubility and stability of probe

The solubility of ML291 was measured in phosphate buffered saline (PBS) at room temperature (23 °C). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4 (See Table 3 “Summary of in vitro ADME Properties …”) Probe ML291 (CID 52940465, SID 134228465) was found to have a solubility measurement of 3.9 μg/mL, or 9.4 μM, under these conditions.

Table 3. Summary of in vitro ADME Properties of UPR CHOP Activator probe ML291.

Table 3

Summary of in vitro ADME Properties of UPR CHOP Activator probe ML291.

Stability was measured under two distinct conditions with ML291 (CID 52940465, SID 134228465, Figure 1). Stability, depicted as closed circles in the graph, was assessed at room temperature (23 °C) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%). Stability, illustrated with closed squares in the graph, was also assessed with 50% acetonitrile added to account for challenges with solubility of the compound in PBS alone. Stability data in each case is depicted as a graph showing the loss of compound with time over a 48 hr period with a minimum of 6 time points and providing the percent remaining compound at the end of the 48 hr. With no additives (closed circles), 11.44% of ML291 remains after 48 hours; however, this data is dependent on and misleading due to the solubility limitations in PBS buffer. With the addition of 50% acetonitrile to account for solubility (closed squares), 100% of ML291 remained after 48 hours.

Figure 1. Stability of ML291 over 48 h in PBS and 1:1 PBS:acetonitrile.

Figure 1

Stability of ML291 over 48 h in PBS and 1:1 PBS:acetonitrile. Apparent instability in PBS is actually due to poor solubility of ML291.

To assess the chemical stability of ML291 and its susceptibility to nucleophilic addition, the probe was exposed to a five-fold excess of 50 μM glutathione (GSH), or 50 μM dithiothreitol (DTT) over 8 h. ML291 was dissolved at 10 μM in 50% acetonitrile/50% PBS at pH 7.4 (1% DMSO) and incubated at room temperature with either no nucleophile (control), 50 μM GSH, or 50 μM DTT. The mixtures were sampled every hour for eight hours and analyzed by LCMS. The analytical LCMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity HSS T3 C18 column (2.1 × 50mm, 1.8um) and elution with a linear gradient of 1% water to 100% CH3CN at 0.6 mL/min flow rate. Peaks on the 214nm chromatographs were integrated using the Waters OpenLynx software. Absolute areas under the curve were compared at each time point to determine relative percent remaining. The masses of potential adducts were searched for in the final samples to determine if any detectable adduct formed. All samples were prepared in duplicate. Ethacrynic acid, a known Michael acceptor, was used as a positive control and showed ~ 55% and ~ 25% ethacrynic acid remaining at 8 h with GSH and DTT, respectively (control plots not shown for clarity).

For each of the conditions described in Figure 2, the percent remaining of ML291 after 8 h was determined to be between 99–100% (Figure 2). LCMS analysis of aliquots of each sample from each condition did not reveal detectable DTT or GSH adduct masses. This experiment demonstrated that ML291 was not prone to alteration by thiol nucleophiles over an 8 h period.

Figure 2. Chemical stability of ML291, represented by percent of parent remaining over time, in the presence of five-fold molar equivalent of GSH or DTT, incubated at room temperature in 1:1 ACN:PBS, pH 7.4, 1% (v/v) DMSO.

Figure 2

Chemical stability of ML291, represented by percent of parent remaining over time, in the presence of five-fold molar equivalent of GSH or DTT, incubated at room temperature in 1:1 ACN:PBS, pH 7.4, 1% (v/v) DMSO.

MLS# of probe molecule and five related samples that were submitted to the SMR collection

Five analogues were selected for the support of probe ML291. All five compounds, including the probe, were synthesized by the KU SCC and are summarized in Table 2.

2.3. Probe Preparation

Synthesis and Structural Verification Information of probe ML291, SID 123083137 corresponding to CID 52940465 (See Scheme 1).

