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Probe Report for PME-1 Inhibitors

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

Received: ; Last Update: October 20, 2010.

Recent findings have identified protein phosphatase methylesterase-1 (PME-1) as a protector of sustained ERK pathway activity in malignant gliomas. PME-1 is a protein methylesterase that functions in the regulation of protein phosphatase 2A (PP2A) by reversible methylation. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors. The probe compound ML136 (CID-44607965), containing a sulfonyl acrylonitrile core, represents the first potent, selective inhibitor of PME-1. Moreover, the probe does not appear to exhibit cytotoxicity. Thus, ML136 should serve as a useful tool for in vitro and in situ research assays in which it is desirable to specifically block PME-1 activity.

Assigned Assay Grant #: CA132630-01

Screening Center Name & PI: Scripps Research Institute Molecular Screening Center (SRIMSC), H. Rosen

Chemistry Center Name & PI: SRIMSC, H. Rosen

Assay Submitter & Institution: Ben Cravatt, TSRI

PubChem Summary Bioassay Identifier (AID): AID-2143

Image ml136fu1
PME-1 Inhibitor Probe
(Sulfonyl Acrylonitrile Scaffold)
CID 44607965 SID-87457340
MLS002699139
PME-1 IC50: 0.5 μM
Anti-Target (other serine hydrolases) IC50:>100 μM
Cytox CC50: >100 μM
ML136

Probe Structure & Characteristics

As described in the CPDP, the chief goal for this probe development project was to find a selective inhibitor of the protein methylesterase PME-1. The probe compound ML136 (CID 44607965), having a sulfonyl acrylonitrile core, is claimed as a potent and selective inhibitor of PME-1. This probe is highly (>40-fold) selective among other serine hydrolases, as demonstrated by activity in the gel-based proteomic profiling assay. In addition, the probe compound CID 44607965 exhibits no cytotoxicity. This compound is the first selective inhibitor of PME-1.

Please see the probe table on the next page for all results described above.

Probe Selection

Following the uHTS campaign and counterscreening by gel-based competitive activity-based protein profiling (ABPP) in proteomes, a sulfonyl acrylonitrile class of inhibitor was identified for probe development. Following two rounds of SAR, compound CID 44607965 was selected as a probe since it was the most potent and selective compound tested for in situ studies. It was ~20-fold more potent than the top initial hit (CID 5703330) and at least 20-fold more selective.

Recommendations for the Scientific Use of This Probe

This compound is useful for in vitro or in situ research assays in which it is desirable to specifically block PME-1 activity.

1. Scientific Rationale for Project

Reversible protein phosphorylation networks play essential roles in most cellular processes. While over 500 kinases catalyze protein phosphorylation, only two enzymes, PP1 and PP2A, are responsible for >90% of all serine/threonine phosphatase activity[1]. Phosphatases, unlike kinases, achieve substrate specificity through complex subunit assembly and post-translational modifications rather than number. PP2A, for example, typically exists as heterotrimer with diverse subunits that may combinatorially make as many as 70 different holoenzyme assemblies[2]. Mutations in several of these PP2A subunits have been identified in human cancers, suggesting that PP2A may act as a tumor suppressor[3]. Adding further complexity, several residues of the catalytic subunit of PP2A can be reversibly phosphorylated, and the C-terminal leucine residue can be reversibly methylated [4, 5]. PME-1 is specifically responsible for demethylation of the carboxyl terminus [4].

Methylesterification is thought to control the binding of different subunits to PP2A, but little is known about physiological significance of this post-translational modification in vivo [5]. Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas [6]. In order to further elucidate the role of PP2A methylation in vivo, our lab has generated mice that lack PME-1 (PME-1 knockout mice) by targeted gene disruption [7]. Unfortunately, PME-1 deletion resulted in perinatal lethality, underscoring the importance of PME-1 but hindering our biological studies. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors.

