Selective, reversible inhibitors of the AAA ATPase p97

Tsui-Fen Chou; Kelin Li; Brian E. Nordin; Patrick Porubsky; Kevin Frankowski; Mathew P. Patricelli; Jeffrey Aubé; Frank J. Schoenen; Raymond Deshaies

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Selective, reversible inhibitors of the AAA ATPase p97

Tsui-Fen Chou, Kelin Li, Brian E. Nordin, Patrick Porubsky, Kevin Frankowski, Mathew P. Patricelli, Jeffrey Aubé, Frank J. Schoenen, and Raymond Deshaies.

Author Information and Affiliations

Received: April 14, 2011; Last Update: May 1, 2013.

The AAA ATPase p97 is a critical factor in maintaining protein homeostasis in eukaryotic cells, through its roles in promoting degradation of ubiquinated proteins by the proteasome and in maturation of autophagosomes. Starting from a hit obtained by high-throughput screening (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), the team developed probe compounds ML240 and ML241 that both inhibit p97 ATPase activity with an IC₅₀ of approximately 100 nM, and block degradation of p97-dependent proteasome substrate with an IC₅₀ of approximately 900 nM and 3500 nM, respectively. By contrast, these probe compounds were far less potent in blocking degradation of the p97-independent substrate oxygen-dependent degradation domain (ODD)-luciferase (IC₅₀ > 28 μM), underscoring their specificity towards p97. The two probe compounds exhibited markedly different potencies for activating executioner caspases and blocking cell growth. ML240 rapidly induces caspases 3 and 7 in HCT15 and SW403 cells and potently blocked proliferation of these cells, whereas ML241 was >10-fold less efficacious in both assays.

Assigned Assay Grant #: 1 R03 MH085687-01

Screening Center Name & PI: None

Chemistry Center Name & PI: Jeffrey Aubé, University of Kansas Specialized Chemistry Center (KU SCC)

Assay Submitter & Institution: Raymond J. Deshaies, California Institute of Technology

PubChem Summary Bioassay Identifier (AID): 1794

Probe Structure & Characteristics

CID/ML#	Target Name	IC50/EC50 (nM) [SID, AID]	Anti-target Name(s)	IC50/EC50 (μM) [SID, AID]	Fold Selective	Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 49830258 ML240	p97ATPase	110nM [SID 10390416, AID 504650]	170 Kinase Panel	<50% I at 20 μM	N/A	Caspase 3/7(SW403): 29-fold at 10,000 nM [SID 103904169, AID 504653]
CID 49830258 ML240	Ub^G76V-GFP	900nM [SID 10390416, AID 504649]	ODD-Luc	28,000nM [SID103904169, AID 504654]	31-fold

CID 49830260 ML241	p97ATPase	110nM [SID 10390418, AID 504650]	170 Kinase Panel	>50% I at 20 μM (JAK1, PIP5K3, DNAPK)	N/A	Caspase 3/7(SW403): No Activation up to 20,000 nM [SID 103904185, AID 504653]
CID 49830260 ML241	Ub^G76V-GFP	3,500nM [SID 10390418, AID 504649]	ODD-Luc	46,000nM [SID103904185, AID 504654]	13-fold

Recommendations for Scientific Use of the Probe

The broad range of cellular functions for p97 is thought to derive from its ability to unfold proteins or disassemble protein complexes, but the detailed mechanism of how p97 works and is linked to specific cellular processes remains largely unknown. A major limitation to current studies on the biological functions of p97 is that there is no well-validated method to rapidly shut off its ATPase activity. There are existing chemical probes of p97 and they suffer from one or more of the following limitations: relatively low potency, poorly-characterized specificity, irreversibility, or unknown mechanism of action. The probe compounds ML240 and ML241 exceed the specifications for all previously described p97 ATPase inhibitor probes, and can be used in vitro and in cell cultures to study the role of p97 in the ubiquitin-proteasome system and autophagy, and more generally, the role of p97 in protein homeostasis. Researchers interested in using these probes should note that the two molecules exhibited markedly different potencies for activating executioner caspases and blocking cell growth. ML240 rapidly induces caspases 3 and 7 in HCT15 and SW403 cells and potently blocked proliferation of these cells, whereas ML241 was >10-fold less efficacious in both assays. The reasons for this discrepancy are unknown and are the subject of ongoing investigation.

2. Materials and Methods

Chemicals used in this study were MG132 (Enzo Life Sciences), and staurosporine (LC Laboratories), cycloheximide (EMD Bioscience) and bortezomib (LC Laboratories). Cells were grown on a 96-well cellstar black μclear bottom plate (ISC Bioexpress) for live-cell imaging on an automated microscope (ImageXpress Micro; Molecular Devices). Luciferase intensity was determined on an Analyst AD plate reader (Molecular Devices) or Synergy HT Microplate Reader (BioTek). All buffers were prepared in deionized water and filtered through a 0.2-μm filter.