The probe was prepared using the following protocols:

Image ml291fu3

4-chloro-1-((4-nitrophenyl)sulfonyl)piperidine: To a vial was added 4-nitrobenzenesulfonyl chloride (0.32 g, 1.4 mmol), pyridine (0.11 g, 1.4 mmol) and THF (1.5 mL). The reaction was stirred at room temperature while 4-chloropiperidine (0.13 g, 1.0 mmol) was added dropwise over 10 minutes. The reaction was subsequently heated to 60 °C for 20 minutes and monitored by TLC. Upon completion the reaction was cooled to room temperature, diluted with EtOAc (10 mL) and washed with saturated aq. NaHCO3 (10 mL). The EtOAc layer was separated, dried with MgSO4, filtered, adsorbed to silica and purified by silica gel flash column chromatography (15 min, 0 – 30% v/v EtOAc/hexanes) to produce pure 4-chloro-1-((4-nitrophenyl)sulfonyl)piperidine (0.29 g, 0.96 mmol, 96% yield). 1H NMR (400 MHz, CDCl3): δ 8.40 (d, J = 9.0 Hz, 2H), 7.96 (d, J = 9.0 Hz, 2H), 4.22 (m, 1H), 3.29 (m, 2H), 3.18 (m, 2H), 2.16 (m, 2H), 1.97 (m, 2H).

Image ml291fu4

4-((4-chloropiperidin-1-yl)sulfonyl)aniline: To a vial containing 4-chloro-1-((4-nitrophenyl)sulfonyl)piperidine (0.29 g, 0.96 mmol) was added 1:1 MeOH:CH2Cl2 (3 mL:3 mL), and the reaction was cooled to 0°C. Raney nickel (0.006 g, 0.096 mmol) was added followed by portionwise addition of sodium borohydride (0.073 g, 1.9 mmol). Once addition was complete, the reaction mixture was stirred at 0°C for 30 minutes and was then diluted with CH2Cl2 (10 mL) and filtered slowly. The CH2Cl2 layer was washed with water (10 mL), separated, dried with MgSO4, filtered, adsorbed to silica and purified by silica gel flash column chromatography (15 min, 0 – 5 % v/v MeOH/DCM) to produce pure 4-((4-chloropiperidin-1-yl)sulfonyl)aniline (0.23 g, 0.82 mmol, 86% yield). 1H NMR (400 MHz, CDCl3): δ 7.66 (d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H), 7.03 (s, 1H), 5.32 (s,1H), 4.12 (m, 1H), 3.16 (m, 2H), 3.10 (m, 2H), 2.13 (m, 2H), 1.95 (m, 2H).

Image ml291fu5

N-(4-((4-chloropiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide: ML291, SID 134228465; CID 52940465. To a microwave vial was added 4-((4-chloropiperidin-1-yl)sulfonyl)aniline (0.23 g, 0.82 mmol), 5-nitro-2-furoyl chloride (0.16 g, 0.90 mmol) and acetonitrile (3 mL). The vial was sealed and heated to 150 °C in the microwave for 20 minutes. The reaction then cooled to room temperature and was diluted with CH2Cl2 (10 mL) and washed with saturated NaHCO3 (10 mL). The CH2Cl2 layer was separated, dried with MgSO4, filtered and adsorbed to silica gel. The crude product was purified by silica gel flash column chromatography (20 min, 0 – 5% v/v MeOH/CH2Cl2) to produce pure N-(4-((4-chloropiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide, ML291 (0.11 g, 0.26 mmol, 31% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 8.03 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 3.9 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 3.9 Hz, 1H), 4.27 (m, 1H), 3.17 (m, 2H), 2.87 (m, 2H), 2.10 (m, 2H), 1.79 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 154.9, 151.9, 147.3, 142.2, 130.4, 128.6, 120.4, 117.2, 113.4, 56.1, 43.4, 33.9. LCMS retention time: 3.147 min. LCMS Purity at 214 nm: 97.5%. HRMS: m/z calcd for C16H17ClN3O6S (M + H+) 414.0521, found 414.0522. Melting point: 228 – 232 °C.

General experimental and analytical details:1H and 13C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500 and 126 MHz respectively) in CDCl3 with 0.03% TMS as an internal standard or DMSO-d6. The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, br. s = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet and m = multiplet. The LCMS analysis was performed on an Agilent 1200 RRL chromatograph with photodiode array UV detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the following parameters: a Waters Acquity BEH C-18 2.1 × 50mm, 1.7 um column; UV detection wavelength = 214 nm; flow rate = 0.4ml/min; gradient = 5 – 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the following parameters: an Agilent multimode source which simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H,1H,3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist ionization. Melting points were determined on a Stanford Research Systems OptiMelt apparatus.

