A robust antibody response is essential for efficient identification and eradication of pathogenic microbes and toxins, whereas dysregulation of the antibody response can lead to autoimmune disease. Specific antigen encounter by B lymphocytes induces clonal expansion that encompasses several distinct stages of differentiation. During this differentiation process, a critical cell fate decision is made wherein some B cells will undergo terminal differentiation into antibody-producing cells, while a separate cohort will assume a distinct pathway of differentiation to become long-lived memory B cells. An exciting new development in the field is the revelation of a novel chemotactic axis involving the recognition of oxysterol compounds by the orphan G-protein-coupled receptor (GPCR), Epstein-Barr virus-induced gene 2 (EBI2). EBI2 is expressed on B cells and is highly induced upon activation. Recent gene targeting experiments revealed that EBI2^-/- B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses. Most recently, two research teams made the unlikely discovery that oxysterol compounds, previously known to bind nuclear receptors, are the physiologic ligands for EBI2. In order to investigate the importance of EBI2 in immune processes and potential as a drug target, selective and potent antagonists with good in vivo pharmacokinetics need to be developed. This probe report describes a potent functional antagonist of EBI-2, ML401 (CID 73169083, SID 173333998), which is potent (IC₅₀ ∼ 1 nM), displays activity in a chemotaxis assay (IC₅₀ ∼ 6 nM), and has a clean profile in a Eurofins/Ricerca panel as well as excellent rodent pharmacokinetics. As such ML401 should be a valuable tool to explore the in vivo effects of EBI-2 inhibition.

Probe Structure & Characteristics

This Center Probe Report describes ML401, a selective inhibitor of EBI-2. ML401 has a central piperazine core. The chemical structure and data summary are shown in(Table 1).

Chemical Structure of ML401

1. Recommendations for Scientific Use of the Probe

A robust antibody response is essential for efficient identification and eradication of pathogenic microbes and toxins, whereas dysregulation of the antibody response can lead to autoimmune disease. Specific antigen encounter by B lymphocytes induces clonal expansion that encompasses several distinct stages of differentiation. The molecular cues that influence this cell fate decision into antibody-producing or long-lived memory B cells is of great importance for understanding vaccine efficacy and how/where B cell differentiation may go awry to cause autoimmunity. In this context, there is a growing appreciation of microenvironment-specific influences on B cell differentiation within the lymphoid tissue. Recently a novel chemotactic axis involving the recognition of oxysterol compounds by the orphan G-protein-coupled receptor (GPCR), Epstein-Barr virus-induced gene 2 (EBI2) (1-4) was discovered.

EBI2 is expressed on B cells and is highly induced upon activation (5). Recent gene targeting experiments revealed that EBI2^-/- B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses (1, 2). Most recently, two research teams made the unlikely discovery that oxysterol compounds, previously known to bind nuclear receptors, are the physiologic ligands for EBI2 (3, 4). Although other closely related oxysterols showed some potency and binding capability to EBI2, the most potent endogenous EBI2 receptor ligand and activator was 7α,25-dihydroxycholesterol (7α,25-OHC) (4). EBI2^-/- B cells did not bind 7α,25-OHC and mice deficient for cholesterol 25-hydroxylase that is necessary to generate 7α,25-OHC display a phenotype similar to that of EBI2^-/- mice (3). The recent publication in July 2011 of the deorphanizing of this receptor is a major step forward towards understanding the role of EBI2 in immunobiology. In order to investigate the importance of EBI2 in immune processes and potential as a drug target, selective potent chemical probes that antagonize this receptor need to be identified and made freely available to the research community.

The proposed work enlists the MLPCN to screen chemical libraries to identify the first small molecule antagonists of EBI2, the probes sought will be identified and optimized based on their ability to antagonize this receptor in functional cellular systems, with the ultimate objective of generating compounds that will function in vivo. Armed with such tools a full examination of the consequences of antagonizing this receptor in well characterized ex vivo murine and human models of the immune system will be possible. Since EBI2 is required for the generation of antibody-producing cells, antagonists would be anticipated to not only blunt antibody production but may also have more broad reaching effects on inflammation. Prospectively, these probes could be used to investigate the role of this receptor in mouse models of autoantibody-dependent inflammatory disease such as rheumatoid arthritis and lupus, or more broadly applied to examine effects on systemic inflammation. Moreover, production of 7α,25-OHC occurs in many cell types and tissues, consistent a broader role in the regulation of inflammation. Of particular interest, studies in rat and human have revealed polymorphisms in the Ebi2 promoter that are associated with multiple inflammatory disorders and type I diabetes (6).

