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. 2016 Sep 13;7(12):1092–1096. doi: 10.1021/acsmedchemlett.6b00299

Buried Hydrogen Bond Interactions Contribute to the High Potency of Complement Factor D Inhibitors

Chao-Yie Yang †,*, James G Phillips ∥, Jeanne A Stuckey §,‡, Longchuan Bai †, Haiying Sun ⊥, James Delproposto §, William Clay Brown §, Krishnapriya Chinnaswamy §
PMCID: PMC5151139  PMID: 27994744

Abstract

graphic file with name ml-2016-00299d_0006.jpg

Aberrant activation of the complement system is associated with diseases, including paroxysmal nocturnal hemoglobinuria and age-related macular degeneration. Complement factor D is the rate-limiting enzyme for activating the alternative pathway in the complement system. Recent development led to a class of potent amide containing pyrrolidine derived factor D inhibitors. Here, we used biochemical enzymatic and biolayer interferometry assays to demonstrate that the amide group improves the inhibitor potency by more than 80-fold. Our crystal structures revealed buried hydrogen bond interactions are important. Molecular orbital analysis from quantum chemistry calculations dissects the chemical groups participating in these interactions. Free energy calculation supports the differential contributions of the amide group to the binding affinities of these inhibitors. Cell-based hemolysis assay confirmed these compounds inhibit factor D mediated complement activation via the alternative pathway. Our study highlights the important interactions contributing to the high potency of factor D inhibitors reported recently.

Keywords: Paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, complement system, complement factor D inhibitors, serine protease, enzyme inhibition assay, biolayer interferometry assay, crystal structure, quantum chemistry calculations, thermodynamic integration method, computational docking, molecular dynamics simulations, hemolysis assay

The complement system plays important roles as a first line of defense against microbial infection and in the clearance of injured tissues. The efficient activation of the complement system “complements” the relatively slower adaptive immune response to serve as a functional bridge between the innate and adaptive immune system.1 The complement system activation is known to be mediated by three known pathways, including classical pathway (CP, triggered by antigen–antibody complex formation), lectin pathway (LP, mediated by lectin binding to pathogen surface), and alternative pathway (AP, initiated by recognition to the activating pathogenic surface), which lead to inflammation, cell lysis, and opsonization. Through a cascade of proteolytic cleavage and activation, these pathways recruit a group of plasma proteins to assemble a membrane attack complex (MAC)2 on the targeted cell surface which forms a hole in the membrane and causes cell lysis. Uncontrolled complement activation causes disease manifestations including paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration, and neurodegenerative diseases.1,3,4

Among them, PNH is an acquired clonal disease of hematopoietic stem cells due to somatic mutation in the PIG-A gene.5 Mutated PIG-A protein causes deficient biosynthesis of glycophosphatidylinositol (GPI)-anchored surface proteins on the progeny blood cells.6 The GPI-linked CD55 and CD59 deficient erythrocytes lose protection from C5- and C3-involved complement activation,3,7 which triggers intravascular hemolysis. Activated complement system targeting the PIG-A mutant blood cells is also responsible for the cardinal symptoms of the disease, including hemolysis, thrombosis, and bone marrow failure observed in the PNH patients.8 Only one C5 antibody-based complement-targeted therapy (eculizumab) has been approved to treat PNH disease.9 Although eculizumab ameliorates anemia and reduces transfusion-dependence in PNH patients, a recent genetic study showed Japanese patients with C5 variants are resistant to eculizumab treatment.10 Recently, a peptide-based inhibitor (AMY-101)3,7 targeting upstream C3 protein has received orphan drug status from US FDA and European Medicines Agency (EMA) for treating C3 glomerulopathy.

Several enzymes play important roles in complement activation by processing the complement proteins. Among them, the plasma concentration of complement factor D (denoted as factor D) was reported to be the lowest among all complement related proteins and considered a rate-limiting enzyme. Acting on the upstream of AP, factor D cleaves complement factor B bound with C3b into the C3bBb complex.11 The ubiquitous role of factor D in AP activation led to the implication that inhibition of factor D can be an attractive strategy in complement-targeted therapy. Factor D belongs to the serine protease family.12 Previous factor D inhibitor developments have led to the discovery of nonspecific catalytic site inhibitors, including coumarin derived covalent inhibitors13,14 and the noncovalent inhibitor (compound 53) with Ki = 3.7 μM15 (Figures 1A, S1). An antibody (AFD) targeting an exosite in factor D has also been developed to block the binding between factor D and C3bB, with an IC50 value of 430 nM.16 Recently, aurin tricarboxylic acid (ATA), a promiscuous inhibitor that is prone to polymerization,17 was reported to bind to a noncatalytic site in factor D18 and inhibited the complement activation. These inhibitors lacking specificity and adequate potency have limited use in studying the consequence of specific factor D inhibition in disease models.

