The recently discovered apelin receptor (APJ, AGTRL-1, APLNR) system has emerged as a critical mediator of cardiovascular homeostasis and is involved in the pathogenesis of hypertension, heart failure, atherosclerosis, and other cardiovascular diseases. We disclose the first discovery and characterization of a potent (1.7 – 2.2 μM), kojic acid based small molecule APJ functional antagonist in cell-based assays that is >37 fold selective over the closely related angiotensin II type 1 (AT1) receptor, derived from an high throughput screening (HTS) of the ~330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid receptor (<50%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented. This probe molecule provides a useful tool compound for investigators interested in understanding apelin receptor pharmacology and function.

Probe Structure & Characteristics

This Center Probe Report describes the first reported small molecule APJ antagonist, ML221 that is also selective against the AT1 receptor and cell active

1. Recommendations for Scientific Use of the Probe

The recently discovered apelin system is emerging as a critical mediator of cardiovascular homeostasis, whose importance in the pathogenesis of hypertension, heart failure, atherosclerosis, and other cardiovascular diseases, is the subject of intense investigation^1–7. These investigations are limited by the paucity of research tools and reagents needed to fully understand the role of apelin in physiology and pathology. One particular challenge is the lack of a robust apelin receptor deficient (knock-out) mouse. Although this mouse has been created and reported in the literature, it is difficult to breed homozygous, apelin receptor null mice. Therefore a small molecule antagonist of the apelin receptor (a.k.a APJ, AGTRL-1, APLNR) would advance apelin research significantly. While not a replacement of a transgenic apelin receptor deficient mouse, a small molecule antagonist would be a useful tool for understanding the pharmacology of APJ and ultimately to validate the importance of this system in animal models of cardiovascular disease. To date, there are no small antagonists for the apelin receptor. In this report we disclose the discovery and characterization of a potent selective apelin receptor antagonist. Synthetic methodology, SAR, and activity of our most efficacious and selective compound in a cell-based assay of apelin receptor inhibition are presented. The probe molecule can be used as a tool compound by investigators interested in understanding apelin receptor pharmacology and function.

2. Materials and Methods

2.1. Assays

Table 2 summarizes the details for the assays that drove this probe project and can be found in the “Assay Description” section under the listed AIDs in Table 2 as viewable in PubChem Bioassays.

Table 2

Summary of Assays and AIDs.

All assays in Table 2, with the exception of the β-galactosidase counterscreen (AID 485352), utilize the DiscoveRx PathHunter^® β-arrestin assay technology. Unlike imaging or other second messenger assays, the DiscoveRx β-arrestin assay allows for a direct measure of GPCR activation by detection of β-arrestin binding to the APJ, and in the case of the counterscreen, the AT1 receptor. In this system, β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor of EA) and the GPCR of interest is fused to a smaller (42 amino acids), weakly complementing fragment termed ProLink™. In cells that stably express these fusion proteins, ligand stimulation results in the interaction of β-arrestin and the ProLink-tagged GPCR, forcing the complementation of the two β-galactosidase fragments and resulting in the formation of a functional enzyme that converts substrate to detectable luminescent signal.

2.2. Probe Chemical Characterization

Chemical name of probe compound & structure including stereochemistry

The IUPAC name of the probe is 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate. The actual batch prepared, tested and submitted to the MLSMR is archived as SID 103061721 corresponding to CID 7217941. The probe ML221 has no chiral centers (Figure 1). This probe is not commercially available. A 25 mg sample of ML221 synthesized at SBCCG has been deposited in the MLSMR (Bio-Focus DPI).

Figure 1

Chemical structure of ML221.

Synthetic Route is in Scheme 1 and structure proof by 1HNMR & LC-MS is in Figures 2 and 3A and 3B.

Scheme 1

Synthesis of ML221, conditions. a. neat SOCl₂ (17 eq); b. pyrimidine-2-thiol (1 eq), NaOMe (1 eq), MeCN; c. 4-nitrobenzoyl chloride (1.4 eq), Cs₂CO₃ (1 eq), MeCN.

Figure 2

¹H NMR Spectra for ML221.

Figure 3A

LC of LC-MS Data for ML221.

Figure 3B

MS of LC-MS Data for ML221.

Solubility and Stability of probe in PBS at room temperature

The stability and solubility of ML221 was investigated in PBS buffer at room temperature (Figure 4). Initial experiments examining the stability of ML221 at its solubility limit suggested that the probe degrades relatively rapidly in PBS buffer. Thus, we subsequently examined the stability of ML221 with 50% acetonitrile/PBS. In 50% acetonitrile/PBS buffer the compound is soluble, and appears to be stable out to 48 hr (92% remaining).

Fig. 4

Stability of ML221 in 1:1 PBS:ACN at ambient temp.

MLS# of probe molecule and five related samples that were submitted to the SMR collection

Samples of the probe (25 mg) and each of five analogs (20 mg) synthesized at SBCCG were submitted to MLSMR (Table 3).

Table 3

Probe and Analog Submissions to MLSMR (BioFocus DPI) for APJ Antagonists.

3. Results

3.1. Dose Response Curves for Probe

Figure 5Representative dose response curve for ML221 in the DiscoveRx assay for apelin receptor (APJ) antagonist activity

3.2. Cellular Activity

All of the primary and selectivity assays are cell based.

Table 4Cellular Activity of Probe ML221

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Probe	CID	SID	MLS-#	S/P*	Chemical Structure	Potency (μM)a
						APJ	AT1	SI
ML221	CID 7217941	SID 99213078	0437359	P		2.16 ± 0.96	>79	>37
		SID 103061721		S		1.75 ± 0.19	>79	>45

*

S = Synthesized P = purchased

a

Ave. ± S.E.M. (n = 4), if replicates is different than n=4 it is noted in parentheses (AIDs in Table 1). SI = Selectivity Index: (IC₅₀ AT1)/(IC₅₀ APJ)

3.3. Profiling Assays

The probe was evaluated in a detailed in vitro pharmacology panel as shown in Table 5.