As a serine hydrolase, catalytically active PME-1 is readily labeled by fluorescent activity-based protein profiling (ABPP) probes bearing a fluorophosphonate (FP) reactive group [8]. This reactivity can be exploited for inhibitor discovery using a competitive-ABPP platform, whereby small molecule enzyme inhibition is assessed by the ability to out-compete ABPP probe labeling [9]. When used in the context of a complex proteome, competitive-ABPP also offers a means to assess inhibitor selectivity against a wide range of probe-reactive enzymes. Competitive ABPP has also been configured to operate in a high-throughput manner via fluorescence polarization readout, FluoPol-ABPP [10], offering a facile means to screen for PME-1 inhibitors.

Probe or AnalogCID/MLSIDMLSTarget NameTarget IC50 [AID-2366] (μM)Anti-target NameAnti-Target IC50 [AID-2366] (μM)SelectivitySecondary Assay Cytox CC50 [AID-2365] (nM)
Probe44607965/ML13687457340MLS002699139PME-10.5>30 serine hydrolases (SHs)>20>40-fold100000
Analog 14460794987457341MLS002699140PME-10.6>30 SHs>20>33-fold100000
Analog 24460795487457343MLS002699141PME-13.0>30 SHs>20>6-foldNT*
Analog 34460797487457347MLS002699142PME-14.8>30 SHs>20>4.2-foldNT
Analog 44460796987457348MLS002699143PME-16.8>30 SHs>20>2-foldNT
Analog 5570333087457336MLS002699144PME-110.8>30 SHs20 75kDa SH<2-foldNT
*

NT: not tested

2. Project Description

a. Original goal for probe characteristics

The goal of the campaign was to discover compounds with inhibitory activity against PME-1 that are selective among the serine hydrolases in mouse tissue and human cell line proteomes as assessed by gel-based ABPP. Compounds of interest should exhibit an IC50 of <10 μM potency, and preferably <1 μM potency.

b. Information for each Assay Implemented and Screening Run

i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)

PubChem BioAssay Table

AIDAssay NameAssay TypeTargetPowder SampleCompound Concentration
2130Primary biochemical high-throughput screening assay to measure PME-1 inhibitionPrimary Assay (1X, %INH)PME-1No5.9 μM
2268Primary Marybridge biochemical high-throughput screening assay to measure PME-1 inhibitionPrimary Assay (1X, %INH)PME-1No8 μM
2171Confirmation biochemical high-throughput screening assay to measure PME-1 inhibitionConfirmation Assay (3X, %INH)PME-1No5.9 μM
2174Counterscreen for PME1 inhibitors: Primary biochemical high- throughput screening assay to measure LYPLA1 inhibitionPrimary Assay (1X, %INH)LYPLA1No5.9 μM
2233Counterscreen for PME1 inhibitors: Confirmation biochemical high-throughput screening assay to measure LYPLA1 inhibitionConfirmation Assay (3X, %INH)LYPLA1No5.9 μM
2177Counterscreen for PME1 inhibitors: Primary biochemical high- throughput screening assay to measure LYPLA2 inhibitionPrimary Assay (1X, %INH)LYPLA2No5.9 μM
2232Counterscreen for PME1 inhibitors: Confirmation biochemical high-throughput screening assay to measure LYPLA2 inhibitionConfirmation Assay (3X, %INH)LYPLA2No5.9 μM
2369Late stage results from the probe development effort to identify inhibitors of Protein Methylesterase-1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) InhibitionSecondary AssayPME-1Yes20 μM
2366Late stage results from the probe development effort to identify inhibitors of Protein Methylesterase-1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) IC50Secondary AssayPME-1, anti-target serine hydrolasesYes0.1 – 100 μM
2371Late stage results from the probe development effort to identify inhibitors of Protein Methylesterase-1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) IC50: Purified enzymeSecondary AssayPME-1Yes0.1 – 100 μM
2365Late stage results from the probe development effort to identify inhibitors of Protein Methylesterase-1 (PME-1): Luminescence- based counterscreen assay to identify cytotoxic compoundsSecondary AssayHEK 293T cellsYes0.001 – 100 μM
2368Late stage results from the probe development effort to identify inhibitors of Protein Methylesterase-1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) Gel Filtration AssaySecondary AssayPME-1Yes100 M
2363Late stage results from the probe development effort to identify inhibitors of Protein Methylesterase-1 (PME-1): Inhibition of PP2A demethylation in HeLa cellsSecondary AssayPME-1Yes5 μM and 20 μM