Purification of murine p97 from E. coli BL21(DE3) stain containing RDB2122 plasmid is described elsewhere.[1]

2.1. Assays

Table 1List of Assays and AIDs for this Project

PubChem BioAssay Name	AID	Assay Type	Assay Format	Assay Detection and Plate Density
Summary of probe development efforts to identify inhibitors of p97ATPase	AID 1794	N/A	N/A	N/A
p97ATPase dose response - confirmatory	AID 463184	Enzymatic	Biochemical	Bio-luminescent Intensity; 96-well
Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - confirmatory	463185	Protein Expression	Cell-based	Fluorescence Intensity
p97ATPase dose response - hit optimization round 2 -confirmatory	488828	Enzymatic	Biochemical	Bio-luminescent Intensity; 96-well
Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - hit optimization round 2 - confirmatory	488830	Protein Expression	Cell-based	Fluorescence Intensity
p97ATPase dose response - hit optimization round 3 -confirmatory	504649	Enzymatic	Biochemical	Bio-luminescent Intensity; 96-well
Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - hit optimization round 3 - confirmatory	504650	Protein Expression	Cell-based	Fluorescence Intensity
Caspase 3/7 activation in response to p97 inhibition	504653	Enzymatic	Cell-based	Bioluminescent Intensity
Inhibition of p97-independent reporter turnover	504654	Protein Expression	Cell-based	Bio-luminescent Intensity

p97 ATPase Dose Response [463184, AID 488828, AID 504650]: Determine inhibition of purified p97 by small molecules for SAR study.

Assay Protocol

Dispense 20 μL of 2.5× Assay Buffer (50 mM Tris pH 7.4, 20 mM MgCl2, 1 mM EDTA) into each well of 96 well plate. Dilute enzyme (100 μL of 5 mg/mL) in 900 μL 1× Assay Buffer. Dispense 10 μL to each well. Add 10 μL compounds or DMSO control and incubate at room temperature for 10 min. Add 10 μL of 500 μM ATP (pH 7.5) and incubate at 37 °C for 30 min then room temperature for 10 min. Add 50 μL Kinase Glo Plus reagent to each well and incubate at room temperture for 10 min in dark. Luminescence is read on Analyst AD (Molecular Device).

The percent of remaining activity for each compound was calculated using the following mathematical expression: ((Test Compound-High Control)/(Low Control-High Control)) * 100

Where:

Test_Compound is defined as wells containing test compound.

Low_Control is defined as wells containing DMSO.

High_Control is defined as wells containing no p97 protein.

IC50 values were calculated from fitting the percentage of remaining activity (%RA) with various concentrations of compounds to a Langmuir equation [%RA =100/(1 + [Compound]/IC50)] by on-linear regression analysis using JUMP IN program. The result was expressed as mean standard error.”

Compounds with an IC50 equal to or less than 10 microM were considered active.

Activity score was ranked by normalizing to the lowest IC50 compounds (activity score = 100). Activity score for the active compounds of this assay can range from 0 to 100 and all inactive compounds are assigned activity score of zero.

For assays with EerI, Myriad 12, 19, and oxidized Myriad 12, 50 μL of biomol green reagent (Enzo Life Sciences) was added to each well instead of kinase Glo Plus (Promega) and absorbance at 630 nm was measured. This was done because these compounds interfered with luciferase assay.

Critical reagents are listed in Table 2.

Table 2

Critical reagents for the p97 ATPase dose response assay.

Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells [AID 463185, AID 488830, AID 504649]: Determine whether inhibitors obtained from the p97 ATPase Dose Response (KUA10001) are able to block turnover of a p97-dependent proteasome reporter substrate in cells.

Assay Protocol

Ub^G76V-GFP/HeLa cells were trypsinized and 5000 cells aliquoted into each well of a 96-well plate and grown for ~16h. HeLa cells expressing Ub^G76V-GFP were treated with DMEM containing MG132 (4 μM) for 1h and washed with 100 μL PBS twice. DMEM containing 2.5% FBS, cycloheximide (50 μg/mL) and the test compound were added into the well. Five 96-well plates were prepared and one of the plates was imaged on ImageXpress Micro at each time point (70, 90, 110, 130, 150, or 170 min) after washing with 100 μL PBS. Four GFP images with 100ms exposure time per well were acquired and average GFP intensity per area of a HeLa cell was determined by using MetaXpress software. Mean GFP intensity of ~500 cells was calculated using Excel.

Normalized GFP intensity was calculated using the following formula: (Test compound - Background)/(Basal GFP intensity - Background)

Where:

Test compound is defined as Mean GFP intensity of Ub^G76V-GFP/HeLa cells treated with the test compound.

Background is defined as background GFP intensity of HeLa cells, which did not express Ub-^G76V-GFP.

Basal GFP intensity is defined as mean GFP intensity of Ub^G76V-GFP/HeLa cells treated with DMSO.

The degradation rate constant (k) was obtained from the slope of plotting Ln(Normalized GFP intensity) versus time ranging from 90 to 170 min. The percent of remaining k for each compound was calculated using the following formula:

(Test compound/DMSO control) * 100

Where:

Test_compound is defined as k determined from wells containing test compound.

DMSO control is defined as k determined from wells containing DMSO.

IC₅₀ values were calculated from fitting the percentage of remaining k (%k) with various concentrations of compounds to a Langmuir equation [%k =100/(1 + [Compound]/IC₅₀)] by non-linear regression analysis using JUMP IN program. The result was expressed a mean +/− standard error.

Compounds with an IC₅₀ equal to or less than 10 microM were considered active.

Activity score was ranked by normalizing to the lowest IC₅₀ compounds (activity score = 100). Activity score for the active compounds of this assay can range from 0 to 100 and all inactive compounds were assigned activity score of zero.

