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Probe Report for RBBP9 Inhibitors - Probe 2

, , , , , , , , , and .

Received: ; Last Update: October 20, 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

Image ml114fu1
RBBP9 Inhibitor Probe
(Oxime Ester Scaffold)
CID: 5934766SID: 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/MLSIDMLSTarget NameTarget IC50 [AID-2254] (μM)Anti-target NameAnti-Target IC50 [AID-2254] (μM)SelectivitySecondary Assay Cytox CC50 [AID-2243] (nM)
Probe (1)5934766/ML11485098567MLS002473500RBBP90.63>30 serine hydrolases(SHs)>100>100-fold>100000
Analog 1 (2)552457585098568MLS002473501RBBP91.9>30 SHs100 75-kDa SH, >100 all other SHs50-foldNT*
Analog 2 (3)582305385098569MLS002473502RBBP95.7>30 SHs>100>15-foldNT
Analog 3 (4)586051785098570MLS002473503RBBP95.0>30 SHs>20>4-foldNT
Analog 4 (5)552457485098571MLS002473504RBBP99.1>30 SHs20 75-kDa SH, >20 all other SHs2-fold>100000
Analog 5 (6)4414191485098573MLS002473505RBBP966% inhibition at 20 μM, IC50 not determined>30 SHs>20NTNT
Prior Art6603320855836MLS000028478RBBP97.8>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

AIDAssay NameAssay TypeTargetPowder SampleCompound Concentration
1515Primary biochemical high-throughput screening assay to measure RBBP9 inhibitionPrimary Assay (1X, %INH)RBBP9No8 μM
1537Confirmation biochemical high-throughput screening assay to measure RBBP9 inhibitionConfirmation Assay (3X, %INH)RBBP9No8 μM
1947Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the serine hydrolase family member Fam108BCounterscreen AssayFam108bNo9.51 μM
1974Fluorescence 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 AssayGSTO1No5.96 μM
1978Fluorescence polarization-based confirmation biochemical high throughput screening assay for inhibitors of the serine hydrolase family member Fam108bCounterscreen AssayFam108bNo9.51 μM
2176Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1)Counterscreen AssayGSTO1No5.96 μM
2269Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel- based Activity-Based Protein Profiling (ABPP) InhibitionSecondary AssayRBBP9Yes20 μM
2254Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel- based Activity-Based Protein Profiling (ABPP) IC50Secondary AssayRBBP9, anti- target serine hydrolasesYes0.1 – 100 μM
2243Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Luminescence-based counterscreen assay to identify cytotoxic compoundsSecondary AssayHEK 293T cellsYes1 – 10 μM
2248Late 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 AssaySecondary AssayRBBP9Yes100 mM
1790Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9) (emetine)N/AN/AN/AN/A
2299Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9) (ester oxime))N/AN/AN/AN/A

N/A: Not applicable.

ii. Assay Rationale & Description

Table of Assay Rationale and Description

AIDAssay RationaleAssay DescriptionZ′S:B
1515To measure the ability of compounds to inhibit RBBP9 activityIn 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 +/− 0.031.54 +/− 0.03
1537Confirmation of hit activity of compounds identified in the Primary ScreenSame as above except compounds were tested in triplicate.0.78 +/− 0.023.14 +/− 0.03
1947Counterscreen to identify compounds that act as inhibitors Fam108BIn 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 +/− 0.031.46 +/− 0.07
1974Counterscreen to identify compounds that act as inhibitors of GSTO1In 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 +/− 0.052.08 +/− 0.21
1978Confirmation of counterscreen to identify inhibitors of Fam108bIn 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 +/− 0.041.54 +/− 0.01
2176Confirmation of counterscreen to identify inhibitors of GSTO1In 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 +/− 0.043.19 +/− 0.14
2269Confirmation of hit activity of compounds identified in the Primary Screen and the Confirmation ScreenIn 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/AN/A
2254To 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
2243To determine cytotoxicity of inhibitor compounds belonging to the oxime ester scaffoldIn 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/AN/A
2248To assess reversibility of binding of inhibitor compounds belonging to the oxime ester scaffoldIn 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/AN/A
1790To summarize probe development efforts for inhibitors of RBBP9 (emetime)N/AN/AN/A
2299To summarize probe development efforts for inhibitors of RBBP9 (ester oxime)N/AN/AN/A

N/A: Not applicable.