Proton NMR Data for ML291/SID 123083137/CID 52940465: 1H NMR (400 MHz; DMSO-d6): δ (ppm) 11.01 (s, 1H), 8.03 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 3.9 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 3.9 Hz, 1H), 4.27 (m, 1H), 3.17 (m, 2H), 2.87 (m, 2H), 2.10 (m, 2H), 1.79 (m, 2H).

Carbon NMR Data for ML291/SID 123083137/CID 52940465: 13C NMR (100 MHz; DMSO-d6): δ (ppm) 154.9, 151.9, 147.3, 142.2, 130.4, 128.6, 120.4, 117.2, 113.4, 56.1, 43.4, 33.9.

LCMS and HRMS Data for ML291, CID 52940465: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental LCMS and HRMS data are represented as follows:

For SID 123083137: LCMS retention time: 3.074 min. LCMS purity at 214 nm: 95.4%. HRMS: m/z calcd for C16H17ClN3O6S (M + H+) 414.0521, found 414.0527. The experimental LCMS and HRMS spectra are included for reference (Appendix A, Figure A6C and A6D, respectively).

For SID 134228465: LCMS retention time: 3.125 min. LCMS purity at 214 nm: 97.5%. HRMS: m/z calcd for C16H17ClN3O6S (M + H+) 414.0521, found 414.0522. The experimental LCMS and HRMS spectra are included for reference (Appendix A, Figure A6E and A6F, respectively).

3. Results

3.1. Dose Response Curves for Probe

Figure 3 illustrates the reproducibility of the efficacy response of ML291 over 4 replicates and it’s selectivity for activating the apoptotic (CHOP) but not the adaptive (XBP1) UPR subpathways in this CHO-K1 cell luciferase reporter system.

Figure 3. Activation of luciferase reporters driven by UPR sub-pathway promoters for the apoptotic (CHOP) and adaptive (XBP1) arms.

Figure 3

Activation of luciferase reporters driven by UPR sub-pathway promoters for the apoptotic (CHOP) and adaptive (XBP1) arms. Engineered CHO-K1 cell were incubated with ML291 for overnight (~16 hr) and luciferase induction measured with SteadyGlo™ (more...)

3.2. Cellular Activity

If our putative activator of the apoptotic arm of the unfolded protein response (UPR) selectively activates these apoptotic pathways in cell, we would expect the ML291 would induce cell death (cytotoxicity as measured by ATPLite® - production of ATP from living cells) in cells where these pathways are operant, but no activate them in the absence of the pathways. This is exactly the result obtained (Figure 4) from mouse embryonic fibroblast (MEF) cell lines engineered to have the wild-type apoptotic pathways intact (MEF w/wt-CHOP) compared to MEF cells where this pathway has been knocked-out (MEF w/CHOP KO). ML291 does not non-specifically kill MEFs; however, ML291 does potently kill MEFs in the presence of an intact CHOP pathway (implying activation of apoptotic pathways). Dr. Fribley’s laboratory replicated (n=4) this result and estimates the activator potency in MEF wt-CHOP as ~ 4.8 μM EC50, which compares favorably (~ 6-fold shift) with its potency in the CHO-K1 cell reporter assays (762 nM EC50)

Figure 4. Cellular Efficacy of ML291 for Activating the CHOP Pathway Induced cytotoxicity in MEF with wt-CHOP vs. CHOP-KO.

Figure 4

Cellular Efficacy of ML291 for Activating the CHOP Pathway Induced cytotoxicity in MEF with wt-CHOP vs. CHOP-KO. (After 16 hr exposure to probe)

3.3. Profiling Assays

The nominated probe ML291 was evaluated in a detailed in vitro pharmacology screen as shown in Table 3.

ML291 has poor to modest solubility ranging from 8.7 – 9.7 μM (3.6 – 4.0 μg/mL) in aqueous buffers between a pH range of 5.2–7.4. Solubility is highest at the physiological of pH 7.4. This solubility is about 11 – 13 fold over its EC50 (762 nM), so its apparent potency is not severely limited by its solubility.

