C&EN: SCIENCE & TECHNOLOGY

For about the past decade, researchers in government, academic, and industrial labs have been pursuing compounds that activate or inhibit adenosine A₃ receptors. These cell-membrane proteins have a wide range of physiological and disease-related effects and are thus considered promising drug targets.


	COURTESY OF KEN JACOBSON, DOV BARAK, AND STEFANO MORO BOUND TO FIT View of the A₃ receptor from above shows the arrangement of its seven transmembrane -helices and a bound agonist, IB-MECA [N⁶-(3-iodobenzyl)-adenosine-5´-N-methyluronamide]. The receptor's van der Waals surface and loops connecting the seven transmembrane helices are omitted.

Those efforts are now beginning to come to fruition, as a number of A₃activators and inhibitors (agonists and antagonists, respectively) enter clinical trials for several human diseases. And such A₃ligands are also of interest as tools that can help scientists learn more about the role of A₃ receptors in the body--functions that have not yet been fully characterized.

A₃ proteins are G-protein-coupled receptors that are normally activated by adenosine. In addition to being the main component of adenosine triphosphate, the energy currency of cells, adenosine is a neuromodulator in that it affects nervous system function but does not act as a neurotransmitter per se. A₃ receptors also can be activated by inosine, a major metabolite of adenosine.

The receptors, which are expressed in a variety of body tissues, have functional effects that are surprisingly contradictory. When activated only moderately, they have a cytoprotective role--such as reducing damage to heart cells from lack of oxygen or protecting cells from apoptosis (programmed cell death). But at high levels of stimulation they can actually cause cell death. A₃ receptor agonists and antagonists are thus being tested for treatment of a number of conditions, ranging from heart disease to cancer.

THE A₃RECEPTOR is actually part of a family of four related adenosine receptor types, and its three siblings also play important functional roles. The A₁ and A_2a receptor subtypes protect organs such as the heart and brain under conditions of stress. And the A_2b subtype, which is expressed on mast cells in inflamed tissues and tends to increase intracellular calcium levels, is considered a promising target for asthma drugs.

Ligands for the A₃ receptor were designed and synthesized by government and academic groups before the receptor's biological functions were at all well defined, and the availability of these ligands has greatly facilitated studies on the receptor's biochemistry and function. The ligand development research thus exemplifies how fundamental biochemical studies can help lead to future biomedical advances.

One of the most active research groups among those that have developed A₃-targeted ligands is that of Kenneth A. Jacobson, chief of the Molecular Recognition Section of the Laboratory of Bioorganic Chemistry at the National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK), Bethesda, Md. Jacobson and coworkers have developed both agonists and antagonists that are potent and selective for these receptors.

But a team led by Pier Giovanni Baraldi, director of the department of pharmaceutical science at Ferrara University, in Italy, made the most recent advance in the A₃ field last December when it reported the most potent and selective human A₃ adenosine antagonist found to date [J. Med. Chem., 43, 4768 (2000)]. Professor of medicinal chemistry Ad P. IJzerman and coworkers at Leiden University, in the Netherlands, have also synthesized a number of A₃-selective agonists and antagonists.

The A₃ receptor was first cloned from a rat brain cDNA library in 1992 by Gary L. Stiles, chief of the Division of Cardiology at Duke University Medical Center, and the first human A₃ receptor was cloned the next year by Marlene A. Jacobson of the department of pharmacology at Merck Research Laboratories, West Point, Pa., and coworkers. Merck has a patent on the human A₃ receptor.

IN THE EARLY '90S, Ken Jacobson (no relation to Marlene) also started working on A₃, at a time when nobody had any idea what physiological functions the A₃ receptor had in the body. Jacobson explains that he and his coworkers "began by making selective ligands, hoping pharmacologists would use them to establish a role for the receptor." His team found selective A₃ agonists in 1994, and "these are still the principal selective agonists used in many labs that study adenosine receptors."

The usual antagonists for adenosine receptors in general "are the xanthine drugs, of which caffeine and theophylline are probably the best known," Ken Jacobson says. "These block most subtypes of adenosine receptors but don't block A₃receptors very well."

The first nonxanthine A₃ antagonists were discovered by the Merck group. The NIDDK team subsequently found other nonxanthine antagonists by "going to molecular diversity to get leads," Ken Jacobson says. "We identified a bunch of heterocycles, including flavonoids, pyrazoloquinazolines, 1,4-dihydropyridines, 1,3-diacylpyridines, and 1-alkylpyridinium salts. We optimized some of those and eventually ended up with selective A₃ antagonists of close to nanomolar potency."

Ken Jacobson and coworkers recently characterized the preferred conformation of pyridine derivatives in the binding site of A₃ receptors. They synthesized ring-constrained analogs, superimposed structurally diverse A₃ antagonists to arrive at a unified model, synthesized combinatorial libraries of potential A₃ligands, and carried out site-directed mutagenesis on the related A_2a receptor to determine the mechanism of A₃ binding.

Like the NIDDK group, Baraldi and coworkers have synthesized a number of A₃antagonists, including the most potent and selective ones identified so far, although some found by Ken Jacobson's team are not far behind. The Italian group also prepared the first radiolabeled A₃ adenosine antagonist as a tool for further characterizing the A₃ receptor subtype and clarifying its functional role in the body.

IJzerman and coworkers have synthesized a variety of A₃-active antagonists as well as some "partial" agonists--ligands that activate human A₃ receptors in a limited way. This actually might be a desirable property, IJzerman says, because of the A₃receptor's tendency to cause severe side effects when overstimulated. He believes chronic low-level stimulation of the receptor could be just the ticket to bring out the receptor's desirable cerebroprotective and cardioprotective properties.

