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.

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.

2. Project Description

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 33–48) 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.

In our next synthetic efforts, we modified the identity of the sulfonamide. We discovered that changing the size of this group (compounds 27–30) led to complete loss of activity. Encouragingly, we also discovered that more electron-withdrawing sulfonamides (compounds 3–5, 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. 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

b. Probe chemical structure including stereochemistry

h. Detailed synthetic pathway

(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.

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].

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.

4. Appendices

Appendix 1. PME-1 Inhibitors SAR Table

PME-1 Inhibitors SAR Table

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								Probe Development Assays
Compound	Scripps ID	Structure	CID	SID	MLS ID	Vendor	Vendor Catalog ID	PME-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]
PROBE	SR-02000000252-1		44607965	87457340	MLS002699139			90	0.5	Irreversible	100000	98
Analog 1	SR-02000000253-1 SR-		44607949	87457341	MLS002699140			See below*	0.64	Irreversible	100000	95
Analog 2	02000000255-1		44607954	87457343	MLS002699141			See below*	3.0	See below†	See below‡	See below§
Analog 3	SR-02000000259-1		44607974	87457347	MLS002699142			See below*	4.8	See below†	See below‡	See below§
Analog 4	SR-02000000260-1		44607969	87457348	MLS002699143			See below*	6.8	See below†	See below‡	See below§
Analog 5	SR-01000639085-3		5703330	87457336	MLS002699144	Maybridge	SEW03242	See below *	10.8	See below†	See below‡	See 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 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)

5. References

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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]

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Janssens V, Goris J, Van Hoof C. PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 2005;15(1):34–41. [PubMed: 15661531]

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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]

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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]

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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]

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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]

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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]

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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]

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Robertson JG. Mechanistic basis of enzyme-targeted drugs. Biochemistry. 2005;44(15):5561–71. [PubMed: 15823014]

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Egan WJ, Merz KM Jr, Baldwin JJ. Prediction of drug absorption using multivariate statistics. J Med Chem. 2000;43(21):3867–77. [PubMed: 11052792]

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