ii. Assay Rationale & Description

Table of Assay Rationale and Description

AIDAssay RationaleAssay DescriptionZ′S:B
2130To measure the ability of compounds to inhibit PME-1 activityIn this assay, a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family is used to label PME-1 in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as PME-1 inhibitors will prevent PME-1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization in the well. Compounds were tested in singlicate at a final nominal concentration of 5.9 micromolar.0.761.72
2268To measure the ability of compounds to inhibit PME-1 activity (Maybridge Library)Same as above except compounds were tested at 8.0 micromolar.0.783.17
2171Confirmation of hit activity of compounds identified in the Primary ScreenSame as 2130 except compounds were tested in triplicate.0.732
2174Counterscreen for PME1 inhibitors: To measure the ability of compounds to inhibit LYPLA1In this assay, a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family is used to label LYPLA1 in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as LYPLA1 inhibitors will prevent LYPLA1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization in the well. Compounds were tested in singlicate at a final nominal concentration of 5.9 micromolar.0.862.12
2233Counterscreen for PME1 inhibitors: Confirmation of activity of compounds identified in 2174Same as above except compounds were tested in triplicate.0.892.3
2177Counterscreen for PME1 inhibitors: To measure the ability of compounds to inhibit LYPLA2In this assay, a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family is used to label LYPLA2 in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as LYPLA2 inhibitors will prevent LYPLA2-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization in the well. Compounds were tested in singlicate at a final nominal concentration of 5.9 micromolar.0.771.69
2232Counterscreen for PME1 inhibitors: Confirmation of activity of compounds identified in 2177Same as above except compounds were tested in triplicate.0.781.69
2369To confirmation activity of compounds in proteomesIn this assay, a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family is used to label PME-1 in mouse brain soluble lysates in the presence of test compounds. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as PME-1 inhibitors will prevent PME-1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe, leading to low fluorescence intensity in the band in the gel. Percent inhibition of PME- 1 in mouse brain soluble lysates (compound at 20 μM) was determined.N/AN/A
2366To determine selectivity of compounds in proteomesSame as above except IC50 values are determined from dose-response curves from three trials at each inhibitor concentration (0.1–100 mM).N/AN/A
2371To confirm activity ofcompounds with purified enzymeSame as above except purified PME-1 (not in lysates) was tested. IC50 values are determined from dose-response curves from three trials at each inhibitor concentration (0.1–100 mM).N/AN/A
2365To determine cytotoxicity of inhibitor compoundsIn this assay, HEK cells are incubated with test compounds, followed by determination of cell viability. The assay utilizes the CellTiter- Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in triplicate in a 7-point 1:10 dilution series starting at a nominal test concentration of 100 micromolar.N/AN/A
2368To assess reversibility of binding of inhibitor compoundsIn this assay, a fraction of the enzyme-inhibitor mixture is passaged over a Sephadex G-25M column (GE Healthcare) before reaction with a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as irreversible PME-1 inhibitors will prevent PME-1-probe interactions after gel filtration, thereby increasing the proportion of free (unbound) fluorescent probe, leading to low fluorescence intensity in the band in the gel. The compound’s reversibility of inhibition of PME-1 was assessed.N/AN/A
2363To confirm that compounds inhibit the ability of PME-1 to demethylate PP2A in HeLA cellsThe purpose of this assay is to monitor the demethylation of PP2A by endogenous PME-1 that occurs during inhibitor incubation with cells in culture (e.g., HeLa) at 37 °C. Following incubation with compound, cells are homogenized, and proteins are separated by SDS- PAGE. PP2A demethylation is visualized by chemi-luminescent dectection using HRP-antibody specific to C-terminal demethylated PP2A. As designed, test compounds that act as PME-1 inhibitors will inhibit demethylation of PP2A, resulting in a decrease in the demethylated PP2a signal.N/AN/A

N/A: Not applicable.