The PAMPA (Parallel Artificial Membrane Permeability Assay) assay is used as an in vitro model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. ML291 exhibits good permeability at pHs of 5.0, 6.2 and 7.4 in the donor compartment, with highest permeability at pH 6.2.

Plasma protein binding is a measure of a drug’s efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. ML291 was highly plasma protein bound to human plasma proteins (>99%), though it is somewhat less tightly bound to mouse plasma proteins (~84%), which will be a consideration in translating any future mouse model data to humans.

Plasma stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which strongly can influence the in vivo efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. ML291 appears to be moderately stable in human plasma (~84% remaining), but less so in mouse plasma (~52% remaining).

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. ML291 is almost completely metabolized in both human and mouse liver homogenates within 1 hour.

ML291 shows some toxicity (~11 μM LC50) toward human hepatocytes.

Profiling against other GPCRs

ML291 was submitted to Ricerca Biosciences LLC (http://www.ricerca.com/discovery-pharmacology.asp Bothell, WA, USA) to evaluate it in radioligand binding assays against activity against a panel of 67 GPCRs, ion channels and transporters at 10 μM. ML291 only scored as having significant activity (68% inhibition) against the dopamine transporter (DAT), so it does not appear to be generally promiscuous compound with respect to these receptors. See Appendix B: Ricerca LeadProfiling® Report for ML291.

Profiling against the NCI-60 Panel

ML291 was submitted to the NCI DPT for screening against the NCI-60 panel of tumor cell lines (http://dtp.cancer.gov/branches/btb/ivclsp.html) and in summary ML291 has broad growth inhibitory activity and has greater than average anti-tumor cell cytotoxicity against the colon, melanoma, and renal cancer cell lines that comprise the NCI-60 panel. See Appendix C: NCI-60 panel profiling Report for ML291.

4. Discussion

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

In November of 2010, at time of our initial scaffold selection to advance into probe development we found no chemical matter for UPR apoptotic arm activation described in the published literature and patents using SciFinder and Prous Integrity as search engines. Additional recent SciFinder searches uncovered no significant prior art, using the search terms: Unfolded Protein Response, CHOP activator, CHOP agonist, CHOP, PERK-eIF2α-CHOP, Apoptotic UPR.

Aside from unpublished results from the assay providers’ laboratories (Dr. Fribley and Dr. Kaufman), there are no patents or publications describing advanced small molecule activators of the apoptotic arm of the UPR. Dr. Fribley has some preliminary data describing two hydrazide containing thiuram compounds, disulfram and NSC-1771. These two compounds while active in both reporter assays and cell based apoptosis assays, represented very simple, unattractive and non-pharmacophoric scaffolds. ML291 provides an improvement in potency and selectivity and provides a more valuable pharmacologic scaffold.

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APPENDIX A. Quality Control Data for Probe ML291 (SID 123083137, CID 52940465)

1H-NMR and 13C-NMR spectra

Figure A6A. Proton 1H NMR spectra for ML291 (SID 123083137, CID 52940465).

Figure A6AProton 1H NMR spectra for ML291 (SID 123083137, CID 52940465)

Figure A6B. Carbon 13C-NMR data for ML291 (SID 123083137, CID 52940465).

Figure A6BCarbon 13C-NMR data for ML291 (SID 123083137, CID 52940465)

LCMS Purity data: HRMS and HPLC

Figure A6C. LCMS purity data at 214 nm for ML291 1st batch (SID 123083137, CID 52940465); LCMS retention time: 3.074 min; purity at 214 nm = 95.4%.

Figure A6CLCMS purity data at 214 nm for ML291 1st batch (SID 123083137, CID 52940465); LCMS retention time: 3.074 min; purity at 214 nm = 95.4%

Figure A6D. HRMS data for ML291 1st batch (SID 123083137, CID 52940465); HRMS m/z calculated for C16H17ClN3O6S [M + H+]: 414.0521, found 414.0527.

Figure A6DHRMS data for ML291 1st batch (SID 123083137, CID 52940465); HRMS m/z calculated for C16H17ClN3O6S [M + H+]: 414.0521, found 414.0527

Figure A6E. LCMS purity data at 214 nm for ML291 2nd batch –submitted to the MLSMR (SID 134228465, CID 52940465); LCMS retention time: 3.125 min; purity at 214 nm = 97.5%.

