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 Lectin Pathway Extrinsic Pathway C5 C5a Lysis C5b6789 C5b MAC C9 C8 C7 C6 Pathway Terminal

Figure 1. Simplified overview of the Complement system.

8 The lectin pathway is similar to the classical pathway, differing only in the initiator protein complex and the triggers.4, 8, 17, 18 It becomes activated in response to complex pattern recognition molecules, such as host secreted damage associated molecules and those present on surfaces of invading microorganisms.4, 8, 17, 18 So far, various molecules such as mannose- binding lectin, ficolins (M-, L-, H-) and collectin 11 have been identified to initiate the lectin pathway.20 The lectin pathway initiator complex consists of the specific recognition molecule and Mannan-Associated Serine Proteases (MASPs) that initiate the cleavage of C2-C4b complex producing the convertase C4bC2a and C3, which is further processed into C3a and

C3b (Fig. 1).

The alternative pathway is activated by microbial and foreign surfaces and is independent of antibodies or specific recognition molecules that trigger classical and lectin pathways. 4, 8, 17,

18 Slow spontaneous hydrolysis of C3 to C3(H2O) is thought to maintain cycling (‘tick over’) of the alternative pathway. Homoeostatic regulator molecules such as decay acceleration

(DAF), properdin and Factor H tightly control aberrant activation of C3 and ensure the alternative pathway is directed towards pathogen or damage molecules and does not damage healthy host tissue. However, when C3(H2O) binds to Factor B, it renders this complex susceptible to cleavage by Factor D, producing C3bBb sometimes referred to as

4, 8, 17, 18 C3(H2O)Bd. C3bBd behaves as a C3-convertase, cleaving and processing C3 to C3a and C3b (Fig. 2).

An amplification loop is a distinctive consequence of the production of C3b by all four pathways (Fig. 1). Regardless of the specific upstream activator, once C3b is generated in the right circumstances, then the amplification loop is engaged and contributes to enhancing the inflammatory response. Factors B and D are thus integrated components of the alternative

9 pathways. Upon production of C3b on a foreign surface or cell membrane (Fig. 2), it combines with Factor B that is processed through the alternative pathway by Factor D to

C3bBb, a second C3 convertase. This then also cleaves C3 near the membrane to C3a and

C3b (Fig. 2). C3b can thus substantially increase the inflammatory response via this amplification loop, but stepwise amplification is normally controlled tightly at all stages of the cascade.4, 8, 17, 18

Figure 2: Schematic showing protein structures (Table 1) involved in the amplification loop of the alternative pathway mediated by C3b at the membrane via all pathways.

In addition to these canonical pathways, C3 and C5 have also been reported to be cleaved by proteases from outside of these three pathways. For example, there is limited but unequivocal in vitro and ex vivo evidence to suggest that some proteases in the coagulation cascade, and others secreted from bacteria, can cleave C3 to C3a and C3b and/or C5 to C5a and C5b.19, 21

Coagulation proteases, such as thrombin, Factor IXa (FIXa), Factor Xa (FXa), Factor XIa

(FXIa) and plasmin, have all been reported to cleave Complement C3 and C5 to form

C3a/C3b and C5a/C5b independent of the three canonical Complement activation pathways,

10 albeit under unnaturally high conditions (Fig. 1).19, 21 Apart from C3 and C5 cleavage, an ex vivo study suggested that FXa can also continue complement activation to form the MAC and cell lysis.21 However, further in vivo validation of this extrinsic pathway leading to MAC formation in various physiological and disease settings is necessary.

The terminal stage of Complement activation begins after cleavage of C3 to C3a and C3b.

Human C3a is a 77-residue inflammatory anaphylatoxin protein, that degranulates immune cells and induces chemotaxis and spasmogenesis, after binding to the cell surface via a G- protein coupled receptor (GPCR) called C3aR.22, 23 On the other hand, C3b, is a key opsonin that coats bacterial surfaces, increasing recognition and destruction by phagocytes.4 C3b additionally is also part of a Complement component 5 (C5) convertase by binding to either

C4b and C2a (C4b2a3b complex) or to another C3b molecule (C3bBb3b complex). Their actions lead to the production of Complement protein 5a (C5a), another potent anaphylatoxin, chemotaxin and immunostimulant, that can bind to and activate two GPCRs, C5aR1 and

C5aR2.24 C3a and C5a are involved in recruiting immune cells to sites of infection, activating granulocytes through degranulation and stimulating other innate and adaptive immune cells.

C3b together with C5 continue the terminal stage of the Complement cascade by facilitating the assembly of Complement components C5b, C6, C7, C8, C9 to form the conglomerate membrane attack complex (MAC), which forms holes in invading microorganisms and infected or otherwise damaged cells.4 This terminal or effector stage leading to MAC is vital for Complement-mediated bactericidal activity and for destroying damaged cells. The complex interplay between all of these proteins has limited our understanding of their precise functional roles, but new protein crystal structures over the last decade in particular (Table 1) have brought fresh insights to protein-protein interactions and structure-function consequences.

11

Table 1. Structures of key Complement proteins

a Complement Target PDB code Resolution Ligand Reference

(Å)

Classical Pathway

25 C1q globular head 1PK6 1.85 -

26 2JG9 1.9 -

27 2WNV 1.25 deoxyribose

28 5HKJ 1.35 -

29 C1r zymogen catalytic domain 1GPZ 2.9 -

30 C1r active catalytic domain 1MD8 2.8 -

31 C1s 1ELV 1.7 -

32 C1s zymogen 4J1Y 2.66 -

33 C1s-CUB2-CCP1 4LOS 2.0 -

33 C1s-CUB2-CCP1-CCP2 4LOT 2.92 -

Lectin Pathway

34 L-Ficolin 2J3G 2.5 -

34 H-Ficolin 2J64 2.2 -

35 L-Ficolin 4NYT 2.25 Phosphocholine

36 L-Ficolin 4R9T 2.25 sulphates

37 MASP-1 3GOV 2.55 -

38 MASP-2 1ZJK 2.18 -

39 1Q3X 2.23 -

12 40 MASP-3 4IW4 3.2 ecotin

41 MASP-3 4KKD 2.6

Classical/Lectin Pathways

42 C2a 2I6Q 2.1 -

43 2ODP 1.9 -

44 C2b 3ERB 1.8 -

45 C4 with MASP-2 5JPM 3.75 -

46 C4b 4XAM 4.2 -

Alternative Pathway

47 Factor B 2OK5 2.3 -

48 Factor B-cobra venom factor 3HRZ 2.2 - complex

49 Factor D 1DFP 2.4 diisopropyl

fluorophosphate

50 Factor D 1DIC 1.8 dichlorocoumarin

51 Factor D- ligand 5FCK 1.86 Compound 5

52 Factor D 5TCA 3.15 JH3

Central Component and/or Extrinsic Pathway

53 C3 2A73 3.3 -

54 Bovine C3 2B39 3.0 -

55 C3b 2I07 4.0 -

56 C3c-compstatin 2QKI 2.4 -

57 C3b in complex with CRIG 2ICF 4.1 -

58 C3b in complex with Factor B 2XWJ 4.0 -

13 58 C3b in complex with Factor B 2XWB 3.49 - and D

59 C3bB EMB-1583 28 -

60 C3b in complex with factor I 5O32 4.21 - and regulatory factor H

61 C3 convertase with properdin 5M6W 6.0 -

62 C3 convertase with scin 2WIN 3.9 -

Complement Proteins and Receptors

63 C3a 4HW5 2.25 -

64 4I6O 2.14 -

65 C5a 1C5A NA -

66 CR1 1GKN NA -

67 CR2-C3D complex 1GHQ 2.04 -

68 3OED 3.16 -

57 CRIg bound to C3b 2ICF 4.1 -

Terminal Pathway

69 C5b-6 4E0S 4.21 -

70 C5b-6 4A5W 3.5 -

71 C6 3T5O 2.87 -

72 C8 3OJY 2.51 -

73 poly-C9 5FMW 6.7 -

b 74 MAC EMD-3135 8.5 - a Ligand name as in PDB. b Electron microscopy databank code

14 3. COMPLEMENT ACTIVATION IN DISEASE

Complement activation is important for immune defense, resolving immuno-compromised states, and maintaining physiological homeostasis. However, even small disruptions to this tightly regulated proteolytic cascade can result in protracted Complement activation, leading to prolonged inflammation and, eventually, inflammatory and autoimmune diseases.5, 7 There is increasing appreciation that Complement dysregulation lies at the heart of numerous immune-mediated and inflammatory disorders and that these detrimental actions become more pronounced with age and are exacerbated by a variety of genetic factors and autoimmune responses.4-7 The catalogue of chronic human diseases in which Complement has an important pathological role, either primary or secondary to other triggers, is long and expanding (Table 2). Complement activation is associated with the development of many autoimmune, inflammatory, respiratory, gastrointestinal, metabolic, cardiovascular, hematological and neurodegenerative diseases, as well as many cancers, ischemia/reperfusion

(I/R) injuries and sepsis.4-7, 75 Further, inherited deficiencies in some Complement proteins predispose individuals to bacterial infections and/or immune complex mediated autoimmune diseases.4-7 These deficiencies, such as gene mutations or polymorphisms in Complement proteins, regulators or receptors, are relatively rare but play direct roles in inducing dysregulation and disease.4-7 In most other disease conditions listed above, Complement is not the primary cause of disease induction, but is activated in response to tissue damage and serves to amplify and exacerbate inflammatory and other signaling responses to infection and injury.4-7 Nonetheless, the exact involvement of Complement still needs to be carefully investigated for each disease condition, as therapeutic modulation of Complement activity emerges as an attractive target for upstream inhibition of these pathological inflammatory processes.7 Knockout mouse phenotypes for key Complement proteins provide important

15 clues for potential pathological roles in many human diseases (Table 3). On the other hand, the Complement system is also important for the clearance of metabolic, apoptotic and oxidative waste products and so inhibiting Complement could potentially also disrupt homeostasis.4 The availability of potent, selective, and bioavailable small molecule

Complement probes and therapeutics to study responses, especially in vivo, will greatly increase our understanding of the roles of Complement in various human inflammatory disease states, while also assessing the viability of the Complement system as a drug target in humans.

Table 2. Pathological role for key Complement proteins in inflammatory, autoimmune and rare diseases.

Disease conditions Complement Proteins Reference

Complement Proteins in Rare Human Diseases

76, 77 Paroxysmal Nocturnal DAF, CD59, C3, C5, MAC

Hemoglobinuria (PNH)

4, 78-80 Atypical Haemolytic Uremic Alternative pathway, Factor H

Syndrome (aHUS)

81 Neuromyelitis Optica C5

82 Hereditary Angioedema C1, C1-INH

Complement Proteins in Animal Models of Inflammatory Diseases

83-86 Age-related Macular Alternative pathway, C3a, C5a,

Degeneration (AMD) MAC

87 Asthma C3, C3a, C5a, C3aR, C5aR1,

16 C5aR2

88-91 Systemic Lupus Alternative pathway, Factor D,

Erythematosus (SLE) C3, CR2, C3d

92-95 Rheumatoid Arthritis (RA) Alternative pathway, C5aR1,

C3aR, MAC, CR2, C3d

96 Neurodegenerative Disorders C1q, C3

97 Alzheimer’s Disease C5a, C5aR1

98 Amyotrophic Lateral C1q, C3, C4, C3d, C4d, C5a,

Sclerosis (ALS) C5aR1

98 Huntington’s and Parkinson’s C3, C4, C7, C9, Factor H, C1q,

Diseases C5aR1, C3, C5

98 Stroke C3, CR2, Factor H, MBL, C3aR,

C5aR1, C5

99 Type 1 Diabetes Mellitus CRIg

100 Obesity and Metabolic C3a, C3aR, C5a, C5aR1

Diseases

101 Sepsis and Acute C3

Inflammatory Disorders

102, 103 Cancers CR2, C5a, C5aR1

104, 105 I/R and Transplantation C3adesArg, C5a, C5aR1

Related Diseases

106 Periodontal Disease C3, C5a, C5aR1

107 Kidney Diseases MBLs, Alternative pathway, C3,

Factor D, C5

17

Table 3. Knockout mouse phenotype for key Complement proteins in acute and chronic inflammatory and autoimmune diseases.

Complement protein Disease (knockout phenotype) Reference

Classical Pathway

108-112 C1q Potentiates SLE, glomerulonephritis,

nephritis, decreases phagocytic

clearance; induces epilepsy

Lectin Pathway

113 MASP1/3 Prevents thrombus formation

114-117 MASP2 Prevents cerebral, myocardial, renal

gastrointestinal ischemia/reperfusion

injury; Improves pneumococcal

meningitis outcomes

Classical/Lectin Pathways

118 C4 Potentiates SLE, glomerulonephritis

and splenomegaly

Alternative Pathway

92 Factor B Prevents antibody induced arthritis;

prevents SLE, glomerulonephritis and

vasculitis

89, 119 Factor D Prevents proliferative kidney disease;

Neuroprotective in light-induced

photoreceptor degeneration.

18 Central Component and Extrinsic Pathway

8, 17 C3 Prevents various autoimmune,

neurodegenerative diseases and many

acute and chronic inflammatory

diseases

Complement Proteins and Receptors

120 CRIg Prevents phagocytosis, increases

infection and mortality

121, 122 CR1/CR2 Increases autoantibody production and

SLE symptoms

123, 124 CR3 Prevents clearance of opsonized

immune complexes in liver; prevents

severe Ross River Virus-induced

disease

8, 17 C3aR Prevents various autoimmune,

neurodegenerative diseases and many

acute and chronic inflammatory

diseases

8, 125 17 C5aR1 Prevents various autoimmune,

neurodegenerative diseases and many

acute and chronic inflammatory

diseases

19 Targeting Complement with potential therapeutic agents in vivo in preclinical models of human inflammatory diseases has been explored for decades.4-7 Despite this long history, relatively few compounds have entered Phase I clinical trials in healthy people and fewer still have progressed in clinical trials for the many inflammatory and age-related Complement mediated diseases.4-7 The complex multifaceted nature of the Complement system, both its activation cascade and its involvement in immune defense and disease, has been a barrier to rational development of Complement-directed therapeutics. More recently, evidence from many human studies, including biomarker and pathological findings, strong genetic evidence from genome-wide association studies (GWAS) and gene linkage studies, and successful use of a few Complement therapeutics in humans (Table 4),4-7 has culminated in renewed interest from academia and industry in developing novel Complement directed therapeutics. Rational drug design and development will require a more detailed understanding of the Complement system in different diseases and the validation of optimal therapeutic intervention points.

Ahead we summarize the diverse Complement protein targets, their protein structures and mechanisms of drug action, current small molecules and some peptide-based leads for each target and the available evidence for their therapeutic efficacy in cells, animals and humans.

Table 4. Genome-Wide Association Studies (GWAS) implicate roles for key

Complement proteins.

GWAS Implicated Disease/Phenotype Reference

Complement Gene/Protein

Classical/Lectin Pathways

126 C4 Increased risk for schizophrenia

Alternative Pathway

20 127, 128 Factor B Increased risk for age-related macular

degeneration (AMD)

129-132 Factor H Increased risk for AMD, dense deposit

disease (DDD), atypical haemolytic uremic

syndrome (aHUS)

Central Component and

Extrinsic Pathway

133 C3 Increased risk for AMD, DDD, aHUS

Complement Proteins and

Receptors

134 CR1 Increased risk for Alzheimer’s disease

Terminal Pathway

135-137 C5 Increased risk for rheumatoid arthritis, coeliac

disease, renal allograft survival

138 Carboxpeptidase B Decreased risk of rheumatoid arthritis

139 C9 Reduced risk for AMD

4. COMPLEMENT PROTEINS AS TARGETS

A number of promising small molecule or peptide-based chemical probes for inhibition or activation of Complement proteins have been developed and are presented in the following sections. We have cataloged representative experimental Complement therapeutics, which have been either in clinical trials or preclinical development, as important clues for design and development of Complement-based medicines. Complement can be targeted at the following intervention points in the Complement system (Fig. 3): (a) the binding and

21 assembly of the initiating complex (eg. C1q); (b) the enzymes of the initiating complex (eg.

C1r, C1s, MASPs); (c) the central Complement component (eg. C3); (d) proteases involved in convertase formation (eg. Factor B, Factor D, C2); (e) peptide anaphylatoxins and their receptors (eg. C3a, C3aR, C5a, C5aR); and (f) proteins of the terminal pathway (eg. C5, C6,

C9, MAC).

Figure 3: Therapeutic intervention in the Complement system.

4.1 Complement Component 1q (C1q)

C1q is the pattern recognition molecule of the classical pathway and, in association with serine proteases C1r and C1s, forms the initiator complex for the Complement cascade.140

22 Therefore, therapeutics that target C1q could be attractive in inhibiting activation of the classical pathway. C1q has long been known to play an important role in the clearance of pathogens, apoptotic cells and subsequent B cell tolerance.140 C1q is capable of directly identifying various substances on microbial surfaces or apoptotic cells, and indirectly via antibodies and C-reactive protein that trigger activation of the Complement system.140 C1q is a 460 kDa hexameric glycoprotein composed of 18 polypeptide chains.140 The crystal structure at 1.9Å resolution of the globular gC1q domain that is responsible for its recognition properties reveals an almost spherical (50Å diameter), dense heterotrimer associated by non-polar forces with Ca2+ bound at the top, and a classical jellyroll topology

(Table 1). C1q can bind to IgG, IgM, HIV-1, phosphatidylserine (PS), HTLV-1, CRP, and many other ligands via this gC1q domain that usually involves recognition of charged patterns or clusters.140 However, due to the complex nature of this recognition feature of gC1q, very little progress has been reported in developing small molecule inhibitors for this protein target. To date, only three small molecules, all peptides, have been reported as C1q inhibitors.

Compound 1 (known as 2J) is a 15-mer cyclic peptide that was discovered as a moderately potent and selective C1q inhibitor from a selection of 42 peptides, previously identified from phage-displayed peptide libraries that bind to C1q.141 In a C1q-dependent hemolytic assay,

o compound 1 was shown to selectively inhibit C1q (IC50 ~2 µM, pH 9.6, 37 C, rabbit erythrocytes), without affecting the alternative pathway function.141 Further, using blocking antibodies that specifically target the different domains of C1q, it was established that 1 binds to the globular head domain of C1q, thereby preventing IgG binding and subsequent activation of C4 and C3 and formation of C5b-9 via the classical pathway.141 Interestingly, compared to cyclic peptide 1, linear analogues did not exhibit any C1q inhibition up to 100

23 µM concentrations,141 suggesting that conformation might be important for binding.