Table of Reagents and Source

AIDReagent (Source)
PME-1 Inhibition Assays (AID-2130, AID-2269, AID-2171)Recombinant PME-1 enzyme (supplied by Assay Provider)
FP-Rh probe (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)
DTT (Invitrogen 15508-013)
PME-1 Counterscreen Assays: LYPLA1 (AID-2174, AID-2233)Recombinant LYPLA1 enzyme (supplied by Assay Provider)
Substrate (FP-Rh probe) (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)
DTT (Invitrogen, part 15508-013)
PME-1 Counterscreen Assays: LYPLA2 (AID-2177, AID-2232)Recombinant LYPLA2 enzyme (supplied by Assay Provider)
Substrate (FP-Rh probe) (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)
DTT (Invitrogen, part 15508-013)
Cytotoxicity Assay (AID-2365)HEK 293T cells
Cell Titer-Glo (Promega, part G75729)
96-well plates
ABPP % INH Assay (AID-2369)Mouse brain cytosol
FP-Rh probe
Sodium Chloride (Fisher, part 980597)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
ABPP IC50 Assay (AID-2366)Mouse brain cytosol
FP-Rh probe
Sodium Chloride (Fisher, part 980597)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
ABPP Assay: Purified enzyme (AID-2371)Recombinant PME-1 enzyme
FP-Rh probe
Sodium Chloride (Fisher, part 980597)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
Demethylation (AID-2363)Hela cells
HRP-antibody specific to C-terminal demethylated PP2a (Millipore 05-577)
Sodium Chloride (Fisher, part 980597)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
Gel Filtration Assay (AID-2368)Recombinant PME-1
FP-Rh probe
Sodium Chloride (Fisher, part 980597)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
Sephadex G-25 (GE Healthcare, part 17-0851-01)

iii. Summary of Results

Following Primary screening in singlicate (AID-2130 and AID-2269), confirmation of hit activity in triplicate (AID-2171), counterscreening by gel-based ABPP in proteomes to determine selectivity (AID-2369 and AID-2366), we identified an sulfonyl acrylonitrile class of inhibitor for probe development

CID 44607965 discovered from these efforts represents the first selective covalent PME-1 inhibitor. We anticipate it will be useful to determine the in vivo function of PME-1.

c. Probe Optimization

i. Description of SAR & chemistry strategy (including structure and data) that led to the probe

We speculated that this sulfonyl acrylonitrile core from our hit compound 10 (Appendix 2, Table 1) was covalently reacting with the active site serine of PME-1. An initial round of SAR by purchase and synthetic efforts in the Cravatt lab indeed revealed that this chemical moiety is critical for PME-1 inhibitory activity. Specifically, oxidation of either the olefin (compound 20) or the nitrile (compound 21) or replacement of the sulfonyl with a ketone (compound 32) resulted in completely inactive compounds. Further, the bis-sulfonyl compound 19 was completely inactive and the bis-nitrile compounds 17 and 18 resulted in a 2-fold loss in potency. Once we had determined that the sulfonyl acrylonitrile was important for activity, we investigated the sulfonamide portion of the compound. Our initial round of SAR by purchase (compounds 3348) showed that a wide-range of pyrroles have no activity if not conjugated to a deactivating sulfonamide. Through synthetic efforts, we confirmed and furthered these results by showing that pyrrole itself (compound 24) is not active, nor is a benzyl group (compound 22) or even the electron-withdrawing para-nitrobenzyl group (compound 23). From these two studies, we determined both the sulfonyl acrylonitrile and the sulfonamide are critical for activity.

Table 1. Comparative data on similar compound structures establishing SAR.

Table 1

Comparative data on similar compound structures establishing SAR.

