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Selective UBC 13 Inhibitors

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Received: ; Last Update: May 13, 2014.

This probe report describes the identification and development of a potent, sub-micromolar (IC50 = 781 nM), first-in-class, small molecule inhibitor of Ubc13 enzyme activity, ML307. The activity of the probe compound was assessed by its ability to suppress Ubc13-mediated formation of heteropolymeric poly-ubquitination chains comprised of a mix of terbium-labeled and fluorescein-labeled ubiquitin molecules, as monitored by a reduction of the time-resolved fluorescent resonance energy transfer (TR-FRET) signal arising from this chains when synthesized in vitro in collaboration with the Ubc13 cofactor UEV1a. ML307 has excellent solubility and stability in buffer and is not a general cysteine protease inhibitor as it is >128-fold selective against Caspase-3, nor a TR-FRET artifact as it is not inhibitory a TR-FRET assay for Bfl-1, an out of class target. ML307 resulted from an extensive elucidation of the structure-activity relationship (SAR) through the purchase and synthesis of over 90 compounds representing 5 related scaffolds. The level of in vitro potency achieved is unprecedented in the current state of art, thus representing a significant advance and providing a chemical tool for assessing the biological roles and biochemical mechanisms of Ubc13.

Assigned Assay Grant #: 1R03MH-085677-01

Screening Center Name & PI: Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute & John C. Reed, M.D., Ph.D.

Chemistry Center Name & PI: Same as Screening Center

Assay Submitter & Institution: John C. Reed, M.D., Ph.D. & Sanford-Burnham Medical Research Institute

PubChem Summary Bioassay Identifier (AID): 485343

Probe Structure & Characteristics

This Center Probe Report describes a small molecule inhibitor of Ubc13, compound ML307, CID 56639556 (SID 134958980). Potency, and selectivity characteristics are summarized for this probe in the next section, while the Absorption, Distribution, Metabolism, Excretion/Toxicity (ADME/T) are profiled in Table 8.

Table 8. Summary of in vitro ADME Properties of UBC13 inhibitor probe ML307.

Table 8

Summary of in vitro ADME Properties of UBC13 inhibitor probe ML307.

ML307.

ML307

CID/ML#Target NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (nM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 56639556
CID ML307
UBC13781 ± 10 (n=2)

[SID 134958980; AID 602404]
Caspase 3

SH- Protease Counter-screen
>100,000 (n=2) [SID 134958980; AID 602401]>128 XBfl-1: IC50>100,000 (n=2)
TR-FRET artifact[SID 134958980; AID 602400]

Recommendations for scientific use of the probe

Currently, no publically available small molecule Ubc13 Probes exist that have submicromolar potency (IC50) against the target protein as measured by in vitro biochemical assays and are at least 10-fold selective for Ubc13 over irrelevant targets or functionally similar targets such as other E2 enzymes. ML307 will be useful for elucidating the enzymology of Ubc13, the effects of specific inhibition of Ubc13 versus other E2, and mapping the active site of Ubc13 or the Ubc13-Uev1a interface. Such probes will be useful for specifically modulating Ubc13-dependent cellular functions. Ubc13 is the only mammalian E2 enzyme known that catalyzes exclusively the formation of lysine 63 (K63)-linked polyubiquitin chains on substrates. Such K63-linked polyubiquitination modifications are associated with novel cellular functions, including innate immunity (inflammation) signaling and DNA damage repair. The probes therefore will be very useful to researchers exploring immunomodulation and inflammation as well as those in the fields of cancer biology and radiobiology. Finally, the larger research community who studies Ubc13 and ubiquitination-regulated pathways will certainly be beneficiaries of such chemical inhibitors.

1. Introduction

This probe project aimed to generate small molecule antagonists of Ubc13 and to distinguish compounds that attack at the active site from those that interrupt the Ubc13-Uev1a interface.

Specific Aims

Aim #1. Screen a library of chemicals employing a TR-FRET-based ubiquitination assay. An in vitro ubiquitination assay based on the TR-FRET assay principle was established for Ubc13. Ubiquitination reactions contain purified recombinant proteins (E1, Ubc13 and Uev1a) mixed with labeled ubiquitin (terbium- and fluorescein-labeled) and ATP-regenerating system to trigger polyubiquitin chain synthesis in vitro. The reaction product is mixed chains of terbium- (fluorescence donor) and fluorescein- (fluorescence acceptor) conjugated Ubiquitin, thus creating the basis for robust TR-FRET signals.

Aim #2. Perform counter-screens to eliminate false-positive compounds from screening hits. Redox modulating compounds could intervene with ubiquitination reaction process by their potential to bind the active site thiol-containing cysteine residue of Ubc13. Hence, primary hits will be re-tested in with a GAPDH assay (an enzyme that also possesses a catalytic cysteine) to eliminate redox-active compounds. Next, a TR-FRET previously established in our laboratory for an entirely different target (Bcl-XL) will be used to eliminate compounds that non-specifically interfere with the FRET-based reaction. Then, an in vitro ubiquitination assay for an alternative E2 and E3 system (Siah/UbcH5) will be counter-screened, thus eliminating compounds that interfere with E1 or with classical E2 enzymes. Finally, compounds will be tested by micro-isothermal titration calorimetry, high throughput surface plasmon resonance (SPR), for 1D-NMR to establish that they bind directly to either Ubc13 or its cofactor Uev1a.

Aim #3. Perform cell-based secondary assays to validate chemical hits. Two types of secondary cell-based assays will be performed to validate the hits. First, it has been determined by RNA interference (RNAi) experiments that Interleukin-1β stimulates NF-κB activity through a Ubc13-dependent mechanism in U2OS cells. We have stably integrated a NF-κB-driven luciferase reporter gene into U2OS cells and will use these cells to determine the cellular activity of the Ubc13 inhibitors. Second, we are developing a cell-based, secondary assay using bioluminescent resonance energy transfer (BRET), in which fusion proteins Rluc-TRAF6 and YFP-Ubiquitin are co-expressed in cells. Stimulation with cytokines or other agents that activate TRAF6 induce formation of K63-linked polyubiquitin chains on TRAF6. Addition of luciferase substrate generates light, which then excites the YFP, producing the signal for the assay.