Table of Reagents and Source

AIDReagent (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 1922) 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

Image ml114fu2

c. Structural Verification Information of probe SID

  1. 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%.
  2. 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

Image ml114fu3

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 CID5934766
PubChem SID85098567
IUPAC Name[1-(1,3-thiazol-2-yl)ethylideneamino] cyclohexanecarboxylate
MLSNone
MFC12H16N2O2S
MW252.33264
Formal Charge0
H Acceptor4
H Donor0
Heavy Atom Count17
Rotatable Bonds4
Rings2
Stereoatoms0
AlogP2.448
logD2.439
XLogP3-AA3.4
Polar surface area79.79
Aqueous solubilitya26.2µM
Aqueous stabilityahalf-life of 45 hours
Reactivity with Glutathioneanone
Mechanism of ActionIrreversible (covalent) inhibitor of RBBP9
ADMET BBBb−0.1400
ADMET BBB levelc2
ADMET absorption leveld0
ADMET solubilitye−3.302
ADMET solubility levelf3
VendorKey Organics
Vendor Catalog Number9w-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

ADMET_BBB_Level: Ranking of the LogBB values into one of the following levels (see (13, 14) for details):

0: Very High 1: High 2: Medium 3: Low

4: Undefined (molecule is outside the confidence area of the regression model).

d

ADMET Passive Intestinal Absorption properties. A ranking of the molecule into one of the following levels (see (13, 14) for details):

0: Good 1: Moderate 2: Poor 3: Very Poor

e

ADMET_Solubility: Log of the water solubility at 25 degrees, LogSw, in mol/L. See (13, 14) for more information.

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.

Image ml114fu4

4. Appendices

Appendix 1. RBBP9 Inhibitors SAR Table

Table A1.1RBBP9 Inhibitors SAR Table (Oxime Ester Scaffold)

CompoundScripps IDStructureCIDSIDMLS IDVendorVendor Catalog IDProbe 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)
PROBESR-0100030 7014-2
Image ml114fu5.jpg
593476685098567MLS002 473500Key Organics9w- 083763591Irreversible>100000
Analog 1SR-0100068 9006-1
Image ml114fu6.jpg
148189485098568MLS002 473501Key Organics6w- 0842190078See below*See below
Analog 2SR- 0100067 9216-2
Image ml114fu7.jpg
342719285098569MLS002 473502Key Organics9w- 0835570076See below*See below
Analog 3SR- 0200000 0166-1
Image ml114fu8.jpg
586051785098570MLS002 473503500062See below*See below
Analog 4SR- 0100030 7018-3
Image ml114fu9.jpg
552457485098571MLS002 473504Key Organics6w- 0841910060See below*>100000
Analog 5SR-0200000 0168-1
Image ml114fu10.jpg
4414191485098573MLS002 473505See below66See 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)