Figure 1.

Figure 1

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(A) Examples of the structures of the covalent and noncovalent factor D inhibitors. (B) Structures of JH1–JH4, their IC50, and binding kinetic data with factor D. For the enzymatic assay, the IC50 values were determined by the average of at least two independent triplicate experiments. Binding kinetic data were obtained from the averages of two independent experiments, and the standard deviations are shown in the parentheses. (C) Enzymatic activity parameters24 of factor D obtained from Complement Technology and our recombinant protein as determined from the enzymatic assay.

Recently, Novartis reported two classes of potent noncovalent factor D inhibitors based on the pyrrolidine and the aminomethyl-biaryl core structures (Figures 1A, S1) to treat age-related macular degeneration and diabetic retinopathy.19,20 Further, patents filed by Achillion pharmaceuticals (WO 2015/130784 A1, WO 2015/130795 A1, WO 2015/130806 A1, WO 2015/130830 A1, WO 2015/130838 A1, WO 2015/130842 A2, WO 2015/130845 A1, and WO 2015/130854 A1) focused on extensive optimization of the derivatives of a similar pyrrolidine core as factor D inhibitors. In the Novartis patent,19 an amide group coupled to the pyrazolopyridine group appears in multiple compounds but its contribution to the inhibitory activity is not clear nor claimed (see Figure 1). The amide group is not known to be a critical group for serine protease inhibitors reported previously.21 To elucidate the role of this chemical group, we combined chemical synthesis, in vitro biochemical and cell-based assays, crystal structure determination, and computational calculations to provide detailed insights into the interaction between factor D and the new class of inhibitors.

We first evaluated the enzymatic inhibition activity of four compounds (JH1–JH4 in Figure 1) based on two related core scaffolds using a well-known substrate, Z-Lys-thiobenzyl ester (Z-Lys-Sbzl). JH1 and JH3 were included in the Novartis patent.19 Factor D extracted from normal human serum purchased from Complement Technology was used in the assay. Our enzymatic assay showed the IC50 values for JH1–4 are 31.95 nM, 6.60 μM, 27.31 nM, and 2.18 μM, respectively. The data showed a 207-fold change in IC50 values between JH1 and JH2. Using a different thiazolidine core, we found a lesser yet significant 80-fold change in IC50 values between JH3 and JH4. The amide group indeed plays an important role in the inhibitory activity of these inhibitors.

We next determined the direct binding constants of the inhibitors to factor D by employing Biolayer interferometry (BLI) experiments. Because a larger amount of biotinylated proteins are required in this assay, recombinant human factor D expressed from Sf9 insect cells was used in the BLI experiments. The insect cell expression system has also been reported to produce matured human factor D previously.22 To confirm the enzymatic activity of our recombinant factor D, we determined and compared the Km, Vmax, and kcat of our recombinant factor D with those using the factor D purchased from Complement Technology. As shown in Figure 1C, both factor D proteins gave similar Vmax, Km, and kcat using Z-Lys-Sbzl as the substrate when the data were fitted to the Michaelis–Menten equation. Our Km and kcat values are comparable to the previously reported kcat = 1.30 s–1 and Km = 3.79 mM using the wild-type human factor D23 despite the differences of DMSO concentrations (4% versus 9%23). We also tested the inhibition activity of JH3 to the recombinant factor D and obtained an IC50 value of 19 nM (Figure S2). We concluded that our recombinant factor D has similar activity as the factor D purchased from Complement Technology.