Compound 1 also inhibited Complement protein C4 and C3 deposition on porcine cells exposed to human serum, suggesting possible efficacy in xenotransplantation applications.141

Furthermore, although 1 was selected from a phage display library on the basis of binding to human C1q, 1 displayed similar inhibitory potency (IC50 ~2-6 µM) in a hemolytic assay against C1q from other species such as chimpanzee, rhesus monkey, rat and mouse.141 This comparable potency suggests that 1 binds to a specific region on C1q that has a high degree of conservation or similarity among species, a feature that potentially could be utilized to investigate in vivo preclinical properties of this or derived molecules.141 Although this compound was reported in 2001,141 it does not appear to have been further developed into more effective inhibitors.

1

A 15-residue acyclic peptide, compound 2 (IALILEPICCQERAA), was reported to be a potent inhibitor of Complement C1q by binding to its collagen-like region.142 Compound 2 is a rationally designed C1q inhibitor based on clues from previously identified binding modes of the recombinant human astrovirus coat protein (CP), the human neutrophil defensin 1 and

24 a 30 residue peptide (CP) derived from this coat protein that binds to C1q.142 Compound 2 inhibited Complement as assessed by serum C4 suppression and inhibition of hemolytic activity in NHS and Factor B depleted serum.142 Using SPR studies, compound 2 was shown

142 to bind to purified C1q (Kd 33 nM, 22°C). In an in vitro model of Complement-mediated disease (ABO incompatibility), 2 dose-dependently inhibited lysis of AB erythrocytes treated with mismatched human O serum.143 Importantly, when administered in vivo to rats, 2 prevented rat serum from lysing antibody-sensitized erythrocytes.143 This in vivo

Complement suppression with 2 lasted up to 24 h post-injection.143 Further, in an attempt to improve the solubility, this peptide was synthetically modified by appending a PEG linker consisting of 24 PEG units on either the N terminus (dPEG24-2), C terminus (2-dPEG24) or both the N and C termini of 2 (dPEG24-2-dPEG24).143 Among these, the C terminus modified compound, 2-dPEG24, not only inhibited Complement activity in Factor B-depleted human serum, but also displayed inhibitory activity in serum from rats, mice and non-human primates, important for use in preclinical studies.144 Importantly, after intravenous injection

(20 mg/mL dose) in rats, 2-dPEG24 inhibited Complement activation in the blood by 90% after 30 seconds with a functional half-life of 4 h, demonstrating very rapid activity in vivo.144

No overt toxicity was observed in these rats up to 48 h after administration.144 No further pharmacokinetic, pharmacodynamics or toxicity studies for 2-dPEG24 have been reported.

A 1988 patent (WO1988007054-A1) reports several small peptides derived by truncation of the amino acid sequence of the IgG CH2 domain known to recognize and bind to C1q. The binding motif comprised residues E318, K320 and K322, the preferred sequence for the invention being compound 3 (Ac-AEAKAKA-NH2). This peptide was able to inhibit guinea

o pig Complement mediated lysis of sheep red blood cells (IC50 166 µM, 37 C).

25 4.2 Complement Component 1r (C1r) and 1s (C1s)

C1r and C1s are serine proteases that are part of the classical pathway initiator complex.

They are essential for Complement activation via the classical pathway.145, 146 Both C1r and

C1s have a six-domain structure and assemble together into a Ca2+-dependent C1s-C1r-C1r-

C1s tetramer.145, 146 These proteases bind to the classical pathway recognition molecule C1q, together referred to as the C1 complex. This assembled complex cleaves both C2 and C4 to propagate Complement activation.147 Targeting these proteases is attractive, as inhibitors should switch off classical pathway activation at the very first stage. The C-terminal catalytic region of both C1r and C1s consists of two Complement control protein (CCP) modules and a trypsin-like serine protease domain.145, 147 This C1 complex circulates preassembled in the blood,7 so an inhibitor that targets the protease domain could be effective in blocking

Complement activation. A few small molecule inhibitors of these proteases are known and are described below.

Compound 4 (FUT-175, Nafamostat, Futhan) is a synthetic inhibitor of serine proteases,

o 148 o 148 including C1r (IC50 800 nM, pH 7.4, 37 C) and C1s (IC50 29 nM, pH 7.4, 37 C) , and is currently approved for use in human pancreatitis and disseminated intravascular coagulation.149 Compound 4 inhibited Complement activation and ameliorated hyperacute rejection in a guinea pig to rat ex vivo xenogeneic lung perfusion model.149 Further, administration of 4 prior to reperfusion effectively attenuated injury to the myocardium in a rabbit model of IR injury.150 In a more recent study, compound 4 was administered in vivo in a rat model of stroke, reducing infarct size, improving behavioral functions, decreasing expression of proinflammatory mediators, together with preventing infiltration of macrophages, neutrophils and T lymphocytes.151, 152 However, 4 is well known to have broad-

26 spectrum inhibitor activity for multiple serine proteases including Factor B, Factor D, kallikrein, trypsin and other coagulation enzymes.153 Hence, it is not clear whether the therapeutic responses observed in vivo in these studies were a direct consequence of

Complement inhibition or if they were mediated through broader inhibition of multiple inflammatory proteases.

4

Another low molecular weight compound, 5 (BCX-1470, BioCryst Pharmaceuticals), has been reported as a potent C1s inhibitor in an in vitro esterolytic activity assay (IC50 1.6 nM,

0.1 mM HEPES), measured against the human C1s enzyme.154 Although potent, 5 may lack selectivity. In the same esterolytic activity assay, 5 inhibited the activity of other proteases, such as Factor D (IC50 96 nM, 0.1 mM HEPES) and trypsin (IC50 326 nM, 0.1 mM

HEPES).154 However, in an in vitro hemolytic assay, 5 inhibited activation of the classical pathway (IC50 ~46 nM, pH 7.3, rabbit Ab-sensitized sheep erythrocytes) more effectively (7- fold) than the alternative pathway (IC50 ~330 nM, pH 7.3, rabbit Ab-sensitized sheep erythrocytes).154 Furthermore, when administered intravenously (i.v.) as either a single dose or a 1 h infusion, 5 significantly reduced edema and serum hemolytic activity in vivo in a rat model of reverse passive Arthus reaction.154 Lastly, a safety evaluation has been performed for compound 5 in a phase I clinical trial involving healthy volunteers,155, 156 but the clinical outcomes have not been released.

27 5

More recently, other attempts have been made to develop novel, potent and selective small molecular C1s inhibitors.157, 158 A series of C1s inhibitors based on a benzimidazole scaffold led to the synthesis of arylsulfonylthiophene-2-carboxamidine derivatives.157 Among these, the most potent compound 6 displayed Ki 10 nM against C1s in in vitro assays.157 Further, compound 6 was reported to be very selective for C1s over other proteases, such as urokinase-type plasminogen activator, tissue plasminogen activator, Factor Xa, thrombin, and plasmin.157 However, there were some concerns regarding potential in vivo toxicity for 6.147

Nonetheless, a series of peggylated biphenyl sulfonyl thiophene derivatives have demonstrated potent and selective C1s inhibition together with a good pharmacokinetic profile in vivo.159

6

Peptidomimetic inhibitors of C1r and C1s have also been developed based on known thrombin inhibitors. The original BASF patent (WO200061608-A2/US6683055-B1) described over 800 compounds, but provided C1r/C1s enzyme inhibition data for only 50 examples. The most potent compound inhibited C1r and C1s with similar inhibitory activity

(IC50 0.6 and 0.9 µM respectively, pH 7.5, 25 °C), with compound 7 described as a preferred

28 and active Complement inhibitor although no data was provided. Analogues such as 8, where reductive alkylation of the N-terminal amino acid with L-rhamnose or other carbohydrates, were later claimed to make these compounds active when administered orally to rats and dogs (US2004048815-A1).

7 8

Subsequently, a low molecular weight (MW 520.5) candidate compound 9 (C1s-INH-248,

BASF Pharma, structure not disclosed) was reported as a derivative of a known thrombin inhibitor (D-Phe-Pro-Arg).160 Compound 9 displayed potent inhibitory activity against both

160 human (IC50 2 nM) and rabbit (IC50 0.7 nM) C1s enzymes in vitro. Furthermore, 9 was reported to be 1000-fold more selective for C1s than for C1r, MASP-1 and thrombin160.

Compound 9 lacked any in vitro inhibitory effect against other serine proteases, such as kallikrein, Factors XIa and XIIa, when examined up to 10 µM concentration.160 In an erythrocyte hemolytic assay, 9 was a potent inhibitor (IC50 0.19 nM, sheep red blood cell) of rabbit serum induced SRBC hemolysis and Complement activation.160 Intravenous administration of compound 9 just before reperfusion dose-dependently (0.1, 0.5, 1 mg/kg body weight) attenuated myocardial injury and inflammation in a rabbit model of ischemia- reperfusion injury.160 However, 9 does not appear to be in further development.

Furan, pyrrole and thiophene amidines containing a thiazole ring linker have been disclosed

29 (WO2000047194A2) as inhibitors of C1s (e.g. 10, Ki 30 nM, pH 7.5, 37oC). Structurally simpler and more potent thiophene amidines were subsequently disclosed (WO2003099805-

A1) by the same group, the most potent inhibitor being 11 (Ki 6 nM). Furthermore, 44 racemic or homochiral thiophene sulfoximines were reported (WO2006101860-A1) as inhibitors of Cls, exemplified by 12 (Ki 11 nM, pH 7.5, 37oC), which also inhibited MASP-2

(Ki 440 nM, pH 7.5, 37oC). Most of these compounds were also found to inhibit thrombin.

The compounds were assayed for cleavage of substrate Z-Gly-Arg-S-Bzl by purified C1s

(Calbiochem) with detection of released thiol by DTNB. Compounds were claimed to be stable, but no in vivo data were reported.

10 11 12

4.3 Mannan-Associated Serine Proteases (MASPs)

Similar to the classical pathway proteases, MASPs are trypsin-like serine proteases that regulate Complement activation via the lectin pathway.161 Recognition of pathogen or related molecules by MBL or ficolins results in activation of MASPs. There are three known MASP isoforms, MASP-1, MASP-2 and MASP-3.161 MASP-2 is the key protease that cleaves C4 and C2 to enable assembly of the C3 convertase, C4b2a, which continues the activation of the

Complement system.161 Until recently, the physiological roles for MASP-1 and MASP-3 were unknown.20, 161 Selective mouse knockout studies suggest that MASP-1 and MASP-3 may be important in activating both the lectin and alternative pathways of the Complement

30 system. Both these proteases seem to be involved in converting Factor D from a proenzyme to a catalytic form.161 Further, until recently MASP-2 was thought to autoactivate itself to initiate the proteolytic cascade.20, 162, 163 However, in normal human serum, MASP-1 directly activates MASP-2 to produce the bulk of C2a that is necessary to drive C3 cleavage.20

Recently, a MASP-2 targeting antibody was awarded US Food and Drug Administration

(FDA) Orphan Drug status for use in aHUS and other thrombotic angiopathies and it is in

Phase II/III trials (NCT02222545).7 Thus, the development of potent and selective chemical inhibitors of MASP proteases could potentially interfere with the Complement cascade at multiple levels and inhibit various Complement-mediated human diseases.

Some broad-spectrum small molecule nonpeptidic serine protease inhibitors, such as compound 4, have been shown to non-specifically inhibit MASP enzymatic activity.149 No

MASP-specific small molecule nonpeptidic inhibitors have been reported. A few peptide inhibitors have been characterized and provide clues for developing potent and selective

MASP inhibitors.164 Two potent 14-residue peptidic inhibitors, compounds 13 (sunflower

MBL-associated SP inhibitor (SFMI-1, GICSRSLPPICIPD) and 14 (SFMI-2,

GYCSRSYPPVCIPD), were initially developed by phage display against MASP-1 and

MASP-2.164 In in vitro assays, compound 13 inhibits both MASP-1 (Ki 65 nM, pH 7.6) and

MASP-2 (Ki 1030 nM, pH 7.6) enzymatic activities.164 Compound 14 displayed some selectivity in inhibiting only MASP-2 (Ki 180 nM). In the Wieslab COMPL 300 (WiELISA kit) in vitro assay, both inhibitors selectively blocked the lectin pathway in human blood samples, without affecting either the classical or alternative pathways.164 Further, both peptides potently inhibited serum C4 deposition (13: IC50 0.23 µM, 14: IC50 2.7 µM) and C3

164 deposition (13: IC50 0.040 µM, 14: IC50 0.22 µM). More recently, the same group reported more potent peptidic inhibitors, 15 (SGMI-1), a 34 residue peptide

31 VTCEPGTTFKDKCNTCRCGSDGKSAFCTRKLCYQ (where the sequence that spans P4-

P4’ is underlined) that is specific for MASP-1; and 16 (SGMI-2), a 36 residue peptide

EVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ (where the sequence that spans

P4-P4’ is underlined) that is specific for MASP-2.165 This study also reports the first

Michaelis-like complex crystal structures of MASP-1 and MASP-2 in complex with compounds 15 and 16, respectively.165 Compound 15 (Ki 7 nM, MASP-1, pH 7.6) and 16

(MASP-2; Ki 6 nM, pH 7.6) seem to be highly selective for MASP-1 and MASP-2 respectively, with no inhibitory effect on the closely related MASP-3 protease or other coagulation proteases.166 These compounds completely suppressed the lectin-pathway at 1µM

153 concentration in the in vitro WiELISA assay (15, IC50 42 nM; 16, IC50 66 nM), while leaving the classical and alternative pathways unaffected.165, 166 Compounds 15 and 16 also effectively inhibited MASP-1 and lectin pathway activation in the C3 deposition assay, irrespective of whether the recognition complexes contained MBL or ficolins.166 No in vivo animal studies have been reported for these MASP inhibitors.

The molecular mechanisms by which proteases, involved in the activation of the classical and lectin pathways, interact with their protein substrates are significantly affected by the specificity of the active site of the enzymes.167-169 Recent insights have been gained into the manner in which proteases of the classical32, 170 and lectin171-173 pathways interact with their primary substrate C4 to effect cleavage and activation. Novel exosites on the surface of the proteases that bind to the substrate have been identified,171-173 and these may represent target sites for therapeutic intervention in these pathways.

4.4 Complement Component 2 (C2)

32 C2 plays an important role in Complement activation via both the classical and lectin pathways.174 When C2 binds to C4b, it is cleaved by either classical (C1s) or lectin (MASP2) pathway proteases to produce C4bC2a, a very short-lived C3 convertase (t1/2 ~2 min at 37°C) that in turn cleaves C3 to C3a and C3b.174 C2 has some structural homology (39%) with

Factor B, each consisting of N-terminal triplet repeat Complement control protein modules

(CCP 1−3), a von-Willebrand Factor type A (vWFA) domain (possible binding site for C4b), and a C-terminal serine-protease (SP) domain.174 C2 was thought to be an inactive zymogen that possesses the same serine protease domain as C4bC2a, requiring a cofactor-induced conformational change for activity.174 This complexity has discouraged studies on developing inhibitors for C2.174

C2 was found to have appreciable enzymatic activity and stability under alkaline conditions

(pH 7.5 to 10).174 This finding led to the discovery of the first small molecule C2 inhibitor in

174 vitro, the simple heptapeptide aldehyde 17 (Ac-SHLGLAR-H). Compound 17 (IC50 ~4.2

µM, pH 9.5) was derived from short peptide substrates (each modified as a para-nitroaniline, pNA) optimized for cleavage by C2.174 A hexapeptide aldehyde 18 (Ac-RLLLAR-H) with an arginine at position 1 increased inhibitory potency against C2 by 12-fold.174 In the same C2 enzyme activity assay, 18 was found to be much more potent, competitive, and also a

174 reversible inhibitor of C2 (IC50 ~330 nM, pH 9.5). Compound 18 potently (IC50 ~0.5-1

µM) inhibited processing of C3 by C2 in vitro.174 Compound 18 also efficiently inhibited C3 convertase activity, terminal MAC formation (IC50 ~7.3 µM), and the hemolysis of

174 sensitized sheep erythrocytes (IC50 ~28 µM). Although 17 and 18 were not reported to have been profiled in animal models of disease, this work was an important advance in understanding how to develop small molecule C2 inhibitors, although none are reported yet.

33

17

18

4.5 Complement Component 3 (C3)

Human C3 protein is now becoming recognized as a useful biomarker for a range of human chronic inflammatory and other diseases.8 From an evolutionary perspective, the C3 protein has been highly conserved, with its occurrence predating all jawed vertebrates.8 C3 is also an attractive target for therapeutic intervention due to its central position in the Complement network and its pivotal role in mediating MAC formation.4 C3 also regulates the amplification loop that can potentially magnify the outcome from any of the Complement initiation pathways leading to MAC formation, as well as controling the production of the peptide anaphylatoxins that can recruit immune cells to further amplify inflammatory responses.4

34

Various attempts have been made to make small molecule inhibitors that directly bind to C3, as distinct from blocking proteolytic events upstream of C3 or inhibiting proteases that cleave

C3. Compound 19 (Compstatin) is a 13-residue disulfide bonded cyclic peptide, cyclo-(2,12)-

175 I[CVVQDWGHHRC]T-NH2, derived from a peptide phage library. It inhibits binding of

o C3 to the convertase of the classical (IC50 63 µM, 37 C, hemolytic assay) and alternative

o 175 (IC50 12 µM, 37 C, hemolytic assay) pathways. Further, 19 bound to C3, C3b and C3c, but not to C3d, suggesting that it binds to the C3c domain of C3.176 Compound 19 showed efficacy in the treatment of age-related macular degeneration (AMD) in non-human primates and has entered clinical trials.177 An N-terminal acetylated analogue of 19 with amino acid substitutions at positions 4 and 9 (Ac-cyclo-(2,12)-I[CVWQDWGAHRC]T-NH2) was reported to be 45-fold more potent than 19.178 The crystal structure of 19 bound to C3c revealed56 the binding site, comprising the macroglobulin (MG) domains 4 and 5 of

C3. Based on this information, 19 was speculated to sterically prevent recognition of the substrate C3 by the convertase complexes, thereby blocking initiation and amplification steps of Complement activation.56 These structural insights have provided potential clues for further development.