Critical reagents are listed in Table 3.

Table 3

Critical reagents for the inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells assay.

Caspase 3/7 activation in response to p97 inhibition [AID 504653]: The purpose of this assay was to evaluate the ability of specific p97 inhibitors to induce apoptosis in cells by monitoring activation of caspase 3/7 with Caspase 3/7 Glo kit from Promega after 7 hour incubation. This assay was used to classify p97 inhibitors into either caspase-activating compounds, which may be useful for developing anti-cancer agents, or non-activating compounds, which may have other therapeutic potentials. For a compound to be considered as a caspase-activating agent, it should activate caspase 3/7 more than 5-fold at a concentration less than 20 μM after 7 h treatment.

Assay protocol

HCT15 or SW403 cells were seeded onto a 384 well white clear bottom plate (3000 cells per well). Cells were treated with compounds for 7 h and caspase-3/7 Glo (Promega) was added into each well and the contents of the well were mixed by shaking at 500 rpm for 1 min. Luminescence signal was determined after incubation at room temperature for 1 h with 0.1 ms integration time on the Synergy HT Microplate Reader (BioTek). Normalized Caspase3/7 activity was calculated as luminescence of test compound/luminescence of DMSO (1 %).

Score = maximal fold activation of each compound/maximal fold activation observed for all compounds × 100.

For a compound to be considered as caspase activating agent, its score should be more than 15.

Critical reagents are listed in Table 4.

Table 4

Caspase 3/7 activation in response to p97 inhibition assay.

Inhibition of p97-independent reporter turnover [AIDS: 554654]: Inhibition of ODD-luciferase turnover is used as a counterscreen for KUA10002 to eliminate compounds that inhibited general components of the UPS (e.g. E1 enzyme, proteasome) other than p97. The reporter was assayed by employing the same approach as for Ub^G76V-GFP (KUA10002) We evaluated the degradation rate constant for ODD-luciferase disappearance in the absence or presence of various concentrations of test compound. From these values, we determined an IC₅₀. For a compound to be considered a specific probe, it should inhibit ODD-luciferase with an IC₅₀ of ≥20 μM.

Assay Protocol

HeLa cells stably expressing ODD-luciferase were seeded onto a 96-well white solid bottom plate (5000cells/well) and cells were grown for 16h. Cells were treated with DMEM containing MG132 (4 μM) for 1h and washed with 100 μL PBS twice. DMEM containing 2.5% FBS, cycloheximide (50 μg/mL) and the test compound were added into the well. Four 96-well plates were prepared and one of the plates was taken out from incubator at each time point (70, 90, 120, or 150 min). Luciferin (50 μL of 1 mg/mL in PBS) was added into each well containing 50 μL of medium and incubated at room temperature with shaking at 500 rpm for 5 min. Luminescence intensity was determined with 0.1 ms integration time on the Synergy HT Microplate Reader (BioTek).

Normalized Luciferase intensity was calculated using the following formula: (Test compound – Background)/(Basal intensity – Background).

Where:

Test compound is defined as luciferase intensity of cells treated with the test compound. Background is defined as background intensity of HeLa cells that did not express ODD-luciferase.

Basal intensity is defined as luciferase intensity of cells treated with DMSO, The degradation rate constant (k) was obtained from the slope of plotting Ln (Normalized intensity) versus time ranging from 90 to 150 min. The percent of remaining k for each compound was calculated using the following formula: (Test compound/DMSO control) * 100.

Where:

Test_compound is defined as k determined from wells containing test compound, DMSO control is defined as k determined from wells containing DMSO. IC₅₀ values were calculated from fitting the percentage of remaining k (%k) with various concentrations of compounds to a Langmuir equation [%k =100/(1 + [Compound]/IC50)] by non-linear regression analysis using JUMP IN program. The result was expressed a mean ± standard error.

IC₅₀ of ≥20 μM was considered inactive (i.e., desirable) in this assay.

Critical reagents are listed in Table 5.

Table 5

Inhibition of p97-independent reporter turnover.

2.2. Probe Chemical Characterization

Probe Chemical Structure, Physical Parameters, and Probe Properties

Figure 1Property summary of probe compound SID 103904169 (CID 49830258)

Figure 2Property summary of probe compound SID 103904185 (CID 49830260)

Structure verification and Purity: ¹H NMR, ¹³C NMR, LCMS and HRMS

For probe ML240

Proton and carbon NMR data for SID 103904169 (CID 49830258): Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The experimental proton and carbon data are presented in Figures 3 and 4.

Figure 3

Proton NMR spectrum for SID 103904169 (CID 49830258).

Figure 4

Carbon NMR spectrum for SID 103904169 (CID 49830258).

SID 103904169 (CID 49830258) Proton NMR Data:¹H NMR (400 MHz, DMSO) δ 9.33 (t, J = 5.9 Hz, 1H), 8.31 – 8.05 (m, 3H), 7.93 (d, J = 7.5 Hz, 1H), 7.50 – 7.39 (m, 3H), 7.39 – 7.30 (m, 3H), 7.25 (t, J = 7.3 Hz, 1H), 7.15 (d, J = 7.1 Hz, 1H), 7.03 (td, J = 1.2, 7.6 Hz, 1H), 6.87 – 6.76 (m, 1H), 4.92 (d, J = 5.8 Hz, 2H), 3.98 (s, 3H).