In our next synthetic efforts, we modified the identity of the sulfonamide. We discovered that changing the size of this group (compounds 2730) led to complete loss of activity. Encouragingly, we also discovered that more electron-withdrawing sulfonamides (compounds 35, 7) led to an increase in potency. Specifically, compound 3 with a meta-cyano group had an IC50 of 3 μM and was selective for PME-1 relative to other serine hydrolases in the mouse brain soluble proteome. However, the addition of electron-withdrawing nature of the sulfonamide could only increase potency to a point, as the strongly electron-withdawing compounds 25 and 26 had no PME-1 inhibitory activity. We next altered the size and electronics of the sulfonyl moiety. Relative to the parent compound 10, we discovered that both increasing the size at this position (compound 9, IC50 = 6.8 μM) and making it more electron-withdrawing (compound 8, IC50 = 4.8) led to an increase in potency. At this point, we combined the best modifications on both sides of the molecule, namely meta-cyano substituted sulfonamide and the para-fluoro sulfonyl, and discovered an additive effect resulting in the most potent compound yet (2) with an IC50 of 640 nM. From this new scaffold we made several minor modifications, including halogen substitution (compounds 11, 14) and replacement of the meta-cyano (compounds 1, 12, 13, 15, 16), to discover the probe compound 1, which has a meta-nitro group instead of a meta-cyano group. This compound has an IC50 for PME-1 of 500 nM and was selective for PME-1 relative to other serine hydrolases by >40-fold.

As a class, the sulfonyl acrylonitrile compounds were highly selective as assessed by their anti-target reactivity by competitive-ABPP. Of the compounds tested at 20µM, only compounds 7, 10, 19, and 30 showed evidence of anti-target reactivity with a 75kDa serine hydrolase (Table 1 Appendix 2, Figure 1d Appendix 2).

Figure 1. Low throughput assays to characterize probe.

Figure 1

Low throughput assays to characterize probe. A. Selective inhibition of PME-1 in the mouse brain soluble proteome as determined by gel-based ABPP. B. Compounds 1 and 2 covalently inhibit PME-1, retaining inhibitory activity after gel filtration. C. Hela (more...)

To date, the lead hit compound 10 (Appendix 2, Table 1) from this inhibitor class has been tested in 152 other bioassays deposited in PubChem and has shown activity in no other systems—further evidence that this compound class does not possess promiscuous activity against other targets.

3. Probe

a. Chemical name of probe compound

(E)-2-(4-fluorophenylsulfonyl)-3-(1-(3-nitrophenylsulfonyl)-1H-pyrrol-2-yl)acrylonitrile [ML136]

b. Probe chemical structure including stereochemistry

Image ml136fu2

c. Structural Verification Information of probe SID-87457340

  1. 1H NMR (500 MHz, CDCl3) δ 8.78 (d, J = 0.6 Hz, 1H), 8.68 (dd, J = 2.0, 2.0 Hz, 1H), 8.58-8.53 (m, 1H), 8.29-8.26 (m, 1H), 8.03 (dd, J =8.7, 4.9 Hz, 2H), 7.87 (dd, J =8.1, 8.1 Hz, 1H), 7.77-7.75 (m, 1H), 7.67-7.64 (m, 1H), 7.30 (dd, J = 8.4, 8.4 Hz, 2H), 6.59 (dd, J = 3.9, 3.3 HZ, 1H), purity >95%
  2. high-res MS analysis (ESI-TOF): M-H expected: 460.0079, M-H observed: 460.0096

d. PubChem CID (corresponding to the SID)

CID 44607965

e. Availability from a Vendor

Not available

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

MLS002699139 (see table p.2)

g. Mode of action for biological activity of probe

We have shown that CID 44607965 (referred as compound 1 in Appendix 2) forms a covalent adduct with PME-1 by demonstrating that blockade of FP-rhodamine labeling is not reversed by gel filtration (Appendix 2, Figure 1B). Even though 1 contains a reactive chemical moiety, this compound selectively inhibits PME-1 in the mouse brain soluble proteome (Appendix 2, Figure 1A). Further, we showed that the probe inhibited the ability of PME-1 to demethylate PP2A in HeLa cells as evidenced by a decrease in demethylated PP2A after incubation of cells with compound (Appendix 2, Figure 1C).

