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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.
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
PubChem Summary Bioassay Identifier (AID): AID-1790, AID-2299

RBBP9 Inhibitor Probe | |
(Oxime Ester Scaffold) | |
CID: 5934766 | SID: 85098567 |
MLS: MLS002473500 | |
RBBP9 IC50: 0.63 μM | |
Anti-Target (other serine hydrolases) IC50: >100 μM | |
Cytox CC50: >100 μM | |
ML114 |
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.
Probe Selection
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.
Probe or Analog (# in SAR Table) | CID/ML | SID | MLS | Target Name | Target IC50 [AID-2254] (μM) | Anti-target Name | Anti-Target IC50 [AID-2254] (μM) | Selectivity | Secondary Assay Cytox CC50 [AID-2243] (nM) |
---|---|---|---|---|---|---|---|---|---|
Probe (1) | 5934766/ML114 | 85098567 | MLS002473500 | RBBP9 | 0.63 | >30 serine hydrolases(SHs) | >100 | >100-fold | >100000 |
Analog 1 (2) | 5524575 | 85098568 | MLS002473501 | RBBP9 | 1.9 | >30 SHs | 100 75-kDa SH, >100 all other SHs | 50-fold | NT* |
Analog 2 (3) | 5823053 | 85098569 | MLS002473502 | RBBP9 | 5.7 | >30 SHs | >100 | >15-fold | NT |
Analog 3 (4) | 5860517 | 85098570 | MLS002473503 | RBBP9 | 5.0 | >30 SHs | >20 | >4-fold | NT |
Analog 4 (5) | 5524574 | 85098571 | MLS002473504 | RBBP9 | 9.1 | >30 SHs | 20 75-kDa SH, >20 all other SHs | 2-fold | >100000 |
Analog 5 (6) | 44141914 | 85098573 | MLS002473505 | RBBP9 | 66% inhibition at 20 μM, IC50 not determined | >30 SHs | >20 | NT | NT |
Prior Art | 6603320 | 855836 | MLS000028478 | RBBP9 | 7.8 | >30 SHs | >1000 | >100-fold | ~100 |
- *
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 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)
PubChem BioAssay Table
AID | Assay Name | Assay Type | Target | Powder Sample | Compound Concentration |
---|---|---|---|---|---|
1515 | Primary biochemical high-throughput screening assay to measure RBBP9 inhibition | Primary Assay (1X, %INH) | RBBP9 | No | 8 μM |
1537 | Confirmation biochemical high-throughput screening assay to measure RBBP9 inhibition | Confirmation Assay (3X, %INH) | RBBP9 | No | 8 μM |
1947 | Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the serine hydrolase family member Fam108B | Counterscreen Assay | Fam108b | No | 9.51 μM |
1974 | Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the oxidoreductase glutathione S- transferase omega 1(GSTO1) | Counterscreen Assay | GSTO1 | No | 5.96 μM |
1978 | Fluorescence polarization-based confirmation biochemical high throughput screening assay for inhibitors of the serine hydrolase family member Fam108b | Counterscreen Assay | Fam108b | No | 9.51 μM |
2176 | Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1) | Counterscreen Assay | GSTO1 | No | 5.96 μM |
2269 | Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel- based Activity-Based Protein Profiling (ABPP) Inhibition | Secondary Assay | RBBP9 | Yes | 20 μM |
2254 | Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel- based Activity-Based Protein Profiling (ABPP) IC50 | Secondary Assay | RBBP9, anti- target serine hydrolases | Yes | 0.1 – 100 μM |
2243 | Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Luminescence-based counterscreen assay to identify cytotoxic compounds | Secondary Assay | HEK 293T cells | Yes | 1 – 10 μM |
2248 | Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel- based Activity-Based Protein Profiling (ABPP) Gel Filtration Assay | Secondary Assay | RBBP9 | Yes | 100 mM |
1790 | Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9) (emetine) | N/A | N/A | N/A | N/A |
2299 | Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9) (ester oxime)) | N/A | N/A | N/A | N/A |
N/A: Not applicable.