SID 103904169 (CID 49830258) Carbon NMR Data:¹³C NMR (101 MHz, DMSO, APT pulse sequence) δ 160.8, 154.7, 153.5, 153.4, 142.8, 140.0, 138.5, 131.5, 128.5, 126.9, 126.6, 124.9, 122.5, 118.7, 114.8, 114.6, 114.1, 113.0, 112.7, 56.1, 44.5.

LCMS and HRMS data for SID 103904169 (CID 49830258): Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. Purity assessment by LCMS at 214 nm for SID 103904169 (CID 49830258) revealed purity at 215 nm of 100 % (retention time = 3.13 minutes). The experimental LCMS and HRMS spectra are included in Figures 5 and 6.

Figure 5

LCMS purity data at 215 nm for SID 103904169 (CID 49830258); LCMS retention time: 3.132; purity at 215 nm = 100%.

Figure 6

HRMS data for SID 103904169 (CID 49830258); HRMS (ESI) m/z calcd for C₂₃H₂₁N₆O ([M+H]⁺), 397.1777, found 397.1776.

For probe ML241

Proton and carbon NMR data for SID 103904185 (CID 49830260): Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The experimental proton and carbon data are presented in Figures 7 and 8.

Figure 7

Proton NMR spectrum for SID 103904185 (CID 49830260).

Figure 8

Carbon NMR spectrum for SID 103904185 (CID 49830260).

SID 103904185 (CID 49830260) Proton NMR Data:¹H NMR (400 MHz, CDCl₃) δ 8.09 – 8.01 (m, 1H), 7.42 – 7.30 (m, 5H), 6.92 – 6.86 (m, 2H), 6.80 – 6.73 (m, 1H), 4.79 (s, 1H), 4.69 (d, J = 5.6 Hz, 2H), 4.28 (dd, J = 3.1, 5.0 Hz, 2H), 4.23 (dd, J = 3.1, 5.0 Hz, 2H), 2.67 (t, J = 5.7 Hz, 2H), 2.32 (t, J = 5.7 Hz, 2H), 1.93 – 1.77 (m, 4H).

SID 103904185 (CID 49830260) Carbon NMR Data:¹³C NMR (101 MHz, CDCl₃, APT pulse sequence) δ 162.5, 160.6, 145.9, 139.5, 128.6, 128.4, 127.5, 127.3, 123.9, 122.6, 119.5, 116.6, 103.8, 65.9, 45.0, 42.2, 32.2, 22.6, 22.5, 21.9.

LCMS and HRMS data for SID 103904185 (CID 49830260): Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. Purity assessment by LCMS at 214 nm for SID 103904169 (CID 49830258) revealed purity at 215 nm of 98 % (retention time = 3.76 minutes). The experimental LCMS and HRMS spectra are included in Figures 9 and 10.

Figure 9

LCMS purity data at 215 nm for SID 103904185 (CID 49830260); LCMS retention time: 3.757; purity at 215 nm = 98%.

Figure 10

HRMS data for SID 103904185 (CID 49830260); HRMS (ESI) m/z calcd for C₂₃H₂₅N₄O ([M+H]⁺), 373.2028, found 373.2030.

Solubility

For probe ML240

Aqueous solubility 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. The solubility of probe SID 103904169 (CID 49830258) was determined to be 0.27 μg/mL at pH 7.4.[2]

For probe ML241

The solubility of probe SID 103904185 (CID 49830260) was measured exactly as for ML240 and was determined to be 0.20 μg/mL at pH 7.4.[2]

Stability

For probe ML240

Aqueous stability was measured at room temperature (23 °C) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%). Stability data are depicted as a graph showing the loss of compound versus time over a 48 hour period, with a minimum of 6 time points and the percent compound remaining at the end of 48 hours (Figure 11). Since the starting compound concentration for this procedure used the kinetic solubility as determined, it is not uncommon to observe apparent compound instability, which, in reality, may be more appropriately assigned to compound insolubility. For this reason, the compound stability measurement was repeated with the addition of acetonitrile (50% v/v final). The stability data for these two experiments are depicted as a graph showing the loss of compound versus time over a 48 hour period with a minimum of 6 time points and the percent compound remaining at the end of 48 hours.

Figure 11

Stability for compound SID 103904169 (CID 49830258) in 100% aqueous and 50% acetonitrile/aqueous.

The apparent stability of probe compound SID 103904169 (CID 49830258), determined as the percent of compound remaining after 48 hours, was 3% in 100% aqueous solution versus 100% in 50% acetonitrile/aqueous solution.[2] We propose that the difference observed for these measurements is due to gradual compound precipitation in 100% aqueous solution.

For probe ML241

Aqueous stability (see Figure 12) was measured exactly as for ML240.

Figure 12

Stability for compound SID 103904185 (CID 49830260) in 100% aqueous and 50% acetonitrile/aqueous.

The apparent stability of probe compound SID 103904185 (CID 49830260), determined as the percent of compound remaining after 48 hours, was 2% in 100% aqueous solution, versus 100% in 50% acetonitrile/aqueous solution.[2] We propose that the difference observed for these measurements is due to gradual compound precipitation in 100% aqueous solution.