h. Detailed synthetic pathway

Image ml136fu3

(E)-2-(4-fluorophenylsulfonyl)-3-(1H-pyrrol-2-yl)acrylonitrile. Powdered 4A molecular sieves were charged into a 25mL round bottom flask and flame dried under vacuum. After replacing the atmosphere with nitrogen, ethanol (12.5mL) was added, followed by pyrrole-2-carboxaldehyde (200mg, 2.1mmol, 1eq) the sulfonyl acetonitrile (419mg, 2.1mmol, 1eq), and triethylamine (1.25mL, 9.0mmol, 4.3eq). The reaction was refluxed for 4 hr, cooled to room temperature and then quenched with water (5mL). The aqueous layer was acidified with 1N HCl (2mL) and the product extracted into DCM (3 x 20mL). The combined organic layers were then washed with water (10mL), brine (10mL), dried over MgS04 and concentrated in vacuo. Flash chromatography (SiO2, 10-50% ethyl acetate/hexanes) afforded the free pyrrole product (498 mg, 1.81mmol, 86%).

(E)-2-(4-fluorophenylsulfonyl)-3-(1-(3-nitrophyeylsulfonyl)-1H-pyrrol-2-yl)acrylonitrile (PROBE AMZ30). Under nitrogen atmosphere, a 5mL flame-dried round bottom flask was charged with the free pyrrole (10mg, 0.036mmol, 1eq). The solid was diluted with dimethylformamide (0.580mL, 0.05M). Triethylamine (21μL, 0.145mmol, 5eq) was then added, followed by 3-nitrobenzenesulfonyl chloride (9.6mg, 0.043mmol, 1.2eq). The reaction was allowed to stir overnight at which point the solvent was removed in vacuo. Purification by preparatory TLC (SiO2, 1000μm, 50% ethyl acetate/hexanes) afforded the product (14.1mg, 0.030mmol, 84%).

i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

ADMET BBB, undefined; ADMET BBB level, undefined; ADMET absorption level, 2; ADMET solubility, −5.031; ADMET solubility level, low.

Solubility of the probe in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4) at room temperature was determined to be 28.4 µM. The probe has a half-life of >48 hours in PBS at room temperature (tested at 10 µM, Figure 3 Appendix 2).

Figure 3. Stability of ML136 (Compound 1) in PBS indicates a half-life of >48 hours.

Figure 3

Stability of ML136 (Compound 1) in PBS indicates a half-life of >48 hours.

The probe compound showed no reactivity with glutathione (100 µM), indicating that it is not generally cysteine reactive, but rather has a tempered electrophilicity and specific structural elements that direct reactivity towards GSTO1. An irreversible probe has some distinct advantages over reversible analogs. Targets can be readily characterized by methods such as mass spectrometry and click chemistry-ABPP, required dosing is often lower, irreversible compounds are not as sensitive to pharmacokinetic parameters, and administration can induce long-lasting inhibition [11]. In the case of the EGFR inhibitor PD 0169414, its irreversibility and high selectivity were credited with producing prolonged inhibition of the target, alleviating concerns over short plasma half-lives and reducing the need for high peak plasma levels, thus minimizing potential nonspecific toxic effects [12].