Based on the BLI experiments, we observed that the dissociation constants, Kd, between JH1–4 and factor D are 16.98 nM, 12.00 μM, 18.85 nM, and 2.21 μM, respectively (Figure 1). The Kd values are consistent with and close to the IC50 values measured using the biochemical enzymatic inhibition assay. Comparison of the Kd values indicated that JH2 is weaker than JH1 by 707-fold whereas JH4 is weaker than JH3 by 117-fold. Data from two biochemical assays concluded that the contribution of the amide group to the inhibitors activities is more profound when the (1R,5S)-2-azabicyclo[3.1.0]hexane core is used. Despite the differences of the cores, JH1 and JH3 have similar potencies to factor D. Further analysis of the BLI data indicated that koff and kon of JH1 are similar to those of JH3 (0.003605 versus 0.003274 1/s and 211677.5 versus 173400.0 1/Ms). Differences in the core structures of JH1 and JH3 have little influence on their binding kinetics with factor D. When comparing JH1 with JH2 versus JH3 with JH4, the greater differential factor to their affinities is their kon values. The ratio of the kon values for JH1 and JH2 is 13, which is much larger than the ratio of 1.3 between JH3 and JH4. In contrast, the ratio of koff for JH1 and JH2 is 52, which is similar to the ratio of 89 between JH3 and JH4.

To gain structural insights, we obtained cocrystal structures of JH3 and JH4 with factor D at 3.15 and 3.36 Å resolution, respectively. The electron density at the binding site was high enough to allow determination of the atomic positions of both inhibitors and interpretation of their interactions with factor D (see Figure 2). Figure 2 showed that a carbonyl group of JH1 or JH3 interacts with the backbone amide groups of G196 and S198, representing the typical oxyanion hole interaction found in serine proteases,21 and H56 in the catalytic triad also adopts the atypical conformation.14 Furthermore, the amide group forms buried hydrogen bonds with the side chain of R217, the backbone carbonyl group of T213, and the hydroxyl group of S198. The crystal structure also indicates that the guanidine group of R217 orients similarly to R217 found in the DIC-bound 3,4-dichloroisocoumarin(DIC)-bound factor D crystal structure (PDBID: 1DIC(13)). Besides the interactions involving the amide group, the bromine atom of the pyridine group in JH3 is at a distance of 3.5 Å to the carbonyl group of W143, representing a halogen bond interaction between the electron deficient σ-hole of the bromine atom and the electron-rich oxygen atom of the carbonyl group.25 An additional cation−π interaction can be identified between the pyridine group and R152. Alignment of both inhibitor-bound factor D structures showed little backbone structural changes in factor D, as the root-mean-square-deviation of the backbone atoms between them is 0.16 Å. The structures also indicated that the indazole group (without the amide group) in JH3 and JH4 orients similarly whereas the amide group in JH3 extends deeper into the binding site. Comparison of the structures between JH4 and DIC showed that an aromatic group at the binding site close to the catalytic S198 location is favored. However, polar amino acids, including R217 and T213 and potentially a mediating water molecule, establish a hydrophilic environment deeper in the binding site to interact with the amide group of the pyrazolo[3,4-b]pyridine group in JH3, unknown previously (Figure 2).

Figure 2.

Figure 2

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JH3 (A) and JH4 (B) are colored in yellow while factor D is colored in green. The mesh maps correspond to the 3σ level differences when the electron density maps were fitted to factor D without the inhibitors. Hydrogen bond interaction is shown as a cyan dashed line. The binding pockets formed by H39, W143, I145, R152, K195, and G196 are shown in a surface representation colored in gray. (C–D) Depiction of the hydrogen bond, the cation−π, and the halogen bond interactions between JH3, JH4, and factor D identified from the cocrystal structures.

To investigate the role of water molecules at the binding site unresolved in our cocrystal structures because of low resolution, we examined all available crystal structures of factor D. We found one water molecule interacting with R217 and T213 is resolved in multiple crystal structures (PDBIDs: 1DIC, 1BIO, 1FDP, 1DSU). Placement of this water molecule (Wat387 in the factor D-DIC cocrystal structure) in the binding site of factor D with JH3 showed that this water molecule can form hydrogen bonds with the amide group of JH3, the Nω1 of R217, and the hydroxyl group of T213. Thus, Wat387 was included in our computational analysis.

To dissect the network of hydrogen bonds between the pyrazolo[3,4-b]pyridine group and neighboring amino acids in factor D, we performed the fragment population analysis based on the quantum chemistry calculations. First, we optimized a simplified model of the binding site represented by the side chain of R217, T213, the side chain of S198 (approximated by the isobutanol), and Wat387 extracted from the crystal structure (see Figure S4). To approximate the electric fields in protein,26 the extracted fragments were embedded in diethyl ether, which has a dielectric constant of 4.24 used by the polarizable continuum model.27 The optimized structure showed that the local crystal structure was mostly preserved despite a substantial change of the hydrophobic side chain of R217 due to the lack of backbone constraint (Figure S4).