Based on structural interactions in the crystal structure of the 19-C3c complex, further backbone N-methylation studies and exploration of extra binding sites were conducted.56, 179

These led to more potent C3 peptidic inhibitors with improved stability and pharmacokinetic properties.180 A series of analogues with tryptophan at position 4 of compound 19 led to a high binding affinity inhibitor 20 (also called 4(1MeW), POT-4, Potentia or AL-78898A) with Kd 15 nM and potent inhibitory activity (IC50 205 nM, pH 7.4, 22°C) against C3 in vitro.181 Compound 20 entered Phase II clinical trials for AMD supported by Alcon (a

35 division company of Novartis) (NCT01157065; NCT01603043). A re-formulated version of

20 (APL-2, Apellis Pharmaceuticals) with a longer half life is in a Phase II clinical trial for supplementing standard of care in paroxysmal nocturnal hemoglobinuria (PNH,

NCT02503332).7

N-methylation studies also led to 21 (Cp20) that displayed improved in vitro potency (IC50 62 nM, pH 7.4, 22°C) and binding affinity (Kd 2.3 nM, pH 7.4, 22°C) for C3.179 Later, it was shown that N-methylation at the N-terminus, by substituting the acetyl group with a sarcosine

(Sar) to give 22 (Cp30), enhanced binding affinity (Kd ~1.6 nM) for C3 and improved peptide solubility.182 The most recent analogue of 19, compound 23 (Cp40 or AMY-101) is a

14-residue cyclic peptide (D-YI[CV(1-MeW)QDWSarAHRC]-MeI) that binds to C3 and

C3b to selectively inhibit Complement activation in both non-human primates and humans.5,

183 Compound 23 showed efficacy in a variety of in vivo disease models, including PNH, hemodialysis-induced inflammation and periodontal disease.184-186 Compound 23 (Amyndas

Pharmaceuticals) gained approval from European Medicines Agency (EMA) and US FDA for Orphan Drug status for use in PNH and C3 glomerulopathy.5 It is under further development for human inflammatory conditions, including transplantation, age-related macular degeneration, hemodialysis and ischemia/reperfusion injuries (John D. Lambris, personal communication, November 2016).

36

19

20

37

21

22

38

23

4.6 Complement Factor B and Factor D

Human Complement proteins Factor B and Factor D are putative therapeutic targets since they are upstream of C3bBb (a C3 convertase enzyme) that drives the alternative pathway including the amplification loop.187, 188 Factor B is a serine protease that circulates in human serum as an inactive zymogen.187, 188 Factor D (or adipsin) is also a trypsin-like serine protease that cleaves this inactive zymogen into functional Factor B that processes C3 into

C3a and C3b.187, 188 Crystal structures are available for Factor B but only either in the inactive state or for the various fragments.187, 188 Small molecule and peptidic inhibitors of both Factor

B and D could be valuable probes that selectively target the alternative pathway and may aid in development of therapeutics targeting Complement mediated immunity and disease.

Although an inactive zymogen, Factor B has also been found to have appreciable enzymatic activity in its own right, but under alkaline conditions (pH 7.4 to 9.5), hinting that the

39 catalytic domain needs the cofactor Bb to alter conformation for activity at pH 7.187 This finding is similar to the pH dependence of serine proteases of Dengue and West Nile viruses, which also require a cofactor domain at pH 7 to stabilize a catalytic conformation.189, 190 This finding enabled the development of robust chromogenic enzyme assays and rational inhibitor development. In 2007, we reported the first competitive and reversible substrate-based Factor

B inhibitor, the heptapeptide aldehyde 24 (Ac-SHLGLAR-H), which was based on optimized peptide substrates for Factor B.187 Compound 24 had moderate Factor B inhibitor potency

187 (IC50 19 µM; pH 9.5) in vitro in enzyme-based assays. In further in vitro studies, 24 dose- dependently prevented Factor B-induced cleavage of the native substrate C3 into C3a and

C3b.187 Introducing an aldehyde moiety enabled a reversible covalent interaction with the catalytic Ser hydroxyl side chain in Factor B.187 This leads to the formation of a tetrahedral intermediate that mimics the transition state during enzyme catalysis.187 Further investigation of structure-activity relationships for 63 substrate-based peptidic inhibitors, all with a C- terminal aldehyde, led to the discovery of hexapeptide aldehyde 25 (Ac-RL(Tba)LAR-H)187 that was a more potent inhibitor (IC50 250 nM, pH 9.5) of Factor B in an in vitro chromogenic enzyme assay.187 Furthermore, at pH 7 compound 25 also blocked cleavage of

C3 and MAC formation via the alternative pathway,187 and attenuated human Complement- mediated hemolysis of rabbit erythrocytes in vitro.187 To date, no in vivo studies have been performed with Factor B inhibitors derived from these clues.

24

40

25

Three series of small molecule Factor B inhibitors have been disclosed by Novartis for treating conditions and diseases related to alternative pathway Complement activation, including age-related macular degeneration, obesity and liver fibrosis. One series featured imidazopyridines (WO2014143638-A1), among which 26 was reported to potently (IC50 30 nM, pH 7.4) inhibit human recombinant Factor B enzymatic activity in vitro. A second series involved 2-benzylbenzimidazoles (WO2015066241-A1) exemplified by 27 (IC50 23 nM, pH

7.4). Although the compounds were resolved, the absolute stereochemistry of the active enantiomer was not determined. The third series of compounds featured dimethoxyquinazolines (WO2013192345-A1), the most potent among 130 examples was 28

(IC50 1 nM, pH 7.4). No development of these small molecule inhibitors of Factor B has been reported. Further, along with Factor B, complement regulatory proteins such as properdin could serve as therapeutic targets to prevent alternate pathway activation. For example, properdin promotes the association of C3b with Factor B and aids in the assembly of C3bBb on a surface. Although, no small molecule or peptidic ligands have been identified to date, recent patent claims for properdin antibodies (WO2013093762) suggest therapeutic potential.

41

26 27 28

Small molecule inhibitors have also been obtained for Factor D. Until recently, the specific structural features of this enzyme, which are uncommon to the S1 serine protease family, has hampered the development of potent and selective Factor D inhibitors. Compound 29 was recently developed as one of the first potent (IC50 0.03 μM against Factor D enzyme), selective, non-covalent, and reversible inhibitors using a structure-based design approach.51

Compound 29 prevented alternative pathway activation and MAC formation in vitro in

o human whole blood (IC50 144 nM, pH 9.0, 37 C). Oral administration to Factor D-humanized mice dose-dependently (1, 3 and 10 mg/kg) inhibited lipopolysaccharide-induced alternative

51 pathway activation both systemically and in ocular tissues. Further, 29 inhibited (IC50 0.07

μM, pH 7.4, 37oC) C3 deposition on, and lysis of, normal human erythrocytes in an in vitro assay that mimics lytic sensitivity of PNH erythrocytes.51 These findings were supported using erythrocytes from PNH patients treated ex vivo with an analogue of 29.51

A crystal structure of 29 bound to human Factor D revealed that 29 extends from S1 to

S2ʹ positions in the protein, with the self-inhibitory loop of the protein shielding the non- prime side of the inhibitor.51 Further, this led to the catalytic residue His57 adopting an unusual conformation. The terminal carboxamide moiety of 29 is in proximity to Arg218 and forms several water-bridged hydrogen bonds with S1 residues Ser190 and Ile227, the backbone NH of Gly193 and the carbonyl of Leu41.51 The meta chloro substituent occupied

42 S2ʹ and formed a halogen–carbonyl bond with Trp141.51 These structural insights suggest an opportunity for Factor D tailored drug design. Further analysis by these authors of the active site of Factor D (PDB code: 1DIC) using consensus Sitemap and FTMap identified additional potential hot spots (sub pocket of latent Factor D) for ligand binding.191 These studies were instrumental for understanding the binding requirements for the generation of potent noncovalent reversible Factor D inhibitors. For example, structure based focused library

screening led to the identification of several active hits (30 IC50 14 μM, 31 IC50 17 μM in the factor D thioesterolysis assay, pH 7.5, 22°C) and a molecule with a novel binding mechanism

(32, Kd 1600 μM on Factor D) was reported.191 A co-crystal structure of this compound with

Factor D, suggested for the first time that a neutral molecule can interact and form H-bond interactions with key residues in the S1 subpocket of Factor D. A Novartis library of fluorinated fragments (LEF) was screened using 19F NMR spectroscopy, resulting in the identification of five relatively weak Factor D inhibitors.191 Further chemical modifications led to 33 (a racemic mixture of diastereomers) and 34 (Kd ~ 500 μM)191. Compounds 33 and

34 were among the first non-covalent reversible inhibitors of Factor D activity.191 Novartis

(WO2015009977) disclosed structurally distinct aminomethyl-biaryl derivates as potent

factor D inhibitors. Compound 35 potently inhibited recombinant human factor D (IC50 4 nM, pH 7.4, 22°C).

More recent potent small molecule Factor D inhibitors such as 36 (ACH-4471), Achillion

Pharmaceuticals) were recently reported as being in human clinical trials.192 Compound 36

bound tightly to human Factor D (Kd < 1 nM) and blocked its proteolytic activity (IC50 0.015

µM, 280 nM C3b, 400 nM factor B, 0.8 nM factor D; 37oC) in the presence of purified human Factor B and C3b.192 Compound 36 potently inhibited Complement-mediated

o 192 hemolysis (IC50 0.0040 µM, pH 6.4, 37 C) in PNH patient samples. Compound 36 also

43 potently inhibited C3 deposition in vitro in PNH erythrocytes (IC50 0.031 µM; IC90 0.089 µM, pH 6.4, 37oC).192 Further, the potential clinical utility of 36 was investigated following oral delivery (200 mg/kg) to non-human primates (cynomolgus monkeys), which tolerated 36 well and showed no clinical abnormalities.192 Based on promising pre-clinical studies (in vivo pharmacology, pharmacokinetic properties, safety and toxicology) and successful passage through human Phase I trials, 36 has progressed to a Phase II clinical trial in PNH patients.192

A 2016 Novartis patent (WO2016088082-Al) disclosed 21 examples of biarylacetamides as inhibitors of human recombinant Factor D. The most effective compound 37 competitively displaced (IC50 23 nM) a known Factor D inhibitor labeled with the fluorophore Cy5, monitored using TR-FRET analysis, but no other biological data was provided. Dimers of acetylsalicyclic acid (but not aspirin itself), including 38 and 39 and other regioisomers, have been reported (WO2015070354-Al) to block human erythrocyte hemolysis by zymogen- activated serum from human, rat, cat, and dog with IC50 0.1 µM. These compounds were shown by Western blot analyses of human red blood cell membranes exposed to zymogen- activated serum to inhibit Factor D processing of C3bB, and therefore formation of the C3 convertase. Additionally 38 and 39 blocked the binding of C9 to C5b678 and therefore formation of the membrane attack complex. Compounds 38 and 39 were shown to be selective for Factor D and C9 and did not bind to C2, C3, C4, C5, C6, C7, C8, Properdin, or

Factor B.

29 30 31

44

32 33 34

35 36

37 38 39

4.7 Complement Protein C3a Receptor (C3aR)

Human C3a is a 77-residue (9 kDa) inflammatory protein that causes chemotaxis, spasmogenesis and degranulation of granulocytes and phagocytes (neutrophils, monocytes,

45 macrophages, mast, dendritic cells).18 C3a exerts its effects through binding to a 100 kDa

GPCR (C3aR) expressed on immune cells (eosinophils, basophils, mast cells, neutrophils, monocytes, macrophages, T and B cells, NK cells) and non-immune cells (e.g. platelets, fibroblasts, adipocytes, endothelial/epithelial cells).193, 194 It induces Ca2+ influx, morphological changes, degranulation, release of reactive oxygen species in macrophages, neutrophils and eosinophils, and release of histamine, vasoactive and inflammatory mediators.8, 18 C3a regulates production of eosinophil cationic protein, adhesion to endothelial cells and migration.193 C3a regulates vasodilation, increases permeability of blood vessels, induces smooth muscle contraction,8, 18 and stimulates or inhibits TNF, IL1β or IL6 in PBMCs depending on conditions.116 C3a suppresses TNF and IL6 release from human B- cells,195 suppresses T-cell proliferation and antibody generation by B-cells,116, 195 and may also have roles in hematopoiesis, bone metabolism, angiogenesis and tissue repair.8, 100

Overexpression of C3a/C3aR or sustained activation of the receptor can lead to inflammatory diseases, including allergies, asthma, arthritis, sepsis, lupus, diabetes, psoriasis, nephropathy, ischaemia-reperfusion injury, obesity, and metabolic and cardiovascular dysfunction.22 C3a is reportedly elevated in lung inflammation, allergic asthma, idiopathic pulmonary fibrosis,8, 17

COPD, rheumatoid arthritis, ulcerative colitis, sepsis, ischemia-reperfusion, organ transplants, dermatomyositis, psoriasis, aneurysmal subarachnoid haemorrhage, acute Lyme disease after tick-bite, age-related macular degeneration, endometriosis, pregnancy issues, stroke, heart failure, cerebral arteriovenous malformations, gestational diabetes, SLE, multiple sclerosis, nephritis, dermatitis.8, 195 C3aR also has roles in adipose inflammation and metabolic dysfunction.100 C3aR knockout mice support roles in lung inflammation, allergic asthma, pulmonary fibrosis, RA, colitis, sepsis and transplant diseases, reperfusion injury

46 following ischemic insult, and graft tolerance.8, 196-198 C3a also reportedly has antimicrobial and antifungal activities that are unrelated to its effector C-terminal region.22

Despite extensive studies, the actions of C3a in vivo have remained uncertain because C3a is synthesized at the cell surface and degraded very rapidly by extracellular carboxypeptidases, which cleave off the C-terminal Arg to form C3ades-Arg that does not bind strongly to C3aR and has a completely different pharmacological profile from C3a.22 This instability has heavily compromised some conclusions drawn about the detection and properties of C3a in vivo. Synthetic agonists that act through C3aR, but do not degrade like C3a or short peptide analogues,199 may have chemotactic, degranulating and immunostimulating activities, whereas stable antagonists may have anti-inflammatory properties. However, despite decades of effort by academia and the pharmaceutical industry, it is only recently that very potent and

C3aR-selective small molecule agonists and antagonists have been derived.22

N2-[(2,2-diphenylethoxy)acetyl]-L-arginine (40, SB290157) was the first small molecule

97 reported to be an effective C3aR antagonist with receptor binding affinity (IC50 200 nM) measured by competitive displacement of radioligand 125I-C3a from rat basophilic leukemia

97 (RBL)-2H3 cells expressing human C3aR (RBL-hC3aR, IC50 200 nM), and from

199 199 differentiated U937 cells (IC50 140 nM). Compound 40 did not bind to C5aR1 and was reported to be a functional C3aR antagonist in inhibiting C3a-induced intracellular calcium

200 mobilization in RBL-hC3aR cells (IC50 28 nM) and human PMNs (28 nM), but much less

201 effective (IC50 1.3 µM) on human monocyte derived macrophages (HMDM). Compound

40 was found to be a weak antagonist in some animal models of inflammatory diseases,202-204 however it has also been reported to be a C3aR agonist in vitro in some cell types, such as transfected RBL and CHO cells, and may also bind to other receptors.199, 205

47

40 41

Compound 41 was reported to antagonize C3aR with greater potency than 40 and to show some efficacy in an ovalbumin-triggered model of airway inflammation in Balb-c mice following i.p. administration at 30 mg/kg or via an aerosol.206 Compound 41 was subsequently found to be equipotent with 40 in inhibiting Ca2+ release in HMDM.201

Compound 42 was discovered from small molecule screening by the same group and represented the first C3aR antagonist (pIC50 5.8, HMC-1 cells, against 5 nM C3a agonist) without an arginine residue.207 The potency was measured using HMC-1 cell membranes stably expressing both the Ca2+ sensitive protein aequorin and the human C3a receptor and, after treatment with coelenterazine, the emitted light was quantitated. Further SAR studies based on 42 led to improved ligand binding affinity, however all new analogues including the commercially available compound 43 (EC50 2 µM) proved to be agonists.

42 43 44

A series of 2-oxo-1,2-dihydropyridine-3-carbonyl-arginine compounds, such as 44 (IC50 0.05

µM, binding) were reported to bind to the C3a receptor (WO2008079371-Al). Binding

48 affinity was evaluated using cell membranes from HEK293 cells stably expressing recombinant C3aR, but no functional activity or other biological data was reported.

A more recent study involved an innovative design strategy, which was used to downsize the human C3a protein to small molecules that were found to have functional activities that were equipotent with C3a.22 A peptide sequence (GLAR) corresponding to the effector C-terminus of C3a was conformationally constrained by incorporating a heterocyclic ring. The nature of the heterocyclic component influenced the binding affinity over a 4 log unit range,18 an effect attributed to optimal positioning of a key hydrogen bond accepting heteroatom. Optimising

2+ the N-terminal capping group led to 45, which induced Ca release in HMDM (EC50 7 nM) and showed comparable potency and reactivity profiles to human C3a. Unlike C3a, 45 and its analogues were stable in vivo.22, 23 The nature of the heterocycle also dictated the ligand conformation, which in turn influenced functional activity. For example, thiazole regioisomers 46 and 47, differing only in the respective positions of nitrogen and sulfur atoms, displayed opposite functions as a C3aR agonist and antagonist, respectively.208 DFT calculations and NMR analyses revealed that the dihedral angle that orients the amide bond and hence the Arg residue was responsible for the opposing agonist verses antagonist functions. Further synthesis of the conformationally rigid bicyclic fused ring systems 48

2+ 2+ (agonist EC50 15 nM, Ca , HMDM) and 49 (antagonist IC50 320 nM, Ca , HMDM) confirmed these above findings.208 The potent C3aR agonist 50 (BR103)23, 201, 208 and antagonist 51 (BR111)23, 201, 208 have been investigated in vivo in a range of animal models of human disease, which confirmed pro-inflammatory activity for the agonist and anti- inflammatory activity for the antagonist.23 These structures have since been modified for enhanced in vitro and in vivo potency, selectivity and stability (Reid RC, unpublished data) through greater knowledge of the ligand binding site obtained from site-directed mutagenesis,

49 molecular modeling and structure-activity relationships (Rowley J, PhD dissertation,

University of Queensland, 2017).

45 46 X=N, Y=S

47 X=S, Y=N

48 49

50 51

50 4.8 Soluble receptors (CR1-4 and CRIg) for Complement Protein 3b (C3b)

Apart from its key role in forming the MAC, C3b can form additional fragments, which bind to cognate receptors such as CR1, CR2, CR3, CR4 and CRIg present on a range of leukocytes that mediate pathogen clearance and inflammation.209 Very little progress has been reported in developing small molecule inhibitors or antagonists of C3b or these important

Complement receptors, with CR3 being the exception. CR3 is a transmembrane heterodimer composed of an alpha subunit (CD11b) and a beta chain (CD18).209 CR3 is involved in adhesion to the extracellular208 matrix and to other cells, as well as in recognition of iC3b.209

Along with CR4, this receptor belongs to the integrin family and plays an important role not only in phagocytosis, but also in leukocyte trafficking and migration, synapse formation and co-stimulation.209 To date, two small molecules 52 and 53 have been reported as CR3

208, 210 antagonists. Compounds 52 and 53 were reported to have IC50 0.14 and 0.33 µM, respectively, against CR3 activation in vitro in an assay that measured direct binding of C3bi- alkaline phosphatase to CR3.210 Both 52 and 53 also inhibited CR3-mediated adhesion of human polymorphonuclear cells to fibrinogen when stimulated with either TNF or PMA

210 (IC50 ~2.5-10 µM). To date, no in vivo studies have been reported for these two CR3 antagonists. Inhibition of ligand binding by 52 and 53 was not easily reversed and requires light, suggesting the possible formation of a covalent adduct through photo-activation.