Background and Significance

Innate immunity signaling events are often initiated by Toll-like receptors (TLRs) and Tumor Necrosis Factor (TNF) family receptors. The intracellular signaling pathways activated by TLRs and TNFRs converge on TRAF family adaptor proteins (1). TRAFs bind to the cytosolic tails of TNFRs or are recruited to TNFRs and TLR (and IL-1Rs) through other adapter proteins (25). Importantly, TRAFs are also involved in signaling by T-cell and B-cell antigen receptors (TCR/BCR) in a pathway involving Carma1, Bcl-10, and paracaspase (MALT1) to induce nuclear factor κB (NF-κB) (68). TRAFs are known to activate specific protein kinases, which in turn activate NF-κB, AP-1, and NFAT proteins (nuclear factor of activated T cells), which are required for induction and expression of lymphokines, lymphokine receptors, and other mediators of immunity (7,913). It was shown, in autoimmune diseases, that TNF-receptor associated factors (TRAFs) are crucial participants in cytokine signaling pathways leading to NF-κB activation and proinflammatory cytokine production (13,14).

TRAFs undergo post-translational modification via an unusual ubiquitination process that is a requirement for triggering activation of downstream kinases (1). Like many other E3 ligases, most TRAFs (five of six in humans and mice) possess RING domains and auto-ubiquitinate themselves. The RING domain of TRAFs binds a specific ubiquitin-conjugating enzyme, Ubc13, which catalyzes the synthesis of polyubiquitin chains on TRAFs (15,16,1719), in addition to protein kinase substrates of TRAFs. However, the polyubiquitin chains created by Ubc13 do not target TRAFs or their substrates for proteasomal degradation. Instead, the ubiquitin modifications activate the proteins, inducing downstream signaling events, including activation of NF-κB and MAPK-family kinases.

In eukaryotic cells, the chemistry of ubiquitination relies on the dynamic interaction of ubiquitin (Ub) with three different classes of enzymes, termed ubiquitin-activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) (20). The canonical pathway of ubiquitination leading to degradation of ubiquitinylated proteins by the proteasome involves a covalent attachment of Gly76 at the C-terminus of Ub with the ɛ-amino group of LYS48 of another Ub to form a polyubiquitin chain. However, in the case of Ubc13, LYS63, instead of LYS48, is used as the site for linking Ub chains. This K63-linked polyubiquitinylation reaction is catalyzed by Ubc13 in complex with its non-enzymatic cofactors Mms2 or Uev1a (15,21). Mms2 is predominantly nuclear while Uev1a is predominantly cytosolic. NMR and crystallography studies have revealed the structures of Ubc13, its catalytic site, and its interface with these obligatory co-factor proteins (13,2225). In this regard, structural studies of Ubc13-Mms2 and Ubc13-Uev1a complexes show a deep, narrow and long hydrophobic binding pocket at the interface of Ubc13-Uev1a. However, Mms2 and Uev1a differ in their sequences at the N-terminus end and are known to differentially regulate cellular processes (26). While the non-covalent interaction of Ubc13-Mms2 is necessary for cellular mechanisms involving DNA damage repair, the Ubc13-Uev1a complex regulates inflammatory and immune signaling pathways. However, Ubc13 also mediates NF-κB activation induced by DNA damage, a process for which the co-factor has not be defined.

Recently, gene ablation studies in mice have validated Ubc13, as a candidate target for autoimmune and inflammatory diseases (27). Homozygous disruption of the Ubc13 gene results in embryonic lethality. Also, tissue-specific homozygous ablation of both alleles of the Ubc13 gene in B-cells results in defective development of marginal B and B1 cells along with impairment of humoral response. However, the Assay Provider’s laboratory has produced hemizygous Ubc13 mice (Ubc13+/−), observing that reduced levels of Ubc13 protein are associated with significantly impaired signaling by TNF and lipopolysaccharide (LPS), resulting in reduced TRAF ubiquitination in vivo, and reduced activation of NF-κB and stress kinases (p38 MAPK and JNK) in vivo (28). These hemizygous Ubc13+/− mice are phenotypically normal in all respects, including various lymphocyte subpopulations. Because the TNFR, TLR, and TCR/BCR signaling pathways all depend on TRAFs, targeting the TRAF-associated Ubc13-Uev1a complex would be expected to interrupt inflammatory signaling and quiet the immune system. Biochemical and structural studies of Ubc13 and Ubc13-Uev1a complex lay the foundation for design of small molecule inhibitors that are expected to have potent anti-inflammatory and immunosuppressive activity for treatment of immune diseases. In addition, because of its role in double-strand DNA break repair and NF-κB signaling induced by DNA damage (which stimulates transcription of multiple anti-apoptotic, pro-survival genes), inhibition of Ubc13 would also potentially be beneficial for cancer therapy.

Prior Art

The ideal probe has a potency ≤100 nM for the biochemical Ubc13 assay and has at least a 10 μM potency in a Ubc13-dependent cell assay. Additionally it would be desirable for the probe to be ≥ 10-fold selective for Ubc13 over other E2 enzymes and similar classes of cysteine-dependent enzymes. No publically available and potent Ubc13 Probes exist that meet these criteria.

A non-drug-like, natural product compound that inhibits the formation of a complex composed of the ubiquitin E2 enzyme Ubc13 and Uev1A was isolated from the marine sponge Leucetta Microrhaphis (Figure 1). The compound was identified as leucettamol A (1) by spectroscopic analysis. Its inhibition of Ubc13-Uev1A interaction was tested by the ELISA method, revealing an IC50 value of 50 μg/mL. The compound is the first reported inhibitor of Ubc13-Uev1A interaction, and that of the E2 activity of Ubc13. The inhibition of Ubc13-Uev1A interaction was tested in an ELISA assay according to standard procedures (see Bioorganic & Medicinal Chemistry Letter (2008) 18: 6319–6320) using purified recombinant Ubc13 and FLAG-Uev1A proteins and a primary anti-FLAG antibody (SIGMA, F3165) and found to be very weak for compound 1 with an estimated IC50 value of 50 μg/mL (~110 μM; m.w. 472). The isolated compounds 1 and 3 are not stable and isomerize readily. This publication also notes that hydrogenation of 1 increases its potency to ~ 4 μg/mL (~8 μM), while 3 is completely inactive. However, testing of the reported compound in the Assay Provider’s laboratory failed to reveal detectable activity against Ubc13.