Prior Art: A SciFinder search on April 27, 2012 showed one reference disclosing the structure and biological activity of several EBI2/GPR183 inverse agonists from a group at GSK (Benned-Jensen, et. al., Journal of Biological Chemistry, 2011, 29292-29302). The lead structure is shown in Figure 1. Although GSK682753A is quite potent as an inverse agonist on its constitutive endogenous activation, it has very poor microsomal and plasma stability (vide infra) and thus is unsuitable for in vivo proof of concept studies. Several structurally similar compounds are also disclosed in this paper. There are no other reports of EBI2/GPR183 antagonists.

Figure 1

EBI2 antagonist GSK682753.

It is noteworthy that three pharmaceutical companies (Euroscreen, Novartis and Johnson & Johnson) have reported on the identification of 7α,25-OHC as the physiologic ligand of EBI2. Certainly, it would be expected that efforts are underway at these companies to develop EBI2 antagonists. However, compounds that they identify are unlikely to be published and available to academic investigators until intellectual property is secured and this typically creates a lag of 3-4 years, where potentially useful pharmacological tools would be unavailable to the research community, that could be used to elucidate the pharmacology and functional biology of this very newly deorphanzied GPCR. Although a compound functioning as an EBI2 inverse agonist on its constitutive endogenous activation has been reported by GSK (7), no EBI2 antagonists that antagonize the binding and/or activation of EBI2 by 7α,25-OHC have been published. Indeed we have requested this compound GSK682753A from GSK, and are still awaiting a response. The antagonists that we identify will become available to the research community in 18 months and thereby will accelerate knowledge and the participation of new researchers on this important new receptor, and which would seed further discoveries of additional chemical probes and validation of their utility. As GPCRs are one of the most “druggable” target classes, we would fully expect that some of the probes arising from the MLPCN will become starting points for the development of potential therapies for immunological and inflammatory diseases.

2. Materials and Methods

2.1. Assays

Table 2 summarizes details for the assays that enabled this probe discovery project. A detailed description of the Primary and all assays can be found in the PubChem AIDs listed.

Table 2

Summary of Assays and AIDs.

2.2. Probe Chemical Characterization

Chemical name of probe compound. The IUPAC name of the probe is (E)-3-(4-bromophenyl)-1-(4-(4-chlorobenzyl)-piperazin-1-yl)prop-2-en-1-one. The actual batch prepared, tested and submitted to the MLSMR is archived as SID173333998 corresponding to CID73169083. The probe ML401 does not have any chiral centers.

Figure 2Structure of ML401

Solubility and Stability of ML401 in PBS at room temperature. The stability of ML401 was investigated (Figure 3) in aqueous buffers at room temperature by monitoring the amount of starting ML401 apparently remaining after incubation at room temperature in either PBS (pH 7.4) or 1:1 PBS:acetonitrile (v/v). ML401 was stable in PBS:acetonitrile with 100% remaining after 48 hrs. The apparently lower stability (48.74%) in neat PBS is a reflection of low solubility. Thus, the stability value is artificially low due to compound precipitation vs. degradation. As noted in the Summary of in vitro ADME/T properties (Section 3.3, Table 5) ML401 has low solubility (0.76/0.53/0.49 μg/mL) at pH 5.0, 6.2 and 7.4 in pION buffer and comparable solubility in PBS at pH 7.4. While the scaffold structure represented by ML401 contains a Michael acceptor, it shows no reactivity when incubated with 1 mM glutathione for 20 hrs at 37°C in PBS buffer (data not shown, but there is essentially no loss of ML401 or presence of a GSH adduct; the control ethacrynic acid was completely reacted as expected). We also note that the probe and close analogs display excellent plasma and microsomal stability as well as rodent pharmacokinetics and thus it appears that the compounds are not reactive in physiological conditions. Table 3 summarizes the deposition of the Probe and 5 analogs.

Figure 3

Stability of ML401 in 1×.

Table 3

Probe and Analog Submissions to MLSMR (Evotec) for EBI2inhibitors.

2.3. Probe Preparation

This probe is not commercially available. A 27 mg sample of ML401 synthesized at SBCCG according to Scheme 1, has been deposited in the MLSMR (Evotec) (see Probe Submission Table 3).

Scheme1

Synthesis of ML401, conditions: a. CDI, THF 2 to 3 hrs at RT (79%); b. DCM, TFA 0 °C; c. 4-Cl benzyl bromide, K₂CO₃, Acetone, 50°C 2 hours (58%).