Synthetic Route

For probe ML240

The probe compound SID 103904169 (CID 49830258) and numerous analogues were synthesized by the reaction sequence shown in Figure 13. Thus, 2,4,-dichloro-8-methoxyquinazoline was treated with the benzylamine to afford N-benzyl-2-chloro-8-methoxyquinazolin-4-amine. N-Benzyl-2-chloro-8-methoxyquinazolin-4-amine was heated, under microwave conditions, with 1H-benzo[d]imiazol-2-amine to afford N-benzyl-2-(2-imini-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-8-methoxyquinazolin-4-amine.

Figure 13

General synthetic route for preparing probe compound SID 103904169 (CID 49830258) and associated analogues.

For probe ML241

The probe compound SID 103904185 (CID 49830260) and numerous analogues were synthesized by the reaction sequence shown in Figure 14. Thus, 2,4-dichloro-5,6,7,8-tetrahydroquinazoline was treated with the benzylamine to afford N-benzyl-2-chloro-5,6,7,8-tetrahydroquinazolin-4-amine. N-Benzyl-2-chloro-8-methoxyquinazolin-4-amine was heated, under microwave conditions, with 3,4-dihydro-2H-benzo[b][1,4]oxazine to afford 2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8-tetrahydroquazolin-4-amine.

Figure 14

General synthetic route for preparing probe compound SID 103904185 (CID 49830260) and associated analogues.

Submission of Probe and Five Related Analogues to the MLSMR

For probe ML240 see Table 6.

Table 6

Probe ML240 and analog submissions to MLSMR (BioFocus DPI) for reversible inhibitors of p97.

For probe ML241 see Table 7.

Table 7

Probe ML241 and analog submissions to MLSMR (BioFocus DPI) for reversible inhibitors of p97.

2.3. Probe Preparation

General synthesis and analysis experimental details: All reagents were used as received from the following suppliers: Alfa Aesar, Ark Pharm, Aldrich, D-L Chiral Chemicals, OChem corporation and Fisher Scientific. Acetonitrile and THF were purified using the Innovative Technology PureSolv solvent purification system. The ¹H and ¹³C spectra were recorded on a Bruker Avance 400 MHz or 500 MHz spectrometer. Chemical shifts are reported in parts per million and were referenced to residual proton solvent signals. Flash column chromatography separations were performed using the Teledyne Isco CombiFlash R_F using RediSep R_F silica gel columns. TLC was performed on Analtech UNIPLATE silica gel GHLF plates (gypsum inorganic hard layer with fluorescence). TLC plates were developed using iodine vapor. Automated preparative RP HPLC purification was performed using an Agilent 1200 Mass-Directed Fractionation system (Prep Pump G1361 with gradient extension, make-up pump G1311A, pH modification pump G1311A, HTS PAL autosampler, UV-DAD detection G1315D, fraction collector G1364B, and Agilent 6120 quadrapole spectrometer G6120A). The preparative chromatography conditions included a Waters X-Bridge C18 column (19 × 150 mm, 5 um, with 19 × 10-mm guard column), elution with a water and acetonitrile gradient, which increases 20% in acetonitrile content over 4 min at a flow rate of 20 mL/min (modified to pH 9.8 through addition of NH₄OH by auxiliary pump), and sample dilution in DMSO. The preparative gradient, triggering thresholds, and UV wavelength were selected according to the analytical RP HPLC analysis of each crude sample. The analytical method used an Agilent 1200 RRLC system with UV detection (Agilent 1200 DAD SL) and mass detection (Agilent 6224 TOF). The analytical method conditions included a Waters Aquity BEH C18 column (2.1 × 50 mm, 1.7 um) and elution with a linear gradient of 5% acetonitrile in pH 9.8 buffered aqueous ammonium formate to 100% acetonitrile at 0.4 mL/min flow rate. Compound purity was measured on the basis of peak integration (area under the curve) from UV/vis absorbance (at 214 nm), and compound identity was determined on the basis of mass analysis. All compounds used for biological studies have purity >90%.

The probe compounds were synthesized using the following protocols:

For probe ML240

N-Benzyl-2-chloro-8-methoxyquinazolin-4-amine: 2,4-Dichloro-8-methoxyquinazoline (500 mg, 2.18 mmol) was suspended in 10 mL of acetonitrile. Triethylamine (0.5 mL) was added, followed by benzylamine (0.24 mL, 2.18 mmol). The mixture was stirred at room temperature for 16 h. The precipitate was filtered and washed with acetontrile to give a white solid (531 mg, 81%). ¹H NMR (400 MHz, CDCl₃) δ 7.38 – 7.22 (m, 6H), 7.12 (d, J = 7.5 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 5.97 (s, 1H), 4.78 (d, J = 5.2 Hz, 2H), 3.94 (s, 3H).