UBC13 Prior Art

Figure 1

Natural Product Prior Art compounds. Compound (1) is registered in PubChem – See SIDs therein

A patent (WO2008/009758) describes compounds claimed to inhibit UBC13-UEV interactions and for use in the production of pharmaceutical compositions intended for antitumor therapy or the treatment and/or prophylaxis of diseases associated with metabolic routes involving the UBC13 enzyme, metabolic routes involving transcriptional factor NF-κB or routes involving PCNA or RAD6. The compounds from this patent are listed (Figure 2.). However, the patents do not provide any potency information:

UBC13 Prior Art 2

Figure 2

Prior Art compounds described in Patent for UBC13/UEV. PubChem CID for the described compounds are noted

2. Materials and Methods

The details of the primary HTS and additional assays can be found in the “Assay Description” section in the PubChem BioAssay view under the AIDs as listed in Table 1. Additionally the details for the primary HTS are provided in the Appendix at the end of this probe report.

Table 1. Summary of Assays and AIDs.

Table 1

Summary of Assays and AIDs.

2.1. Assays

Table 1 summarizes the details for the assays that drove this probe project.

2.2. Probe Chemical Characterization

a. Chemical name of probe compound

The IUPAC name of the probe ML307 is (R)-N-((1-(3-chlorobenzyl)piperidin-4-yl)methyl)-1-(6,7,8,9-tetrahydropyrido[1,2-e]purin-4-yl)piperidine-3-carboxamide. The actual batch prepared, tested and submitted to MLSMR is SID 134958980 corresponding to CID 56639556.

b. Probe chemical structure including stereochemistry if known

The probe molecule CID 56639556 has one chiral center and that center has the R conformation (Figure 3).

Figure 3. Structure of ML307.

Figure 3

Structure of ML307. Note R-configuration stereochemistry as indicated around the 3-position of the piperdine ring.

c. Synthesis and Structural Verification Information of probe SID 134958980 corresponding to CID 56639556 (See Scheme 1)

Scheme 1. Synthesis of ML307, conditions.

Scheme 1Synthesis of ML307, conditions

a. DMF, 113 °C, overnight (46%); b. Triethylorthoformate, DMF, 150 °C, overnight (58%); c. POCl3, CH3CN, 90 °C (59%); d. DIPEA, DCM, rt, overnight; e.4M HCl, Dioxane (60%, 2 steps); f. EDCI, HOBT, DIPEA, DMF, rt, overnight (53%); g. 10% TFA, DCM (100%); h. K2CO3, CH3CN, 80 °C (54%).

d. If available from a vendor, please provide details

This probe is not commercially available. A 25 mg sample of ML307 synthesized at SBCCG has been deposited in the MLSMR (Evotec) (see Probe Submission Table 4).

e. Solubility and Stability of probe in PBS at room temperature

The solubility of ML307 was investigated in aqueous buffers at room temperature. As noted in the Summary of in vitro ADME/T properties (see Table 8), ML307 has excellent solubility relative to its potency against UBC13 in aqueous buffer at all pH’s tested: 348, 351 and 373 μM, at pH 5, 6.2, and 7.4 in pION buffer, and 352μM in PBS, which are 446-, 449-, 478- and 451-fold its potency (0.781 μM) against UBC13. To evaluate its potential hydrolytic instability an aliquot of ML307 was prepared in PBS or a acetonitrile:PBS (1:1) mixture and incubated at room temperature, and the amounts of the parent compound remaining at various times were analyzed by LC/MS (Figure 4 time course and Table 2). The results here and in Table 8 indicate that ML307 is very stable with 80.5% and 100% remaining after incubation for 48 hrs at ambient temperatures in PBS or 1:1 PBS:acetonitrile, respectively.

Figure 4. Stability of ML307 in (△) PBS or (●) 1:1 acetonitrile:PBS at room temperature.

Figure 4

Stability of ML307 in (△) PBS or (●) 1:1 acetonitrile:PBS at room temperature.

Table 2. Hydrolytic stability of ML307 at ambient temperature.

Table 2

Hydrolytic stability of ML307 at ambient temperature.

f. Calculated and known probe properties: are shown in Table 3

Table 3CID 56639556 [ML307] MLS-0471611

Molecular Weight [g/mol]568.11012
Molecular FormulaC29H38ClN7O3
AlogP1.079
H-Bond Donor2
H-Bond Acceptor8
Rotatable Bond Count6
Exact Mass567.272466
MonoIsotopic Mass567.272466
Topological Polar Surface Area117
Heavy Atom Count40
Formal Charge0
Complexity775
Isotope Atom Count0
Defined Atom StereoCenter Count1
Undefined Atom StereoCenter Count0
Defined Bond StereoCenter Count0
Undefined Bond StereoCenter Count0
Covalently-Bonded Unit Count2

g. Table 4 summarizes the deposition of the Probe and 5 analogs

Table 4Probe and Analog Submissions to MLSMR (BioFocus DPI) for UBC13 Inhibitors

Probe ML307 - CID56639556
Probe/AnalogMLS_ID (BCCG)MLS_ID (MLSMR)CIDSIDSource (vendor or syn)Amt (mg)Date ordered/submitted
Probe ML307MLS-0471611MLS00425664456639556134958980Syn31.83/31/2012
Analog 1MLS-0471622MLS00425664320912564134958967Syn26.23/31/2012
Analog 2MLS-0471621MLS00425664256832464134958966Syn23.73/31/2012
Analog 3MLS-0471627MLS00425664156832476134958972Syn21.33/31/2012
Analog 4MLS-0471620MLS00425664056832461134958964Syn24.63/31/2012
Analog 5MLS-0469614MLS00425663920912547134958963Syn20.13/31/2012

2.3. Probe Preparation

The preparation of the probe molecule ML307 was moderately difficult. Compound 5 is commercially available but the cost of the starting material $5000 for 1 gram with a 12-week delivery date, forced us to synthesize this molecule in our laboratories. The synthetic route to this molecule is straightforward but the intermediates compounds 3 and 4 are very insoluble molecules. Purification of these molecules by either silica gel or C-18 HPLC liquid chromatography resulted in the isolation of miniscule amounts of the desired intermediates. Based upon these findings, it was decided to carry through the impure compounds 3 and 4 and purify at a later stage of the synthesis. It should be noted that in the preparation of compound 4, triethylorthoformate was used as a co-solvent otherwise the reaction would not proceed to completion. Fortunately for us, Compound 5 was isolated cleanly as an orange solid after trituration of the crude product with acetone. The synthesis of ML307 proceeded smoothly with known chemistry after we synthesized compound 5.

Experimental: (compounds are numbers as in Scheme 1)

Preparation of 3-amino-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-2-carboxamide [3]

3.