52 53

51

4.9 Complement Component 5 (C5)

Formation of either the C4b2a3b or C3bBb3b (also a C5 convertase) from C3b eventually leads to cleavage of C5.5 C5 is thought to play an important role in inflammatory and cell killing processes.5 C5 is composed of an alpha and a beta polypeptide chain that are linked by a disulfide bridge.5 Over the past few years, Alexion’s antibody, eculizumab (Soliris) has helped to validate C5 inhibition as a clinically relevant therapeutic target,5 with encouraging results for treating the human blood disorder PNH, the rare kidney disorder aHUS, and it has orphan-drug status for the neurological disorder neuromyelitis optica. This antibody directly binds to C5 and thereby inhibits the cleavage of C5 to C5a and C5b.5 However, the principal therapeutic property is thought to be due to inhibition of C5b, and subsequently MAC, formation.5 Like many treatments for rare diseases, this antibody is among the world’s most expensive drugs for patients, an economic factor that influences pharmaceutical development decisions.

So far, no small molecule C5 inhibitor has been disclosed. A compound referred to as

RA101495 (structure not disclosed) is evidently a C5 blocking peptide that was developed by

Ra Pharma as a self-administrated subcutaneous therapy for human patients with PNH.211

Evidently, this compound has been successfully assessed for safety, tolerability, pharmacokinetics and pharmacodynamics in a Phase I clinical trial in healthy volunteers

(rapharma.com). Further, it has been claimed to almost completely suppress ex vivo hemolysis and Complement activity in human blood from healthy volunteers after a single s.c. dose. This response was robustly maintained with daily s.c. dosing within an acceptable safety and tolerability profile and no serious adverse events. Ra Pharma indicate that this compound is in two Phase II trials for safety, tolerability, efficacy, pharmacokinetics and

52 pharmacodynamics in human PNH patients, including those patients that are eculizumab- naïve, eculizumab-switch and eculizumab-inadequate responders (NCT03030183;

NCT03078582).

4.10 Complement Protein 5a Receptor (C5aR1)

Cleavage of C5 produces Complement component peptides C5a and C5b. C5a is a potent pro-inflammatory and chemotactic factor that primarily signals via a GPCR, the C5a receptor

C5aR that was recently renamed C5aR1.212 A second receptor, C5aR2 (previously known as

C5L2), that is not coupled to G proteins and has largely non-signaling properties, appears to modulate C5aR1 function although its significance remains controversial.213 C5aR1 is expressed widely on immune cells, including neutrophils, monocytes, macrophages, eosinophils and T cells, but also on other cells including of the liver, kidney, adipose, and central nervous system.212 C5aR1 activation is also now implicated in many functions besides immunity and inflammation, such as metabolic functions and dysfunction, crosstalk with

TLR signalling, developmental biology, and cancer metastasis and progression.212 Thus, it may be desirable to modulate C5a mediated Complement activation using therapeutic interventions that target C5aR1. Most importantly, unlike targeting C5 inhibition, antagonizing the responses of C5a-C5aR1 does not prevent downstream formation of MAC that is necessary for immunity against pathogens.212 Instead, C5aR1 antagonism dampens the recruitment of infiltrating and differentiating immune cells and inhibits their activation, degranulation and proinflammatory actions.

Over the past two decades, very few potent and selective low molecular weight peptidomimetic and small molecule C5aR1 antagonists (e.g. 54-62) have been reported.201-212

Some of these C5aR1 antagonists have shown anti-inflammatory activity in vitro in human

53 immune cells and efficacy in vivo in animal models of inflammatory disease.213 However, despite successes in pre-clinical models few compounds have so far progressed beyond Phase

I clinical trials. Among these, the cyclic peptide compound 54 (3D53, PMX53) was discovered in our laboratory among a class of designed macrocyclic antagonists (54: IC50 30 nM, PMNs; 20 nM HMDM) of C5aR1,213-216 with negligible binding to C5aR2. Such macrocyclic C5aR1 antagonists, in particular 54 had efficacy in a wide variety of human immune cells and in over 20 different animals models of human inflammatory disease.213

Compound 54 was licensed for clinical development to the former company Promics Ltd

(subsumed successively into Peptech, Arana Therapeutics, Cephalon and TEVA).213

Compound 54 was well tolerated in humans, passed through Phase I clinical trials,217 and was evaluated in Phase II for rheumatoid arthritis and psoriasis.218 A close analogue 55 (3D624,

PMX205) had a slightly improved oral pharmacokinetic profile and has also been evaluated in mouse models of neurodegenerative diseases97, 219 and allergic asthma.220 An interesting property of these macrocyclic antagonists of C5aR1 is their insurmountable antagonism and long residence time on this receptor,212 exemplified in a comparison between 54 with 56 and

57 as described below.

Other small molecule C5aR1 antagonists have been reported to have potent inhibitory activity, such as 56 (W54011) and 57 (JJ47). Racemic compound 56, discovered from a high throughput screening and lead optimization,221 is a potent competitive antagonist of C5aR1

221 (IC50 2 nM against 0.1nM rhC5a on human neutrophils; IC50 3 nM against 1 nM rhC5a on

HMDM212) with good oral bioavailability (74%, 10 mg/kg, rats).212 It inhibited C5a-induced calcium mobilization, chemotaxis and generation of reactive oxygen species in human neutrophils.221 Compound 57 was discovered from a series of synthetic aniline-substituted tetrahydroquinolines as a competitive C5a receptor antagonist (IC50 7 nM versus 1.5 nM

54 rhC5a, U937 cells).222 The antagonists 54, 56 and 57 were compared for rule-of-five compliance drug-likeness, receptor affinity and antagonist potency in human macrophages

(HMDM), and for anti-inflammatory efficacy in rats. Only the least rule-of-five compliant antagonist 54 maintained potency in HMDM against increasing C5a concentrations and had a

212 much longer duration of action (t1/2 ~ 20 h) than 56 or 57 (t1/2 ~ 1–3 h). Despite very low oral bioavailability (F ≤ 2%, rat, 10 mg/kg), a single dose of 54 was much more orally efficacious than the more orally bioavailable 56 or 57 in preventing agonist-induced rat paw oedema with effects lasting 24 h. Molecular dynamics simulations supported an unusually long residence time on C5R1 for 54 due to long-lasting molecular interactions that trap the antagonist within the receptor.212 These results highlight a growing realization in the pharmaceutical industry that residence time on a receptor can trump rule-of-five drug- likeness in determining efficacy, even oral efficacy, of pharmacological agents.

Compound 58 (NDT9520492) is a species-specific C5aR1 antagonist in primates and gerbils221 and inhibited [35S]-GTPγS binding to human C5aR1 (Ki 15.2 nM). NGD 2000-1, an analogue of 58 with an undisclosed structure, did not show therapeutic efficacy in a Phase

II asthma trial and inhibited cytochrome P450 3A4.213 This compound was also tested in human clinical trials for the treatment of rheumatoid arthritis but its development has been

125 o halted. Compound 59 (CP-447697) is a C5aR1 antagonist (IC50 31 nM, 37 C against 1nM rhC5a), but had low bioavailability and a short half-life.223 Compound 60 (NDT 9513727) is a competitive inverse agonist disclosed by Neurogen.224 In cell-based assays, it inhibited a broad range of C5a-mediated functions, including [35S]-GTPγS binding, calcium mobilization, chemotaxis, degranulation, oxidative burst, and CD11b cell–surface expression

(IC50 1–9 nM). Recently, an allosteric inhibitor of C5aR1 (61, DF2593A) was designed by targeting the minor pocket of the 7TM helix bundle (between TM1, 2, 3, 6 and 7) that is

55 highly conserved in class A GPCR proteins.225 Oral administration reduced mechanical hyperalgesia in mouse models of acute and chronic inflammatory and neuropathic pain, a comparison of C5aR1-/- mice with WT suggesting C5aR1 specificity for 61.

Compound 62 (CCX-168, Avacopan) is a competitive C5aR antagonist recently reported to enter a Phase III clinical trial (NCT02994927). It inhibited C5a-mediated calcium mobilization, migration, and CD11b upregulation in U937 cells and human neutrophils.

Compound 62 efficiently attenuated migration in vitro and in ex vivo chemotaxis assays, and inhibited C5a-induced neutrophil vascular endothelial margination and migration in cynomolgus monkeys. It was well tolerated across a wide dose range (1 to 100 mg) and demonstrated dose-dependent pharmacokinetics. In anti-neutrophil cytoplasmic antibody

(ANCA)-associated vasculitis, 62 was able to replace oral glucocorticoids to treat the disease without compromising efficacy. 226

54 R=NHAc

55 R=H

56

56 57

58 59

60 61

57

62

4.11 Terminal Pathway Complement Proteins and Membrane Attack Complex (MAC)

Besides targeting the initiation and amplification of the proteolytic cascade, effective

Complement inhibition in various diseases might be achievable by interfering with the terminal pathway.211 Serum Complement components C5b, C6, C7, C8, C9 become important after C5 processing by leading to assembly of the MAC complex that forms pores in the lipid bilayers of invading microorganisms and infected or damaged cells.211 MAC has a key surveillance role in pathogen detection and cell clearance, but also plays a very important role in many chronic human disease conditions including neurodegenerative diseases.211 For example, MAC can be detected on nerve-muscle junctions in patients suffering from ALS.

Unpublished studies from some pharmaceutical companies (e.g. Regenesance) suggest that

MAC inhibition could be effective in delaying nerve degeneration and accelerating nerve regeneration (US8703136 B2).

Inhibiting any of the terminal pathway proteins could potentially prevent MAC formation.211

However, so far no effective agents have been reported to inhibit C5b, C7 or C8.

Regenesance has targeted C6 with both anti-C6 antibody and antisense approaches, an antisense C6 inhibitor being effective in vivo in some animal models.227 In terms of small

58 molecules, Regenesance also described a low molecular weight C6 inhibitor in development for Guillain–Barré syndrome, however chemical structures or information on further development has not been disclosed.211 In relation to C9, aurin/carboxylic acid-based compounds 63 (aurin tricarboxylic acid), 64 (aurin quadracarboxylic acid) and 65 (aurin hexacarboxylic acid) were shown to block addition of C9 to C5b-8 and prevent MAC formation.228 All three compounds 63-65 potently and dose-dependently inhibited in vitro

Complement-mediated hemolysis of human (IC50 ~500 nM, pH 7.4), rat, and mouse erythrocytes.228 Ex vivo, compound 63 was also effective in preventing Complement- mediated hemolysis of human erythrocytes from patients with PNH.229 Further, in an in vivo mouse model of Alzheimer’s disease, oral administration (~100mg/kg/day) of a mixture containing 63, 64 and 65 (78:15:7 ratio) prevented MAC formation in serum and improved memory retention.228 New advances in cryo-electron microscopy has enabled us to understand the molecular details of the MAC assembly and mechanism of action of pore formation.74 This cryo-EM structure was refined to 8.5 Å resolution.74 Careful analysis of this complex structure has helped to identify a network of interfacial interactions that may determine the MAC assembly mechanism, providing insight into how MAC forms and potentially how it could be targeted through structure-based drug discovery.

63 64 65

59 5. CONCLUSIONS AND FUTURE PROSPECTS

Complement has unique activation mechanisms involving complex coordination of protein- protein interactions and proteolytic enzymes, originating both from successive steps of the

Complement cascade and from other proteolytic networks. Complement proteins have numerous pathological roles in many inflammatory, respiratory, gastrointestinal, metabolic, cardiovascular, neurological disorders and cancers, so the development of potent and selective Complement drugs is expected to produce valuable new therapeutic agents across a wide range of diseases. The identification of the importance of Complement proteins in rare diseases has also recently provided some additional impetus for Complement intervention and pharmaceutical development.

Traditional medicinal chemistry drug discovery programs aim to develop small molecules that are potent, selective, cheaper to synthesize and with potential for oral administration.

However, many of the critical steps in the Complement system are fundamentally based on large and complex, but also often shallow, interacting protein surfaces that can be challenging to modulate using conventional drug-like small molecules. Historically, complement- directed modulators have had to be derived without knowledge of protein structures or how they interact, and with limited understanding of molecular immunology. Instead most compounds have been derived from high-throughput random screening programs, followed by painstaking structure-activity medicinal chemistry studies to enable hit-to-lead development. Recent advances in biochemistry, protein crystallography, pharmacology and immunology, together with genetic evidence for the importance of Complement activation pathways, have provided important new insights to structures and functions of Complement proteins. These advances are beginning to provide new opportunities and new approaches for

60 rational design and development of more effective drugs to modulate Complement-mediated human diseases. A number of promising small molecule or peptide-based chemical probes for modulating Complement-mediated physiology have been identified and are outlined in this perspective review. We have catalogued the few known Complement therapeutics for which there is preclinical or clinical evidence of efficacy, as well as some early examples of peptides or small molecules that modulate Complement activation and that could serve as clues for developing new Complement-targeted medicines.

61 AUTHOR INFORMATION

Corresponding Author

Professor David Fairlie, Institute for Molecular Bioscience, The University of Queensland,

Brisbane, QLD 4072, Australia. E: [email protected],.

Notes

The University of Queensland owns multiple patents, associated with Complement C3a and

C5a receptor agonists and antagonists, on which RR and DF were inventors. The authors declare no other competing financial interests.

Biographies

Abishek Iyer received his BSc from Bangalore University, India and his M Mol Biol. and

Ph.D. from The University of Queensland, Australia. He is currently a senior postdoctoral researcher in Professor David Fairlie’s laboratory at the Institute for Molecular Bioscience,

The University of Queensland, Australia. His research interests are diverse, including understanding biological processes and disease development in the area of immunometabolism. His studies also focus on drug discovery and identifying mechanisms of novel drug action.

Weijun Xu is a postdoctoral researcher at the University of Queensland. He was previously a lecturer at the School of Chemical and Life Sciences (Singapore Polytechnic), and an undergraduate (B.Sc. Hons. Biochemistry, 2006) and postgraduate student at the University

62 of Queensland (PhD, 2013-2017). His interests are in computer-aided molecular modeling of protein-ligand and protein-protein interactions, involving discovery of ligands for GPCRs, proteases, enzymes and other proteins involved in human immune systems.

Robert Reid is an organic chemist trained in the total synthesis of natural products (Sydney and Oxford Universities). In addition to modern synthetic methodology his interests are diverse including medicinal chemistry, drug design, computational chemistry and spectroscopy all applied to discovery of new enzyme inhibitors and ligands for GPCRs.

David Fairlie studied at Adelaide, Australian National, New South Wales, Stanford and

Toronto universities. He is located at the University of Queensland where was in its Centre for Drug Design and Development (3D Centre) and is now Head of the IMB Division of

Chemistry and Structural Biology. Interests are medicinal chemistry, molecular and experimental pharmacology, immunology, particularly of agonists/antagonists/inhibitors of

GPCRs, PPIs and enzymes in inflammation, infection, neurodegeneration and cancer. He studies mechanisms of chemical, immunological and biological reactions, disease development and drug action.

ACKNOWLEDGEMENTS

Complement research in our laboratories has been supported by the National Health and

Medical Research Council, for example in recent grants (1028423, 1084018, 1117017, Senior

Principal Research Fellowship 1027369 to DPF), an Australian Research Council grant

(DP130100629), the Australian Research Council Centre of Excellence in Advanced

Molecular Imaging (CE140100011) for imaging studies on the molecular basis of immunity,

63 and the Queensland Government (CIF grant). We thank Dr. Andrew Lucke (University of

Queensland) for his contributions to Figure 2, the University of Queensland for a PhD scholarship to WX and for a UQ Postdoctoral Research Fellowship to AI.

NON-STANDARD ABBREVIATIONS

MAC - Membrane Attack Complex; C1q - Complement component 1q; C1r - Complement component 1r; C1s - Complement component 1s; Ag/Ab - antigen-antibody; C2 -

Complement component 2; C4 - Complement component 4; MBL - Mannose Binding Lectin;

MASPs - Mannan-Associated Serine Proteases; FIXa - Factor IXa; FXa - Factor Xa; FXIa -

Factor Xia; C3 - Complement component 3; C5 - Complement component 5; GPCR - G- protein coupled receptor; C3a - Complement protein 3a; C5a - Complement protein 5a; I/R - ischemia/reperfusion; PNH - Paroxysmal Nocturnal Hemoglobinuria; aHUS - Atypical

Haemolytic Uremic Syndrome; AMD - Age-related Macular Degeneration; SLE - Systemic

Lupus Erythematosus; RA - Rheumatoid Arthritis; ALS - Amyotrophic lateral Sclerosis; CR

– Complement receptor; C3aR – C3a receptor; C5aR1 – C5a receptor; GWAS – Genome

Wide Association Studies; C6 - Complement component 6; C9 - Complement component, CP

- coat protein; CCP = Complement control protein; US FDA – United States Food and Drug

Administration; WiELISA - Wieslab COMPL 300; vWFA - von-Willebrand Factor type A;

EMA - European Medicines Agency; PBMCs – Peripheral Blood Mononuclear Cells; PMNs

– Polymorphonuclear cells; HMDM - Human Monocyte Derived Macrophages.

64 REFERENCES

1. Nathan, C. Points of control in inflammation. Nature 2002, 420, 846-852.

2. Iyer, A.; Brown, L.; Whitehead, J. P.; Prins, J. B.; Fairlie, D. P. Nutrient and immune

sensing are obligate pathways in metabolism, immunity, and disease. FASEB J. 2015,

29, 3612-3625.

3. Medzhitov, R. Origin and physiological roles of inflammation. Nature 2008, 454, 428-

435.

4. Ricklin, D.; Hajishengallis, G.; Yang, K.; Lambris, J. D. Complement: A key system

for immune surveillance and homeostasis. Nat. Immunol. 2010, 11, 785-797.

5. Ricklin, D.; Reis, E. S.; Lambris, J. D. Complement in disease: A defence system

turning offensive. Nat. Rev. Nephrol. 2016, 12, 383-401.

6. Holers, V. M. Complement and its receptors: New insights into human disease. Annu.

Rev. Immunol. 2014, 32, 433-459.

7. Morgan, B. P.; Harris, C. L. Complement, a target for therapy in inflammatory and

degenerative diseases. Nat. Rev. Drug Discov. 2015, 14, 857-877.

8. Klos, A.; Wende, E.; Wareham, K. J.; Monk, P. N. International union of basic and

clinical pharmacology. [corrected]. Lxxxvii. Complement peptide C5a, C4a, and C3a

receptors. Pharmacol. Rev. 2013, 65, 500-543.