ii. Assay Rationale & Description
Table of Assay Rationale and Description
AID | Assay Rationale | Assay Description | Z′ | S:B |
---|---|---|---|---|
1515 | To measure the ability of compounds to inhibit RBBP9 activity | In this assay, a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family is used to label RBBP9 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 RBBP9 inhibitors will prevent RBBP9-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 7.94 micromolar. | 0.75 +/− 0.03 | 1.54 +/− 0.03 |
1537 | Confirmation of hit activity of compounds identified in the Primary Screen | Same as above except compounds were tested in triplicate. | 0.78 +/− 0.02 | 3.14 +/− 0.03 |
1947 | Counterscreen to identify compounds that act as inhibitors Fam108B | In this biochemical assay, recombinant Fam108B protein is incubated with test compounds and FP-Rh probe. 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 Fam 108B inhibitors will prevent Fam 108B-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds were tested in singlicate at a final nominal concentration of 9.51 micromolar. | 0.83 +/− 0.03 | 1.46 +/− 0.07 |
1974 | Counterscreen to identify compounds that act as inhibitors of GSTO1 | In this assay, recombinant GSTO1 protein is incubated with test compounds and a Rh-conjugated sulfonate ester (SE) activity-based probe. 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 GSTO1 inhibitors will prevent GSTO1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a final nominal concentration of 5.96 micromolar. | 0.80 +/− 0.05 | 2.08 +/− 0.21 |
1978 | Confirmation of counterscreen to identify inhibitors of Fam108b | In this biochemical assay, recombinant Fam108b protein is incubated with test compounds and FP-Rh probe. 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 Fam 108b inhibitors will prevent Fam 108b-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds were tested in triplicate at a final nominal concentration of 9.51 micromolar. | 0.70 +/− 0.04 | 1.54 +/− 0.01 |
2176 | Confirmation of counterscreen to identify inhibitors of GSTO1 | In this assay, recombinant GSTO1 protein is incubated with test compounds and a Rh-conjugated sulfonate ester (SE) activity-based probe. 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 GSTO1 inhibitors will prevent GSTO1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in triplicate at a final nominal concentration of 5.96 micromolar. | 0.84 +/− 0.04 | 3.19 +/− 0.14 |
2269 | Confirmation of hit activity of compounds identified in the Primary Screen and the Confirmation Screen | In this assay, a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family is used to label RBBP9 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 RBBP9 inhibitors will prevent RBBP9-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe, leading to low fluorescence polarization in the band in the gel. Percent inhibition of recombinant RBBP9 (compound at 20 μM) was determined. | N/A | N/A |
2254 | To determine selectivity of compounds in proteomes | Same as above except IC50 values are determined from dose-response curves from three trials at each inhibitor concentration (0.1–100 mM). | N/A | N/A |
2243 | To determine cytotoxicity of inhibitor compounds belonging to the oxime ester scaffold | In 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 10-point 1:3 dilution series starting at a nominal test concentration of 40 micromolar. | N/A | N/A |
2248 | To assess reversibility of binding of inhibitor compounds belonging to the oxime ester scaffold | In 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 is used to label RBBP9 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 irreversible RBBP9 inhibitors will prevent RBBP9-probe interactions after gel filtration, thereby increasing the proportion of free (unbound) fluorescent probe, leading to low fluorescence polarization in the band in the gel. The compound’s reversibility of inhibition of RBBP9 was assessed. | N/A | N/A |
1790 | To summarize probe development efforts for inhibitors of RBBP9 (emetime) | N/A | N/A | N/A |
2299 | To summarize probe development efforts for inhibitors of RBBP9 (ester oxime) | N/A | N/A | N/A |
N/A: Not applicable.