In the fragment population analysis, Wat387, T213, the side chain of S198, the side chain of R217, and JH3 were treated as fragments. The hydrogen bond interactions between fragments were further verified via visual inspection of molecular orbitals. After examining the 30 highest occupied molecular orbitals of the optimized structure, we identified four types of hydrogen bond interactions (see Figure 3). The first type of interaction is between the side chain of R217 and JH3 as seen in the molecule orbital 204 (MO204) and MO206 (Figure 3A). While JH3 contributes 79% of electrons to MO204, R217 and JH3 equally participate in MO206. The electron densities also revealed a long-range resonance contribution of the lone pair electron from N7 on JH3 to both molecular orbitals. The second type of interaction is between S198 and JH3, as seen in MO189 and MO190 in Figure 3B. Our analysis shows that JH3/MO107 engages S194/LUMO and S194/HOMO–2 to form MO189 and MO190, respectively. The third type of hydrogen bond interaction involving T213, S198, and JH3 is shown in Figure 3C. In this case, T213/HOMO–1 and T213/HOMO–2 interact with S198/HOMO and JH3/MO118 to form MO210 and MO211, respectively, where T213 appears to be a mediator between S198 and JH3. The fourth type of interaction is between T213/HOMO–1 and JH3/MO118, as shown in Figure 3D. The p-type orbital on the amide group of JH3/MO118 can be found to participate in multiple hydrogen bond interactions, as demonstrated in Figures 3C–3D. The third and fourth types of interactions appear to involve only the amide group of JH3 whereas the electron delocalization in JH3 plays a prominent role in the first two types of interactions. Among the 30 highest occupied molecular orbitals analyzed, we found Wat387 plays no role in hydrogen bonding. We conclude Wat387 does not contribute directly to the binding affinity of JH3 or JH1 with factor D.

Figure 3.

Figure 3

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Molecular orbital analysis of four types of hydrogen bond interactions between the 1H-pyrazolo[3,4-b]pyridine-3-carboxamide in JH3 and the neighboring residues. The percentage of electron density contributed from each fragment is shown in the parentheses. Energy levels of the molecular orbitals in reference to HOMO and LUMO are labeled with a plus and a minus number corresponding to the number of energy levels higher and lower than HOMO or LUMO.

Because no cocrystal structures between JH1, JH2, and factor D were obtained, we modeled the binding poses of JH1 and JH2 using the JH3-bound factor D structure (Figure S5) and computed the binding free energy changes of JH1–JH4 via alchemical transformations to provide additional supporting evidence of their binding affinity differences. We first confirmed the stability of factor D in complex with JH3 and JH4 from the MD simulations (discussed in the SI) and calculated their binding free energy differences using the thermodynamic integration method.28 Between JH3 and JH4, our calculations gave a difference of 3.10 ± 0.54 kcal/mol in ΔΔG (Table S2), which is comparable to the 2.82 kcal/mol estimated from BLI experiments. For JH1 and JH2, we obtained ΔΔG = 5.63 ± 0.20 kcal/mol (versus 3.89 kcal/mol from BLI), which is higher than that between JH3 and JH4. Thus, the binding free energy differences in JH1–JH4 obtained from our computational calculations are consistent with those calculated based on the Kd values of the BLI experiments and the IC50 values from the enzymatic assay.

To demonstrate these compounds inhibit complement activation by targeting factor D in AP, we evaluated them in the standard rabbit erythrocyte hemolysis assay. Activation of AP was induced by introducing human serum to rabbit erythrocytes in the presence of Mg2+ while depleting Ca2+ ions via EGTA. Figure 4 showed the most potent inhibitor to the hemolysis is JH1 followed by JH3. The IC50 values of JH2 and JH4 are at micromolar ranges (77 and 19 μM). Although JH3 has similar enzymatic IC50 and Kd values as JH1, yet the IC50 value of JH3 (205 nM) observed in the hemolysis assay is 4-fold higher than that of JH1 (47 nM) (Figure 4). Whether serum protein binding plays a role to decrease the free JH3 concentration leading to the reduced IC50 value in the hemolysis assay remains to be determined.

Figure 4.

Figure 4

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Inhibition of factor D by JH1–4 in the hemolysis assay using the normal human serum and rabbit erythrocytes. The IC50 values are shown as the mean ± SE from triplicate (n = 3) experiments.