3

2-amino-2-cyanoacetamide 1 (6.1 g, 62 mmol) and o-methylvalerolactam 2 (7.0 g, 62 mmol) was dissolved in 200 ml of DMF. The resulting mixture was heated at 113 °C overnight. When the reaction was determined to be complete by HPLC, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting oil was triturated with ethanol or acetone and filtered to yield 5.1 g of dark brown crude product 3 (46 % yield). MS(EI) m/z 181 (M+1).

Preparation of 6,7,8,9-tetrahydropyrido[1,2-e]purin-4-ol [4]

4.

4

3-amino-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-2-carboxamide 3 (5.1 g, 28.3 mmol) and triethylorthoformate (100 g, 0.68 mol) was dissolved in 100 ml of DMF. The resulting mixture was heated at 150 °C overnight. When the reaction was determined to be complete by HPLC, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting oil was triturated with ethanol or acetone and filtered to yield 3.1 g of a dark brown crude product 4 (58 % yield). MS(EI) m/z 191 (M+1).

Preparation of 4-chloro-6,7,8,9-tetrahydropyrido[1,2-e]purine [5]

5.

5

Phosphorus oxychloride POCl3 (50 mL, 0.20 mol) was added drop wise to a solution of 6,7,8,9-tetrahydropyrido[1,2-e]purin-4-ol 4 (3.1 g, 16.3 mmol) in dry acetonitrile (50 mL) to give an dark solution, then several drops of DMF was added if necessary. The reaction was heated at 90 °C overnight. When the reaction was determined to be complete by HPLC, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting oil was basified with 1N NaOH solution, extracted with ethyl acetate and dried over magnesium sulfate. The organic layer was concentrated under reduced pressure, triturated with acetone, filtered and dried to give 2.0 g of compound 5 (59% yield). 1H NMR (400 MHz, CDCl3) δ 2.08 (m, 2H), 2.15 (m, 2H), 3.17(t, 2H), 4.23 (t, 2H), 8.66 (s, 1H). MS(EI) m/z 209 (M+1).

Preparation of (1-(3-chlorobenzyl)piperidin-4-yl)methanamine [8]

8.

8

To a solution of 4-Boc-(aminomethyl)piperdine 6 (2.14 g, 1.0 mmol) and 3-chlorobenzyl bromide 7 (2.1 g, 1.0 mmol) in anhydrous dichloromethane(100 ml) was added 2.0 mL of DIPEA. The reaction mixture was stirred at room temperature overnight. The resulting mixture was washed with water, dried over magnesium sulfate and concentrated under reduced pressure to give an oil, which was purified by preparative C-18 reverse phase HPLC to give a white solid. The solid was deprotected with 4 M HCl in dioxane to 0.4 g of compound 8 (HCl salt, 60% yield, 2 steps). 1H NMR (400 MHz, DMSO-d) δ 1.37 (m, 2H), 1.77 (m, 1H), 1.88 (m, 2H), 2.48 (m, 1H), 2.66 (m, 2H), 2.89 (m, 2H), 3.10 (m, 1H), 3.33 (m, 2H), 4.24 (s, 2H), 7.40 (m, 3H), 7.55 (m, 1H). MS(EI) m/z 239 (M+1).

Preparation of (R)-N-((1-(3-chlorobenzyl)piperidin-4-yl)methyl)piperidine-3-carboxamide [10]

10.

10

To a solution of (1-(3-chlorobenzyl)piperidin-4-yl)methanamine 7 (1.2 g, 5.0 mmol) and (R)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid (1.2 g, 5.2 mmol) in anhydrous DMF(50 ml) was added 1.0 mL of DIPEA, EDCI (1.6 g, 8.4 mmol) and HOBT (1.1 g, 8.1 mmol). The reaction mixture was stirred at room temperature overnight. When the reaction was determined to be complete by HPLC, the reaction solution was concentrated under reduced pressure to give an oil. The oil was dissolved in 100 ml of ethyl acetate and washed with 1N NaOH solution and water. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to get 1.2 g of a white solid (53% yield), which was deprotected with 10% TFA in dichloromethane to afford the compound 10 (TFA salt). 1H NMR (400 MHz, DMSO-d) δ 1.35 (m, 2H), 1.60 (m, 1H), 1.63 (m, 2H), 1.75 (m, 3H), 1.79 (m, 1H), 2.49 (m, 1H), 2.88 (m, 5H), 2.90 (m, 1H), 3.16 (m, 2H), 3.32 (m, 2H), 4.27 (s, 2H), 7.47 (m, 3H), 7.60 (m, 1H). MS(EI) m/z 350 (M+1).

Preparation of (R)-N-((1-(3-chlorobenzyl)piperidin-4-yl)methyl)-1-(6,7,8,9-tetrahydropyrido-[1,2-e]purin-4-yl)piperidine-3-carboxamide [11]

11.

11

Potassium carbonate (1.5 g, 11 mmol)) was added to a solution of 4-chloro-6,7,8,9-tetrahydropyrido[1,2-e]purine 5 (500 mg, 2.4 mmol) and (R)-N-((1-(3-chlorobenzyl)piperidin-4-yl)methyl)piperidine-3-carboxamide 10 (0.95 g, 2.7 mmol) in acetonitrile (50 ml). The reaction was heated at 80 °C overnight. When the reaction was determined to be complete by HPLC, the reaction mixture was cooled to room temperature and filtered to give a solution, which was purified by preparative C-18 reverse phase HPLC to afford 0.67 g of compound 11 (54% yield). 1H NMR (400 MHz, CDCl3) δ 1.32 (m, 2H), 1.50 (m, 1H), 1.63 (m, 4H), 1.84 (m, 2H), 2.02 (m, 6H), 2.22 (m, 2H), 2.54 (m, 1H), 2.95 (m, 4H), 3.10 (m, 2H), 3.55 (m, 2H), 4.10 (m, 4H), i4.32 (m, 2H), 7.32 (m, 4H), 8.27 (s, 1H). MS(EI) m/z 522 (M+1).

3. Results

The NIH collection was screened against the Ubc13 TR-FRET assay and confirmed hits were screened against Caspase-3 to remove general cysteinyl protease inhibitors, and Bfl-1 to weed out those artifacts inhibiting the TR-FRET assay irrespective of the Ubc13 target. Ultimately, from this hit triage, five related scaffolds were advance for further chemical optimization and SAR elucidation.