Preparation of (E)-tert-butyl 4-(3-(4-bromophenyl)acryloyl)piperazine-1-carboxylate (3)

4-Bromo Cinnamic acid 1 (200 mg, 0.880 mmol) was dissolved in 10 ml of THF and after the addition of CDI (213.9 mg, 1.3 mmol), the mixture was stirred at room temperature for 30 minutes. Boc-Piperazine 2 (196.6 mg 1.055 mmol) was added to the resulting mixture and the reaction mixture was stirred for 2 hours at room temperature. When the reaction was determined to be complete by HPLC, the reaction mixture was concentrated under reduced pressure and the resulting solid was dissolved in EtOAc. The solution was washed with saturated aqueous NaHCO₃, washed with saturated aqueous NaCl and dried over Na₂SO₄. The resulting organic layer was concentrated under reduced pressure afforded the title compound 3 (275 mg, 79%) as a white solid which was used directly into the next step without purification. ¹H NMR (400 MHz, DMSO-d₆) δ 1.42 (s, 9H), 3.75 (m, 4H), 4.45 (m, 2H), 7.87 (d, 2H), 8.15 (d, 2H), MS (EI) m/z 397 (M+1).

Preparation of (E)-3-(4-bromophenyl)-1-(4-2, 2, 2-trifluroacetyl) piprazin-1-yl) prop-2-en-1one (4)

(E)-tert-butyl4-(3-(4-bromophenyl)acryloyl)piperazine-1-carboxylate 3 (200 mg, 0.505 mmol) was dissolved in dichloromethane (3 ml) and cooled to 0 °C using an ice-water bath. Trifluoro acetic acid (1.2 mL) was slowly added to the resulting mixture and the reaction was stirred for one hour at room temperature. When the reaction was determined to be complete by HPLC, the reaction mixture was concentrated under reduced pressure. The resulting mixture was washed with toluene twice to remove excess of TFA and the organic layer was concentrated under reduced pressure to yielded the TFA salt of product 4 (379 mg), MS (EI) m/z 297 (M+1).

Preparation of (E)-3-(4-bromophenyl)-1-(4-(4-chlorobenzyl)piperazin-1-yl)prop-2-en-1one (5)

(E)-3-(4-bromophenyl)-1-(4-2,2,2-trifluroacetyl)piprazin-1-yl)prop-2-en-1one 4 (100 mg, 0.244 mmol) was dissolved in acetone (5 mL), potassium carbonate (112 mg, 0.812 mmol) was added followed by 4-chlorobenzyl bromide (50 mg, 0.244 mmol). The resulting reaction mixture was stirred at 50 °C for about 2 hours. 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 precipitate was dissolved in EtOAc (20 ml). Water (15 mL) was added and the two layers were separated. The aqueous phase was extracted with EtOAc (3 × 20 mL), the collected organic phases were washed with brine (10 mL), dried over Na₂SO₄ and the solvent was evaporated to give crude product which was purified using HPLC with acetonitrile and water (20:100 gradient) to yield the desired product 5 (60 mg, 58 %). ¹H NMR (400 MHz, DMSO-d₆) δ 2.35 (d, 4H), 3.49 (s, 2H), 3.55 (s, 2H), 3.68 (s, 2H), 7.35 (m, 6H), 7.58 (d, 2H), 7.66 (d, 2H), ¹³C NMR (400 MHz, DMSO-d₆) δ(ppm) 41.8, 45.0, 52.1, 53.1, 60.8, 119.1, 122.7, 128.2, 130.0, 130.7, 131.60, 134.4, 136.9, 140.2, 164.2, MS (EI) m/z 420 (M+1).

Figure 4A¹H NMR Spectrum of ML401 (500 MHz, CDCl₃)

Figure 4B¹³C NMR Spectrum of ML401 (125 MHz, CDCl₃)

Figure 4CLC/MS for ML401

Figure 4DMS for ML401

3. Results

3.1 & 3.2. Response Curves for Probe & Cellular Activity

The dose response curves of ML401 for antagonism in the β-arrestin and chemotaxis assays are shown in Figure 5. There is only a 5-fold loss of potency in chemotaxis.

Figure 5

Potency of ML401 inhibition in EBI2 beta-arrestin and RS11846 cell chemotaxis assays. Error bars are standard deviation of quadruplicate runs tested on at least two separate days.

3.3. Profiling Assays

As a pro forma activity, the SBCCG is committed to profiling all final probe compounds 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. ML401 was evaluated in a detailed in vitro pharmacology screen as shown in Table 5:

Table 5Summary of in vitro ADME Properties of EBI2 inhibitor probe ML401 (MLS-0472527)

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Aqueous Solubility in pION's buffer (μg/mL) pH 5.0/6.2/7.4			0.76/0.53/0.49
Aqueous Solubility in 1× PBS, pH 7.4 (μg/mL)			0.04
Chemical Stability in 1× PBS pH 7.4 / with 50% ACN (% remaining after 48 hrs)			100
PAMPA Permeability, Pe (×10-6 cm/s) Donor pH: 5.0 / 6.2 / 7.4 Acceptor pH: 7.4			1858/1180/1577
Plasma Protein Binding (% Bound)		Human 1 μM / 10 μM	98.89/99.15
		Mouse 1 μM / 10 μM	98.91/99.30
Plasma Stability (% Remaining at 3 hrs) Human/Mouse			73.55/83.64
Hepatic Microsome Stability (% Remaining at 1hr) Human/Mouse			51.87/36.56
Cytotoxicity	Fa2N-4 Immortalized Human Hepatocytes LC50 (μM)		>50

ML401 achieved modest concentrations 18-20 × IC₅₀ in aqueous buffer between a pH range of 5.0-7.4. The solubility was comparable in PBS.

