Introduction
In the face of infection, tissue damage or acute inflammation, humans and other mammals undergo a series of biochemical and physiological changes termed the acute-phase response. This response is critical for innate immunity and for limiting tissue injury. One of its key components is altered hepatic synthesis of a wide array of proteins that are involved in the coagulation and complement systems1. The acute-phase reaction is rapid, and in humans CRP concentration can increase up to 1,000-fold within 48 h after infection or tissue injury. Chronic systemic inflammation promotes much more modest increases in CRP concentration that nevertheless might increase the risk for atherosclerosis. Indeed, clinical studies have shown that elevated concentrations of circulating CRP predict the risk of cardiovascular disease independently of traditional factors like cholesterol2, but the role of this acute-phase protein in disease progression is not known. In an important new study, Pepys and colleagues have synthesized an inhibitor of CRP and demonstrated that it limits myocardial infarction in rats3. This observation provides strong evidence that CRP not only is a marker for cardiovascular disease but also might be pathogenic.
CRP binds phosphocholine and other extracellular components of invading pathogens in a calcium-dependent manner4. When human CRP is ligand bound, it triggers activation of the complement pathway, a key microbicidal component of the innate immune system. CRP (so named because it reacts with the C-polysaccharide protein of Streptococcus pneumoniae, a common bacterial cause of pneumonia) belongs to the pentraxin family of proteins, in which five noncovalently linked building blocks (or protomers) surround a central pore. CRP also binds to ligands produced in damaged tissue and to Fc receptors for antigen-antibody complexes and aggregated immunoglobulins. These processes may facilitate the uptake and clearance of dead and dying cells during the acute-phase response.
The pathophysiological role of CRP in humans is unknown because mutations and deficiencies of the protein have yet to be identified. CRP has been proposed to contribute to the development of atherosclerosis by promoting vascular injury5, 6. In this view, inflammatory proteins directly affect biological processes in the arterial wall or regulate atherogenic processes at other sites. The hypothesis that CRP might directly mediate atherosclerosis through sustained systemic inflammation has been controversial. For example, much of our current understanding of CRP's potential atherogenic mechanisms is based on studies of cultured cells. It is important to note that many of these studies used high concentrations of the protein, often failed to use relevant controls and did not assess the purity of the CRP that was used. These are important issues because CRP can be contaminated with other factors that exert potent biological effects. Moreover, expression of CRP in animal models has had variable effects on atherosclerosis7, suggesting that even high concentrations of the protein might have little effect on vascular disease. Thus it remains to be determined whether CRP is a marker or a mediator of cardiovascular disease.
A specific inhibitor of CRP now provides a valuable tool to probe these open mechanistic issues. Using the crystal structure of CRP bound to phosphocholine, Pepys and colleagues designed a palindromic small molecule that they termed bis(PC)H (ref. 3). This inhibitor consists of two negatively charged phosphate groups linked by a six-carbon chain and terminated at each end by a positively charged choline group. Thus, it resembles a snake with two heads—a phosphocholine dimer interrupted by a flexible hydrocarbon chain (Fig. 1a). The underlying hypothesis was that suitably juxtaposed phosphate groups in the inhibitor might interact with calcium ions bound to CRP, preventing CRP from interacting with ligands.
Figure 1: Structure of bis(PC)H complexed with CRP.
(a) bis(PC)H and its proposed CRP-binding domains. (b) Schematic view of bis(PC)H bound to CRP. The figure is based on the crystal structure of Pepys et al.3. CRP is composed of five identical, noncovalently associated protomers assembled around a central pore. Each bis(PC)H is interposed between two molecules of CRP. Note that the individual CRP protomers contain two calcium ions, which bind to the phosphate group of bis(PC)H. CRP also interacts with the choline groups of bis(PC)H.
Ann Thomson
Full size image (38 KB)Supporting this proposed binding mechanism, the crystal structure of CRP complexed with bis(PC)H revealed five of the small molecules bound at the interface of two pentameric CRP protomers3. Each bis(PC)H was interposed between calcium ions on different faces of two CRP protomers (Fig. 1b). The structure of the complex was very similar to that of CRP bound to phosphocholine, validating the overall synthetic strategy.
To determine whether bis(PC)H could serve as a physiological inhibitor of CRP, Pepys and co-workers took advantage of their rodent model of myocardial infarction, in which human CRP can function to activate rat complement3. When the researchers occluded a coronary artery, the animals suffered a myocardial infarction, and the damaged area of the heart enlarged after human CRP was administered. Remarkably, bis(PC)H completely prevented the increase in infarct size induced by human CRP. Importantly, the inhibitor did not restrict lesion size in control mice that suffered a myocardial infarction but did not receive CRP. Collectively, these observations provide strong evidence that bis(PC)H can block the deleterious effects of human CRP in an animal model of acute myocardial infarction.
Thrombosis is centrally important in triggering heart attacks in humans8, and CRP colocalizes with activated complement in infarcted human myocardial tissue7. The pioneering studies of Pepys and colleagues demonstrate that human CRP promotes myocardial infarction in a rat model of human disease3. Bis(PC)H will provide a powerful tool for mechanistic studies of CRP in cardiovascular disease, and it lays the groundwork for the rational design of CRP inhibitors. If a safe, orally available small-molecule inhibitor of CRP becomes available, it would offer the opportunity to determine whether the acute-phase–response protein has a pathogenic role in humans, perhaps by pathways involving the complement and coagulation systems.
