Binding kinetics, structure-activity relationship, and biotransformation of the complement inhibitor compstatin

We have previously identified a 13-residue cyclic peptide, Compstatin, that binds to complement component C3 and inhibits complement activation. Herein, we describe the binding kinetics, structure-activity relationship, and biotransformation of Compstatin. Biomolecular interaction analysis using surface-plasmon resonance showed that Compstatin bound to native C3 and its fragments C3b and C3c, but not C3d. While binding of Compstatin to native C3 was biphasic, binding to C3b and C3c followed the 1:1 Langmuir binding model; the affinities of Compstatin for C3b and C3c were 22- and 74-fold lower, respectively, than that of native C3. Analysis of Compstatin analogs synthesized for structure-function studies indicated that 1) the 11-membered ring between disulfide-linked Cys2-Cys12 constitutes a minimal structure required for optimal activity; 2) retro-inverso isomerization results in loss of inhibitory activity; and 3) some residues of the type I beta-turn segment also interact with C3. In vitro studies of Compstatin in human blood indicated that a major pathway of biotransformation was the removal of Ile1, which could be blocked by N-acetylation of the peptide. These findings indicate that acetylated Compstatin is stable against enzymatic degradation and that the type I beta-turn segment is not only critical for preservation of the conformational stability, but also involved in intermolecular recognition.     

Structure‐kinetic relationship analysis of the therapeutic complement inhibitor compstatin

Compstatin is a 13‐residue peptide that inhibits activation of the complement system by binding to the central component C3 and its fragments C3b and C3c. A combination of theoretical and experimental approaches has previously allowed us to develop analogs of the original compstatin peptide with up to 264‐fold higher activity; one of these analogs is now in clinical trials for the treatment of age‐related macular degeneration (AMD). Here we used functional assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to assess the effect of modifications at three key residues (Trp‐4, Asp‐6, Ala‐9) on the affinity and activity of compstatin and its analogs, and we correlated our findings to the recently reported co‐crystal structure of compstatin and C3c. The K_D values for the panel of tested analogs ranged from 10^?6 to 10^?8 M. These differences in binding affinity could be attributed mainly to differences in dissociation rather than association rates, with a >4‐fold range in k_on values (2–10 × 10⁵ M^?1 s^?1) and a k_off variation of >35‐fold (1–37 × 10^?2 s^?1) being observed. The stability of the C3b‐compstatin complex seemed to be highly dependent on hydrophobic effects at position 4, and even small changes at position 6 resulted in a loss of complex formation. Induction of a β‐turn shift by an A9P modification resulted in a more favorable entropy but a loss of binding specificity and stability. The results obtained by the three methods utilized here were highly correlated with regard to the activity/affinity of the analogs. Thus, our analyses have identified essential structural features of compstatin and provided important information to support the development of analogs with improved efficacy. Copyright © 2009 John Wiley & Sons, Ltd.     

Thermodynamic studies on the interaction of the third complement component and its inhibitor, compstatin

Compstatin is a 13-residue cyclic peptide that inhibits complement activation by binding to complement component, C3. Although the activity of compstatin has been improved severalfold using combinatorial and rational design approaches, the molecular basis for its interaction with C3 is not yet fully understood. In the present study, isothermal titration calorimetry was employed to dissect the molecular forces that govern the interaction of compstatin with C3 using four different compstatin analogs. Our studies indicate that the C3-compstatin interaction is an enthalpy-driven process. Substitution of the valine and histidine residues at positions 4 and 9 with tryptophan and alanine, respectively, resulted in the increase of enthalpy of the interaction, thereby increasing the binding affinity for C3. The data also suggest that the interaction is mediated by water molecules. These interfacial water molecules could be the source for unfavorable entropy and large negative heat capacity changes observed in the interaction. Although part of the negative heat capacity changes could be accounted for by the water molecules, the rest might be resulting from the conformational changes in C3 and/or compstatin up on binding. Finally, we propose based on the pK(a) values determined from the protonation studies that histidine on compstatin participates in protonation changes and contributes to the specificity of the interaction between compstatin and C3. These protonation changes vary significantly between the binding of different compstatin analogs to C3.     

Species specificity of the complement inhibitor compstatin investigated by all‐atom molecular dynamics simulations

The development of compounds to regulate the activation of the complement system in non‐primate species is of profound interest because it can provide models for human diseases. The peptide compstatin inhibits protein C3 in primate mammals and is a potential therapeutic agent against unregulated activation of complement in humans but is inactive against nonprimate species. Here, we elucidate this species specificity of compstatin by molecular dynamics simulations of complexes between the most potent natural compstatin analog and human or rat C3. The results are compared against an experimental conformation of the human complex, determined recently by X‐ray diffraction at 2.4‐Å resolution. The human complex simulations provide information on the relative contributions to stability of specific C3 and compstatin residues. In the rat simulations, the protein undergoes reproducible conformational changes, which eliminate or weaken specific interactions and reduce the complex stability. The simulation insights can be used to design improved compstatin‐based inhibitors for human C3 and active inhibitors against lower mammals. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition

Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compstatin in complex with C3c, a major proteolytic fragment of C3. The structure reveals that the compstatin-binding site is formed by the macroglobulin (MG) domains 4 and 5. This binding site is part of the structurally stable MG-ring formed by domains MG 1-6 and is far away from any other known binding site on C3. Compstatin does not alter the conformation of C3c, whereas compstatin itself undergoes a large conformational change upon binding. We propose a model in which compstatin sterically hinders the access of the substrate C3 to the convertase complexes, thus blocking complement activation and amplification. These insights are instrumental for further development of compstatin as a potential therapeutic.     