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 its solubility data, ML401 exhibited excellent permeability across the pH range of the donor compartment.

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. ML401 was highly plasma protein bound.

Plasma stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which can strongly influence the in vivo efficacy of a test compound. Drug candidates are exposed to enzymatic processes (proteinases, esterases) in plasma, and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. ML401 showed very good stability in both human plasma 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. ML401 showed moderate stability in both human and mouse liver microsomes after 1 hour.

ML401 showed no toxicity (>50 μM) towards immortalized Fa2-N4 human hepatocytes and non-cytotoxic in MTT (CellTiter96, Promega) in both LnCap and IMR-32 cells (>50 μM).

Counterscreening

Lastly, we also subjected ML401 to a lead profiling panel, looking at its cross reactivity across a range of enzymes and receptors (Figure 6). ML401 was found to have minimal promiscuity, with no activity on 5 CYPs that were tested. Modest activity (in the high μM range) was found across several targets but this was not viewed as an issue with single digit nM activity on EBI-2.

Figure 6

Eurofins Panel for ML401.

4. Discussion

5. References

1.

Gatto D, Paus D, Basten A, Mackay CR, Brink R. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. Immunity. 2009;31(2):259–69. [PubMed: 19615922]

2.

Pereira JP, Kelly LM, Xu Y, Cyster JG. EBI2 mediates B cell segregation between the outer and centre follicle. Nature. 2009;460(7259):1122–6. [PMC free article: PMC2809436] [PubMed: 19597478]

3.

Hannedouche S, Zhang J, Yi T, Shen W, Nguyen D, Pereira JP, et al. Oxysterols direct immune cell migration via EBI2. Nature. 2011;475(7357):524–7. [PMC free article: PMC4297623] [PubMed: 21796212]

4.

Liu C, Yang XV, Wu J, Kuei C, Mani NS, Zhang L, et al. Oxysterols direct B-cell migration through EBI2. Nature. 2011;475(7357):519–23. [PubMed: 21796211]

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Chan TD, Gardam S, Gatto D, Turner VM, Silke J, Brink R. In vivo control of B-cell survival and antigen-specific B-cell responses. Immunological Reviews. 2010;237(1):90–103. [PubMed: 20727031]

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Heinig M, Petretto E, Wallace C, Bottolo L, Rotival M, Lu H, et al. A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk. Nature. 2010;467(7314):460–4. [PMC free article: PMC3657719] [PubMed: 20827270]

7.

Benned-Jensen T, Smethurst C, Holst PJ, Page KR, Sauls H, Sivertsen B, et al. Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2: IDENTIFICATION OF A POTENT AND EFFICACIOUS INVERSE AGONIST. The Journal of Biological Chemistry. 2011;286(33):29292–302. [PMC free article: PMC3190735] [PubMed: 21673108]

8.

Lapinsh M, Gutcaits A, Prusis P, Post C, Lundstedt T, Wikberg JE. Classification of G-protein coupled receptors by alignment-independent extraction of principal chemical properties of primary amino acid sequences. Protein science : a publication of the Protein Society. 2002;11(4):795–805. [PMC free article: PMC2373523] [PubMed: 11910023]

9.

Birkenbach M, Josefsen K, Yalamanchili R, Lenoir G, Kieff E. Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors. Journal of Virology. 1993;67(4):2209–20. [PMC free article: PMC240341] [PubMed: 8383238]

10.

Joost P, Methner A. Phylogenetic analysis of 277 human G-protein-coupled receptors as a tool for the prediction of orphan receptor ligands. Genome biology. 2002;3(11):RESEARCH0063. [PMC free article: PMC133447] [PubMed: 12429062]

11.

Yoshida R, Imai T, Hieshima K, Kusuda J, Baba M, Kitaura M, et al. Molecular cloning of a novel human CC chemokine EBI1-ligand chemokine that is a specific functional ligand for EBI1, CCR7. The Journal of Biological Chemistry. 1997;272(21):13803–9. [PubMed: 9153236]

12.

Norregaard K, Benned-Jensen T, Rosenkilde MM. EBI2, GPR18 and GPR17--three structurally related, but biologically distinct 7TM receptors. Curr Top Med Chem. 2011;11(6):618–28. [PubMed: 21261596]