Figure A6ELCMS purity data at 214 nm for ML291 2nd batch –submitted to the MLSMR (SID 134228465, CID 52940465); LCMS retention time: 3.125 min; purity at 214 nm = 97.5%

Figure A6F. HRMS data for ML291 2nd batch –submitted to the MLSMR (SID 134228465, CID 52940465); HRMS m/z calculated for C16H17ClN3O6S [M + H+]: 414.0521, found 414.0522.

Figure A6FHRMS data for ML291 2nd batch –submitted to the MLSMR (SID 134228465, CID 52940465); HRMS m/z calculated for C16H17ClN3O6S [M + H+]: 414.0521, found 414.0522

APPENDIX B. Ricerca LeadProfiling® Report for ML291

STUDY OBJECTIVE

To evaluate, in Radioligand Binding assays, the activity of compound SID123083137.

METHODS

Methods employed in this study have been adapted from the scientific literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained. Assays were performed under conditions described in the accompanying “Methods” section of this report. Where presented, IC50 values were determined by a non-linear, least squares regression analysis using MathIQTM (ID Business Solutions Ltd., UK). Where inhibition constants (Ki) are presented, the Ki values were calculated using the equation of Cheng and Prusoff (Cheng, Y., Prusoff, W.H., Biochem. Pharmacol. 22:3099–3108, 1973) using the observed IC50 of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the KD of the ligand (obtained experimentally at Ricerca Biosciences, LLC). Where presented, the Hill coefficient (nH), defining the slope of the competitive binding curve, was calculated using MathIQTM. Hill coefficients significantly different than 1.0, may suggest that the binding displacement does not follow the laws of mass action with a single binding site. Where IC50, Ki, and/or nH data are presented without Standard Error of the Mean (SEM), data are insufficient to be quantitative, and the values presented (Ki, IC50, nH) should be interpreted with caution.

RESULTS

A summary of results meeting the significance criteria is presented in the following sections. Complete results are presented under the section labeled “Experimental Results”. Individual responses, if requested, are presented under the section labeled “Individual Responses”.

SUMMARY/CONCLUSION

Significant results are displayed in the following table(s) in rank order of potency for estimated IC50 and/or Ki values.

Summary of Significant Results

Biochemical assay results are presented as the percent inhibition of specific binding or activity throughout the report. All other results are expressed in terms of that assay’s quantitation method (see Methods section).

  • For primary assays, only the lowest concentration with a significant response judged by the assays’ criteria, is shown in this summary.
  • Where applicable, either the secondary assay results with the lowest dose/concentration meeting the significance criteria or, if inactive, the highest dose/concentration that did not meet the significance criteria is shown.
  • Unless otherwise requested, primary screening in duplicate with quantitative data (e.g., IC50 ± SEM, Ki ± SEM and nH) are shown where applicable for individual requested assays. In screening packages, primary screening in duplicate with semi-quantitative data (e.g., estimated IC50, Ki and nH) are shown where applicable (concentration range of 4 log units); available secondary functional assays are carried out (30 mM) and MEC or MIC determined only if active in primary assays >50% at 1 log unit below initial test concentration.
  • Please see Experimental Results section for details of all responses. Significant responses (≥ 50% inhibition or stimulation for Biochemical assays) were noted in the primary assays listed below:
Cat #Assay NameSpeciesConc.% Inh.
220320Transporter, Dopamine (DAT)hum10 μM68

