Small molecule analogues (SMAs) of an immunomodulatory helminth product provide a novel approach to dissecting macrophage signal transduction pathways

The basic unit of life is the cell and all living organisms on Earth are made up of one or more of these. In order to survive and function a cell must be able to communicate with its environment and respond to the signals that it receives. Within the human body, cells respond to signals that they receive either when they make contact with other cells or when they interact with molecules such as proteins and hormones, present in fluid components of the body such as blood. These interactions can trigger changes in cells that may ultimately translate into altered functional capability. This is because the interactions activate biochemical pathways ('signal tranduction pathways') in cells and these pathways promote changes in the cell's molecular composition. For example, the cell may start to produce a new protein that bestows on it properties, previously unpossessed. The response of a cell to a signal is dependent on the nature and quantity of the signal and the signal transduction network of the cell responds to and deciphers each signal to produce an appropriate response. Although it is possible for a cell to produce thousands of new proteins, the signal transduction network uses a resticted number of components to facilitate this. Thus the key to understanding how the cell responds to signals is to elucidate which members of the signal transduction pathway are activated in any particular case. The macrophage is a cell that is involved in fighting disease. As a consequence of this, not only does it have to respond to the types of signal referred to above, it responds to signals it receives from infectious agents attempting to invade the body. The signals come in the form of molecules of the pathogens that bind to receptors on the surface of the macrophage. A good example of a type of receptor is the Toll-like family of receptors. These respond to pathogen products by activating signal transduction pathways that ultimately result in the production within the macrophage of a group of molecules called pro-inflammatory cytokines. These molecules are secreted and have an important role in combatting infectious agents. However, their production must be carefully regulated as left unchecked they have the potential to cause more harm than good, as overproduction of these molecules is associated with many chronic inflammatory diseases. The key question that we wish to address is how the macrophage signal transduction network regulates production of individual inflammatory mediators. We have found that a worm pathogen product that we discovered (ES-62) inhibits certain signal transduction pathways in macrophages and certain other cells of the immune system. We have investigated a number of small molecule analogues (SMAs) of ES-62 and found that although some of them possess inhibitory activity, this is often more focussed than that associated with the parent molecule and also differs in target amongst SMAs. Thus it appears to be possible to inhibit production of a particular cytokine and this offers the opportunity to then establish the signalling events underlying regulation of this cytokine. The aim of this project is thus to make a larger number (a 'library') of related SMAs and to test their ability to inhibit pro-inflammatory cytokine responses and thus understand their associated regulation. If successful we will provide novel and important information on how macrophages respond to pathogens. Furthermore, as signalling pathways are highly conserved in evolution, we will advance knowledge at the fundamental level in the fields of cell biology and biochemistry as a whole.

We have discovered a molecule secreted by filarial nematodes (ES-62) that can induce an anti-inflammatory phenotype similar to that observed during natural infection with the worms. When investigating the mechanism of action of ES-62, it was observed that it modulated signal transduction pathways that were involved in the response to infection of various cells of the immune system. Although of importance to the immunology of filariasis, it quickly became apparent that by determining the molecular targets of ES-62 in the cell, it was also possible to obtain information of relevance to understanding signal transduction per se. Furthermore when specifically investigating control of the inflammatory cytokine response of macrophages to pathogen products, it was possible to find small molecule analogues (SMAs) of ES-62 that were capable of selective inhibition of differential cytokine production thereby offering the opportunity to investigate regulation of production at the level of the individual cytokine. We thus plan to generate a library of SMAs that differentially modulate lL-12, TNFalpha, IL-6 and IL-10 secretion by macrophages in response to toll-like receptor (TLR) ligation by conserved pathogen molecular patterns. We will then define whether such SMAs differentially target Erk, Jnk and p38 MAPkinase and NF-kappaB signalling cassettes to exhibit their differential effects on cytokine secretion. Next, we intend to is to characterise novel targets of such SMAs by gene array and signalsome proteomic technology in order to identify specific regulatory elements in the differential signalling pathways controlling individual cytokine release. Finally, we aim to define whether SMAs act as TLR receptor agonists, antagonists or whether they enter cells and directly inhibit signalling activities. Overall, we will generate novel information on macrophage biology that will have implications not only for immunology but also for all aspects of cell biology and biochemistry.