Table of Reagents and Source
AID | Reagent (Source) |
---|---|
RBBP9 Inhibition Assays (AID-1515 and AID-1537) | Recombinant RBBP9 (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) |
RBBP9 Follow-up Assays (AID-2269 and AID-2254) | Recombinant RBBP9 protein (supplied by Assay Provider) FP-rhodamine (supplied by Assay Provider) Sodium Chloride (Fisher, part 980597) 1M Tris, pH 8.0 (Invitrogen, part T-3038) |
Cytotoxicity Assay (AID-2243) | HEK cells (provided by Assay Provider) Cell Titer-Glo (Promega, part G75729) 96-well plates (Corning, 3704) |
RBBP9 Gel Filtration Assay (AID-2248) | Recombinant RBBP9 protein (provided by Assay Provider) FP-rhodamine (provided by Assay Provider) 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-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
Potency
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).
Selectivity
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.
3. Probe
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
CID 5934766
e. Vendor
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
Properties Computed from Structure
PubChem CID | 5934766 |
PubChem SID | 85098567 |
IUPAC Name | [1-(1,3-thiazol-2-yl)ethylideneamino] cyclohexanecarboxylate |
MLS | None |
MF | C12H16N2O2S |
MW | 252.33264 |
Formal Charge | 0 |
H Acceptor | 4 |
H Donor | 0 |
Heavy Atom Count | 17 |
Rotatable Bonds | 4 |
Rings | 2 |
Stereoatoms | 0 |
AlogP | 2.448 |
logD | 2.439 |
XLogP3-AA | 3.4 |
Polar surface area | 79.79 |
Aqueous solubilitya | 26.2µM |
Aqueous stabilitya | half-life of 45 hours |
Reactivity with Glutathionea | none |
Mechanism of Action | Irreversible (covalent) inhibitor of RBBP9 |
ADMET BBBb | −0.1400 |
ADMET BBB levelc | 2 |
ADMET absorption leveld | 0 |
ADMET solubilitye | −3.302 |
ADMET solubility levelf | 3 |
Vendor | Key Organics |
Vendor Catalog Number | 9w-0837 |
- a
determined according to NIH guidelines
- b
ADMET_BBB: Log of Brain/Blood partition coefficient (LogBB). See (13) for details on this method.
- c
- d
- e
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.

4. Appendices
Appendix 1. RBBP9 Inhibitors SAR Table
Table A1.1RBBP9 Inhibitors SAR Table (Oxime Ester Scaffold)
Compound | Scripps ID | Structure | CID | SID | MLS ID | Vendor | Vendor Catalog ID | Probe Development Assays | |||
---|---|---|---|---|---|---|---|---|---|---|---|
RBBP9ABBP IC50 [AID-2254] (nM) | Inhibition of purified RBBP9 (20 μM) [AID-2269] (%INH) | Gel Filtration Assay [AID-2248] | Cytotoxicity Assay (CC50) [AID-2243] (nM) | ||||||||
PROBE | SR-0100030 7014-2 | ![]() | 5934766 | 85098567 | MLS002 473500 | Key Organics | 9w- 0837 | 635 | 91 | Irreversible | >100000 |
Analog 1 | SR-0100068 9006-1 | ![]() | 1481894 | 85098568 | MLS002 473501 | Key Organics | 6w- 0842 | 1900 | 78 | See below* | See below† |
Analog 2 | SR- 0100067 9216-2 | ![]() | 3427192 | 85098569 | MLS002 473502 | Key Organics | 9w- 0835 | 5700 | 76 | See below* | See below† |
Analog 3 | SR- 0200000 0166-1 | ![]() | 5860517 | 85098570 | MLS002 473503 | 5000 | 62 | See below* | See below† | ||
Analog 4 | SR- 0100030 7018-3 | ![]() | 5524574 | 85098571 | MLS002 473504 | Key Organics | 6w- 0841 | 9100 | 60 | See below* | >100000 |
Analog 5 | SR-0200000 0168-1 | ![]() | 44141914 | 85098573 | MLS002 473505 | See below‡ | 66 | See below* | See below† |
- *
Analogs 1 through 5 were not tested in the Gel Filtration Assay because this is a mechanistic assay and these related compounds will all interact covalently in the same way with RBBP9.