3.1. Summary of Screening Results

The flow chart (Figure 5) summarizers the screening process used for the Ubc13 inhibitor campaign. Initial active compounds were identified following primary HTS of approximately 330,000 Molecular Libraries Small Molecules Repository (MLSMR) compounds at 20 μM using the Ubc13 TR-FRET Assay (AID 485273), 1,547 initial actives (~0.47% hit rate) were obtained. Compounds were deemed to be positive if they had 45% activity and an acceptable fluorescence profile defined as an F-Ratio >0.5 AND <1.5. This ratio allows for the analysis of normalized fluorescence (F) in a plate well with a compound comparing to control wells. It is calculated as a fluorescence ratio of the reference channel in TR-FRET. This filter effectively eliminates compounds that inherently alter the fluorescent properties of TR-FRET.

Figure 5. Screening results and Hit validation.

Figure 5

Screening results and Hit validation.

Fresh stock solutions “cherry picks” of these 1,547 hits were requested from the MLSMR (Biofocus DPI) and 1,430 were received (92.4%). These were then retested in the Ubc13 TR-FRET confirmatory assay (AID 488859) at 20 μM. For the single point re-confirmatory assay, a cutoff of 45% inhibition and an F-Ratio >0.5 AND <1.5. yielded an 87% reconfirm rate resulting in a total of 1,245 compounds. To triage this hit set these hits were subjected to a sulfhydryl (Caspase-3) counterscreen (AID 488856), this filter is designed to remove promiscuous compounds for the common site of activity for Ubc13. This filter excluded a total of 35 compounds. The activities (IC50) of the remaining 1,210 compounds were confirmed in full 10-point dose-response titrations (AID 493155, AID 493182). Of the 1,210 compounds tested a total of 296 compounds displayed activity with IC50 values below 20 μM. These compounds were then further scrutinized for potential promiscuous LanthaScreen assay interference using an unrelated protein, Bfl-1 (AID 504689), this further excluded 59 compounds. This collection of 251 compounds comprised of 43 scaffolds, with 2 or more examples and 83 singletons. Continued investigation focused on the most potent compounds with IC50 values less than 5 μM. Of these 72 compounds, several of them contained chemical liabilities, i.e. Michael acceptor, frequent hitters, quinones, detergents, etc. and were eliminated from consideration. Based on these experimental results and informatic analysis, dry powder compounds were selected for follow up studies and additional, commercially available analogues were ordered.

3.2. Dose Response Curves for Probe

Representative Dose Response of ML307 in the Ubc13 TR-FRET, Caspase-3 and Bfl-1 Assays.

Representative Dose Response of ML307 in the Ubc13 TR-FRET, Caspase-3 and Bfl-1 Assays

3.3. Scaffold/Moiety Chemical Liabilities

Neither the scaffold of the selected probe, nor the related scaffolds (A, B, C, D & E) contain any groups associated with any chemical liabilities or reactivities.

3.4. SAR Tables

Identification of 7, 8, 9, 10-tetrahydro-6H-azepino[1,2-e]purine Scaffolds

We initially tested 1210 compounds in dose response format and found:

Number of compoundsPotency thresholds (μM)
72< 5
87< 10
194< 20
857> 20
12.

12

(CID 20912511) IC50 = 3.44 μM

Of the 72 members of the most potent scaffolds classes, several of the them contained chemical liabilities, i.e. Michael acceptor, frequent hitters, quinones, detergents, etc. and were eliminated from consideration. Compound 12 (CID 20912511) was identified as the most potent member of the 7, 8, 9, 10-tetrahydro-6H-azepino[1,2-e]purine scaffold in the initial hit series, although Compound 13 is almost as potent in our assay with an IC50 = 3.87 μM. This scaffold does not contain any standard chemical liabilities.

Representative examples of other members of the initial screening hit set for this scaffold are compounds 2 to 23 in Table 4.

After confirming the initial results, the hit-to-probe process was initiated both an analog by catalog approach and an internal medicinal chemistry effort. Using an analog by catalog approach, 37 additional analogs were purchased in the 7, 8, 9, 10-tetrahydro-6H-azepino[1,2-e]purine, 7- membered ring scaffolds A and B. It should be noted that few (if any) compounds with the 6- membered ring scaffolds are available commercially.

One of the purchased compounds entry 13, CID 20912362 (see Table 5: SAR Around Scaffolds A & B), is significantly more potent (IC50 = 0.89 μM) than the original DPI plated compound. We synthesized entry 34, in which the pendant amide of entry 13 was moved to the 4-position of the piperidine ring (Scaffold B) and activity of this molecule was significantly decreased, IC50 = 40.1 μM.

Table 5. SAR Around Scaffolds A & B.

Table 5

SAR Around Scaffolds A & B.

Looking closely at Scaffolds A and B, we examined analogs in which other groups replace the substituents on the phenyl ring. For example, when the chlorine atom of entry 13 is moved from the meta position to the para position on the aromatic ring, we observe a decrease in activity, entry 15. Comparable decreases in activity are observed, if the chloride atom is replaced by a fluorine atom (entries 18 and 19), a methyl group (entry 17), and a di-methyl group (entry 23). When we examine the lengthening of the carbon linker chain in entry 18 by 2 carbon atoms entry 16 the activities were similar. Another modification we made was replacing the piperidine ring of entry 19 (IC50 = 9.91 μM) with a piperazine ring entry 31, this modification resulted in the complete loss of activity, IC50 > 100 μM.

Entry 13, CID 20912362 has one chiral center on the piperidine ring. We synthesized both the R isomer entry 29, IC50 = 3.37 μM and the S isomer entry 30, IC50 = 15.0 μM. We were disappointed that the compounds were both less potent than the commercial racemic compound entry 13.

Next, we investigated the effects of modifying the aryl amide substituents in Scaffold B and we found the most potent compound in this series of molecules was entry 35, IC50 = 3.73 μM. This compound contained a 3-chloro-4-methylphenyl ring. We observed small changes in substituents on the phenyl ring greatly altered the activity. For example, when the 2-chlor-3-methylphenyl ring was substituted, entry 42, the compound was completely inactive, IC50 > 100 μM. Any subsequent changes to the original combination of atoms on the phenyl ring resulted in the decrease in activity of the series. (See entries 35 – 47, 57). In addition we observed that replacement of the phenyl ring with a benzyl, substituted benzyl or phenyethyl moieties (See entries 48 – 55). resulted in compounds that are less potent than entry 34.

