The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. The enzyme RB binding protein 9 (RBBP9) is widely expressed in different tissues and upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. We previously reported a modestly potent, RBBP9 reversible inhibitor, ML081 (CID-6603320). However, ML081 exhibits high cytotoxicity. We, therefore, have now identified a newer probe, ML114 (CID-5934766), which is 10-fold more potent than ML081, exhibits no cytotoxicity, and is from an entirely different structural and mechanistic class of compounds that covalently inhibit RBBP9. This new probe will be useful for in vitro assays in which it is desirable to specifically block RBBP9 activity for primary research purposes.
Assigned Assay Grant #: R01 CA087660-05
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
Probe Structure & Characteristics and Prior Art
As described in the CPDP, the chief goal for this probe development project was to find a selective inhibitor of the retinoblastoma binding protein 9 (RBBP9). In collaboration with the SRIMSC, we previously reported a modestly potent 8 µM reversible inhibitor that was selective RBBP9 (emetine, CID 6603320, SID-855836). However, emetine exhibited high cytotoxicity (CC50 < 100 nM, see Figure A2.2 Appendix 2). As such, we optimized a second scaffold from the uHTS screening hits to achieve greater potency towards RBBP9 and reduced cytotoxicity. The probe (ML114) detailed in this report is 10-fold more potent than emetine (IC50 0.63µM), exhibits no cytotoxicity up to 100 µM concentration, and is from an entirely different structural and mechanistic class of inhibitors. Unlike emetine, ML114 covalently inhibits RBBP9. This probe is highly selective (>100-fold), as demonstrated by lack of serine-hydrolase anti-targets by competitive activity-based protein profiling (ABPP) assays. No other selective RBBP9 inhibitors have been reported to date.
Please see the probe table on the next page for all results described above.
Following the uHTS campaign and counterscreening by gel-based competitive activity-based protein profiling (ABPP) in proteomes, an oxime ester class of inhibitor was identified for probe development. Following two rounds of SAR, ML114 (Compound 1, Table A2.1 Appendix 2) was selected as a probe, as it was the most potent and selective analog and exhibited no cytotoxicity up to 100 µM. ML114 is ~3-fold more potent than the top initial hit (compound 2, Table A2.1 Appendix 2).
Recommendations for the Scientific Use of This Probe
This compound is useful for in vitro assays in which it is desirable to specifically block RBBP9 activity for primary research purposes.
1. Scientific Rationale for Project
The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F (1), which activates genes essential for DNA replication. Due to the critical role of RB in regulating the cell cycle, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. One such protein, RB binding protein 9 (RBBP9), is widely expressed in different tissues and upregulated in certain tumors (2, 3). The RBBP9 protein contains an alpha/beta hydrolase fold, which belongs to the DUF1234 domain superfamily of unknown function. Although an enzymatic activity for RBBP9 has not been reported, this protein does react with activity-based probes that target serine hydrolases, suggesting that it is a functional enzyme. Also consistent with this premise, the crystal structure of RBBP9 was recently solved and revealed a well-structured active site with a properly arranged catalytic triad indicative of serine hydrolase activity (4). A role for RBBP9 in cellular transformation is supported by studies showing that RBBP9 mRNA expression is increased in transformed rat liver cell lines and human liver tumor biopsies (3). Furthermore, RBBP9-overexpressing cells form tumors when implanted into immuno-deficient mice (3), and RBBP9 overexpression confers resistance to TGF-β1-induced growth inhibition through its interaction with RB and displacement of E2F (3, 5). RBBP9 is also suggested to play a role in gender-related differential responses to radiation-induced cell proliferation (6). As a result, the identification of compounds that selectively inhibit RBBP9 activity may provide valuable probes for the study of apoptosis, cell cycle, and tumorigenesis.
As a serine hydrolase, catalytically active RBBP9 is readily labeled by fluorescent activity-based protein profiling (ABPP) probes bearing a fluorophosphonate (FP) reactive group (7). 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 (8). 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 been configured to operate in a high-throughput manner via fluorescence polarization readout, FluoPol-ABPP (9). Given the lack of substrate assays for RBBP9, the FluoPol-ABPP platform offers a unique opportunity to develop inhibitors for this important enzyme.