Table 5

Summary of in vitro ADME Properties of APJ Antagonist probe ML221.

ML221 is poorly soluble in aqueous media at pH 5.0/6.2/7.4, although the solubility appears pH-dependent as it is almost three-fold higher in pH 7.4 than in either pH6.2 or pH5.0. We note that the aqueous solubilities obtained at physiological pH are 5 – 14 fold higher than the obtained potency of the probe.

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, ML221 exhibits moderate permeability that increased with pH.

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. ML221 was undetectable in the plasma protein binding assay indicating that the compound is likely rapidly metabolized in plasma.

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. ML221 shows very poor stability in human plasma (<1% remaining) after 3 hr. This data explains the lack of compound detected in the plasma protein binding assay, as that assay involves an 18h incubation.

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. ML221 shows poor stability (4.2 % & 4.9% remaining at 60 min) in both human and mouse liver homogenates. Neither the plasma nor the microsomal stability assay results are surprising given the ester linkage in this probe. Attempts to replace the ester with a more stable functional group resulted in inactive compounds, although replacements were not widely investigated. Ultimately this limits the utility of this probe to in vitro studies or apelin receptor or in vivo studies using acute intravenous doses to avoid metabolism.

ML221 shows no toxicity (>50 μM) toward human hepatocytes.

Profiling against other GPCRs. The probe, ML221 (CID7217941), was submitted to the Psychoactive Drug Screening Program (PDSP) at the University of North Carolina (PDSP, Bryan Roth, PI) and the data against a GPCR binding assay panel is shown in Figure 6. Overall, the compound shows a relatively clean binding profile with the only significant activity at the kappa opioid receptor.

Figure 6

GPCR profiling panel for ML221.

4. Discussion

Currently there are no small molecule tools to investigate the biological functions of apelin and its receptor. Apelin is the endogenous peptide ligand⁸ for the G-protein coupled receptor (GPCR) APJ (angiotensin II receptor-like 1, AGTRL-1 and APLNR). Until the discovery of apelin, APJ was an orphan GPCR. APJ is coupled to Gαi, and has been shown in cell culture to inhibit adenylate cyclase. The APJ gene encodes a receptor that most closely resembles the angiotensin receptor AT1. However, the APJ receptor does not bind angiotensin II⁹. Underscoring the emerging importance of the apelin/APJ system, recent studies have shown that apelin reduces the extent of atherosclerotic lesions in ApoE−/− mice (7), and opposes the development of abdominal aortic aneurysms¹⁰. Additionally, work in Dr. Smith’s lab has revealed that APJ forms a heterodimer with the Ang II receptor AT1¹¹, and that this complex facilitates antagonism of Ang II signaling by apelin. Despite these exciting results, there remains a multitude of unanswered questions regarding the role of apelin and APJ in the physiology and pathology, so chemical biological tools would be an advance.

5. References

1.

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2.

Kleinz MJ, Davenport AP. Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells. Regul Pept. 2004;118:119–125. [PubMed: 15003827]

3.

Kleinz MJ, Skepper JN, Davenport AP. Immunocytochemical localization of the apelin receptor, APJ, to human cardiomyocytes, vascular smooth muscle and endothelial cells. Regul Pept. 2005;126:233–240. [PubMed: 15664671]

4.

Quazi R, Palaniswamy C, Frishman WH. The emerging role of apelin in cardiovascular disease and health. Cardiol Rev. 2009;17:283–286. [PubMed: 19829178]

5.

Sorli SC, van den Berghe L, Masri B, Knibiehler B, Audigier Y. Therapeutic potential of interfering with apelin signaling. Drug Discov Today. 2006;11:1100–1106. [PubMed: 17129829]

6.

Ashley EA, Powers J, Chen M, Kundu R, Finsterbach T, Caffarelli A, Deng A, Eichhorn J, Mahajan R, Agrawal R, et al. The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo. Cardiovasc Res. 2005;65:73–82. [PMC free article: PMC2517138] [PubMed: 15621035]

7.

Chun HJ, Ali ZA, Kojima Y, Kundu RK, Sheikh AY, Agrawal R, Zheng L, Leeper NJ, Pearl NE, Patterson AJ, et al. Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis. J Clin Invest. 2008;118:3343–3354. [PMC free article: PMC2525695] [PubMed: 18769630]

8.

Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, Osmond DH, George SR, O’Dowd BF. Characterization of apelin, the ligand for the APJ receptor. J Neurochem. 2000;74:34–41. [PubMed: 10617103]

9.

O’Dowd BF, Heiber MM, Chan AA, Heng HHH, Tsui LLC, Kennedy JJL, Shi XX, Petronis AA, George SSR, Nguyen TT. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene. 1993;136:355. [PubMed: 8294032]

10.

Leeper NJ, Tedesco MM, Kojima Y, Schultz GM, Kundu RK, Ashley EA, Tsao PS, Dalman RL, Quertermous T. Apelin prevents aortic aneurysm formation by inhibiting macrophage inflammation. Am J Physiol Heart Circ Physiol. 2009;296:H1329–1335. [PMC free article: PMC2685356] [PubMed: 19304942]

11.

Lee DK, Lanca AJ, Cheng R, Nguyen T, Ji XD, Gobeil F Jr, Chemtob S, George SR, O’Dowd BF. Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors. J Biol Chem. 2004;279:7901–7908. [PubMed: 14645236]