Indeed, over a third of enzymatic drug targets are irreversibly inhibited by currently marketed drugs [13]. Examples of covalent enzyme-inhibitor pairs include serine type D-Ala-D-Ala carboxypeptidase, which is covalently modified by all beta-lactam antibiotics, acetylcholinesterase, whose active site serine undergoes covalent modification by pyridostigmine, prostaglandin-endoperoxide synthase, which is the target of the ubiquitously prescribed aspirin, aromatase, which is irreversibly modified by exemestane, monoamine oxidase, which is covalently modified by L-deprenyl, thymidylate synthase, which is covalently modified by floxuridine, H+/K+ ATPase, which undergoes covalent modification by omaprazole, esmoprazole, and lanoprazole, and triacylglycerol lipase, whose serine nucleophile is targeted by orlistat [13].

j. Probe properties

Properties Computed from Structure

PubChem CIDCID 44607965
PubChem SIDSID-87457340
IUPAC Name(E)-2-(4-fluorophenylsulfonyl)-3-(1-(3-nitrophenylsulfonyl)-1H-pyrrol-2-yl)acrylonitrile
MLSMLS002699139
MFC19H12FN3O6S2
MW461.44
Formal Charge0
H Acceptor6
H Donor0
Atom Count43
Rotatable Bonds6
Rings3
Stereoatoms1
AlogP3.825
logD3.825
Polar surface area155.21
Aqueous solubilitya28.4 µM
Aqueous stabilityahalf-life >48 hours
Reactivity with GlutathioneaNone
Mechanism of ActionIrreversible (covalent) inhibitor of PME-1
ADMET BBBbUndefined
ADMET BBB levelcUndefined
ADMET absorption leveld2 (Low)
ADMET solubilitye−5.031
ADMET solubility leveleLow
VendorNone
Vendor Catalog NumberNone
a

Determined according to NIH guidelines.

b

ADMET_BBB: Log of Brain/Blood partition coefficient (LogBB) [14].

c

ADMET_BBB_Level: Ranking of the LogBB values into one of the following levels: 0: Very High, 1: High, 2: Medium, 3: Low, 4: Undefined (molecule is outside the confidence area of the regression model) [14, 15].

d

ADMET Passive Intestinal Absorption properties. Ranking of the molecule into one of the following levels: 0: Good, 1: Moderate, 2: Poor, 3: Very Poor [14, 15].

e

ADMET_Solubility: Log of the water solubility at 25 degrees, LogSw, in mol/L [14, 15].

k. Dose Response Curve for Probe

Below is the IC50 Curve for Probe Compound as determined by gel-based competitive-ABPP with FP-Rh. Calculated IC50 = 0.50 µM.

Image ml136fu4

4. Appendices

Appendix 1. PME-1 Inhibitors SAR Table

PME-1 Inhibitors SAR Table

Probe Development Assays
CompoundScripps IDStructureCIDSIDMLS IDVendorVendor Catalog IDPME-1 ABPP % INH (20μM) [AID-2369]PME-1 ABPP IC50 (μM) [AID-2366]Gel Filtration Assay [AID-2368]Cytotoxicity Assay (CC50) (nM) [AID-2365]Demethylation Assay % INH (20 μM) [AID-2363]
PROBESR-02000000252-1
Image ml136fu5.jpg
4460796587457340MLS002699139900.5Irreversible10000098
Analog 1SR-02000000253-1 SR-
Image ml136fu6.jpg
4460794987457341MLS002699140See below*0.64Irreversible10000095
Analog 202000000255-1
Image ml136fu7.jpg
4460795487457343MLS002699141See below*3.0See belowSee belowSee below§
Analog 3SR-02000000259-1
Image ml136fu8.jpg
4460797487457347MLS002699142See below*4.8See belowSee belowSee below§
Analog 4SR-02000000260-1
Image ml136fu9.jpg
4460796987457348MLS002699143See below*6.8See belowSee belowSee below§
Analog 5SR-01000639085-3
Image ml136fu10.jpg
570333087457336MLS002699144MaybridgeSEW03242See below *10.8See belowSee belowSee below§
*

Only the more precise IC50 value is reported.

These compounds were not tested in this assay because this is a mechanistic assay, and these related compounds are expected to all interact in the same covalent manner with PME-1.

Not Tested.

§

Not Tested.