Table 6SAR Around Scaffold B

R1 = N-Aryl amide substituents
Image ml307fu32.jpg
EntryCIDSIDSBCCG IDS/PScaffoldR1nIC50 (μM)
35
Screen Hit
2091256149724193MLS-0362591PB
Image ml307fu33.jpg
39.87±5.32
104223056MLS-0362591P63.73±0.51
3620912445104223052MLS-0365095PB
Image ml307fu34.jpg
65.95±1.24
3720912440125317312MLS-0469607PB
Image ml307fu35.jpg
47.69±2.38
3820912446125317314MLS-0367408PB
Image ml307fu36.jpg
1>100
3916021815125317315MLS-0469609PB
Image ml307fu37.jpg
429.6±3.74
4020912454125317316MLS-0469610PB
Image ml307fu38.jpg
1>100
4120912477125317317MLS-0469611PB
Image ml307fu39.jpg
1>100
4220912496125317318MLS-0469612PB
Image ml307fu40.jpg
1>100
4320912528125317319MLS-0469613PB
Image ml307fu41.jpg
437.6±12.9
4420912547125317320MLS-0469614PB
Image ml307fu42.jpg
428.7±29.2
4520912558125317321MLS-0469615PB
Image ml307fu43.jpg
1>100
4620912563125317322MLS-0469616PB
Image ml307fu44.jpg
448.2±2.46
4716021599125317323MLS-0469617PB
Image ml307fu45.jpg
418.6±7.92
4820912444134415624MLS-0469608SB
Image ml307fu46.jpg
224.5±0.17
4956832461134958964MLS-0471620SB
Image ml307fu47.jpg
1>100
5056832464134958966MLS-0471621SB
Image ml307fu48.jpg
1>100
5120912564134958967MLS-0471622SB
Image ml307fu49.jpg
258.7±0.42
5256832458134958968MLS-0471623SB
Image ml307fu50.jpg
1>100
5356832477134958969MLS-0471624SB
Image ml307fu51.jpg
1>100
5420912342134958970MLS-0471625SB
Image ml307fu52.jpg
276.1±4.59
5516021755134958971MLS-0471626SB
Image ml307fu53.jpg
272.8±2.33
5656832452134958973MLS-0471628SB
Image ml307fu54.jpg
236.8±1.17
5756639568134415625MLS-0471608SB
Image ml307fu55.jpg
1>100
5856639549134415626MLS-0471609SB
Image ml307fu56.jpg
223.8±0.47

Finally, we investigated the effects of the changing the 7-membered ring scaffolds A and B, to 6-membered ring scaffolds C, D, E and F. To this end, a select group of nineteen (19) analogs were synthesized and the results are presented in Table 4C. A comparison of the activities shows that Scaffolds C and D when compared to their counterparts in Scaffolds A and B, all compounds displayed similar activity. For example entry 58 (Scaffold B) has an IC50 = 23.8 μM and when compared to its counterpart entry 68 (Scaffold D) IC50 = 27.2 μM.

The most interesting compound in the series of synthesized molecules is entry 60 (CID 56593285, IC 50 = 0.962 μM).

Image ml307fu57

The comparable compound in which the chlorine atom is moved to the ortho position entry 59 (CID 56593287) has similar activity IC 50 = 1.04 μM. Moving the chlorine atom to the para position entry 61 (CID 56593276) results in a decrease in activity, IC 50 = 6.79 μM. Replacing the chlorine atom with methyl, di-chloro, dimethyl, fluoro, etc. atoms results in a decrease in activity. In Scaffold E, entry 60 (CID 56593285, IC 50 = 0.962 μM) when the meta-chlorphenyl substituent is attached to Scaffold F, entry 69, the activity of the molecule greatly diminishes, IC 50 = 12.6 μM. Generally, Scaffold C and E are preferred over Scaffold D and F.

From these results we focused on Entry 60, which has one chiral center. We thought it would be interesting to synthesize both the R and S isomers of this molecule. To this end we synthesized the R-isomer entry 70 (CID 56639556, IC 50 = 0.781 μM ) and the S-isomer entry 71 (CID 56639543, IC50 = 2.66 μM). For the probe molecule we identified entry 70, CID 56639556 (SID 134958980) as our most potent molecule screened in both the purchased and synthesized molecular set. However this 3-fold improvement in potency indicates that there isn’t a very strong stereochemical dependence for inhibition.

Table 7SAR Around Scaffolds C, D, E and F

R1 = N-Aryl amide & alkyl N-benzyl substituents
Image ml307fu58.jpg
EntryCIDSIDSBCCG IDS/PScaffoldR1IC50 (μM)
5956593287134225056MLS-0471521SE
Image ml307fu59.jpg
21.04±0.04
6056593285134225057MLS-0471522SE
Image ml307fu60.jpg
20.962±0.04
6156593276134225058MLS-0471523SE
Image ml307fu61.jpg
26.79±0.21
6256639566134415614MLS-0471524SE
Image ml307fu62.jpg
25.74±0.99
6356639564134415615MLS-0471600SD
Image ml307fu63.jpg
252.6±7.28
6456639540134415616MLS-0471601SF
Image ml307fu62.jpg
1>100
6556639551134415617MLS-0471602SD
Image ml307fu64.jpg
272.3±9.82
6656639552134415618MLS-0471603SD
Image ml307fu55.jpg
279.5±0.3
6756639559134415619MLS-0471604SD
Image ml307fu65.jpg
232.8±1.5
6856639554134415620MLS-0471605SD
Image ml307fu66.jpg
227.2±0.71
6956639532134415621MLS-0471606SF
Image ml307fu67.jpg
212.6±0.49
7056639543134415622MLS-0471607
S-Isomer
SE
Image ml307fu67.jpg
22.66±0.22
7156639556134415628MLS-0471611
R-Isomer
SE
Image ml307fu67.jpg
20.781±0.01
7256832457134958974MLS-0471629SC
Image ml307fu62.jpg
291.2±2.44
7356832460134958975MLS-0471630SC
Image ml307fu68.jpg
242.6±0.74
7456832462134958976MLS-0471631SC
Image ml307fu69.jpg
232.2±1.53
7556832454134958977MLS-0471632SC
Image ml307fu42.jpg
232.6±1.09
7656832471134958978MLS-0471633SC
Image ml307fu70.jpg
277.1±0.48
7756832468134958979MLS-0471634SC
Image ml307fu60.jpg
1>100

3.5. Cellular Activity

Cytotoxicity assays in immortalized human cell lines will be used to assess LD50. ML307 will be tested for inhibition of known Ubc13 ubiquitination targets. One approach will be to transiently transfect TRAF6, a known target of Ubc13, and monitor the ubiquitination level of this assay with Western blot analysis. To determine the selectivity of ML307 for inhibition of K-63 specific ubiquitination and therefore Ubc13 specificity a cell based p53 ubiquitination assay will be performed. The ubquitination of p53 is a K-48 mediated event, which is mediated by other E2s (not Ubc13) and therefore should not be affected by ML307.