- †
Analogs 1, 2, 3, and 5 were not tested in the Cytotoxicity Assay.
- ‡
Analog 5 was not tested because it was less selective than the lead compound.
Appendix 2. Assay Provider/Probe Development Assays
Table A2.1Comparative data on similar compound structures establishing SAR (right-hand derivatization)
Number | Structure | Chemist or Vendor | Catalog/Notebook Number | PubChem CID | PubChem SID | MLS Number | IC50 (nM) ABPP | %Inhibition purified RBBP9 (20 μM) | Anti-targets□ (20 μM) |
---|---|---|---|---|---|---|---|---|---|
1 | ![]() | Key Organics | 9w-0837 | 5934766 | 85098567 | MLS002473500 | 635 | 91 | 0 |
2 | ![]() | Key Organics | 6w-0842 | 1481894 | 85098568 | MLS002473501 | 1900 | 78 | 0 |
3 | ![]() | Key Organics | 9w-0835 | 3427192 | 85098569 | MLS002473502 | 5700 | 76 | 0 |
4 | ![]() | DAB | DAB-II- 296 | 5860517 | 85098570 | MLS002473503 | 5000 | 62 | 0 |
5 | ![]() | Key Organics | 6w-0841 | 5524574 | 85098571 | MLS002473504 | 9100 | 60 | 75-kDa SH |
6 | ![]() | DAB/MW | MW-I-18 | 44141913 | 85098572 | - | See below* | 15 | 0 |
7 | ![]() | DAB/MW | MW-I-11 | 44141914 | 85098573 | MLS002473505 | See below* | 66 | 0 |
8 | ![]() | Key Organics | 9w-0836 | 6280709 | 85098574 | - | See below* | 63 | 0 |
9 | ![]() | Key Organics | 6w-0843 | 1481895 | 85098575 | - | See below* | 64 | 0 |
10 | ![]() | Key Organics | 6w-0840 | 6346382 | 85098576 | - | See below* | 66 | 0 |
11 | ![]() | DAB | DAB-II- 297 | 44141915 | 85098577 | - | See below* | 4 | 0 |
12 | ![]() | Key Organics | 9w-0838 | 9632052 | 85098578 | - | See below* | 46 | 0 |
13 | ![]() | Maybridge | CD 11516 | 2806061 | 85857710 | - | See below * | 0 | NT |
14 | ![]() | DAB/MW | MW-I-45 | 44521938 | 85857715 | - | See below* | 42 | 0 |
15 | ![]() | DAB | DAB-IV- 40 | 44521939 | 85857716 | - | See below* | 0 | NT |
16 | ![]() | Key Organics | 9w-0871 | 9655388 | 25817285 | - | See below* | 17 | 0 |
17 | ![]() | Key Organics | 9w-0873 | 9638786 | 25817287 | - | See below* | 14 | NT |
- *
These compounds were not tested in ABPP dose response assays because they were less selective than the lead compound as determined using the Gel-based ABPP Inhibition assay.