9. Melis, J. P.; Strumane, K.; Ruuls, S. R.; Beurskens, F. J.; Schuurman, J.; Parren, P. W.

Complement in therapy and disease: Regulating the complement system with antibody-

based therapeutics. Mol. Immunol. 2015, 67, 117-130.

10. Reis, E. S.; Mastellos, D. C.; Yancopoulou, D.; Risitano, A. M.; Ricklin, D.; Lambris,

J. D. Applying complement therapeutics to rare diseases. Clin. Immunol. 2015, 161,

225-240.

65 11. Gros, P.; Milder, F. J.; Janssen, B. J. Complement driven by conformational changes.

Nat. Rev. Immunol. 2008, 8, 48-58.

12. Kemper, C.; Pangburn, M. K.; Fishelson, Z. Complement nomenclature 2014. Mol.

Immunol. 2014, 61, 56-58.

13. Meyer, S.; Leusen, J. H.; Boross, P. Regulation of complement and modulation of its

activity in monoclonal antibody therapy of cancer. MAbs. 2014, 6, 1133-1144.

14. Walport, M. J. Complement. First of two parts. N. Engl. J. Med. 2001, 344, 1058-1066.

15. Liszewski, M. K.; Kolev, M.; Le Friec, G.; Leung, M.; Bertram, P. G.; Fara, A. F.;

Subias, M.; Pickering, M. C.; Drouet, C.; Meri, S.; Arstila, T. P.; Pekkarinen, P. T.; Ma,

M.; Cope, A.; Reinheckel, T.; Rodriguez de Cordoba, S.; Afzali, B.; Atkinson, J. P.;

Kemper, C. Intracellular complement activation sustains T cell homeostasis and

mediates effector differentiation. Immunity 2013, 39, 1143-1157.

16. Satyam, A.; Kannan, L.; Matsumoto, N.; Geha, M.; Lapchak, P. H.; Bosse, R.; Shi, G.

P.; Dalle Lucca, J. J.; Tsokos, M. G.; Tsokos, G. C. Intracellular activation of

complement 3 is responsible for intestinal tissue damage during mesenteric ischemia. J.

Immunol. 2017, 198, 788-797.

17. Verschoor, A.; Karsten, C. M.; Broadley, S. P.; Laumonnier, Y.; Kohl, J. Old dogs-new

tricks: Immunoregulatory properties of C3 and C5 cleavage fragments. Immunol. Rev.

2016, 274, 112-126.

18. Forneris, F.; Wu, J.; Gros, P. The modular serine proteases of the complement cascade.

Curr. Opin. Struct. Biol. 2012, 22, 333-341.

19. Kenawy, H. I.; Boral, I.; Bevington, A. Complement-coagulation cross-talk: A potential

mediator of the physiological activation of complement by low pH. Front. Immunol.

2015, 6, 215.

66 20. Heja, D.; Kocsis, A.; Dobo, J.; Szilagyi, K.; Szasz, R.; Zavodszky, P.; Pal, G.; Gal, P.

Revised mechanism of complement lectin-pathway activation revealing the role of

serine protease MASP-1 as the exclusive activator of MASP-2. Proc. Natl. Acad. Sci.

U. S. A. 2012, 109, 10498-10503.

21. Amara, U.; Flierl, M. A.; Rittirsch, D.; Klos, A.; Chen, H.; Acker, B.; Bruckner, U. B.;

Nilsson, B.; Gebhard, F.; Lambris, J. D.; Huber-Lang, M. Molecular

intercommunication between the complement and coagulation systems. J. Immunol.

2010, 185, 5628-5636.

22. Reid, R. C.; Yau, M. K.; Singh, R.; Hamidon, J. K.; Reed, A. N.; Chu, P.; Suen, J. Y.;

Stoermer, M. J.; Blakeney, J. S.; Lim, J.; Faber, J. M.; Fairlie, D. P. Downsizing a

human inflammatory protein to a small molecule with equal potency and functionality.

Nat. Commun. 2013, 4, 2802.

23. Lohman, R. J.; Hamidon, J. K.; Reid, R. C.; Rowley, J. A.; Yau, M. K.; Halili, M. A.;

Nielsen, D. S.; Lim, J.; Wu, K.-C.; Loh, Z.; Do, A.; Suen, J. Y.; Iyer, A.; Fairlie, D. P.

Exploiting a novel conformational switch to control innate immunity mediated by

complement protein C3a. Nat. Comm. 2017, 8, 351.

24. Seow, V.; Lim, J.; Iyer, A.; Suen, J. Y.; Ariffin, J. K.; Hohenhaus, D. M.; Sweet, M. J.;

Fairlie, D. P. Inflammatory responses induced by lipopolysaccharide are amplified in

primary human monocytes but suppressed in macrophages by complement protein C5a.

J. Immunol. 2013, 191, 4308-4316.

25. Gaboriaud, C.; Juanhuix, J.; Gruez, A.; Lacroix, M.; Darnault, C.; Pignol, D.; Verger,

D.; Fontecilla-Camps, J. C.; Arlaud, G. J. The crystal structure of the globular head of

complement protein C1q provides a basis for its versatile recognition properties. J.

Biol. Chem. 2003, 278, 46974-46982.

67 26. Paidassi, H.; Tacnet-Delorme, P.; Garlatti, V.; Darnault, C.; Ghebrehiwet, B.;

Gaboriaud, C.; Arlaud, G. J.; Frachet, P. C1q binds phosphatidylserine and likely acts

as a multiligand-bridging molecule in apoptotic cell recognition. J. Immunol. 2008,

180, 2329-2338.

27. Garlatti, V.; Chouquet, A.; Lunardi, T.; Vives, R.; Paidassi, H.; Lortat-Jacob, H.;

Thielens, N. M.; Arlaud, G. J.; Gaboriaud, C. Cutting edge: C1q binds deoxyribose and

heparan sulfate through neighboring sites of its recognition domain. J. Immunol. 2010,

185, 808-812.

28. Moreau, C.; Bally, I.; Chouquet, A.; Bottazzi, B.; Ghebrehiwet, B.; Gaboriaud, C.;

Thielens, N. Structural and functional characterization of a single-chain form of the

recognition domain of complement protein C1q. Front Immunol. 2016, 7, 79.

29. Budayova-Spano, M.; Lacroix, M.; Thielens, N. M.; Arlaud, G. J.; Fontecilla-Camps, J.

C.; Gaboriaud, C. The crystal structure of the zymogen catalytic domain of complement

protease C1r reveals that a disruptive mechanical stress is required to trigger activation

of the C1 complex. EMBO J. 2002, 21, 231-239.

30. Budayova-Spano, M.; Grabarse, W.; Thielens, N. M.; Hillen, H.; Lacroix, M.; Schmidt,

M.; Fontecilla-Camps, J. C.; Arlaud, G. J.; Gaboriaud, C. Monomeric structures of the

zymogen and active catalytic domain of complement protease C1r: Further insights into

the C1 activation mechanism. Structure 2002, 10, 1509-1519.

31. Gaboriaud, C.; Rossi, V.; Bally, I.; Arlaud, G. J.; Fontecilla-Camps, J. C. Crystal

structure of the catalytic domain of human complement C1s: A serine protease with a

handle. EMBO J. 2000, 19, 1755-1765.

32. Perry, A. J.; Wijeyewickrema, L. C.; Wilmann, P. G.; Gunzburg, M. J.; D'Andrea, L.;

Irving, J. A.; Pang, S. S.; Duncan, R. C.; Wilce, J. A.; Whisstock, J. C.; Pike, R. N. A

68 molecular switch governs the interaction between the human complement protease C1s

and its substrate, complement C4. J. Biol. Chem. 2013, 288, 15821-15829.

33. Venkatraman Girija, U.; Gingras, A. R.; Marshall, J. E.; Panchal, R.; Sheikh, M. A.;

Gal, P.; Schwaeble, W. J.; Mitchell, D. A.; Moody, P. C.; Wallis, R. Structural basis of

the C1q/C1s interaction and its central role in assembly of the C1 complex of

complement activation. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 13916-13920.

34. Garlatti, V.; Belloy, N.; Martin, L.; Lacroix, M.; Matsushita, M.; Endo, Y.; Fujita, T.;

Fontecilla-Camps, J. C.; Arlaud, G. J.; Thielens, N. M.; Gaboriaud, C. Structural

insights into the innate immune recognition specificities of L- and H-ficolins. EMBO J.

2007, 26, 623-633.

35. Vassal-Stermann, E.; Lacroix, M.; Gout, E.; Laffly, E.; Pedersen, C. M.; Martin, L.;

Amoroso, A.; Schmidt, R. R.; Zahringer, U.; Gaboriaud, C.; Di Guilmi, A. M.;

Thielens, N. M. Human L-ficolin recognizes phosphocholine moieties of pneumococcal

teichoic acid. J. Immunol. 2014, 193, 5699-5708.

36. Laffly, E.; Lacroix, M.; Martin, L.; Vassal-Stermann, E.; Thielens, N. M.; Gaboriaud,

C. Human ficolin-2 recognition versatility extended: An update on the binding of

ficolin-2 to sulfated/phosphated carbohydrates. FEBS Lett. 2014, 588, 4694-4700.

37. Dobo, J.; Harmat, V.; Beinrohr, L.; Sebestyen, E.; Zavodszky, P.; Gal, P. MASP-1, a

promiscuous complement protease: Structure of its catalytic region reveals the basis of

its broad specificity. J. Immunol. 2009, 183, 1207-1214.

38. Gal, P.; Harmat, V.; Kocsis, A.; Bian, T.; Barna, L.; Ambrus, G.; Vegh, B.; Balczer, J.;

Sim, R. B.; Naray-Szabo, G.; Zavodszky, P. A true autoactivating enzyme. Structural

insight into Mannose-binding lectin-Associated Serine Protease-2 activations. J. Biol.

Chem. 2005, 280, 33435-33444.

69 39. Harmat, V.; Gal, P.; Kardos, J.; Szilagyi, K.; Ambrus, G.; Vegh, B.; Naray-Szabo, G.;

Zavodszky, P. The structure of MBL-associated serine protease-2 reveals that identical

substrate specificities of C1s and MASP-2 are realized through different sets of

enzyme-substrate interactions. J. Mol. Biol. 2004, 342, 1533-1546.

40. Gaboriaud, C.; Gupta, R. K.; Martin, L.; Lacroix, M.; Serre, L.; Teillet, F.; Arlaud, G.

J.; Rossi, V.; Thielens, N. M. The serine protease domain of MASP-3: Enzymatic

properties and crystal structure in complex with ecotin. PLoS. One 2013, 8, e67962.

41. Yongqing, T.; Wilmann, P. G.; Reeve, S. B.; Coetzer, T. H.; Smith, A. I.; Whisstock, J.

C.; Pike, R. N.; Wijeyewickrema, L. C. The x-ray crystal structure of Mannose-binding

lectin-Associated Serine Proteinase-3 reveals the structural basis for enzyme inactivity

associated with the carnevale, mingarelli, malpuech, and michels (3MC) syndrome. J.

Biol. Chem. 2013, 288, 22399-22407.

42. Milder, F. J.; Raaijmakers, H. C.; Vandeputte, M. D.; Schouten, A.; Huizinga, E. G.;

Romijn, R. A.; Hemrika, W.; Roos, A.; Daha, M. R.; Gros, P. Structure of complement

component C2a: Implications for convertase formation and substrate binding. Structure

2006, 14, 1587-1597.

43. Krishnan, V.; Xu, Y.; Macon, K.; Volanakis, J. E.; Narayana, S. V. The crystal

structure of C2a, the catalytic fragment of classical pathway C3 and C5 convertase of

human complement. J. Mol. Biol. 2007, 367, 224-233.

44. Krishnan, V.; Xu, Y.; Macon, K.; Volanakis, J. E.; Narayana, S. V. The structure of

C2b, a fragment of complement component C2 produced during C3 convertase

formation. Acta. Crystallogr. D. Biol. Crystallogr. 2009, 65, 266-274.

45. Croll, T. I.; Andersen, G. R. Re-evaluation of low-resolution crystal structures via

interactive molecular-dynamics flexible fitting (imdff): A case study in complement

C4. Acta. Crystallogr. D. Struct. Biol. 2016, 72, 1006-1016.

70 46. Mortensen, S.; Kidmose, R. T.; Petersen, S. V.; Szilagyi, A.; Prohaszka, Z.; Andersen,

G. R. Structural basis for the function of complement component C4 within the

classical and lectin pathways of complement. J. Immunol. 2015, 194, 5488-5496.

47. Milder, F. J.; Gomes, L.; Schouten, A.; Janssen, B. J.; Huizinga, E. G.; Romijn, R. A.;

Hemrika, W.; Roos, A.; Daha, M. R.; Gros, P. Factor B structure provides insights into

activation of the central protease of the complement system. Nat. Struct. Mol. Biol.

2007, 14, 224-228.

48. Janssen, B. J.; Gomes, L.; Koning, R. I.; Svergun, D. I.; Koster, A. J.; Fritzinger, D. C.;

Vogel, C. W.; Gros, P. Insights into complement convertase formation based on the

structure of the factor B-Cobra Venom Factor complex. EMBO J. 2009, 28, 2469-2478.

49. Cole, L. B.; Chu, N.; Kilpatrick, J. M.; Volanakis, J. E.; Narayana, S. V.; Babu, Y. S.

Structure of diisopropyl fluorophosphate-inhibited factor D. Acta. Crystallogr. D. Biol.

Crystallogr. 1997, 53, 143-150.

50. Cole, L. B.; Kilpatrick, J. M.; Chu, N.; Babu, Y. S. Structure of 3,4-

dichloroisocoumarin-inhibited factor D. Acta. Crystallogr. D. Biol. Crystallogr. 1998,

54, 711-717.

51. Maibaum, J.; Liao, S. M.; Vulpetti, A.; Ostermann, N.; Randl, S.; Rudisser, S.;

Lorthiois, E.; Erbel, P.; Kinzel, B.; Kolb, F. A.; Barbieri, S.; Wagner, J.; Durand, C.;

Fettis, K.; Dussauge, S.; Hughes, N.; Delgado, O.; Hommel, U.; Gould, T.; Mac

Sweeney, A.; Gerhartz, B.; Cumin, F.; Flohr, S.; Schubart, A.; Jaffee, B.; Harrison, R.;

Risitano, A. M.; Eder, J.; Anderson, K. Small-molecule factor D inhibitors targeting the

alternative complement pathway. Nat. Chem. Biol. 2016, 12, 1105-1110.

52. Yang, C. Y.; Phillips, J. G.; Stuckey, J. A.; Bai, L.; Sun, H.; Delproposto, J.; Brown,

W. C.; Chinnaswamy, K. Buried hydrogen bond interactions contribute to the high

potency of complement factor D inhibitors. ACS Med. Chem. Lett. 2016, 7, 1092-1096.

71 53. Janssen, B. J.; Huizinga, E. G.; Raaijmakers, H. C.; Roos, A.; Daha, M. R.; Nilsson-

Ekdahl, K.; Nilsson, B.; Gros, P. Structures of complement component C3 provide

insights into the function and evolution of immunity. Nature 2005, 437, 505-511.

54. Fredslund, F.; Jenner, L.; Husted, L. B.; Nyborg, J.; Andersen, G. R.; Sottrup-Jensen,

L. The structure of bovine complement component 3 reveals the basis for thioester

function. J. Mol. Biol. 2006, 361, 115-127.

55. Janssen, B. J.; Christodoulidou, A.; McCarthy, A.; Lambris, J. D.; Gros, P. Structure of

C3b reveals conformational changes that underlie complement activity. Nature 2006,

444, 213-216.

56. Janssen, B. J.; Halff, E. F.; Lambris, J. D.; Gros, P. Structure of compstatin in complex

with complement component C3c reveals a new mechanism of complement inhibition.

J. Biol. Chem. 2007, 282, 29241-29247.

57. Wiesmann, C.; Katschke, K. J.; Yin, J.; Helmy, K. Y.; Steffek, M.; Fairbrother, W. J.;

McCallum, S. A.; Embuscado, L.; DeForge, L.; Hass, P. E.; van Lookeren Campagne,

M. Structure of C3b in complex with CRIg gives insights into regulation of

complement activation. Nature 2006, 444, 217-220.

58. Forneris, F.; Ricklin, D.; Wu, J.; Tzekou, A.; Wallace, R. S.; Lambris, J. D.; Gros, P.

Structures of C3b in complex with factors B and D give insight into complement

convertase formation. Science 2010, 330, 1816-1820.

59. Torreira, E.; Tortajada, A.; Montes, T.; Rodriguez de Cordoba, S.; Llorca, O. 3D

structure of the C3bB complex provides insights into the activation and regulation of

the complement alternative pathway convertase. Proc. Natl. Acad. Sci. U. S. A. 2009,

106, 882-887.

60. Xue, X.; Wu, J.; Ricklin, D.; Forneris, F.; Di Crescenzio, P.; Schmidt, C. Q.;

Granneman, J.; Sharp, T. H.; Lambris, J. D.; Gros, P. Regulator-dependent mechanisms

72 of C3b processing by factor i allow differentiation of immune responses. Nat. Struct.

Mol. Biol. 2017, 24, 643-651.

61. Pedersen, D. V.; Roumenina, L.; Jensen, R. K.; Gadeberg, T. A.; Marinozzi, C.; Picard,

C.; Rybkine, T.; Thiel, S.; Sorensen, U. B.; Stover, C.; Fremeaux-Bacchi, V.;

Andersen, G. R. Functional and structural insight into properdin control of complement

alternative pathway amplification. EMBO J. 2017, 36, 1084-1099.

62. Rooijakkers, S. H.; Wu, J.; Ruyken, M.; van Domselaar, R.; Planken, K. L.; Tzekou,

A.; Ricklin, D.; Lambris, J. D.; Janssen, B. J.; van Strijp, J. A.; Gros, P. Structural and

functional implications of the alternative complement pathway C3 convertase stabilized

by a staphylococcal inhibitor. Nat. Immunol. 2009, 10, 721-727.

63. Bajic, G.; Yatime, L.; Klos, A.; Andersen, G. R. Human C3a and C3aDesArg

anaphylatoxins have conserved structures, in contrast to C5a and C5aDesArg. Protein

Sci. 2013, 22, 204-212.

64. Ghassemian, A.; Wang, C. I.; Yau, M. K.; Reid, R. C.; Lewis, R. J.; Fairlie, D. P.;

Alewood, P. F.; Durek, T. Efficient chemical synthesis of human complement protein

C3a. Chem. Commun. 2013, 49, 2356-2358.

65. Williamson, M. P.; Madison, V. S. Three-dimensional structure of porcine C5aDesArg

from 1h nuclear magnetic resonance data. Biochemistry 1990, 29, 2895-2905.

66. Smith, B. O.; Mallin, R. L.; Krych-Goldberg, M.; Wang, X.; Hauhart, R. E.; Bromek,

K.; Uhrin, D.; Atkinson, J. P.; Barlow, P. N. Structure of the C3b binding site of CR1

(CD35), the immune adherence receptor. Cell 2002, 108, 769-780.