NumberStructureChemist or VendorCatalog/Notebook NumberPubChem CIDPubChem SIDMLS NumberIC50 (nM) ABPP%Inhibition purified RBBP9 (20 μM)Anti-targets (20 μM)
1
Image ml114fu11.jpg
Key Organics9w-0837593476685098567MLS002473500635910
2
Image ml114fu12.jpg
Key Organics6w-0842148189485098568MLS0024735011900780
3
Image ml114fu13.jpg
Key Organics9w-0835342719285098569MLS0024735025700760
4
Image ml114fu14.jpg
DABDAB-II- 296586051785098570MLS0024735035000620
5
Image ml114fu15.jpg
Key Organics6w-0841552457485098571MLS00247350491006075-kDa SH
6
Image ml114fu16.jpg
DAB/MWMW-I-184414191385098572-See below*150
7
Image ml114fu17.jpg
DAB/MWMW-I-114414191485098573MLS002473505See below*660
8
Image ml114fu18.jpg
Key Organics9w-0836628070985098574-See below*630
9
Image ml114fu19.jpg
Key Organics6w-0843148189585098575-See below*640
10
Image ml114fu20.jpg
Key Organics6w-0840634638285098576-See below*660
11
Image ml114fu21.jpg
DABDAB-II- 2974414191585098577-See below*40
12
Image ml114fu22.jpg
Key Organics9w-0838963205285098578-See below*460
13
Image ml114fu23.jpg
MaybridgeCD 11516280606185857710-See below *0NT
14
Image ml114fu24.jpg
DAB/MWMW-I-454452193885857715-See below*420
15
Image ml114fu25.jpg
DABDAB-IV- 404452193985857716-See below*0NT
16
Image ml114fu26.jpg
Key Organics9w-0871965538825817285-See below*170
17
Image ml114fu27.jpg
Key Organics9w-0873963878625817287-See below*14NT
*

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 A2.1. Low throughput assays to characterize probe.

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 A2.2. Cytotoxicity of RBBP9 inhibitors against HEK 293T cells after 48h of treatment as determined by the CellTiter-Glo assay (Promega).

Figure A2.2Cytotoxicity of RBBP9 inhibitors against HEK 293T cells after 48h of treatment as determined by the CellTiter-Glo assay (Promega)

Emetine is highly cytotoxic (CC50<100 nM), but the oxime ester compounds, including the probe compound, exhibited no toxicity (CC50>100 μM) at compound concentrations tested. Compound numbers refer to their entry numbers in Tables 1 and 2

Figure A2.3. Stability of ML114 (Compound 1) in PBS indicates a half-life of 45 hours.

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)

EntryStructureVendor or ChemistCatalog NumberPubChem CIDPubChem SIDABPP IC50 μM)% Inhibition (20 μM)Anti- targets (20 μM)
18
Image ml114fu28.jpg
ChemBridge6625888885318174140901.278NT
19
Image ml114fu29.jpg
ChemBridge719598271089935544911.5930
20
Image ml114fu30.jpg
ChemBridge712375871085285857709See below*760
21
Image ml114fu31.jpg
ChemBridge66288507761773493187See below*480
22
Image ml114fu32.jpg
DAB/MWMW-I-504452194185857717See below*42NT
23
Image ml114fu33.jpg
ChemBridge5330467284178385857711See below*50
24
Image ml114fu34.jpg
ChemBridge6151656288784885857708See below*00
25
Image ml114fu35.jpg
ChemBridge6575017573092785857712See below*60
26
Image ml114fu36.jpg
ChemBridge6576173573096685857713See below*280
*

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

NumberStructureVendorCatalog NumberPubChem CIDPubChem SID% Inhibition (20 μM)Anti- targets (20 μM)
27
Image ml114fu37.jpg
EnamineT0502-263854100012505107400
28
Image ml114fu38.jpg
EnamineT0502-26466948141250510810NT
29
Image ml114fu39.jpg
EnamineT0503-653058625092508617557FAAH
30
Image ml114fu40.jpg
EnamineT0503-88255884610250604060FAAH
31
Image ml114fu41.jpg
EnamineT0504-3259445219408585771400
32
Image ml114fu42.jpg
EnamineT0510-115757593082510146700
33
Image ml114fu43.jpg
EnamineT0511-0232584857125093682100
34
Image ml114fu44.jpg
EnamineT0516-604646226852518331200
35
Image ml114fu45.jpg
EnamineT0517-859135911602517297800
36
Image ml114fu46.jpg
EnamineT530036495692632528585200
37
Image ml114fu47.jpg
EnamineT57862091755818928310332150

Anti-targets: ≥ 50 inhibition observed at 20µM compound concentration (mouse brain membrane proteome, 1mg/mL)

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