2. Project Description
a. Original goal for probe characteristics
The goal of the campaign was to discover compounds with inhibitory activity against RBBP9 that are selective among the serine hydrolases in mouse tissue as assessed by gel-based competitive-ABPP. Compounds of interest should exhibit an IC50 of <10 μM, and preferably <1 μM.
b. Information for each Assay Implemented and Screening Run
i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)
iii. Summary of Results
Following Primary screening in singlicate (AID-1515), Confirmation of hit activity in triplicate (AID-1537), and counterscreening by gel-based competitive-ABPP in proteomes to determine selectivity (AID-2269 and AID-2254), we identified an oxime ester class of inhibitor for probe development. Compound ML114 (CID 5934766, compound 1 Table A2.1 Appendix 2) discovered from these efforts represents the first selective irreversible RBBP9 inhibitor (Appendix 2, Fig A2.1b, AID-2248). Unlike the reversible RBBP9 inhibitor emetine, this compound is not cytotoxic (Appendix 2, Fig A2.2, AID-2243) and we anticipate it will be useful to determine the function of this uncharacterized enzyme.
c. Probe Optimization
i. Description of SAR & chemistry strategy (including structure and data) that led to the probe
We identified three related compounds with oxime ester cores: two compounds had a left-hand oxime substituent containing a thiazole (CIDs 1481894 and 44141914, compounds 2 and 5, Appendix 2, Table A2.1), and the third compound (CID 885318, compound 18 Appendix 3, Table A3.1) had a left-hand cyclohexyldienone-containing substituent. An initial round of SAR by purchase on the oxime ester core revealed that large structural modifications, especially to the oxime leaving group, ablated inhibitory activity (Appendix 3, Table A3.2). Both the thiazole and cyclohexyldienone structures are conjugated systems capable of stabilizing negative charge, which likely makes these moieties good leaving groups during covalent modification [the right-hand substituent was shown to remain bound to a serine nucleophile of RBBP9 (Appendix 2, Figure A2.1c)]. The second round of SAR by purchase and synthesis preserved the oxime ester core and the left-hand thiazole containing substituent common to compounds 2 and 3 or the cyclohexyldienone containing substituent of compound 18 (Appendix 3, Tables A3.1 and 2). For the thiazole, some steric bulk and structural rigidity was required on the right-hand side for RBBP9 inhibition, as small substituents (compounds 13, 14) were inactive; however, large groups (compounds 11, 16, 17) were also not tolerated. A variety of electronically neutral (compounds 1, 4), weak electron-withdrawing (compounds 2, 3, 7–10) or electron-donating (compound 5) substituents were allowed; however, molecules with strong electron-withdrawing groups (compounds 6, 12) were inactive, possibly because they predisposed the compound to hydrolysis rather than RBBP9 reactivity. For the cyclohexyldienone, the initial hit (compound 18) and its analogs (compounds 19–22) were less selective and were not extensively pursued. Overall, the probe (compound 1) with the left-hand thiazole and right hand cyclohexyl was the most potent and selective compound tested, and was ~3-fold more potent than the top initial hit (compound 2).
As a class, the oxime esters were highly selective as assessed by their anti-target reactivity by competitive-ABPP. At 20µM, only compounds 5, 29, and 30 showed evidence of anti-target reactivity (Table A2.1 Appendix 2, Tables 2 & A3.3 Appendix 3, Figure A2.1d Appendix 2). Additionally, the highly structurally-related oxime ester compounds screened by uHTS (CIDs 1481894, 5135022, 3427192, 1481895, 885318, 710899, 2841783) all showed very low activity (<2.3%) in other bioassays (each tested in >300 bioassays), indicating that, as a class, this type of oxime ester scaffold is not generally active.
a. Chemical name of probe compound
[1-(1,3-thiazol-2-yl)ethylideneamino] cyclohexanecarboxylate [ML114]
b. Probe chemical structure
c. Structural Verification Information of probe SID
- 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 3.2Hz, 1H), 7.44 (d, J = 3.2Hz, 1H), 2.57–2.51 (m, 1H), 2.54 (s, 3H), 2.10–1.96 (m, 2H), 1.9–1.77 (m, 2H), 1.75–1.65 (m, 1H), 1.65–1.51 (m, 2H), 1.44–1.24 (m, 3H), purity >95%.