67. Szakonyi, G.; Guthridge, J. M.; Li, D.; Young, K.; Holers, V. M.; Chen, X. S. Structure

of complement receptor 2 in complex with its C3d ligand. Science 2001, 292, 1725-

1728.

73 68. van den Elsen, J. M.; Isenman, D. E. A crystal structure of the complex between human

complement receptor 2 and its ligand C3d. Science 2011, 332, 608-611.

69. Aleshin, A. E.; DiScipio, R. G.; Stec, B.; Liddington, R. C. Crystal structure of C5b-6

suggests structural basis for priming assembly of the membrane attack complex. J. Biol.

Chem. 2012, 287, 19642-19652.

70. Hadders, M. A.; Bubeck, D.; Roversi, P.; Hakobyan, S.; Forneris, F.; Morgan, B. P.;

Pangburn, M. K.; Llorca, O.; Lea, S. M.; Gros, P. Assembly and regulation of the

membrane attack complex based on structures of C5b6 and sC5b9. Cell Rep. 2012, 1,

200-207.

71. Aleshin, A. E.; Schraufstatter, I. U.; Stec, B.; Bankston, L. A.; Liddington, R. C.;

DiScipio, R. G. Structure of complement C6 suggests a mechanism for initiation and

unidirectional, sequential assembly of membrane attack complex (MAC). J Biol. Chem.

2012, 287, 10210-10222.

72. Lovelace, L. L.; Cooper, C. L.; Sodetz, J. M.; Lebioda, L. Structure of human C8

protein provides mechanistic insight into membrane pore formation by complement. J.

Biol. Chem. 2011, 286, 17585-17592.

73. Dudkina, N. V.; Spicer, B. A.; Reboul, C. F.; Conroy, P. J.; Lukoyanova, N.; Elmlund,

H.; Law, R. H.; Ekkel, S. M.; Kondos, S. C.; Goode, R. J.; Ramm, G.; Whisstock, J. C.;

Saibil, H. R.; Dunstone, M. A. Structure of the poly-C9 component of the complement

membrane attack complex. Nat. Commun. 2016, 7, 10588.

74. Serna, M.; Giles, J. L.; Morgan, B. P.; Bubeck, D. Structural basis of complement

membrane attack complex formation. Nat. Commun. 2016, 7, 10587.

75. Flyvbjerg, A. The role of the complement system in diabetic nephropathy. Nat. Rev.

Nephrol. 2017, 13, 311-318.

74 76. Risitano, A. M. Paroxysmal nocturnal hemoglobinuria and other complement-mediated

hematological disorders. Immunobiology 2012, 217, 1080-1087.

77. Rother, R. P.; Rollins, S. A.; Mojcik, C. F.; Brodsky, R. A.; Bell, L. Discovery and

development of the complement inhibitor eculizumab for the treatment of paroxysmal

nocturnal hemoglobinuria. Nat. Biotechnol. 2007, 25, 1256-1264.

78. Zipfel, P. F.; Skerka, C.; Hellwage, J.; Jokiranta, S. T.; Meri, S.; Brade, V.; Kraiczy, P.;

Noris, M.; Remuzzi, G. Factor H family proteins: On complement, microbes and

human diseases. Biochem. Soc. Trans. 2002, 30, 971-978.

79. Nester, C. M.; Brophy, P. D. Eculizumab in the treatment of atypical haemolytic

uraemic syndrome and other complement-mediated renal diseases. Curr. Opin. Pediatr.

2013, 25, 225-231.

80. Schmidtko, J.; Peine, S.; El-Housseini, Y.; Pascual, M.; Meier, P. Treatment of atypical

hemolytic uremic syndrome and thrombotic microangiopathies: A focus on eculizumab.

Am. J. Kidney Dis. 2013, 61, 289-299.

81. Pittock, S. J.; Lennon, V. A.; McKeon, A.; Mandrekar, J.; Weinshenker, B. G.;

Lucchinetti, C. F.; O'Toole, O.; Wingerchuk, D. M. Eculizumab in AQP4-IgG-positive

relapsing neuromyelitis optica spectrum disorders: An open-label pilot study. Lancet

Neurol. 2013, 12, 554-562.

82. Cugno, M.; Bergamaschini, L.; Uziel, L.; Cicardi, M.; Agostoni, A.; Jie, A. F.; Kluft,

C. Haemostasis contact system and fibrinolysis in hereditary angioedema (C1-inhibitor

deficiency). J. Clin. Chem. Clin. Biochem. 1988, 26, 423-427.

83. Rohrer, B.; Coughlin, B.; Kunchithapautham, K.; Long, Q.; Tomlinson, S.; Takahashi,

K.; Holers, V. M. The alternative pathway is required, but not alone sufficient, for

retinal pathology in mouse laser-induced choroidal neovascularization. Mol. Immunol.

2011, 48, e1-e8.

75 84. Bora, N. S.; Kaliappan, S.; Jha, P.; Xu, Q.; Sohn, J. H.; Dhaulakhandi, D. B.; Kaplan,

H. J.; Bora, P. S. Complement activation via alternative pathway is critical in the

development of laser-induced choroidal neovascularization: Role of factor B and factor

H. J. Immunol. 2006, 177, 1872-1878.

85. Nozaki, M.; Raisler, B. J.; Sakurai, E.; Sarma, J. V.; Barnum, S. R.; Lambris, J. D.;

Chen, Y.; Zhang, K.; Ambati, B. K.; Baffi, J. Z.; Ambati, J. Drusen complement

components C3a and C5a promote choroidal neovascularization. Proc. Natl. Acad. Sci.

U. S. A. 2006, 103, 2328-2333.

86. Cashman, S. M.; Ramo, K.; Kumar-Singh, R. A non membrane-targeted human soluble

CD59 attenuates choroidal neovascularization in a model of age related macular

degeneration. PLoS. One 2011, 6, e19078.

87. Khan, M. A.; Nicolls, M. R.; Surguladze, B.; Saadoun, I. Complement components as

potential therapeutic targets for asthma treatment. Respir. Med. 2014, 108, 543-549.

88. Bao, L.; Haas, M.; Kraus, D. M.; Hack, B. K.; Rakstang, J. K.; Holers, V. M.; Quigg,

R. J. Administration of a soluble recombinant complement C3 inhibitor protects against

renal disease in MRL/lpr mice. J. Am. Soc. Nephrol. 2003, 14, 670-679.

89. Elliott, M. K.; Jarmi, T.; Ruiz, P.; Xu, Y.; Holers, V. M.; Gilkeson, G. S. Effects of

complement factor D deficiency on the renal disease of MRL/lpr mice. Kidney Int.

2004, 65, 129-138.

90. Sekine, H.; Kinser, T. T.; Qiao, F.; Martinez, E.; Paulling, E.; Ruiz, P.; Gilkeson, G. S.;

Tomlinson, S. The benefit of targeted and selective inhibition of the alternative

complement pathway for modulating autoimmunity and renal disease in MRL/lpr mice.

Arthritis Rheum. 2011, 63, 1076-1085.

76 91. Sekine, H.; Ruiz, P.; Gilkeson, G. S.; Tomlinson, S. The dual role of complement in the

progression of renal disease in NZB/W F(1) mice and alternative pathway inhibition.

Mol. Immunol. 2011, 49, 317-323.

92. Banda, N. K.; Thurman, J. M.; Kraus, D.; Wood, A.; Carroll, M. C.; Arend, W. P.;

Holers, V. M. Alternative complement pathway activation is essential for inflammation

and joint destruction in the passive transfer model of collagen-induced arthritis. J.

Immunol. 2006, 177, 1904-1912.

93. Banda, N. K.; Takahashi, K.; Wood, A. K.; Holers, V. M.; Arend, W. P. Pathogenic

complement activation in collagen antibody-induced arthritis in mice requires

amplification by the alternative pathway. J. Immunol. 2007, 179, 4101-4109.

94. Banda, N. K.; Hyatt, S.; Antonioli, A. H.; White, J. T.; Glogowska, M.; Takahashi, K.;

Merkel, T. J.; Stahl, G. L.; Mueller-Ortiz, S.; Wetsel, R.; Arend, W. P.; Holers, V. M.

Role of C3a receptors, C5a receptors, and complement protein C6 deficiency in

collagen antibody-induced arthritis in mice. J. Immunol. 2012, 188, 1469-1478.

95. Kuhn, K. A.; Cozine, C. L.; Tomooka, B.; Robinson, W. H.; Holers, V. M.

Complement receptor CR2/CR1 deficiency protects mice from collagen-induced

arthritis and associates with reduced autoantibodies to type II collagen and citrullinated

antigens. Mol. Immunol. 2008, 45, 2808-2819.

96. Stevens, B.; Allen, N. J.; Vazquez, L. E.; Howell, G. R.; Christopherson, K. S.; Nouri,

N.; Micheva, K. D.; Mehalow, A. K.; Huberman, A. D.; Stafford, B.; Sher, A.; Litke,

A. M.; Lambris, J. D.; Smith, S. J.; John, S. W.; Barres, B. A. The classical

complement cascade mediates CNS synapse elimination. Cell 2007, 131, 1164-1178.

97. Fonseca, M. I.; Ager, R. R.; Chu, S. H.; Yazan, O.; Sanderson, S. D.; LaFerla, F. M.;

Taylor, S. M.; Woodruff, T. M.; Tenner, A. J. Treatment with a C5aR antagonist

77 decreases pathology and enhances behavioral performance in murine models of

Alzheimer's disease. J. Immunol. 2009, 183, 1375-1383.

98. Brennan, F. H.; Lee, J. D.; Ruitenberg, M. J.; Woodruff, T. M. Therapeutic targeting of

complement to modify disease course and improve outcomes in neurological

conditions. Semin. Immunol. 2016, 28, 292-308.

99. Fu, W.; Wojtkiewicz, G.; Weissleder, R.; Benoist, C.; Mathis, D. Early window of

diabetes determinism in NOD mice, dependent on the complement receptor CRIg,

identified by noninvasive imaging. Nat. Immunol. 2012, 13, 361-368.

100. Lim, J.; Iyer, A.; Suen, J. Y.; Seow, V.; Reid, R. C.; Brown, L.; Fairlie, D. P. C5aR and

C3aR antagonists each inhibit diet-induced obesity, metabolic dysfunction, and

adipocyte and macrophage signaling. FASEB J. 2013, 27, 822-831.

101. Silasi-Mansat, R.; Zhu, H.; Popescu, N. I.; Peer, G.; Sfyroera, G.; Magotti, P.; Ivanciu,

L.; Lupu, C.; Mollnes, T. E.; Taylor, F. B.; Kinasewitz, G.; Lambris, J. D.; Lupu, F.

Complement inhibition decreases the procoagulant response and confers organ

protection in a baboon model of escherichia coli sepsis. Blood 2010, 116, 1002-1010.

102. Elvington, M.; Huang, Y.; Morgan, B. P.; Qiao, F.; van Rooijen, N.; Atkinson, C.;

Tomlinson, S. A targeted complement-dependent strategy to improve the outcome of

mab therapy, and characterization in a murine model of metastatic cancer. Blood 2012,

119, 6043-6051.

103. Markiewski, M. M.; DeAngelis, R. A.; Benencia, F.; Ricklin-Lichtsteiner, S. K.;

Koutoulaki, A.; Gerard, C.; Coukos, G.; Lambris, J. D. Modulation of the antitumor

immune response by complement. Nat. Immunol. 2008, 9, 1225-1235.

104. Strey, C. W.; Markiewski, M.; Mastellos, D.; Tudoran, R.; Spruce, L. A.; Greenbaum,

L. E.; Lambris, J. D. The proinflammatory mediators C3a and C5a are essential for

liver regeneration. J. Exp. Med. 2003, 198, 913-923.

78 105. He, S.; Atkinson, C.; Qiao, F.; Cianflone, K.; Chen, X.; Tomlinson, S. A complement-

dependent balance between hepatic ischemia/reperfusion injury and liver regeneration

in mice. J. Clin. Invest. 2009, 119, 2304-2316.

106. Mastellos, D. C.; Ricklin, D.; Hajishengallis, E.; Hajishengallis, G.; Lambris, J. D.

Complement therapeutics in inflammatory diseases: Promising drug candidates for C3-

targeted intervention. Mol. Oral. Microbiol. 2016, 31, 3-17.

107. Thurman, J. M.; Le Quintrec, M. Targeting the complement cascade: Novel treatments

coming down the pike. Kidney Int. 2016, 90, 746-752.

108. Botto, M. C1q knock-out mice for the study of complement deficiency in autoimmune

disease. Exp. Clin. Immunogenet. 1998, 15, 231-234.

109. Robson, M. G.; Cook, H. T.; Botto, M.; Taylor, P. R.; Busso, N.; Salvi, R.; Pusey, C.

D.; Walport, M. J.; Davies, K. A. Accelerated nephrotoxic nephritis is exacerbated in

C1q-deficient mice. J. Immunol. 2001, 166, 6820-6828.

110. Botto, M.; Dell'Agnola, C.; Bygrave, A. E.; Thompson, E. M.; Cook, H. T.; Petry, F.;

Loos, M.; Pandolfi, P. P.; Walport, M. J. Homozygous C1q deficiency causes

glomerulonephritis associated with multiple apoptotic bodies. Nat. Genet. 1998, 19, 56-

59.

111. Mitchell, D. A.; Pickering, M. C.; Warren, J.; Fossati-Jimack, L.; Cortes-Hernandez, J.;

Cook, H. T.; Botto, M.; Walport, M. J. C1q deficiency and autoimmunity: The effects

of genetic background on disease expression. J. Immunol. 2002, 168, 2538-2543.

112. Chu, Y.; Jin, X.; Parada, I.; Pesic, A.; Stevens, B.; Barres, B.; Prince, D. A. Enhanced

synaptic connectivity and epilepsy in C1q knockout mice. Proc. Natl. Acad. Sci. U. S.

A. 2010, 107, 7975-7980.

113. La Bonte, L. R.; Pavlov, V. I.; Tan, Y. S.; Takahashi, K.; Takahashi, M.; Banda, N. K.;

Zou, C.; Fujita, T.; Stahl, G. L. Mannose-binding lectin-Associated Serine Protease-1 is

79 a significant contributor to coagulation in a murine model of occlusive thrombosis. J.

Immunol. 2012, 188, 885-891.

114. Schwaeble, W. J.; Lynch, N. J.; Clark, J. E.; Marber, M.; Samani, N. J.; Ali, Y. M.;

Dudler, T.; Parent, B.; Lhotta, K.; Wallis, R.; Farrar, C. A.; Sacks, S.; Lee, H.; Zhang,

M.; Iwaki, D.; Takahashi, M.; Fujita, T.; Tedford, C. E.; Stover, C. M. Targeting of

Mannan-binding lectin-Associated Serine Protease-2 confers protection from

myocardial and gastrointestinal ischemia/reperfusion injury. Proc. Natl. Acad. Sci. U.

S. A. 2011, 108, 7523-7528.

115. Kasanmoentalib, E. S.; Valls Seron, M.; Ferwerda, B.; Tanck, M. W.; Zwinderman, A.

H.; Baas, F.; van der Ende, A.; Brouwer, M. C.; van de Beek, D. Mannose-binding

lectin-Associated Serine Protease 2 (MASP-2) contributes to poor disease outcome in

humans and mice with pneumococcal meningitis. J. Neuroinflammation 2017, 14, 2.

116. Asgari, E.; Farrar, C. A.; Lynch, N.; Ali, Y. M.; Roscher, S.; Stover, C.; Zhou, W.;

Schwaeble, W. J.; Sacks, S. H. Mannan-binding lectin-Associated Serine Protease 2 is

critical for the development of renal ischemia reperfusion injury and mediates tissue

injury in the absence of complement C4. FASEB J. 2014, 28, 3996-4003.

117. Orsini, F.; Chrysanthou, E.; Dudler, T.; Cummings, W. J.; Takahashi, M.; Fujita, T.;

Demopulos, G.; De Simoni, M. G.; Schwaeble, W. Mannan binding lectin-Associated

Serine Protease-2 (MASP-2) critically contributes to post-ischemic brain injury

independent of MASP-1. J. Neuroinflammation 2016, 13, 213.

118. Chen, Z.; Koralov, S. B.; Kelsoe, G. Complement C4 inhibits systemic autoimmunity

through a mechanism independent of complement receptors CR1 and CR2. J. Exp.

Med. 2000, 192, 1339-1352.

80 119. Rohrer, B.; Guo, Y.; Kunchithapautham, K.; Gilkeson, G. S. Eliminating complement

factor D reduces photoreceptor susceptibility to light-induced damage. Invest.

Ophthalmol. Vis. Sci. 2007, 48, 5282-5289.

120. Helmy, K. Y.; Katschke, K. J., Jr.; Gorgani, N. N.; Kljavin, N. M.; Elliott, J. M.; Diehl,

L.; Scales, S. J.; Ghilardi, N.; van Lookeren Campagne, M. CRIg: A macrophage

complement receptor required for phagocytosis of circulating pathogens. Cell 2006,

124, 915-927.

121. Boackle, S. A.; Culhane, K. K.; Brown, J. M.; Haas, M.; Bao, L.; Quigg, R. J.; Holers,

V. M. CR1/CR2 deficiency alters IgG3 autoantibody production and IgA glomerular

deposition in the MRL/lpr model of sle. Autoimmunity 2004, 37, 111-123.

122. Wu, X.; Jiang, N.; Deppong, C.; Singh, J.; Dolecki, G.; Mao, D.; Morel, L.; Molina, H.

D. A role for the CR2 gene in modifying autoantibody production in systemic lupus

erythematosus. J. Immunol. 2002, 169, 1587-1592.

123. Yan, J.; Vetvicka, V.; Xia, Y.; Hanikyrova, M.; Mayadas, T. N.; Ross, G. D. Critical

role of kupffer cell CR3 (CD11b/CD18) in the clearance of IgM-opsonized

erythrocytes or soluble beta-glucan. Immunopharmacology 2000, 46, 39-54.

124. Morrison, T. E.; Simmons, J. D.; Heise, M. T. Complement receptor 3 promotes severe

ross river virus-induced disease. J. Virol. 2008, 82, 11263-11272.

125. Lee, H.; Whitfeld, P. L.; Mackay, C. R. Receptors for complement C5a. The

importance of C5aR and the enigmatic role of C5L2. Immunol. Cell Biol. 2008, 86,

153-160.

126. Sekar, A.; Bialas, A. R.; de Rivera, H.; Davis, A.; Hammond, T. R.; Kamitaki, N.;

Tooley, K.; Presumey, J.; Baum, M.; Van Doren, V.; Genovese, G.; Rose, S. A.;

Handsaker, R. E.; Daly, M. J.; Carroll, M. C.; Stevens, B.; McCarroll, S. A.; Grp, S. W.