EXPERIMENTAL RESULTS FOR SID 123083137 ON OTHER TARGETS

Cat #Assay NameBatch*Spec.Rep.Cone.% Inh.
Compound: SID123083137, PT #: 1158789
200510Adenosine A1310992hum210 μM12
200610Adenosine A2A310993hum210 μM8
200720Adenosine A3310976hum210 μM6
203100Adrenergic α1A310901rat210 μM19
203200Adrenergic α1B310902rat210 μM13
203400Adrenergic α1D310903hum210 μM8
203620Adrenergic α2A310904hum210 μM11
204010Adrenergic β1310974hum210 μM1
204110Adrenergic β2310983hum210 μM17
285010Androgen (Testosterone) AR311022rat210 μM8
212510Bradykinin B1311038hum210 μM11
212620Bradykinin B2311061hum210 μM4
214510Calcium Channel L-Type, Benzothiazepine310999rat210 μM6
214600Calcium Channel L-Type, Dihydropyridine310998rat210 μM24
216000Calcium Channel N-Type310899rat210 μM18
217030Cannabinoid CB1311000hum210 μM20
219500Dopamine D1310984hum210 μM−2
219700Dopamine D2S310985hum210 μM21
219800Dopamine D3311010hum210 μM9
219900Dopamine D4.2310959hum210 μM1
224010Endothelin ETA311036hum210 μM−10
224110Endothelin ETB311037hum210 μM3
225510Epidermal Growth Factor (EGF)310884hum210 μM−5
226010Estrogen ERα.310897hum210 μM0
226600GABAA, Flunitrazepam, Central310986rat210 μM21
226500GABAA, Muscimol, Central311011rat210 μM21
228610GABAB1A310896hum210 μM5
232030Glucocorticoid311064hum210 μM19
232700Glutamate, Kainate311034rat210 μM−9
232810Glutamate, NMDA, Agonism311035rat210 μM4
232910Glutamate, NMDA, Glycine310895rat210 μM2
233000Glutamate, NMDA, Phencyclidine311003rat210 μM−3
239610Histamine H1310934hum210 μM−2
239710Histamine H2310975hum210 μM10
239820Histamine H3311012hum210 μM−5
241000Imidazoline I2, Central311006rat210 μM−3
243520Interleukin IL-1311266mouse210 μM−4
250460Leukotriene, Cysteinyl CysLT1311007hum210 μM11
251600Melatonin MT1310969hum210 μM8
252610Muscarinic M1311151hum210μM2
252710Muscarinic M2310988hum210 μM10
252810Muscarinic M3310989hum210 μM13
257010Neuropeptide Y Y1310893hum210 μM7
257110Neuropeptide Y Y2310894hum210 μM4
258590Nicotinic Acetylcholine310971hum210 μM−1
258700Nicotinic Acetylcholine α, Bungarotoxin310972hum210 μM−1
260130Opiate δ1 (OP1, DOP)310879hum210 μM−13
260210Opiate κ(OP2, KOP)310880hum210 μM7
260410Opiate μ(OP3, MOP)310881hum210 μM1
264500Phorbol Ester310990mouse210 μM9
265010Platelet Activating Factor (PAF)310876hum210 μM39
265600Potassium Channel [KATP]310991ham210 μM11
265900Potassium Channel hERG310905hum210 μM32
268420Prostanoid EP4311013hum210 μM−2
268700Purinergic P2X311014rabbit210 μM−12
268810Purinergic P2Y311015rat210 μM9
270000Rolipram311177rat210 μM12
271110Serotonin (5-Hydroxytryptamine) 5-HT2A311017hum210 μM4
271700Serotonin (5-Hydroxytryptamine) 5-HT2B311019hum210 μM8
271910Serotonin (5-Hydroxytryptamine) 5-HT3310891hum210 μM−13
278110Sigma σ1311021hum210 μM38
255520Tachykinin NK1311044hum210 μM3
285900Thyroid Hormone310909rat210 μM7
220320Transporter, Dopamine (DAT)310996hum210 μM68
226400Transporter, GABA311002rat210 μM17
204410Transporter, Norepinephrine (NET)310995hum210 μM28
274030Transporter, Serotonin (5-Hydroxytryptamine) (SERT)311020hum210 μM5

Note: Items meeting criteria for significance (≥50% stimulation or inhibition) are highlighted.

*

Batch: Represents compounds tested concurrently in the same assay(s).

R=See Remarks (if any) at end of this section.

ham=Hamster; hum=Human

Appendix C. NCI-60 panel profiling Report for ML291

Image ml291fu6a
Image ml291fu6b
Image ml291fu7a
Image ml291fu7b

Reported value is an average of values from two separate batches of probe compound, SID123083137, EC50 = 740 nM and for SID 134228465, EC50 = 780 nM, respectively.

Reported value is an average of values from two separate batches of probe compound, SID123083137, EC50 > 80 μM and for SID 134228465, EC50 > 80 μM, respectively.

Batch: Represents compounds tested concurrently in the same assay(s).

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