Chemical Approaches To Modulating Complement-Mediated Diseases Abishek Iyer,†, ‡,§ Weijun Xu,‡, § Robert C. Reid,‡ David P. Fairlie†,‡,* †Centre for Inflammation and Disease Research, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, AUSTRALIA ‡ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, AUSTRALIA § Joint first authors *Correspondence to: Professor David P Fairlie, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. E: [email protected], T: +61-733462989. 1 Abstract Numerous diseases are driven by chronic inflammation, placing major burdens on our health systems. Controlling inflammation is an important preventative and therapeutic goal. Over forty ‘Complement’ proteins are produced in blood or on cell surfaces through activation of the Complement protein network by infection or injury. These proteins complement immune cells and antibodies to identify, tag, destroy and eliminate pathogens and infected or damaged cells, and repair tissues. If the inflammatory stimulus is not removed by localized acute immune responses, Complement activation may be prolonged or misdirected to healthy cells, and chronic inflammation can lead to inflammatory or auto-immune diseases. The formation, structures and interplay between Complement proteins are complex and this has limited our detailed understanding of their roles and importance in physiology and disease. With the availability of new structures for Complement proteins, new knowledge of how they function, and new modulators of Complement-driven signaling, there are also new opportunities to intervene in Complement-mediated disease. Small molecule and peptide- based drug-leads, identified as clues for Complement-directed therapeutic development, are assembled here together with the available evidence for their efficacy in cellular and animal models of human inflammatory disease, and in some human clinical conditions. Word count: 195 words Keywords: protease, inhibitor, antagonist, agonist, inflammation. 2 Graphical Abstract 3 List of Figures and Tables Figure 1. Simplified overview of the Complement system. Figure 2: Schematic showing protein structures involved in the amplification loop of the alternative pathway mediated by C3b formed at the membrane via all pathways. Figure 3: Therapeutic intervention in the Complement system. Table 1. Structures of key Complement proteins. Table 2. Pathological role for key Complement proteins in inflammatory, autoimmune and rare diseases. Table 3. Knockout mouse phenotype for key Complement proteins in acute and chronic inflammatory and autoimmune diseases. Table 4. Genome-Wide Association Studies (GWAS) implicate roles for key Complement proteins. 4 1. INTRODUCTION Inflammation is produced through a wide variety of physiological and pathological processes and is crucial for the survival of an organism.1, 2 It is an important defense mechanism involving a complex set of interactions between soluble factors and immune cells that can arise in any tissue in response to traumatic, infectious, post-ischemic, toxic or autoimmune injury.3 The immune system orchestrates recognition and tagging of foreign surfaces, recruits immune cells to sites of infection or injury, and mounts protective inflammatory responses designed to destroy or remove the inflammatory stimulus and damaged cells.1, 3 However, when localized acute responses by immune cells are not resolved, the inflammation can continue unabated and may lead to a chronic inflammatory disease.2 Various soluble and cell- surface proteins linked by a network of complex proteolytic activation cascades are essential for mounting an immune response and have largely evolved with structural and functional conservation.1, 3 The ‘Complement system’ is an ancient and conserved protein network activated through proteolytic cascades by serine proteases in a highly coordinated and controlled fashion.4, 5 The Complement system is a vital part of host survival and defense that is even found in invertebrate organisms that are incapable of mounting an adaptive immune response.6, 7 It is thought to have played crucial roles in innate immunity even before the evolution of the adaptive immune system in jawed vertebrates.8 In humans, Complement was first described in the 1890s as a heat-labile component of normal plasma that ‘complements’ antibacterial activity of antibodies.9, 10 We now know that the Complement system comprises over 40 proteins, expressed in blood or on surfaces of immune and other cells, which together contribute to both innate and adaptive immune responses in humans.11-14 Originally, it was thought that the liver was the only site where serum Complement proteins were synthesized 5 and that they