81 Schizophrenia risk from complex variation of complement component 4. Nature 2016,

530, 177-183.

127. Gold, B.; Merriam, J. E.; Zernant, J.; Hancox, L. S.; Taiber, A. J.; Gehrs, K.; Cramer,

K.; Neel, J.; Bergeron, J.; Barile, G. R.; Smith, R. T.; Dean, M.; Allikmets, R.; Grp, A.

G. C. S. Variation in factor B (Bf) and complement component 2 (C2) genes is

associated with age-related macular degeneration. Nat. Genet. 2006, 38, 458-462.

128. Harris, C. L.; Heurich, M.; de Cordoba, S. R.; Morgan, B. P. The complotype:

Dictating risk for inflammation and infection. Trends Immunol. 2012, 33, 513-521.

129. Haines, J. L.; Hauser, M. A.; Schmidt, S.; Scott, W. K.; Olson, L. M.; Gallins, P.;

Spencer, K. L.; Kwan, S. Y.; Noureddine, M.; Gilbert, J. R.; Schnetz-Boutaud, N.;

Agarwal, A.; Postel, E. A.; Pericak-Vance, M. A. Complement factor H variant

increases the risk of age-related macular degeneration. Science 2005, 308, 419-421.

130. Edwards, A. O.; Ritter, R.; Abel, K. J.; Manning, A.; Panhuysen, C.; Farrer, L. A.

Complement factor H polymorphism and age-related macular degeneration. Science

2005, 308, 421-424.

131. Klein, R. J.; Zeiss, C.; Chew, E. Y.; Tsai, J. Y.; Sackler, R. S.; Haynes, C.; Henning, A.

K.; SanGiovanni, J. P.; Mane, S. M.; Mayne, S. T.; Bracken, M. B.; Ferris, F. L.; Ott,

J.; Barnstable, C.; Hoh, J. Complement factor H polymorphism in age-related macular

degeneration. Science 2005, 308, 385-389.

132. Pickering, M. C.; de Jorge, E. G.; Martinez-Barricarte, R.; Recalde, S.; Garcia-Layana,

A.; Rose, K. L.; Moss, J.; Walport, M. J.; Cook, H. T.; de Cordoba, S. R.; Botto, M.

Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking

surface recognition domains. J. Exp. Med. 2007, 204, 1249-1256.

133. Habibi, I.; Sfar, I.; Kort, F.; Bouraoui, R.; Chebil, A.; Limaiem, R.; Ayed, S.; Ben

Abdallah, T.; El Matri, L.; Gorgi, Y. Complement component C3 variant (R102G) and

82 the risk of neovascular age-related macular degeneration in a tunisian population.

Klinische. Monatsblatter. Fur. Augenheilkunde. 2017, 234, 478-482.

134. Lambert, J. C.; Heath, S.; Even, G.; Campion, D.; Sleegers, K.; Hiltunen, M.;

Combarros, O.; Zelenika, D.; Bullido, M. J.; Tavernier, B.; Letenneur, L.; Bettens, K.;

Berr, C.; Pasquier, F.; Fievet, N.; Barberger-Gateau, P.; Engelborghs, S.; De Deyn, P.;

Mateo, I.; Franck, A.; Helisalmi, S.; Porcellini, E.; Hanon, O.; de Pancorbo, M. M.;

Lendon, C.; Dufouil, C.; Jaillard, C.; Leveillard, T.; Alvarez, V.; Bosco, P.; Mancuso,

M.; Panza, F.; Nacmias, B.; Bossu, P.; Piccardi, P.; Annoni, G.; Seripa, D.; Galimberti,

D.; Hannequin, D.; Licastro, F.; Soininen, H.; Ritchie, K.; Blanche, H.; Dartigues, J. F.;

Tzourio, C.; Gut, I.; Van Broeckhoven, C.; Alperovitch, A.; Lathrop, M.; Amouyel, P.;

Initiative, E. A. D. Genome-wide association study identifies variants at CLU and CR1

associated with Alzheimer's disease. Nat. Genet. 2009, 41, 1094-1099.

135. Kurreeman, F. A. S.; Padyukov, L.; Marques, R. B.; Schrodi, S. J.; Seddighzadeh, M.;

Stoeken-Rijsbergen, G.; van der Helm-van Mil, A. H. M.; Allaart, C. F.; Verduyn, W.;

Houwing-Duistermaat, J.; Alfredsson, L.; Begovich, B.; Klareskog, L.; Huizinga, T. W.

J.; Toes, R. E. M. A candidate gene approach identifies the TRAF1/C5 region as a risk

factor for rheumatoid arthritis. PloS Med. 2007, 4, e278.

136. Zhernakova, A.; Stahl, E. A.; Trynka, G.; Raychaudhuri, S.; Festen, E.; Franke, L.;

Fehrmann, R. S. N.; Kurreeman, F. A. S.; Thomson, B.; Gupta, N.; Romanos, J.;

McManus, R.; Ryan, A. W.; Turner, G.; Remmers, E. F.; Greco, L.; Toes, R.;

Grandone, E.; Mazzilli, M. C.; Rybak, A.; Cukrowska, B.; Li, Y. H.; de Bakker, P. I.

W.; Gregersen, P. K.; Worthington, J.; Siminovitch, K. A.; Klareskog, L.; Huizinga, T.

W. J.; Wijmenga, C.; Plenge, R. M. Meta-analysis of Genome-Wide Association

Studies in celiac disease and rheumatoid arthritis identifies fourteen non-HLA shared

loci. PLoS Genet. 2011, 7, e1002004.

83

137. Jeong, J. C.; Hwang, Y. H.; Kim, H.; Ro, H.; Park, H. C.; Kim, Y. J.; Kim, M. G.; Ha,

J.; Park, M. H.; Chae, D. W.; Ahn, C.; Yang, J. Association of complement 5 genetic

polymorphism with renal allograft outcomes in Korea. Nephrol. Dialysis Transplant.

2011, 26, 3378-3385.

138. Song, J. J.; Hwang, I.; Cho, K. H.; Garcia, M. A.; Kim, A. J.; Wang, T. H.; Lindstrom,

T. M.; Lee, A. T.; Nishimura, T.; Zhao, L.; Morser, J.; Nesheim, M.; Goodman, S. B.;

Lee, D. M.; Bridges, S. L.; Gregersen, P. K.; Leung, L. L.; Robinson, W. H.;

Evaluation, C. L. Plasma carboxypeptidase B downregulates inflammatory responses in

autoimmune arthritis. J. Clin. Invest. 2011, 121, 3517-3527.

139. Nishiguchi, K. M.; Yasuma, T. R.; Tomida, D.; Nakamura, M.; Ishikawa, K.; Kikuchi,

M.; Ohmi, Y.; Niwa, T.; Hamajima, N.; Furukawa, K.; Terasaki, H. C9-R95X

polymorphism in patients with neovascular age-related macular degeneration. Invest.

Ophthalmol. Vis. Sci. 2012, 53, 508-512.

140. Kouser, L.; Madhukaran, S. P.; Shastri, A.; Saraon, A.; Ferluga, J.; Al-Mozaini, M.;

Kishore, U. Emerging and novel functions of complement protein C1q. Front Immunol.

2015, 6, 317.

141. Roos, A.; Nauta, A. J.; Broers, D.; Faber-Krol, M. C.; Trouw, L. A.; Drijfhout, J. W.;

Daha, M. R. Specific inhibition of the classical complement pathway by C1q-binding

peptides. J. Immunol. 2001, 167, 7052-7059.

142. Gronemus, J. Q.; Hair, P. S.; Crawford, K. B.; Nyalwidhe, J. O.; Cunnion, K. M.;

Krishna, N. K. Potent inhibition of the classical pathway of complement by a novel

C1q-binding peptide derived from the human astrovirus coat protein. Mol. Immunol.

2010, 48, 305-313.

84 143. Mauriello, C. T.; Pallera, H. K.; Sharp, J. A.; Woltmann, J. L., Jr.; Qian, S.; Hair, P. S.;

van der Pol, P.; van Kooten, C.; Thielens, N. M.; Lattanzio, F. A.; Cunnion, K. M.;

Krishna, N. K. A novel peptide inhibitor of classical and lectin complement activation

including ABO incompatibility. Mol. Immunol. 2013, 53, 132-139.

144. Sharp, J. A.; Hair, P. S.; Pallera, H. K.; Kumar, P. S.; Mauriello, C. T.; Nyalwidhe, J.

O.; Phelps, C. A.; Park, D.; Thielens, N. M.; Pascal, S. M.; Chen, W.; Duffy, D. M.;

Lattanzio, F. A.; Cunnion, K. M.; Krishna, N. K. Peptide inhibitor of complement C1

rapidly inhibits complement activation after intravascular injection in rats. PLoS. One

2015, 10, e0132446.

145. Gal, P.; Dobo, J.; Zavodszky, P.; Sim, R. B. Early complement proteases: C1r, C1s and

MASPs. A structural insight into activation and functions. Mol. Immunol. 2009, 46,

2745-2752.

146. Rossi, V.; Bally, I.; Lacroix, M.; Arlaud, G. J.; Thielens, N. M. Classical complement

pathway components C1r and C1s: Purification from human serum and in recombinant

form and functional characterization. Methods Mol. Biol. 2014, 1100, 43-60.

147. Qu, H.; Ricklin, D.; Lambris, J. D. Recent developments in low molecular weight

complement inhibitors. Mol. Immunol. 2009, 47, 185-195.

148. Aoyama, T.; Ino, Y.; Ozeki, M.; Oda, M.; Sato, T.; Koshiyama, Y.; Suzuki, S.; Fujita,

M. Pharmacological studies of FUT-175, nafamstat mesilate. I. Inhibition of protease

activity in in vitro and in vivo experiments. Jpn. J. Pharmacol. 1984, 35, 203-227.

149. Tagawa, T. Protease inhibitor nafamostat mesilate attenuates complement activation

and improves function of xenografts in a discordant lung perfusion model.

Xenotransplantation 2011, 18, 315-319.

150. Schwertz, H.; Carter, J. M.; Russ, M.; Schubert, S.; Schlitt, A.; Buerke, U.; Schmidt,

M.; Hillen, H.; Werdan, K.; Buerke, M. Serine protease inhibitor nafamostat given

85 before reperfusion reduces inflammatory myocardial injury by complement and

neutrophil inhibition. J. Cardiovasc. Pharmacol. 2008, 52, 151-160.

151. Li, C.; Wang, J.; Fang, Y.; Liu, Y.; Chen, T.; Sun, H.; Zhou, X. F.; Liao, H. Nafamostat

mesilate improves function recovery after stroke by inhibiting neuroinflammation in

rats. Brain Behav. Immun. 2016, 56, 230-245.

152. Liu, Y.; Li, C.; Wang, J.; Fang, Y.; Sun, H.; Tao, X.; Zhou, X. F.; Liao, H. Nafamostat

mesilate improves neurological outcome and axonal regeneration after stroke in rats.

Mol. Neurobiol. 2017, 54, 4217-4231.

153. Ikari, N.; Sakai, Y.; Hitomi, Y.; Fujii, S. New synthetic inhibitor to the alternative

complement pathway. Immunology 1983, 49, 685-691.

154. Szalai, A. J.; Digerness, S. B.; Agrawal, A.; Kearney, J. F.; Bucy, R. P.; Niwas, S.;

Kilpatrick, J. M.; Babu, Y. S.; Volanakis, J. E. The arthus reaction in rodents: Species-

specific requirement of complement. J. Immunol. 2000, 164, 463-468.

155. Morikis, D.; Lambris, J. D. Structural aspects and design of low-molecular-mass

complement inhibitors. Biochem. Soc. Trans. 2002, 30, 1026-1036.

156. Pugsley, M. K.; Abramova, M.; Cole, T.; Yang, X.; Ammons, W. S. Inhibitors of the

complement system currently in development for cardiovascular disease. Cardiovasc.

Toxicol. 2003, 3, 43-70.

157. Subasinghe, N. L.; Travins, J. M.; Ali, F.; Huang, H.; Ballentine, S. K.; Marugan, J. J.;

Khalil, E.; Hufnagel, H. R.; Bone, R. F.; DesJarlais, R. L.; Crysler, C. S.; Ninan, N.;

Cummings, M. D.; Molloy, C. J.; Tomczuk, B. E. A novel series of

arylsulfonylthiophene-2-carboxamidine inhibitors of the complement component C1s.

Bioorg. Med. Chem. Lett. 2006, 16, 2200-2204.

158. Subasinghe, N. L.; Ali, F.; Illig, C. R.; Jonathan Rudolph, M.; Klein, S.; Khalil, E.;

Soll, R. M.; Bone, R. F.; Spurlino, J. C.; DesJarlais, R. L.; Crysler, C. S.; Cummings,

86 M. D.; Morris, P. E., Jr.; Kilpatrick, J. M.; Sudhakara Babu, Y. A novel series of potent

and selective small molecule inhibitors of the complement component C1s. Bioorg.

Med. Chem. Lett. 2004, 14, 3043-3047.

159. Subasinghe, N. L.; Khalil, E.; Travins, J. M.; Ali, F.; Ballentine, S. K.; Hufnagel, H. R.;

Pan, W.; Leonard, K.; Bone, R. F.; Soll, R. M.; Crysler, C. S.; Ninan, N.; Kirkpatrick,

J.; Kolpak, M. X.; Diloreto, K. A.; Eisennagel, S. H.; Huebert, N. D.; Molloy, C. J.;

Tomczuk, B. E.; Gaul, M. D. Design and synthesis of polyethylene glycol-modified

biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component

C1s. Bioorg. Med. Chem. Lett. 2012, 22, 5303-5307.

160. Buerke, M.; Schwertz, H.; Seitz, W.; Meyer, J.; Darius, H. Novel small molecule

inhibitor of C1s exerts cardioprotective effects in ischemia-reperfusion injury in

rabbits. J. Immunol. 2001, 167, 5375-5380.

161. Sekine, H.; Takahashi, M.; Iwaki, D.; Fujita, T. The role of MASP-1/3 in complement

activation. Adv. Exp. Med. Biol. 2013, 735, 41-53.

162. Vorup-Jensen, T.; Petersen, S. V.; Hansen, A. G.; Poulsen, K.; Schwaeble, W.; Sim, R.

B.; Reid, K. B.; Davis, S. J.; Thiel, S.; Jensenius, J. C. Distinct pathways of mannan-

binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of

MBL with recombinant MBL-Associated Serine Protease-2. J. Immunol. 2000, 165,

2093-2100.

163. Chen, C. B.; Wallis, R. Two mechanisms for mannose-binding protein modulation of

the activity of its associated serine proteases. J. Biol. Chem. 2004, 279, 26058-26065.

164. Kocsis, A.; Kekesi, K. A.; Szasz, R.; Vegh, B. M.; Balczer, J.; Dobo, J.; Zavodszky, P.;

Gal, P.; Pal, G. Selective inhibition of the lectin pathway of complement with phage

display selected peptides against Mannose-binding lectin-Associated Serine Protease

87 (MASP)-1 and -2: Significant contribution of MASP-1 to lectin pathway activation. J.

Immunol. 2010, 185, 4169-4178.

165. Heja, D.; Harmat, V.; Fodor, K.; Wilmanns, M.; Dobo, J.; Kekesi, K. A.; Zavodszky,

P.; Gal, P.; Pal, G. Monospecific inhibitors show that both Mannan-binding lectin-

Associated Serine Protease-1 (MASP-1) and -2 are essential for lectin pathway

activation and reveal structural plasticity of MASP-2. J. Biol. Chem. 2012, 287, 20290-

20300.

166. Oroszlan, G.; Kortvely, E.; Szakacs, D.; Kocsis, A.; Dammeier, S.; Zeck, A.; Ueffing,

M.; Zavodszky, P.; Pal, G.; Gal, P.; Dobo, J. MASP-1 and MASP-2 do not activate pro-

factor D in resting human blood, whereas MASP-3 is a potential activator: Kinetic

analysis involving specific MASP-1 and MASP-2 inhibitors. J. Immunol. 2016, 196,

857-865.

167. Kerr, F. K.; O'Brien, G.; Quinsey, N. S.; Whisstock, J. C.; Boyd, S.; de la Banda, M.

G.; Kaiserman, D.; Matthews, A. Y.; Bird, P. I.; Pike, R. N. Elucidation of the substrate

specificity of the C1s protease of the classical complement pathway. J. Biol. Chem.

2005, 280, 39510-39514.

168. Kerr, F. K.; Thomas, A. R.; Wijeyewickrema, L. C.; Whisstock, J. C.; Boyd, S. E.;

Kaiserman, D.; Matthews, A. Y.; Bird, P. I.; Thielens, N. M.; Rossi, V.; Pike, R. N.

Elucidation of the substrate specificity of the MASP-2 protease of the lectin

complement pathway and identification of the enzyme as a major physiological target

of the serpin, C1-inhibitor. Mol. Immunol. 2008, 45, 670-677.

169. Wijeyewickrema, L. C.; Yongqing, T.; Tran, T. P.; Thompson, P. E.; Viljoen, J. E.;

Coetzer, T. H.; Duncan, R. C.; Kass, I.; Buckle, A. M.; Pike, R. N. Molecular

determinants of the substrate specificity of the complement-initiating protease, C1r. J.

Biol. Chem. 2013, 288, 15571-15580.

88 170. Duncan, R. C.; Mohlin, F.; Taleski, D.; Coetzer, T. H.; Huntington, J. A.; Payne, R. J.;

Blom, A. M.; Pike, R. N.; Wijeyewickrema, L. C. Identification of a catalytic exosite

for complement component C4 on the serine protease domain of C1s. J. Immunol.

2012, 189, 2365-2373.

171. Duncan, R. C.; Bergstrom, F.; Coetzer, T. H.; Blom, A. M.; Wijeyewickrema, L. C.;

Pike, R. N. Multiple domains of MASP-2, an initiating complement protease, are

required for interaction with its substrate C4. Mol. Immunol. 2012, 49, 593-600.

172. Drentin, N.; Conroy, P.; Gunzburg, M. J.; Pike, R. N.; Wijeyewickrema, L. C.

Investigation of the mechanism of interaction between Mannose-binding lectin-

Associated Serine Protease-2 and complement C4. Mol. Immunol. 2015, 67, 287-293.

173. Kidmose, R. T.; Laursen, N. S.; Dobo, J.; Kjaer, T. R.; Sirotkina, S.; Yatime, L.;

Sottrup-Jensen, L.; Thiel, S.; Gal, P.; Andersen, G. R. Structural basis for activation of

the complement system by component C4 cleavage. Proc. Natl. Acad. Sci. U. S. A.

2012, 109, 15425-15430.