Other groups working on A₃ inhibition and activation include professor Christa E. Müller and coworkers at the Pharmaceutical Institute at the University of Bonn, in Germany, who recently developed a tritiated imidazopurinone derivative as a new A₃ antagonist radioligand. The group is currently preparing a manuscript on the work.

A₃ KNOCKOUT ORGANISMS--mice with deficient expression of the A₃receptor gene--were reported last year by Marlene Jacobson and coworkers at Merck. They currently are being used worldwide in a range of studies on physiological functions of the A₃ receptor and effects of A₃ inhibition on a variety of disease models. For example, the Merck group, in collaboration with research assistant professor of medicine Beverly H. Koller and coworkers at the University of North Carolina, Chapel Hill, has used A₃ knockouts to determine physiological effects of adenosine and inosine on A₃-related changes in blood vessel permeability.

With potent ligands that activate or inhibit A₃ receptors in hand, it wasn't much of a conceptual leap to speculate that some of these compounds might be good leads for drug discovery, and that has turned out to be true. For example, Pnina Fishman, head of the Laboratory of Clinical & Tumor Immunology at Felsenstein Medical Research Center, Petach Tikva, Israel, and coworkers have published a number of papers on the use of A₃ agonists for shrinking tumors. "The differential effect of A₃ agonists on normal and tumor cells is, in my opinion, the most fascinating phenomenon regarding the activation of this receptor," Fishman notes. "We have established a biotech company, Can-Fite Biopharma (also in Petach Tikva), that focuses on the development of A₃ agonists as anticancer and chemoprotective agents."

In addition, Baraldi has been collaborating with Medco Research, Research Triangle Park, N.C., a subsidiary of King Pharmaceuticals, Bristol, Tenn., to develop A₃-active therapeutic agents. IJzerman believes some of his ligands might make good drug candidates as well.

"The hottest area defined so far is cardioprotection for A₃ receptor agonists," Ken Jacobson says. "They really work dramatically well." The concept has been validated now by genetic overexpression of the A₃ receptor in mice and subsequent limitation of damage in models of cardiac ischemia--a decrease in blood supply to the heart owing to obstruction or constriction.

Associate professor of medicine and pharmacology Bruce T. Liang and coworkers at the University of Pennsylvania, working in collaboration with the NIDDK group, were first to show that genetically engineered cardiac myocytes overexpressing human A₃ rece ptors are highly resistant to the deleterious effects of ischemia.


	KEN JACOBSON

The Penn-NIDDK team also has found a synergistic cardioprotective interaction between A₁ and A₃ receptors. "The concept is that agonists coactivating both A₁ and A₃ receptors are likely to provide protection from ischemia at lower doses than those required for selective A₁ or A₃ agonists and could thus have fewer side effects," Liang explains.

A₃ agonists that limit heart attack damage to cardiac muscle cells are currently being studied as possible drug prospects by the Penn-NIDDK group. Such A₃-targeted drugs could be administered either prospectively prior to an operation with a high risk of cardiac ischemia or retrospectively to treat ischemia after a heart attack has already occurred. Studies on the cardiovascular role of the A₃ receptor are being carried out at Merck as well.

A₃ AGONISTS may also have applications in stroke treatment. "We have found that chronic administration of an A₃ agonist is highly cerebroprotective in a model of global cerebral ischemia in gerbils," Ken Jacobson says. "The benefit is seen in preservation of neurons of the hippocampus and in the survival and behavior of the animals following recovery."

There currently is no drug on the market "to limit the spread of excitotoxic damage in the brain during the first few days following a stroke," he adds. "We have evidence that modulating A₃ receptors may be useful in this regard. A₃ agonists would have fewer side effects than A₁ agonists, which may also be cerebroprotective but tend to depress heart function."

He points out that for most G-protein-coupled receptors (GPCRs), antagonists are the principal targets for drug development, whereas for A₃ the agonists appear to have more potential use as therapeutic agents. "But I wouldn't rule out A₃ antagonists" as potential drugs, he says.

A₃ antagonists have been suggested to be potentially useful in lowering intraocular pressure in glaucoma patients, for instance. This proposal is based on studies on the effects of A₃ ligands on chloride transport in ciliary epithelial cells by Penn professor of physiology and medicine Mortimer M. Civan and coworkers. Civan, Ken Jacobson, and coworkers have filed a joint patent application for use of some of the NIDDK group's antagonists for treatment of glaucoma.

And IJzerman has synthesized some human A₃ antagonists that he believes might be useful as anti-inflammatory agents because they impede the release of allergic mediators from blood cells.

Dov Barak, a molecular modeler from the Israel Institute for Biological Research who is currently on sabbatical in Ken Jacobson's group, notes that the GPCR class to which A₃ receptors belong "is the most prevalent paradigm for signal transduction in nature. Any drug designed to target these receptors will affect major signaling pathways in cells"--suggesting why A₃ studies have been so fruitful.

Barak points out that common structural motifs shared by GPCRs--such as their seven transmembrane a-helices--simplify drug design studies to some extent, because the structure and activity of these receptors are well known. However, the commonality among GPCRs also poses special challenges, he says, in that it makes it more difficult to develop agonists and antagonists with the requisite specificity of action. Only the future will tell to what extent Barak and other researchers succeed in exploiting such opportunities and overcoming such roadblocks as they continue to pursue their efforts to target the A₃ receptor.