were then released into the circulation.6 We now know that other important cell types, including mast cells, eosinophils/basophils, dendritic cells, monocytes, macrophages, T and B lymphocytes, epithelial cells, fibroblasts, neuronal cells, adipocytes and endothelial cells can locally produce Complement proteins that play roles in organ/tissue surveillance.6 There is also emerging evidence that complement activation may not be restricted to the extracellular space and can also occur intracellularly to play important roles in both physiology and pathophysiology.15, 16 Compounds that block Complement activation can attenuate innate immune responses, rapidly reducing inflammation and eradicating sources of infection, but also it can attenuate adaptive immune responses to foreign and tissue antigens.5, 7 Although specific mechanisms vary, prolonged Complement activation can cause or exacerbate many diseases with an infectious or inflammatory etiology. Furthermore, our understanding of the roles of Complement has extended far beyond fighting infection, and now encompasses maintenance of homeostasis, tissue regeneration, developmental biology, and pathophysiology of multiple human diseases.5, 7 Nonetheless, the complexity of the proteolytic cascade, protein structures and interplay between Complement proteins have limited our understanding of their roles and significance in physiology and disease. Recent advances in biochemistry, protein crystallography and pharmacology have provided important new insights to structures and functions of many Complement proteins, providing a better understanding of how Complement is involved in disrupting or restoring homeostasis and in clearance of metabolic, apoptotic and oxidative waste products. These advances are now providing new opportunities for medicinal chemists to rationally design and develop novel drugs that can modulate Complement-mediated human diseases. 6 This review highlights key chemical approaches used to control Complement activation using small molecules and polypeptides as important clues to novel Complement-directed drug development. Evidence is assembled for therapeutic efficacy in human cells, innate and adaptive immune cells, animal models of human inflammatory diseases, and some human clinical conditions. This Complement-based chemical biology information can help to catalyze the development of novel drugs for Complement activation in human diseases. 2. COMPLEMENT PROTEINS AND ACTIVATION PATHWAYS The Complement system is activated by a diverse array of stimuli, from infectious organisms, antigen-antibody complexes and carbohydrate-binding lectins to microbial and foreign surfaces, chemical or physical injury, radiation or neoplasia.4, 8, 17, 18 These stimuli catalyze a complex, multi-pathway, cascading series of protein cleavages by serine proteases, themselves generated by Complement activation. Complement activation leads to a diverse family of proteins that effect opsonisation, leukocyte recruitment and activation, and assemble into a protein conglomerate known as the pore forming membrane attack complex (MAC). MAC formation in organisms and infected or damaged cells enables cell lysis leading to death and elimination of debris.4, 8, 17, 18 The purpose of Complement activation is to protect the host but, if not appropriately regulated, it can also lead to deleterious effects. Complement system consists of four separate pathways – the classical, lectin, alternative and extrinsic proteolytic pathways – producing a common event, the cleavage of C3 to C3a and C3b, the latter perpetuating an immune response to fight infection or host injury (Fig. 1). The classical pathway is activated by antigen-antibody complexes. Complement component 1q (C1q), in association with the serine proteases Complement component 1r (C1r) and 1s (C1s), forms an initiator complex that is activated after binding predominantly to IgG and 7 IgM, arranged in antigen-antibody (Ag/Ab) complexes.4, 8, 17, 18 More recently, the classical pathway has been shown also to be activated, independent of Ag/Ab complexes, by binding to other triggers such as C-reactive protein, polyanions, bacterial lipopolysaccharides, viral proteins and pneumolysin.19 Complement component 2 (C2), bound to Complement component 4 (C4), is cleaved by the classical pathway initiator complex protease C1s to produce C4bC2a, itself a very short-lived C3 convertase that in turn cleaves C3 to C3a and C3b.4, 8, 17, 18 Classical Pathway Alternative Pathway Ag/Ab Complexes Bacteria, Foreign Surfaces C4 C1q/C1r-C1s B C3b C4a C2 C3 C4b2a C3bBb D C4b C3bB C4a Ba C2b C3a C4 C3b C4b2b Complex Bacterial, Coagulation Polysaccharides C4b2a3b Proteases