174. Halili, M. A.; Ruiz-Gomez, G.; Le, G. T.; Abbenante, G.; Fairlie, D. P. Complement

component C2, inhibiting a latent serine protease in the classical pathway of

complement activation. Biochemistry 2009, 48, 8466-8472.

175. Sahu, A.; Kay, B. K.; Lambris, J. D. Inhibition of human complement by a C3-binding

peptide isolated from a phage-displayed random peptide library. J. Immunol. 1996, 157,

884-891.

176. Sahu, A.; Soulika, A. M.; Morikis, D.; Spruce, L.; Moore, W. T.; Lambris, J. D.

Binding kinetics, structure-activity relationship, and biotransformation of the

complement inhibitor compstatin. J. Immunol. 2000, 165, 2491-2499.

89 177. Chi, Z. L.; Yoshida, T.; Lambris, J. D.; Iwata, T. Suppression of drusen formation by

compstatin, a peptide inhibitor of complement C3 activation, on cynomolgus monkey

with early-onset macular degeneration. Adv. Exp. Med. Biol. 2010, 703, 127-135.

178. Katragadda, M.; Morikis, D.; Lambris, J. D. Thermodynamic studies on the interaction

of the third complement component and its inhibitor, compstatin. J. Biol. Chem. 2004,

279, 54987-54995.

179. Qu, H.; Magotti, P.; Ricklin, D.; Wu, E. L.; Kourtzelis, I.; Wu, Y. Q.; Kaznessis, Y. N.;

Lambris, J. D. Novel analogues of the therapeutic complement inhibitor compstatin

with significantly improved affinity and potency. Mol. Immunol. 2011, 48, 481-489.

180. Knerr, P. J.; Tzekou, A.; Ricklin, D.; Qu, H.; Chen, H.; van der Donk, W. A.; Lambris,

J. D. Synthesis and activity of thioether-containing analogues of the complement

inhibitor compstatin. ACS Chem. Biol. 2011, 6, 753-760.

181. Katragadda, M.; Magotti, P.; Sfyroera, G.; Lambris, J. D. Hydrophobic effect and

hydrogen bonds account for the improved activity of a complement inhibitor,

compstatin. J. Med. Chem. 2006, 49, 4616-4622.

182. Qu, H.; Ricklin, D.; Bai, H.; Chen, H.; Reis, E. S.; Maciejewski, M.; Tzekou, A.;

DeAngelis, R. A.; Resuello, R. R.; Lupu, F.; Barlow, P. N.; Lambris, J. D. New analogs

of the clinical complement inhibitor compstatin with subnanomolar affinity and

enhanced pharmacokinetic properties. Immunobiology 2013, 218, 496-505.

183. Zhang, Y.; Shao, D.; Ricklin, D.; Hilkin, B. M.; Nester, C. M.; Lambris, J. D.; Smith,

R. J. Compstatin analog Cp40 inhibits complement dysregulation in vitro in C3

glomerulopathy. Immunobiology 2015, 220, 993-998.

184. Maekawa, T.; Abe, T.; Hajishengallis, E.; Hosur, K. B.; DeAngelis, R. A.; Ricklin, D.;

Lambris, J. D.; Hajishengallis, G. Genetic and intervention studies implicating

90 complement C3 as a major target for the treatment of periodontitis. J. Immunol. 2014,

192, 6020-6027.

185. Reis, E. S.; DeAngelis, R. A.; Chen, H.; Resuello, R. R.; Ricklin, D.; Lambris, J. D.

Therapeutic C3 inhibitor Cp40 abrogates complement activation induced by modern

hemodialysis filters. Immunobiology 2015, 220, 476-482.

186. Risitano, A. M.; Ricklin, D.; Huang, Y.; Reis, E. S.; Chen, H.; Ricci, P.; Lin, Z.;

Pascariello, C.; Raia, M.; Sica, M.; Del Vecchio, L.; Pane, F.; Lupu, F.; Notaro, R.;

Resuello, R. R.; DeAngelis, R. A.; Lambris, J. D. Peptide inhibitors of C3 activation as

a novel strategy of complement inhibition for the treatment of paroxysmal nocturnal

hemoglobinuria. Blood 2014, 123, 2094-2101.

187. Le, G. T.; Abbenante, G.; Fairlie, D. P. Profiling the enzymatic properties and

inhibition of human complement factor B. J. Biol. Chem. 2007, 282, 34809-34816.

188. Ruiz-Gomez, G.; Lim, J.; Halili, M. A.; Le, G. T.; Madala, P. K.; Abbenante, G.;

Fairlie, D. P. Structure-activity relationships for substrate-based inhibitors of human

complement factor B. J. Med. Chem. 2009, 52, 6042-6052.

189. Leung, D.; Schroder, K.; White, H.; Fang, N. X.; Stoermer, M. J.; Abbenante, G.;

Martin, J. L.; Young, P. R.; Fairlie, D. P. Activity of recombinant dengue 2 virus NS3

protease in the presence of a truncated NS2B co-factor, small peptide substrates, and

inhibitors. J. Biol. Chem. 2001, 276, 45762-45771.

190. Nall, T. A.; Chappell, K. J.; Stoermer, M. J.; Fang, N. X.; Tyndall, J. D.; Young, P. R.;

Fairlie, D. P. Enzymatic characterization and homology model of a catalytically active

recombinant West Nile virus NS3 protease. J. Biol. Chem. 2004, 279, 48535-48542.

191. Vulpetti, A.; Randl, S.; Rudisser, S.; Ostermann, N.; Erbel, P.; Mac Sweeney, A.;

Zoller, T.; Salem, B.; Gerhartz, B.; Cumin, F.; Hommel, U.; Dalvit, C.; Lorthiois, E.;

Maibaum, J. Structure-based library design and fragment screening for the

91 identification of reversible complement factor D protease inhibitors. J. Med. Chem.

2017, 60, 1946-1958.

192. Yuan, X.; Gavriilaki, E.; Thanassi, J. A.; Yang, G.; Baines, A. C.; Podos, S. D.; Huang,

Y.; Huang, M.; Brodsky, R. A. Small-molecule factor D inhibitors selectively block the

alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and

atypical hemolytic uremic syndrome. Haematologica 2017, 102, 466-475.

193. Klos, A.; Tenner, A. J.; Johswich, K. O.; Ager, R. R.; Reis, E. S.; Kohl, J. The role of

the anaphylatoxins in health and disease. Mol. Immunol. 2009, 46, 2753-2766.

194. Hohenhaus, D. M.; Schaale, K.; Le Cao, K. A.; Seow, V.; Iyer, A.; Fairlie, D. P.;

Sweet, M. J. An mRNA atlas of G protein-coupled receptor expression during primary

human monocyte/macrophage differentiation and lipopolysaccharide-mediated

activation identifies targetable candidate regulators of inflammation. Immunobiology

2013, 218, 1345-1353.

195. Morgan, E. L.; Weigle, W. O.; Hugli, T. E. Anaphylatoxin-mediated regulation of

human and murine immune responses. Fed. Proc. 1984, 43, 2543-2547.

196. Humbles, A. A.; Lu, B.; Nilsson, C. A.; Lilly, C.; Israel, E.; Fujiwara, Y.; Gerard, N.

P.; Gerard, C. A role for the C3a anaphylatoxin receptor in the effector phase of

asthma. Nature 2000, 406, 998-1001.

197. Bautsch, W.; Hoymann, H. G.; Zhang, Q.; Meier-Wiedenbach, I.; Raschke, U.; Ames,

R. S.; Sohns, B.; Flemme, N.; Meyer zu Vilsendorf, A.; Grove, M.; Klos, A.; Kohl, J.

Cutting edge: Guinea pigs with a natural C3a-receptor defect exhibit decreased

bronchoconstriction in allergic airway disease: Evidence for an involvement of the C3a

anaphylatoxin in the pathogenesis of asthma. J. Immunol. 2000, 165, 5401-5405.

198. Sacks, S. H.; Zhou, W. The role of complement in the early immune response to

transplantation. Nat. Rev. Immunol. 2012, 12, 431-442.

92 199. Scully, C. C.; Blakeney, J. S.; Singh, R.; Hoang, H. N.; Abbenante, G.; Reid, R. C.;

Fairlie, D. P. Selective hexapeptide agonists and antagonists for human complement

C3a receptor. J. Med. Chem. 2010, 53, 4938-4948.

200. Ames, R. S.; Lee, D.; Foley, J. J.; Jurewicz, A. J.; Tornetta, M. A.; Bautsch, W.;

Settmacher, B.; Klos, A.; Erhard, K. F.; Cousins, R. D.; Sulpizio, A. C.; Hieble, J. P.;

McCafferty, G.; Ward, K. W.; Adams, J. L.; Bondinell, W. E.; Underwood, D. C.;

Osborn, R. R.; Badger, A. M.; Sarau, H. M. Identification of a selective nonpeptide

antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory

activity in animal models. J. Immunol. 2001, 166, 6341-6348.

201. Reid, R. C.; Yau, M. K.; Singh, R.; Hamidon, J. K.; Lim, J.; Stoermer, M. J.; Fairlie, D.

P. Potent heterocyclic ligands for human complement C3a receptor. J. Med. Chem.

2014, 57, 8459-8470.

202. Hutamekalin, P.; Takeda, K.; Tani, M.; Tsuga, Y.; Ogawa, N.; Mizutani, N.; Yoshino,

S. Effect of the C3a-receptor antagonist SB290157 on anti-OVA polyclonal antibody-

induced arthritis. J. Pharmacol. Sci. 2010, 112, 56-63.

203. Mizutani, N.; Nabe, T.; Yoshino, S. Complement C3a regulates late asthmatic response

and airway hyperresponsiveness in mice. J. Immunol. 2009, 183, 4039-4046.

204. Lim, J.; Iyer, A.; Suen, J. Y.; Seow, V.; Reid, R. C.; Brown, L.; Fairlie, D. P. C5aR and

C3aR antagonists each inhibit diet-induced obesity, metabolic dysfunction, and

adipocyte and macrophage signaling. FASEB J. 2013, 27, 822-831.

205. Mathieu, M. C.; Sawyer, N.; Greig, G. M.; Hamel, M.; Kargman, S.; Ducharme, Y.;

Lau, C. K.; Friesen, R. W.; O'Neill, G. P.; Gervais, F. G.; Therien, A. G. The C3a

receptor antagonist SB290157 has agonist activity. Immunol. Lett. 2005, 100, 139-145.

206. Denonne, F.; Binet, S.; Burton, M.; Collart, P.; Dipesa, A.; Ganguly, T.; Giannaras, A.;

Kumar, S.; Lewis, T.; Maounis, F.; Nicolas, J.-M.; Mansley, T.; Pasau, P.; Preda, D.;

93 Stebbins, K.; Volosov, A.; Zou, D. Discovery of new C3aR ligands. Part 1: Arginine

derivatives. Bioorg. Med. Chem. Lett. 2007, 17, 3258-3261.

207. Denonne, F.; Binet, S.; Burton, M.; Collart, P.; Defays, S.; Dipesa, A.; Eckert, M.;

Giannaras, A.; Kumar, S.; Levine, B.; Nicolas, J. M.; Pasau, P.; Pegurier, C.; Preda, D.;

Van houtvin, N.; Volosov, A.; Zou, D. Discovery of new C3aR ligands. Part 2: Amino-

piperidine derivatives. Bioorg. Med. Chem. Lett. 2007, 17, 3262-3265.

208. Reid, R. C.; Yau, M. K.; Singh, R.; Lim, J.; Fairlie, D. P. Stereoelectronic effects

dictate molecular conformation and biological function of heterocyclic amides. J. Am.

Chem. Soc. 2014, 136, 11914-11917.

209. van Lookeren Campagne, M.; Wiesmann, C.; Brown, E. J. Macrophage complement

receptors and pathogen clearance. Cell Microbiol. 2007, 9, 2095-2102.

210. Bansal, V. S.; Vaidya, S.; Somers, E. P.; Kanuga, M.; Shevell, D.; Weikel, R.; Detmers,

P. A. Small molecule antagonists of complement receptor type 3 block adhesion and

adhesion-dependent oxidative burst in human polymorphonuclear leukocytes. J.

Pharmacol. Exp. Ther. 2003, 304, 1016-1024.

211. Ricklin, D.; Lambris, J. D. New milestones ahead in complement-targeted therapy.

Semin. Immunol. 2016, 28, 208-222.

212. Seow, V.; Lim, J.; Cotterell, A. J.; Yau, M. K.; Xu, W.; Lohman, R. J.; Kok, W. M.;

Stoermer, M. J.; Sweet, M. J.; Reid, R. C.; Suen, J. Y.; Fairlie, D. P. Receptor residence

time trumps drug-likeness and oral bioavailability in determining efficacy of

complement C5a antagonists. Sci. Rep. 2016, 6, 24575.

213. Monk, P. N.; Scola, A. M.; Madala, P.; Fairlie, D. P. Function, structure and therapeutic

potential of complement C5a receptors. Br. J. Pharmacol. 2007, 152, 429-448.

214. Wong, A. K.; Finch, A. M.; Pierens, G. K.; Craik, D. J.; Taylor, S. M.; Fairlie, D. P.

Small molecular probes for G-protein-coupled C5a receptors: Conformationally

94 constrained antagonists derived from the C terminus of the human plasma protein C5a.

J. Med. Chem. 1998, 41, 3417-3425.

215. Finch, A. M.; Wong, A. K.; Paczkowski, N. J.; Wadi, S. K.; Craik, D. J.; Fairlie, D. P.;

Taylor, S. M. Low-molecular-weight peptidic and cyclic antagonists of the receptor for

the complement factor C5a. J. Med. Chem. 1999, 42, 1965-1974.

216. March, D. R.; Proctor, L. M.; Stoermer, M. J.; Sbaglia, R.; Abbenante, G.; Reid, R. C.;

Woodruff, T. M.; Wadi, K.; Paczkowski, N.; Tyndall, J. D.; Taylor, S. M.; Fairlie, D. P.

Potent cyclic antagonists of the complement C5a receptor on human

polymorphonuclear leukocytes. Relationships between structures and activity. Mol.

Pharmacol. 2004, 65, 868-879.

217. Kohl, J. Drug evaluation: The C5a receptor antagonist PMX-53. Curr. Opin. Mol. Ther.

2006, 8, 529-538.

218. Vergunst, C. E.; Gerlag, D. M.; Dinant, H.; Schulz, L.; Vinkenoog, M.; Smeets, T. J.;

Sanders, M. E.; Reedquist, K. A.; Tak, P. P. Blocking the receptor for C5a in patients

with rheumatoid arthritis does not reduce synovial inflammation. Rheumatology

(Oxford) 2007, 46, 1773-1778.

219. Lee, J. D.; Kumar, V.; Fung, J. N.; Ruitenberg, M. J.; Noakes, P. G.; Woodruff, T. M.

Pharmacological inhibition of complement C5a-C5a1 receptor signalling ameliorates

disease pathology in the hSOD1G93A mouse model of amyotrophic lateral sclerosis.

Br. J. Pharmacol. 2017, 174, 689-699.

220. Staab, E. B.; Sanderson, S. D.; Wells, S. M.; Poole, J. A. Treatment with the C5a

receptor/CD88 antagonist PMX205 reduces inflammation in a murine model of allergic

asthma. Int. Immunopharmacol. 2014, 21, 293-300.

221. Waters, S. M.; Brodbeck, R. M.; Steflik, J.; Yu, J.; Baltazar, C.; Peck, A. E.; Severance,

D.; Zhang, L. Y.; Currie, K.; Chenard, B. L.; Hutchison, A. J.; Maynard, G.; Krause, J.

95 E. Molecular characterization of the gerbil C5a receptor and identification of a

transmembrane domain V amino acid that is crucial for small molecule antagonist

interaction. J. Biol. Chem. 2005, 280, 40617-40623.

222. Gong, Y.; Barbay, J. K.; Buntinx, M.; Li, J.; Wauwe, J. V.; Claes, C.; Lommen, G. V.;

Hornby, P. J.; He, W. Design and optimization of aniline-substituted

tetrahydroquinoline C5a receptor antagonists. Bioorg. Med. Chem. Lett. 2008, 18,

3852-3855.

223. Blagg, J.; Mowbray, C.; Pryde, D. C.; Salmon, G.; Schmid, E.; Fairman, D.; Beaumont,

K. Small, non-peptide C5a receptor antagonists: Part 1. Bioorg. Med. Chem. Lett. 2008,

18, 5601-5604.

224. Brodbeck, R. M.; Cortright, D. N.; Kieltyka, A. P.; Yu, J.; Baltazar, C. O.; Buck, M. E.;

Meade, R.; Maynard, G. D.; Thurkauf, A.; Chien, D. S.; Hutchison, A. J.; Krause, J. E.

Identification and characterization of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-

ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a novel, orally

bioavailable C5a receptor inverse agonist. J. Pharmacol. Exp. Ther. 2008, 327, 898-

909.

225. Moriconi, A.; Cunha, T. M.; Souza, G. R.; Lopes, A. H.; Cunha, F. Q.; Carneiro, V. L.;

Pinto, L. G.; Brandolini, L.; Aramini, A.; Bizzarri, C.; Bianchini, G.; Beccari, A. R.;

Fanton, M.; Bruno, A.; Costantino, G.; Bertini, R.; Galliera, E.; Locati, M.; Ferreira, S.

H.; Teixeira, M. M.; Allegretti, M. Targeting the minor pocket of C5aR for the rational

design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief. Proc.

Natl. Acad. Sci. U. S. A. 2014, 111, 16937-16942.

226. Jayne, D. R. W.; Bruchfeld, A. N.; Harper, L.; Schaier, M.; Venning, M. C.; Hamilton,

P.; Burst, V.; Grundmann, F.; Jadoul, M.; Szombati, I.; Tesar, V.; Segelmark, M.;

Potarca, A.; Schall, T. J.; Bekker, P.; Group, C. S. Randomized trial of C5a receptor

96 inhibitor avacopan in ANCA-associated vasculitis. J. Am. Soc. Nephrol. 2017, 28,

2756-2767.

227. Fluiter, K.; Opperhuizen, A. L.; Morgan, B. P.; Baas, F.; Ramaglia, V. Inhibition of the

membrane attack complex of the complement system reduces secondary neuroaxonal

loss and promotes neurologic recovery after traumatic brain injury in mice. J. Immunol.

2014, 192, 2339-2348.

228. Lee, M.; Guo, J. P.; McGeer, E. G.; McGeer, P. L. Aurin tricarboxylic acid self-

protects by inhibiting aberrant complement activation at the C3 convertase and C9

binding stages. Neurobiol. Aging 2013, 34, 1451-1461.

229. Lee, M.; Narayanan, S.; McGeer, E. G.; McGeer, P. L. Aurin tricarboxylic acid protects

against red blood cell hemolysis in patients with paroxysmal nocturnal

hemoglobinemia. PLoS. One 2014, 9, e87316.

97