- □
Anti-targets: ≥ 50 inhibition observed at 20µM compound concentration (mouse brain membrane proteome, 1mg/mL)

Figure A2.1Low throughput assays to characterize probe
a. Selective inhibition of exogenous RBBP9 doped into the mouse brain membrane proteome by oxime ester compounds as determined by gel-based ABPP. b. Unlike emetine, compound 1 covalently inhibits RBBP9, retaining inhibitory activity after gel filtration. c. Identification of the acyl-enzyme adduct of compound 1 by mass spectrometry. d. (next page) Compound selectivity at 20 µM in mouse brain proteome. a, b (top), and d: fluorescent image of SDS-PAGE gels shown in grey scale. Compound numbers refer to their entry numbers in Table A2.1

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
Appendix 3. Supplemental SAR Tables
Table A3.1Additional oxime ester SAR, including analogs of oxime ester 18 (CID 885318)
Entry | Structure | Vendor or Chemist | Catalog Number | PubChem CID | PubChem SID | ABPP IC50 μM) | % Inhibition (20 μM) | Anti- targets□ (20 μM) |
---|---|---|---|---|---|---|---|---|
18 | ![]() | ChemBridge | 6625888 | 885318 | 17414090 | 1.2 | 78 | NT |
19 | ![]() | ChemBridge | 7195982 | 710899 | 3554491 | 1.5 | 93 | 0 |
20 | ![]() | ChemBridge | 7123758 | 710852 | 85857709 | See below* | 76 | 0 |
21 | ![]() | ChemBridge | 6628850 | 776177 | 3493187 | See below* | 48 | 0 |
22 | ![]() | DAB/MW | MW-I-50 | 44521941 | 85857717 | See below* | 42 | NT |
23 | ![]() | ChemBridge | 5330467 | 2841783 | 85857711 | See below* | 5 | 0 |
24 | ![]() | ChemBridge | 6151656 | 2887848 | 85857708 | See below* | 0 | 0 |
25 | ![]() | ChemBridge | 6575017 | 5730927 | 85857712 | See below* | 6 | 0 |
26 | ![]() | ChemBridge | 6576173 | 5730966 | 85857713 | See below* | 28 | 0 |
- *
These compounds were not tested in ABPP dose response assays because they were less selective than the lead compound, as determined using the Gel-based ABPP Inhibition assay.
- □
Anti-targets: ≥ 50 inhibition observed at 20µM compound concentration (mouse brain membrane proteome, 1mg/mL)
Table A3.2Initial round of SAR by purchase with large structural modifications
Number | Structure | Vendor | Catalog Number | PubChem CID | PubChem SID | % Inhibition (20 μM) | Anti- targets□ (20 μM) |
---|---|---|---|---|---|---|---|
27 | ![]() | Enamine | T0502-2638 | 5410001 | 25051074 | 0 | 0 |
28 | ![]() | Enamine | T0502-2646 | 6948141 | 25051081 | 0 | NT |
29 | ![]() | Enamine | T0503-6530 | 5862509 | 25086175 | 57 | FAAH |
30 | ![]() | Enamine | T0503-8825 | 5884610 | 25060406 | 0 | FAAH |
31 | ![]() | Enamine | T0504-3259 | 44521940 | 85857714 | 0 | 0 |
32 | ![]() | Enamine | T0510-1157 | 5759308 | 25101467 | 0 | 0 |
33 | ![]() | Enamine | T0511-0232 | 5848571 | 25093682 | 10 | 0 |
34 | ![]() | Enamine | T0516-6046 | 4622685 | 25183312 | 0 | 0 |
35 | ![]() | Enamine | T0517-8591 | 3591160 | 25172978 | 0 | 0 |
36 | ![]() | Enamine | T5300364 | 9569263 | 25285852 | 0 | 0 |
37 | ![]() | Enamine | T5786209 | 17558189 | 28310332 | 15 | 0 |
- □
Anti-targets: ≥ 50 inhibition observed at 20µM compound concentration (mouse brain membrane proteome, 1mg/mL)
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- PMCPubMed Central citations
- PubChem BioAssay for Chemical ProbePubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter
- PubChem SubstanceRelated PubChem Substances
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- Application of the RBBP9 Serine Hydrolase Inhibitor, ML114, Decouples Human Pluripotent Stem Cell Proliferation and Differentiation.[Int J Mol Sci. 2020]Application of the RBBP9 Serine Hydrolase Inhibitor, ML114, Decouples Human Pluripotent Stem Cell Proliferation and Differentiation.Lim S, Shparberg RA, Coorssen JR, O'Connor MD. Int J Mol Sci. 2020 Nov 26; 21(23). Epub 2020 Nov 26.
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