Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

Complement factor D (FD), a highly specific S1 serine protease, plays a central role in the amplification of the alternative complement pathway (AP) of the innate immune system. Dysregulation of AP activity predisposes individuals to diverse disorders such as age-related macular degeneration, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II, and paroxysmal nocturnal hemoglobinuria. Previously, we have reported the screening efforts and identification of reversible benzylamine-based FD inhibitors (1 and 2) binding to the open active conformation of FD. In continuation of our drug discovery program, we designed compounds applying structure-based approaches to improve interactions with FD and gain selectivity against S1 serine proteases. We report herein the design, synthesis, and medicinal chemistry optimization of the benzylamine series culminating in the discovery of 12, an orally bioavailable and selective FD inhibitor. 12 demonstrated systemic suppression of AP activation in a lipopolysaccharide-induced AP activation model as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD.

The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.

Dysregulation of the alternative complement pathway is a major pathogenic mechanism in age-related macular degeneration. We investigated whether locally synthesized complement components contribute to AMD by profiling complement expression in postmortem eyes with and without AMD.METHODS. AMD severity grade 1 to 4 was determined by analysis of postmortem acquired fundus images and hematoxylin and eosin stained histological sections. TaqMan (donor eyes n = 39) and RNAscope/in situ hybridization (n = 10) were performed to detect complement mRNA. Meso scale discovery assay and Western blot (n = 31) were used to measure complement protein levels. RESULTS.The levels of complement mRNA and protein expression were approximately 15-to 100-fold (P < 0.0001-0.001) higher in macular retinal pigment epithelium (RPE)/choroid tissue than in neural retina, regardless of AMD grade status. Complement mRNA and protein levels were modestly elevated in vitreous and the macular neural retina in eyes with geographic atrophy (GA), but not in eyes with early or intermediate AMD, compared to normal eyes. Alternative and classical pathway complement mRNAs (C3, CFB, CFH, CFI, C1QA) identified by RNAscope were conspicuous in areas of atrophy; in those areas C3 mRNA was observed in a subset of IBA1 + microglia or macrophages. CONCLUSIONS.We verified that RPE/choroid contains most ocular complement; thus RPE/choroid rather than the neural retina or vitreous is likely to be the key site for complement inhibition to treat GA or earlier stage of the disease. Outer retinal local production of complement mRNAs along with evidence of increased complement activation is a feature of GA.