N-Benzyl-2-(2-imino-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-8-methoxyquinazolin-4-amine: To a suspension of N-benzyl-2-chloro-8-methoxyquinazolin-4-amine (20 mg, 67 mmol) in acetonitrile (1 mL) was added 1H-benzo[d]imidazol-2-amine (18 mg, 133 mmol, 2 equiv.). The mixture was heated to 180 °C for 1 h under microwave irradiation. The evaporated residue was purified by reverse phase HPLC to give a white solid (14.6 mg, 55%). ¹H NMR (400 MHz, DMSO) δ 9.33 (t, J = 5.9 Hz, 1H), 8.31 – 8.05 (m, 3H), 7.93 (d, J = 7.5 Hz, 1H), 7.50 – 7.39 (m, 3H), 7.39 – 7.30 (m, 3H), 7.25 (t, J = 7.3 Hz, 1H), 7.15 (d, J = 7.1 Hz, 1H), 7.03 (td, J = 1.2, 7.6 Hz, 1H), 6.87 – 6.76 (m, 1H), 4.92 (d, J = 5.8 Hz, 2H), 3.98 (s, 3H). ¹³C NMR (101 MHz, DMSO, APT pulse sequence) δ 160.8, 154.7, 153.5, 153.4, 142.8, 140.0, 138.5, 131.5, 128.5, 126.9, 126.6, 124.9, 122.5, 118.7, 114.8, 114.6, 114.1, 113.0, 112.7, 56.1, 44.5. LCMS retention time: 3.132; purity at 214 nm = 100%. HRMS (ESI) m/z calcd for C₂₃H₂₁N₆O ([M+H]⁺), 397.1777, found 397.1776.

For probe ML241

N-Benzyl-2-chloro-5,6,7,8-tetrahydroquinazolin-4-amine: 2,4-Dichloro-5,6,7,8 tetrahydroquinazoline (100 mg, 0.49 mmol) was suspended in 2 mL of acetonitrile. Triethylamine (0.1 mL) and then benzylamine (53 mL, 0.49 mmol) were added. The mixture was stirred at room temperature for 16 h. The mixture was evaporated to dryness. The residue was purified by silica gel chromatography (EtOAc:hexanes = 1:2, R_F = 0.6) to give a white solid (38 mg, 28%). ¹H NMR (400 MHz, CDCl₃) δ 7.44 – 7.31 (m, 5H), 4.95 (s, 1H), 4.71 (d, J = 5.4 Hz, 2H), 2.71 (t, J = 5.4 Hz, 2H), 2.28 (t, J = 5.5 Hz, 2H), 1.95 – 1.74 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 164.3, 161.7, 157.5, 138.2, 128.8, 128.1, 127.8, 110.3, 45.4, 31.6, 21.9, 21.9.

2-(2H-Benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8-tetrahydroquinazolin-4-amine: To a suspension of N-benzyl-2-chloro-5,6,7,8-tetrahydroquinazolin-4-amine (10 mg, 0.036 mmol) in CH₃CN (1 mL) was added 3,4-dihydro-2H-benzo[b][1,4]oxazine (9.8 mg, 0.072 mmol, 2 equiv.). The mixture was heated to 180 °C for 1h under microwave irradiation. The solvent was removed under vacuum, the residue was suspended in EtOAc, washed with saturated NaHCO₃, and the layers were separated. The organic layer was dried over MgSO₄ and concentrated under vacuum. The residue was purified by silica gel chromatography (EtOAc:hexanes = 1:2, R_F = 0.6) to give a colorless oil (9 mg, 66%). ¹H NMR (400 MHz, CDCl₃) δ 8.09 – 8.01 (m, 1H), 7.42 – 7.30 (m, 5H), 6.92 – 6.86 (m, 2H), 6.80 – 6.73 (m, 1H), 4.79 (s, 1H), 4.69 (d, J = 5.6 Hz, 2H), 4.28 (dd, J = 3.1, 5.0 Hz, 2H), 4.23 (dd, J = 3.1, 5.0 Hz, 2H), 2.67 (t, J = 5.7 Hz, 2H), 2.32 (t, J = 5.7 Hz, 2H), 1.93 – 1.77 (m, 4H). ¹³C NMR (101 MHz, CDCl₃, APT pulse sequence) δ 162.5, 160.7, 157.3, 145.9, 139.5, 128.6, 128.5, 127.5, 127.3, 123.9, 122.6, 119.5, 116.6, 103.8, 65.9, 45.0, 42.2, 32.2, 22.6, 22.5, 21.9. LCMS retention time: 3.757; purity at 214 nm = 98%. HRMS (ESI) m/z calcd for C₂₃H₂₅N₄O ([M+H]⁺), 373.2028, found 373.2030.

3. Results

3.1. Dose Response Curves for Probe

Figure 15

(A) Titration curves for inhibition of in vitro p97 ATPase activity by ML240 (filled circle) and ML241 (open diamond). (B) Titration curves of ML240 (filled circle) and ML241 (open diamond). for inhibiting Ub^G76V-GFP degradation.

Figure 16

(A) MIN (filled circle and square) and CIN (open circle and square) colon cancer lines were treated with ML240 (1.1, 3,3, 10, or 20 μM) for 7 h prior to determination of caspase 3 plus 7 activities in cell extract. (B) HCT116 cells were were incubated with 10 μM of DBeQ, ML240, STS (staurosporine) or 20 μM of bortezomib for 3, 7, 10 or 20 h prior to determination of caspases-3 plus -7 activities in the cell extract. (C) Same as (B) except HT29 cells were used.

3.2. Cellular Activity

Four cell-based assays were used to characterize in-depth the probe compounds, CID 49830258 [ML240], and CID 49830260 [ML241]. These assays were: (i) stabilization of Ub^G76V-GFP, (ii) stabilization of ODD-luciferase, (iii) activation of caspase 3/7, and (iv) inhibition of proliferation of SW403 cells. The activity for the probe compounds in this suite of cell-based assays is shown in detail in Table 8.

Table 8

Characterization of ML240 and ML241 in cell based assays.