In summary, our data, including the binding between four inhibitors and factor D obtained from in vitro biochemical assays, crystal structures, computational calculations, and the cell-based hemolysis assay, reveal the critical role of the amide group in these inhibitors for their high potencies to factor D. The findings can be useful for developing potent and selective complement factor D inhibitors in the future to assess their therapeutic potential in disease models.

Acknowledgments

We thank Jaroslaw Maciejewski from Cleveland Clinic for introducing us to PNH and for helpful discussions, and Shaomeng Wang from University of Michigan for providing equipment to conduct enzymatic and hemolysis assays. We also thank Liu Liu, Bruce Palfey, and Mou-Chi Cheng for helpful discussions on the enzymatic assay.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.6b00299.

  • Experimental binding data, the docking models, and methods (PDF)

Author Contributions

J.G.P. and H.S. synthesized the compounds; J.A.S. solved the crystal structures; C.-Y.Y. performed the enzymatic assay; J.D. conducted the BLI assay; L.B. performed the hemolysis assay; W.C.B. cloned and expressed the protein; K.C. purified the protein; C.-Y.Y. conducted computational calculations, designed the project, and wrote the manuscript; the manuscript was written through contributions of all authors.

We are grateful for financial support from the Aplastic Anemia MDS international Foundation and from the National Institutes of Health through the University of Michigan Cancer Center Support Grant (P30CA046592) by the use of the following Cancer Center Core: Center of Structural Biology. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (Grant 085P1000817).

The authors declare no competing financial interest.

Supplementary Material

ml6b00299_si_001.pdf (1.4MB, pdf)

