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.

Probe Structure & Characteristics

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.

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.

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.

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.

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. 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.

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).

Scheme 1

The probe was prepared using the following protocols:

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).

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).

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. 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. (After 16 hr exposure to probe)

4. Discussion

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

1H-NMR and ¹³C-NMR spectra

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

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

LCMS Purity data: HRMS and HPLC

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

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

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

Figure A6FHRMS data for ML291 2^nd 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

Appendix C. NCI-60 panel profiling Report for ML291