3.3. Profiling Assays

To uncover potential off-target effects of our two probes on protein kinases, we evaluated them using an activity-based proteomics platform (KiNativ; ActivX Biosciences). This assay employs “mulitple reaction monitoring” (MRM) mass spectrometry to measure the ability of small molecules to inhibit the covalent labeling of protein kinases in native cell lysates by a broadly reactive ATP acyl-phosphate probe.[3] Remarkably, CID 49830260 [ML241] (20 μM) did not appreciably inhibit labeling of any of the ≈170 kinases that were evaluated. In contrast, CID 49830258 [ML240] inhibited labeling of three protein kinase domains by greater than 50% when tested at 20 μM: PIP5K3 (a member of the phosphoinositide-3 kinase family), JAK1-domain 1 (it is unclear whether this domain is regulatory in nature or catalytically active), and DNA-dependent protein kinase (DNAPK). By contrast, a scaffold related to a known protein kinase inhibitor blocked labeling of 56 protein kinases by >50% (data not shown). We conclude that both CID 49830258 [ML240] and CID 49830260 [ML241] are quite specific. Interestingly, CID 49830258 [ML240] presents itself as an agent that may be useful for initiating probe development programs focused on PIP5K3, JAK1, or DNAPK.

In Vitro Pharmacology Profiles of probes CID 49830258 [ML240] and CID 49830260 [ML241] (SeeTable 9.).[2]

Table 9

Summary of in vitro ADMET/T Properties for p97ATPase probe(s).

Probe ML240 had poor solubility at all pH’s tested. ML241 had reasonable solubility at the lowest pH and had poor solubility at the other pHs.

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 was placed between a donor and acceptor compartment. At the start of the test, compound was introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments were measured using UV spectroscopy. In this assay, probe CID 49830258 [ML240] and probe CID 49830260 [ML241] had good permeability.

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. Compounds that exhibit high plasma protein binding are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, compounds that remain largely unbound in plasma are generally available for distribution to other organs and tissues. CID 49830258 [ML240] and CID 49830260 [ML241] were highly bound (> 99%) to both human and mouse plasma.

Plasma Stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which can strongly 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. CID 49830258 [ML240] and CID 49830260 [ML241] showed excellent stability (>99%) in both human and mouse plasma.

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. CID 49830258 [ML240] exhibited acceptable stability upon incubation with human microsomes, and was much more rapidly metabolized by mouse microsomes.

CID 49830258 [ML240] showed a moderate level of toxicity toward human hepatocyctes, while CID 49830260 [ML241] showed no toxicity toward human hepatocyctes.

4. Discussion

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

Currently, one of the major limitations in studying p97 function is the lack of a potent, selective, and reversible small molecule inhibitor that has a defined mechanism of action. During the course of this work, EerI,[4] ML080,[5] and 2-anilino-4-aryl-1,3-thiazole (Myriad 12 and Myriad 19) [6] were reported to be p97 inhibitors. To understand how these compounds compare to DBeQ [7], CID 49830258 [ML240], and CID 49830260 [ML241], we evaluated their properties using our assays, and the results are shown in Table 10.

Table 10

Comparison of known inhibitors of p97ATPase with DBeQ, CID 49830258 [ML240], and CID 49830260 [ML241].

In summary, in contrast to eeyarestatin, the new probe compounds work by a defined mechanism of action, are reversible inhibitors, and have defined activity towards non-p97 ATPase and protein kinase targets. In contrast to the ML080 and the Myriad compounds, within the context of the UPS, the new probe compounds are specific for p97, and are far-better characterized in terms of various p97-dependent processes. Limitations of the new probe compounds are the poor solubility and the relatively low cell-based potency in the Ub-GFP assay.

In our hands, the Myriad compounds were less potent p97 inhibitors both in vitro (IC₅₀ of 20 μM) and in cells (IC50 of 16~26 μM) compared to what has been reported.[7] We do not fully understand the basis for this discrepancy, but would like to note that different in vitro assay methods were used (coupled enzyme assay vs. Biomol Green) and the cell-based assay employed by Bursavich and coworkers [6] involved long exposure to compound and monitored accumulation of an Ub^G76V-luciferase reporter as opposed to degradation. A potential problem with the accumulation format was noted by Alvarez-Castelao and coworkers, who demonstrated that accumulation of three UPS reporters in response to proteasome inhibitors is driven in large measure by up-regulation of the constructs’ cytomegalovirus promoter.[8] It is also worth noting that the Myriad compounds interfere with luciferase read-outs, which is in agreement with a recent report on a related scaffold.[9] In addition, the Myriad compounds partially inhibit turnover of a p97-independent substrate.[10] We identified the same scaffold as the original Myriad hit in the MLSMR HTS, but subsequent assays revealed that this hit is of low potency and it stabilizes both the p97-dependent and independent reporters. Thus, although we confirmed that the top Myriad compounds inhibit p97, they exhibit properties that may compromise their utility. By contrast, EerI is a specific inhibitor of degradation of the p97-dependent reporter in our assays, but this compound exhibits other characteristics, including interference with luciferase read-outs and irreversibility, that circumscribe its use for some applications. Moreover, EerI has been reported to inhibit other cellular components including ataxin 3 [4] and Sec61.[10] The mechanism by which EerI inhibits p97 is not currently understood. Interestingly, EerI did not inhibit ATPase activity of purified p97 and it has been suggested that it needs to be metabolized into an active compound to exert its inhibitory effect.[4] This could explain why high concentrations of EerI are needed to inhibit Sec61-mediated translocation in vitro.[10]