References

  1. Ricklin D.; Hajishengallis G.; Yang K.; Lambris J. D. Complement: a key system for immune surveillance and homeostasis. Nat. Immunol. 2010, 11, 785–797. 10.1038/ni.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Serna M.; Giles J. L.; Morgan B. P.; Bubeck D. Structural basis of complement membrane attack complex formation. Nat. Commun. 2016, 7, 10587. 10.1038/ncomms10587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Mastellos D. C.; Ricklin D.; Yancopoulou D.; Risitano A.; Lambris J. D. Complement in paroxysmal nocturnal hemoglobinuria: exploiting our current knowledge to improve the treatment landscape. Expert Rev. Hematol. 2014, 7, 583–598. 10.1586/17474086.2014.953926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ricklin D.; Lambris J. D. Progress and trends in complement therapeutics. Adv. Exp. Med. Biol. 2013, 735, 1–22. 10.1007/978-1-4614-4118-2_1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kinoshita T.; Takeda J. [GPI-anchored proteins and paroxysmal nocturnal hemoglobinuria]. Nippon Saikingaku Zasshi 1993, 48, 533–539. 10.3412/jsb.48.533. [DOI] [PubMed] [Google Scholar]
  6. Tomita M. Biochemical background of paroxysmal nocturnal hemoglobinuria. Biochim. Biophys. Acta, Mol. Basis Dis. 1999, 1455, 269–286. 10.1016/S0925-4439(99)00068-X. [DOI] [PubMed] [Google Scholar]
  7. Brodsky R. A. Complement in hemolytic anemia. Blood 2015, 126, 2459–2465. 10.1182/blood-2015-06-640995. [DOI] [PubMed] [Google Scholar]
  8. Parker C.; Omine M.; Richards S.; Nishimura J.; Bessler M.; Ware R.; Hillmen P.; Luzzatto L.; Young N.; Kinoshita T.; Rosse W.; Socie G.; International P. N. H. I. G. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 2005, 106, 3699–3709. 10.1182/blood-2005-04-1717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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. 10.1038/nbt1344. [DOI] [PubMed] [Google Scholar]
  10. Nishimura J.-i.; Yamamoto M.; Hayashi S.; Ohyashiki K.; Ando K.; Brodsky A. L.; Noji H.; Kitamura K.; Eto T.; Takahashi T.; Masuko M.; Matsumoto T.; Wano Y.; Shichishima T.; Shibayama H.; Hase M.; Li L.; Johnson K.; Lazarowski A.; Tamburini P.; Inazawa J.; Kinoshita T.; Kanakura Y. Genetic Variants in C5 and Poor Response to Eculizumab. N. Engl. J. Med. 2014, 370, 632–639. 10.1056/NEJMoa1311084. [DOI] [PubMed] [Google Scholar]
  11. 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. 10.1126/science.1195821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Di Cera E. Serine proteases. IUBMB Life 2009, 61, 510–515. 10.1002/iub.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cole L. B.; Kilpatrick J. M.; Chu N.; Babu Y. S. Structure of 3,4-dichloroisocoumarin-inhibited factor D. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1998, 54, 711–717. 10.1107/S0907444997010457. [DOI] [PubMed] [Google Scholar]
  14. Jing H.; Babu Y. S.; Moore D.; Kilpatrick J. M.; Liu X. Y.; Volanakis J. E.; Narayana S. V. Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity. J. Mol. Biol. 1998, 282, 1061–1081. 10.1006/jmbi.1998.2089. [DOI] [PubMed] [Google Scholar]
  15. 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 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. 10.1016/j.bmcl.2004.04.034. [DOI] [PubMed] [Google Scholar]
  16. Katschke K. J. Jr.; Wu P.; Ganesan R.; Kelley R. F.; Mathieu M. A.; Hass P. E.; Murray J.; Kirchhofer D.; Wiesmann C.; van Lookeren Campagne M. Inhibiting alternative pathway complement activation by targeting the factor D exosite. J. Biol. Chem. 2012, 287, 12886–12892. 10.1074/jbc.M112.345082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee M.; Guo J. P.; Schwab C.; McGeer E. G.; McGeer P. L. Selective inhibition of the membrane attack complex of complement by low molecular weight components of the aurin tricarboxylic acid synthetic complex. Neurobiol. Aging 2012, 33, 2237–2246. 10.1016/j.neurobiolaging.2011.12.005. [DOI] [PubMed] [Google Scholar]
  18. 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. 10.1016/j.neurobiolaging.2012.10.023. [DOI] [PubMed] [Google Scholar]
  19. Hommel U.; Lorthiois E. L. J.; Maibaum J. K.; Ostermann N.; Randl S. A.; Vulpetti A.; Rogel O.. Pyrrolidine derivatives and their use as complement pathway modulators. WO 2014/002057 A1, 2014.
  20. Belanger D. B.; Flohr S.; Gelin C. F.; Jendza K.; Ji N.; LIU D.; Lorthiois E. L. J.; Karki R. G.; Mainolfi N.; Powers J. J.; Randl S. A.; Rogel O.; Vulpetti A.; Yoon T.. Aminomethyl-biaryl derivatives as complement factor D inhibitors and uses thereof. WO 2015/009977 A1, 2015.
  21. Hedstrom L. Serine protease mechanism and specificity. Chem. Rev. 2002, 102, 4501–4524. 10.1021/cr000033x. [DOI] [PubMed] [Google Scholar]
  22. Jing H.; Macon K. J.; Moore D.; DeLucas L. J.; Volanakis J. E.; Narayana S. V. Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D. EMBO J. 1999, 18, 804–814. 10.1093/emboj/18.4.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim S.; Narayana S. V.; Volanakis J. E. Crystal structure of a complement factor D mutant expressing enhanced catalytic activity. J. Biol. Chem. 1995, 270, 24399–24405. 10.1074/jbc.270.41.24399. [DOI] [PubMed] [Google Scholar]
  24. Conversion of the OD value to the enzyme concentration was calculated using the extinction coefficient of 13260 M–1 cm–1 for the catalyzed product and the 0.29 cm path correction for 100 μL of solution in each well.
  25. Wilcken R.; Zimmermann M. O.; Lange A.; Joerger A. C.; Boeckler F. M. Principles and applications of halogen bonding in medicinal chemistry and chemical biology. J. Med. Chem. 2013, 56, 1363–1388. 10.1021/jm3012068. [DOI] [PubMed] [Google Scholar]
  26. Kukic P.; Farrell D.; McIntosh L. P.; García-Moreno E B.; Jensen K. S.; Toleikis Z.; Teilum K.; Nielsen J. E. Protein Dielectric Constants Determined from NMR Chemical Shift Perturbations. J. Am. Chem. Soc. 2013, 135, 16968–16976. 10.1021/ja406995j. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tomasi J.; Mennucci B.; Cammi R. Quantum mechanical continuum solvation models. Chem. Rev. 2005, 105, 2999–3093. 10.1021/cr9904009. [DOI] [PubMed] [Google Scholar]
  28. Kollman P. Free energy calculations: Applications to chemical and biochemical phenomena. Chem. Rev. 1993, 93, 2395–2417. 10.1021/cr00023a004. [DOI] [Google Scholar]

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Supplementary Materials

ml6b00299_si_001.pdf (1.4MB, pdf)

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