3.6. Profiling Assays

As a pro forma activity, the SBCCG is committed to profiling all final probe(s) compound(s) and in certain cases key informative analogs in the PanLabs full panel as negotiated by the MLPCN network. Additional commercial profiling services will be considered for funding by SBCCG as deemed appropriate and informative.

The nominated probe was evaluated in a detailed in vitro pharmacology screen as shown in Table 8:

ML307 is very soluble in aqueous media at pH 5.0/6.2/7.4. With solubilities in pION buffer of 348 μM (pH5.0), 351 μM (pH6.2), and 373 μM (pH7.4), which are 446-fold, 449-fold, and 478-fold its in vitro potency of 781 nM. ML307 is also comparably soluble in 1X PBS (pH 7.4) with a solubility of 352 μM, which is 451-fold its potency (IC50 value).

The PAMPA (Parallel Artificial Membrane Permeability Assay) assay is used as an in vitro model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. Consistent with the predicted LogP (see Table 3), ML307 is moderately permeable at pH5.0 and very permeable at pH 6.2 and 7.4 in this assay.

Plasma Protein Binding is a measure of a drug’s efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. ML307 shows high binding to plasma proteins in both mouse and human plasma.

Stability in PBS and in 1:1 PBS Acetonitrile. As ML307 is very soluble in PBS, stability measurement are not confounded by compound solubility issues, and ML307 is apparently fairly stable in PBS and completely stable in 1:1 PBS:Acetonitrile with 80.51% and 100% of parent ML307 remaining after 48 hr of incubation at ambient temperature.

Plasma Stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which strongly can influence the in vivo efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. ML307 shows excellent stability in human and mouse plasma.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. ML307 shows poor stability in both human and mouse liver homogenates, potentially limiting the utility of this probe in in vivo rodent models and in a human therapeutic context. This is one area of needed improvement for future PK optimization approaches. However, it will still be a useful in vitro tool for probing the chemical biology and biochemical mechanism of inhibition, as these are not subject to “first-pass” liver metabolism.

ML307 exhibits no toxicity (LC50 > 50 μM) toward human hepatocyctes.

4. Discussion

Here we report a novel small molecule inhibitor of Ubc13-dependent poly-ubiquitination. This was achieved with the successful collaboration between the Sanford-Burnham Center for Chemical Genomics, and the research laboratory of the assay provider (Dr. John C. Reed). Following significant chemical optimization, we identified ML307 as a potent, sub-μM, inhibitor of the E2 ubquitination enzyme, Ubc13. ML307 offers exciting opportunities as a tool compound to further elucidate the specific role K63-dependent ubiquitination plays in cellular biology.

4.1. Comparison to existing art and how the new probe is an improvement

Previously the most potent inhibitor of Ubc13 was isolated from the marine sponge Leucetta Microrhaphis This non-drug-like compound was identified as leucettamol A (1). with an estimated IC50 value of 50 μg/mL (~110 μM; m.w. 472). This compound is not very stable and isomerizes readily. Hydrogenation of compound 1 produced compound 3, with a potency of ~ 4 μg/mL (~8 μM),

Image ml307fu71

The patent literature (WO2008/009758), describes compounds that effect UBC13-UEV interactions. The patent gives no definitive data on the claimed compounds. We synthesized the compounds but were unable to confirm its activity against Ubc13 using the TR-FRET biochemical assay.

In this report we have identified the most potent Ubc13 inhibitor, in the literature to date, ML307 (CID 56639556) IC 50 = 0.781 μM. ML307 is >10-fold more potent than any known compound in the literature and it is chemically stable and will serve as a unique biochemical probe. This probe will be useful for specifically modulating Ubc13-dependent cellular functions. Indeed, Ubc13 currently performs such novel functions by the formation of differential holoenzymes containing Uev1A or mms2 affecting inflammation and DNA damage repair, respectively; the latter of which holds major implications for cancer intervention. ML307 will be very useful to researchers exploring immunomodulation and inflammation as well as those in the fields of cancer biology. Finally, the larger research community who study UBC13 and ubiquitination-regulated pathways will certainly be beneficiaries of such inhibitors.

4.2. Mechanism of Action Studies

Analogs were also profiled for their ability to suppress TRAF6 ubquitination in HEK293T cells, showing inhibition by active compounds but not compounds that fail to show activity in the biochemical TR-FRET assay. Additionally, analogs of ML307 were identified that failed to suppress K48-linked ubquitination of p53 mediated by the E3 ligase Mdm2, which partners with other E2 enzymes in phenotypic or cell signaling pathway based assays. Planned future studies will determine mechanism of action or provide a plan to biochemically identify and characterize the mechanisms of action.

4.3. Planned Future Studies

The Assay Provider’s future plans for characterization of the probe compound focus on elaborating its biochemical mechanism and on characterizing its cellular activity. For biochemical mechanism, it will be determined whether the compound interferes with cofactor (UEV1a, MMS2) binding to Ubc13. Additional, the approximate binding site of the compound will be mapped by 2D-NMR experiments using 1H-15N-Ubc13. It is possible that the probe compound binds cofactor UEV1a instead of Ubc13, which will be determined by comparing the affinity of the compound for Ubc13 versus UEV1a by isothermal titration calorimetry (ITC) and NMR experiments. For cellular activity, the probe compound will be profiled using the same TRAF6 and p53 ubiquitination assays used for analog compounds. Additionally, cell-based assays for other categories of E2 enzymes are in production, including SUMOylation (Ubc9) and NEDDylation (Ubc12). Inhibitory activity against various Ubc13-mediated signal transduction pathways will also be assessed, including NF-κB and stress kinase activation induced by cytokines, TLR ligands, PKC-activators and DNA-damaging agents. Lastly, it would be advantageous to embark on a SAR campaign oriented to improving microsomal stability.