For a more comprehensive description of the research disclosed in this probe report, please see the associated full paper. [11]

5. References

1.: Chou TF, Deshaies RJ. Quantitative cell-based proteindegradation assays to identify and classify drugs that target the ubiquitin-proteasome system. J Biol Chem. 2011;286:16546–16554. [PMC free article: PMC3089497] [PubMed: 21343295]
2.: Solubility and stability data measurements were performed at the Sanford-Burnham Medical Research Institute, under the direction of Dr. Layton Smith
3.: Patricelli MP, Szardenings AK, Liyanage M, Nomanbohoy TK, Wu M, Weissig H, Aban A, Chun D, Tanner S, Kozarich JW. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry. 2007;46:350–358. [PubMed: 17209545]
4.: Wang Q, Li L, Ye Y. Inhibition of p97-dependent protein degradation by Eeyarestatin I. J Biol Chem. 2008;283:7445–7454. [PMC free article: PMC2276333] [PubMed: 18199748]
5.: Brown SJ, Chou TF, Deshaies R, Roberts E, Guerrero M, Minond D, Mercer BA, Hodder P, Rosen HR. Probe Report for P97/cdc48 Inhibitors. 2010.
6.: Bursavich MG, et al. 2-Anilino-4-aryl-1,3-thiazole inhibitors of valosincontaining protein (VCP or p97). Bioorg Med Chem Lett. 2010;20:1677–1679. [PubMed: 20137940]
7.: Chou TF, Brown SJ, Minond D, Nordin BE, Li K, Jones AC, Chase P, Porubsky PR, Stoltz BM, Schoenen FJ, Patricelli MP, Hodder P, Rosen H, Deshaies RJ. Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. Proc Natl Acad Sci USA. 2011;108:4834–4839. [PMC free article: PMC3064330] [PubMed: 21383145]
8.: Alvarez-Castelao B, Martin-Guerrero I, Garcia-Orad A, Castano JG. Cytomegalovirus promoter up-regulation is the major cause of increased protein levels of unstable reporter proteins after treatment of living cells with proteasome inhibitors. J Biol Chem. 2009;284:28253–28262. [PMC free article: PMC2788877] [PubMed: 19679666]
9.: Auld DS, Thorne N, Nguyen DT, Inglese J. A specific mechanism for nonspecific activation in reporter-gene assays. ACS Chem Biol. 2008;3:463–470. [PMC free article: PMC2729322] [PubMed: 18590332]
10.: Cross BC, McKibbin C, Callan AC, Roboti P, Piacenti M, Rabu C, Wilson CM, Whitehead R, Flitsch SL, Pool MR, High S, Swanton E. Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum. Journal of cell science. 2009;122:4393–4400. [PMC free article: PMC2779136] [PubMed: 19903691]
11.: Chou TF, Li K, Frankowski KJ, Schoenen FJ, Deshaies RJ. Structure-Activity Relationship Study Reveals ML240 and ML241 as Potent and Selective Inhibitors of p97 ATPase. ChemMedChem. 2013 February;8(2):297–312. [PMC free article: PMC3662613] [PubMed: 23316025]

Bookshelf ID: NBK133422PMID: 23658965

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Chou TF, Li K, Nordin BE, et al. Selective, reversible inhibitors of the AAA ATPase p97. 2011 Apr 14 [Updated 2013 May 1]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Structure & Characteristics
Recommendations for Scientific Use of the Probe
Materials and Methods
Results
Discussion
References

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Selective, reversible inhibitors of the AAA ATPase p97 - Probe Reports from the NIH Molecular Libraries Program
SMAD5-AS1 SMAD5 antisense RNA 1 [Homo sapiens]
SMAD5-AS1 SMAD5 antisense RNA 1 [Homo sapiens]
Gene ID:9597
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9597[uid] AND (alive[prop]) (1)
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Probe Reports from the NIH Molecular Libraries Program [Internet].

Selective, reversible inhibitors of the AAA ATPase p97

Authors

Affiliations

Probe Structure & Characteristics

Recommendations for Scientific Use of the Probe

2. Materials and Methods

2.1. Assays

Table 1List of Assays and AIDs for this Project

Assay Protocol

Assay Protocol

Assay protocol

Assay Protocol

2.2. Probe Chemical Characterization

Probe Chemical Structure, Physical Parameters, and Probe Properties

Figure 1Property summary of probe compound SID 103904169 (CID 49830258)

Figure 2Property summary of probe compound SID 103904185 (CID 49830260)

Structure verification and Purity: 1H NMR, 13C NMR, LCMS and HRMS

For probe ML240

For probe ML241

Solubility

For probe ML240

For probe ML241

Stability

For probe ML240

For probe ML241

Synthetic Route

For probe ML240

For probe ML241

Submission of Probe and Five Related Analogues to the MLSMR

2.3. Probe Preparation

For probe ML240

For probe ML241

3. Results

3.1. Dose Response Curves for Probe

Figure 15

Figure 16

3.2. Cellular Activity

3.3. Profiling Assays

4. Discussion

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

5. References

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Structure verification and Purity: ¹H NMR, ¹³C NMR, LCMS and HRMS