Appendix 2. Assay Provider/Probe Development Assays

Figure 2. Cytotoxicity of PME-1 inhibitors against HEK 293T cells after 48h of treatment as determined by the CellTitre assay (Promega).

Figure 2Cytotoxicity of PME-1 inhibitors against HEK 293T cells after 48h of treatment as determined by the CellTitre assay (Promega)

Both compounds 1 (AMZ30) and 2 (DAB8) are cytotoxic only at very high concentrations (CC50~100 μM), well above the concentrations used to generate effects on PP2A methylation (<20 μM)

PMID List

PMID List

RefPMID
19727084
211171037
315661531
48650216
511060018
619293187
718596935
812149457
912740587
1019329999
1120640225
1210663641
1315823014
1411052792
1511922948

5. References

1.
Oliver CJ, Shenolikar S. Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 1998;3:D961–72. [PubMed: 9727084]
2.
Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 2001;353(Pt 3):417–39. [PMC free article: PMC1221586] [PubMed: 11171037]
3.
Janssens V, Goris J, Van Hoof C. PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 2005;15(1):34–41. [PubMed: 15661531]
4.
Lee J, et al. A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc Natl Acad Sci U S A. 1996;93(12):6043–7. [PMC free article: PMC39185] [PubMed: 8650216]
5.
Wu J, et al. Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. EMBO J. 2000;19(21):5672–81. [PMC free article: PMC305778] [PubMed: 11060018]
6.
Puustinen P, et al. PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma. Cancer Res. 2009;69(7):2870–7. [PMC free article: PMC2810347] [PubMed: 19293187]
7.
Ortega-Gutierrez S, et al. Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. PLoS One. 2008;3(7):e2486. [PMC free article: PMC2438471] [PubMed: 18596935]
8.
Jessani N, et al. Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc Natl Acad Sci U S A. 2002;99(16):10335–40. [PMC free article: PMC124915] [PubMed: 12149457]
9.
Leung D, et al. Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat Biotechnol. 2003;21(6):687–91. [PubMed: 12740587]
10.
Bachovchin DA, et al. Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat Biotechnol. 2009;27(4):387–94. [PMC free article: PMC2709489] [PubMed: 19329999]
11.
Johnson DS, Weerapana E, Cravatt BF. Strategies for discovering and derisking covalent, irreversible enzyme inhibitors. Future Med Chem. 2010;2(6):949–964. [PMC free article: PMC2904065] [PubMed: 20640225]
12.
Vincent PW, et al. Anticancer efficacy of the irreversible EGFr tyrosine kinase inhibitor PD 0169414 against human tumor xenografts. Cancer Chemother Pharmacol. 2000;45(3):231–8. [PubMed: 10663641]
13.
Robertson JG. Mechanistic basis of enzyme-targeted drugs. Biochemistry. 2005;44(15):5561–71. [PubMed: 15823014]
14.
Egan WJ, Merz KM Jr, Baldwin JJ. Prediction of drug absorption using multivariate statistics. J Med Chem. 2000;43(21):3867–77. [PubMed: 11052792]
15.
Egan WJ, Lauri G. Prediction of intestinal permeability. Adv Drug Deliv Rev. 2002;54(3):273–89. [PubMed: 11922948]

NT: not tested

Determined according to NIH guidelines.

ADMET_BBB: Log of Brain/Blood partition coefficient (LogBB) [14].

ADMET_BBB_Level: Ranking of the LogBB values into one of the following levels: 0: Very High, 1: High, 2: Medium, 3: Low, 4: Undefined (molecule is outside the confidence area of the regression model) [14, 15].

ADMET Passive Intestinal Absorption properties. Ranking of the molecule into one of the following levels: 0: Good, 1: Moderate, 2: Poor, 3: Very Poor [14, 15].

ADMET_Solubility: Log of the water solubility at 25 degrees, LogSw, in mol/L [14, 15].

Only the more precise IC50 value is reported.

These compounds were not tested in this assay because this is a mechanistic assay, and these related compounds are expected to all interact in the same covalent manner with PME-1.

Not Tested.

Not Tested.

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