5. References

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Chen ZJ. Nat Cell Biol. 2005;7:758–765. [PMC free article: PMC1551980] [PubMed: 16056267]
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Ni C-Z, Welsh K, Leo E, Chiou C-K, Wu H, Reed JC, Ely KR. Proc Natl Acad Sci USA. 2000;97:10395–10399. [PMC free article: PMC27035] [PubMed: 10984535]
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Bidere N, Snow AL, Sakai K, Zheng L, Lenardo MJ. Curr Biol. 2006;16:1666–1671. [PubMed: 16920630]
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Karin M, Lin A. Nat Immunol. 2002;3:221–227. [PubMed: 11875461]
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Ea CK, Sun L, Inoue J, Chen ZJ. Proc Natl Acad Sci U S A. 2004;101(43):15318–15323. [PMC free article: PMC524439] [PubMed: 15492226]
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6. Supplementary Information

6.1. Assay Details

Primary Assay and Secondary Assay Protocol

Materials
  • Assay buffer: 50 mM Hepes (pH 7.5), 0.1mM DTT, 0.005% Empigen, 0.1% BSA, 1.25 mM MgCl2
  • E1: Produced at the Sanford-Burnham Medical Research Institute’s protein production core facility
  • Fl-Ub: Invitrogen
  • Tb-Ub: Invitrogen
  • ATP: Sigma
  • Ubc13: Produced at the Sanford-Burnham Medical Research Institute’s protein production core facility
  • Uev1a: Produced at the Sanford-Burnham Medical Research Institute’s protein production core facility
  • Assay plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Protocol
I. Compound Addition
  1. Using LabCyte Echo, transfer 40 nL from a 2 mM Echo qualified plate containing test compounds into assay plate columns 5 – 48 (final concentration of test compounds is 20 microM, 1.0 % DMSO). Transfer 40 nL of DMSO to positive and negative control wells in columns 1 – 4.
  2. Centrifuge plates at 1000 rpm for 1 min.
  3. Seal the plates and leave them at RT.
    Note: Compounds are added to the plates before reagent addition
II. Set up of Ubc13 assay

Prepare assay buffer

  1. E2: E2 is prepared in two steps. First, prepare E2 stock from Ubc13 and Uev1a at 10 μM. This E2 stock will form a heterodimer after 2 hour incubation at 4 degrees C. After 2 hour incubation at 4 degrees C, dilute E2 stock in assay buffer to make 2 X intermediate solution at 250 nM.
  2. E1/Ub/ATP: 30 minutes before the end of E2 stock incubation at 4 degrees C, prepare 2X intermediate E1/Ub/ATP solution with E1 at 200 nM, Fl-Ub at 150 nM, Tb-Ub at 12 nM, and ATP at 2 mM.
III. Reagent Addition
  1. Add 2 μL assay buffer to columns 1&2
  2. Add 2 μL 2X intermediate E2 to columns 3 – 48
  3. Add 2 μL 2X intermediate E1/Ub/ATP to all columns
  4. Incubate at RT for 90 mins
IV. Reading plates
  1. After 90 minutes of incubation, plates are read
  2. Read plates using BMG PHERAstar FS using Lanthascreen protocol.

Secondary Bfl-1 Assay: Dry Powder Protocol

Materials
  • Assay buffer: 25 mM Bis-Tris, 1mM TCEP, 0.005% Tween 20
  • Bfl-1: Produced at the Sanford-Burnham Medical Research Institute
  • F-Bid: Produced at the Sanford-Burnham Medical Research Institute
  • Tb × GST: Invitrogen (Catalogue #: PV3551)
  • TCEP: Sigma (Catalogue #: 646547)
  • Assay plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Protocol
I. Compound Addition
  1. Using LabCyte Echo, transfer 40 nL from a 10 mM Echo qualified plate containing test compounds into assay plate columns 5 – 48 (final concentration of test compounds is 100 microM, 1.0 % DMSO). Transfer 40 nL of DMSO to positive and negative control wells in columns 1 – 4.
  2. Centrifuge plates at 1000 rpm for 1 min.
  3. Seal the plates and leave them at RT.
    Note: Compounds are added to the plates before reagent addition
II. Set up of Bfl-1 assay
  1. Prepare assay buffer
  2. Bfl-1: Dilute Bfl-1 in assay buffer to make 2X intermediate solution at 10 nM
  3. F-Bib/Tb × GST: Dilute F-Bid and Tb × GST in assay buffer to make 2X intermediate solution at the concentration of 8 nM for F-Bid and 5 nM for Tb × GST.
III. Reagent Addition
  1. Add 2 μL assay buffer to columns 1&2
  2. Add 2 μL 2X intermediate Bfl-1 to columns 3 – 48
  3. Add 2 μL of 2X intermediate F-Bid/Tb × GST to all columns
IV. Reading plates
  1. 10. Read plates using BMG PHERAstar FS using Lanthascreen protocol.”

Secondary Caspase-3 Assay: Dry Powder Protocol

Materials
  • Caspase-Glo® - 3/7 Assay: Promega (Catalogue #: G8092)
  • Assay buffer: 150 mM Hepes (pH 7.8), 4.0 mM DTT, 1.0 mM EDTA, 0.01% Tween, 0.1% BSA
  • Assay plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Protocol
I. Compound Addition
  1. Using LabCyte Echo, transfer 20 nL from a 10 mM Echo qualified plate containing test compounds into assay plate columns 5 – 48 (final concentration of test compounds is 100 microM, 1.0 % DMSO). Transfer 20 nL of DMSO to positive and negative control wells in columns 1 – 4.
  2. Centrifuge plates at 1000 rpm for 1 min.
  3. Seal the plates and leave them at RT.

Note: Compounds are added to the plates before reagent addition

II. Set up of Caspase-3 assay
  1. Prepare assay buffer
  2. Caspase 3: Dilute Caspase 3 in assay buffer to make 2X intermediate solution at 0.046 nM
  3. Caspase Glo substrate: Dilute 1X substrate solution in assay buffer to make 2X intermediate solution at 0.75X
III. Reagent Addition
  1. Add 1 μL assay buffer to columns 1 & 2
  2. Add 1 μL 2X intermediate Caspase 3 to columns 3 – 48
  3. Add 1 μL 2X intermediate Caspase Glo substrate to all columns
  4. Incubate at RT for 60 mins
IV. Reading plates
  1. Read plates using PerkinElmer ViewLux using SENP Luminescence 60 sec protocol.

6.2. 1H NMR and LC-MS spectra of ML307

1H NMR Spectrum of ML307 (500 MHz, CDCl3).

1H NMR Spectrum of ML307 (500 MHz, CDCl3)

LC-MS for ML307.

LC-MS for ML307

(Reverse phase C18 column: isocratic)

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