- high-res MS analysis (ESI-TOF): M+H+ expected: 253.1005, M+H+ observed: 253.1014
d. PubChem CID
Key Organics, catalog number 9w-0837
f. MLS#'s of probe molecule and five related samples that were submitted to the SMR collection
MLS002473500 (see table in section 1).
g. Mode of action for biological activity of probe
We have shown that CID 5934766 (referred to as compound 1 in the Appendix) forms a covalent adduct with RBBP9 by demonstrating that blockade of FP-rhodamine labeling is not reversed by gel filtration and by identifying the acylated RBBP9 active site nucleophile (S75) by mass spectrometry (see Appendix 2, Figures A2.1b and A2.1c). Even though 1 contains a reactive chemical moiety, this compound selectively inhibits RBBP9 in the mouse brain membrane proteome. As the biological function of RBBP9 is unknown, the biological effects of RBBP9 inhibition are currently unknown. We therefore anticipate that this oxime ester inhibitor, and future compounds derived from this chemically tractable scaffold, will assist the assignment of an enzymatic function of RBBP9 in biological systems.
h. Detailed synthetic pathway
1-(thiazol-2-yl)ethanone oxime (B)
Sodium acetate (2.84 g, 34.6 mmol) was added to a solution of hydroxylamine hydrochloride (2.4g, 24.6 mmol) in EtOH (60 mL) at room temperature. After being stirred for 30 minutes, 2-acetylthiazole (A) (3.26 mL, 30.2 mmol) was added dropwise over 1 hour. After 2 additional hours of stirring, the reaction was quenched with water (added dropwise over 30 minutes). The mixture was extracted with EtOAc and the combined organic layer was washed with brine, dried over anhydrous MgSO4, and concentrated by rotary evaporation under reduced pressure. The crude product was purified by flash column chromatography on silica gel in hexane-EtOAc (4:1) to afford 1-(thiazol-2-yl)ethanone oxime (B) (20%).
Aryl/Acyl Oxime Ester (D)
1-(thiazol-2-yl)ethanone oxime (B) (0.128 g, 1 mmol) was added to an aryl or acyl chloride (C) (1.5 mmol) in the presence of Et3N (0.32 mL, 2.3 mmol) at 0 °C. After stirring for 1 hour, the mixture was quenched with 1 N HCl, extracted with CH2Cl2, washed with brine, dried over MgSO4, and concentrated by rotary evaporation under reduced pressure. Purification by flash column chromatography affords the target oxime ester (D) in good yield (>60%).
i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)
ADMET BBB, −0.1400; ADMET BBB leve, l 2; ADMET absorption level, 0; ADMET solubility, −3.302; ADMET solubility level, 3
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 26.2 µM. The probe has a half-life of 45 hours in PBS at room temperature (tested at 10 µM, Figure A2.3 Appendix 2).
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 (10). 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 (11).
Indeed, over a third of enzymatic drug targets are irreversibly inhibited by currently marketed drugs (12). 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 (12).
j. Probe properties
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 = 0.63 µM.
Appendix 1. RBBP9 Inhibitors SAR Table
Appendix 2. Assay Provider/Probe Development Assays
Figure A2.1Low throughput assays to characterize probe
Figure A2.2Cytotoxicity of RBBP9 inhibitors against HEK 293T cells after 48h of treatment as determined by the CellTiter-Glo assay (Promega)
Figure A2.3Stability of ML114 (Compound 1) in PBS indicates a half-life of 45 hours
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DA Bachovchin, AE Speers, SJ Brown, BF Cravatt, V, Fernandez-Vega, T Spicer, BA Mercer, J Ferguson, P Hodder, and HR Rosen.
Received: August 20, 2009; Last Update: October 20, 2010.
National Center for Biotechnology Information (US), Bethesda (MD)
Bachovchin DA, Speers AE, Brown SJ, et al. Probe Report for RBBP9 Inhibitors - Probe 2. 2009 Aug 20 [Updated 2010 Oct 20]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.