Musings on ADME predictions and structure-activity relations

The first part of the paper examines Structure-Activity Relations (SARs) and their components from a very general point of view. The various types of interpretation emerging from statistically valid relations will be examined, namely causal (mechanistic), contextual (empirical), fortuitous, and tautological correlations. Implications for ADME predictions will be seen when discussing the diversity of interactions between active compounds (e.g., drugs) and biological systems. The second part of the paper is more specific and presents the concept of molecular-property space, an all but neglected concept in SARs. Recent results from Molecular Dynamics (MD) simulations and Molecular Interaction Fields (MIF) computations of acetylcholine will be used to illustrate not only the well-known conformational space of this molecule, but also its property space as exemplified by its lipophilicity space. It will be seen that a molecule as small as acetylcholine is able to span a relatively broad property space. Most significantly in an ADME perspective, the molecule is able, within the limits of its property space, to adapt to the medium. This is equivalent to saying that the medium constrains the molecule to resemble it as much as feasible.     

A simple, yet highly accurate, QSAR model captures the complement inhibitory activity of compstatin

Compstatin is a 13-residue cyclic peptide inhibitor of complement activation that was originally identified through phage-mediated presentation of a peptide library to C3b. Recent efforts to improve its activity have led to a rich dataset of complement analogs, with the most active analog being approximately 260 times more active than the parent compstatin. In the present work, a highly transparent quantitative structure-activity relationship model (Radj2=0.89) with four parameters is presented that captures important physico-chemical and geometrical properties of the analog molecules with regard to activity. The number of aromatic bonds and hydrophobicity of the fourth residue of compstatin correlated strongly with activity. Also important were the hydrophobic patch size near the disulfide bond and the solvent-accessible surface area occupied by nitrogen atoms of basic amino acid residues.     

Insecticidal chromenes from the Asteraceae: structure-activity relations

Chromenes (benzopyrans), common to many genera of the Asteraceae, are lethal to the neonate variegated cutworm, Peridroma saucia Hübner, when presented on the inner walls of glass vials at concentrations of 1.0 mol/vial or less. Closely related benzofurans, including some from the same plants, are relatively inactive at these concentrations. The most potent of seven naturally-occurring and four derived chromenes is the well known allatocidin, precocene II, with an LD₅₀ of 0.134 mol/vial or 0.67 g/cm². A more widely distributed chromene, desmethoxyencecalin, is also relatively toxic with an LD₅₀ of 0.98 g/cm².Structure-activity comparisons of the chromenes tested in this study suggest that the presence of a free hydroxyl group at either C-7 or C-11 significantly diminishes activity. Saturation of the 3,4 double bond, which destroys the allatocidal activity of the precocenes, diminishes, but does not alleviate, contact toxicity of chromenes to cutworm larvae. Brief exposure (24 h) of 2-day-old larvae to chromene residues significantly inhibits subsequent larval growth. The degree of chronic growth inhibition (sublethal toxicity) from five different chromenes is highly correlated to their lethal potencies to neonate larvae.

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Zusammenfassung Verschiedene Chromene (Benzopyrane), die in einer Vielzahl von Gattungen aus der Familie der Asteraceae vorkommen, erwiesen sich als insektizid gegenüber frischgeschlüpften Raupen von Peridroma saucia. Die getesteten Substanzen wurden dabei als Film an den Innenwänden von Glass-Scintillationsgefäßen in Konzentrationen bis zu 1 mol/Gefäß appliziert und die Raupen über 24 h in diesen Gefäßen gehalten. Den Chromenen biogenetisch ähnliche Benzofurane, die oft gemeinsam in Asteraceen gefunden werden, weigten im Vergleich keine oder nur schwache insektizide Wirkungen. Von sieben natürlich vorkommenden und vier durch Partialsynthesen getesteten Chromenen erwies sich das bekannte Allatocidin Precocen II als die aktivste Verbindung. Der LD 50-Wert dieses Chromens betrug 0.134 mol/Scintillationsgefäß oder 0.67 g/cm². Das weitaus häufigere Chromen desmethoxyencecalin wies im Vergleich einen LD 50-Wert von 0.98 g/cm² in diesem Biotest auf.Struktur/Aktivitätsuntersuchungen der getesteten Chromene zeigten, daß die Anwesenheit einer OH-Gruppe an den Positionen C-7 und C-11 zu einer deutlichten Aktivitätsminderung führte. Hydrierung der heterocyclischen Doppelbindung, die einen völligen Verlust der allatociden Wirkung von Precocenen bewirkt, verringerte die Kontakttoxizität der Chromene auf 50% der ursprünglichen Aktivität.Ein kurzer Kontakt (24 h) von zwei Tage alten Peridroma-Raupen des ersten Larvenstadiums mit den Chromenen resultierte in einer signifikanten Verringerung des nachfolgenden Wachstums der Raupen auf künstlicher Diät.Die deutlichsten Struktur/Aktivitätsbeziehungen, die in der vorliegenden Arbeit ermittelt wurden, lassen sich wie folgt zusammenfassen: 1) Die Gegenwart eines Furanringes statt eines Pyranringes (Chromen) bewirkt einen zumeist vollständigen Verlust an insektizider Wirkung; 2) die Gegenwart eines OH-Substituenten führt zu einer drastischen Abnahme an Aktivität. Eine Übersicht der bisher vorliegenden Daten sowie laufender Untersuchungen zeigt, daß Chromene ein breites Spektrum an insektizider Wirkung besitzen.

     

Plasminogen is a complement inhibitor

Plasminogen is a 92-kDa single chain glycoprotein that circulates in plasma as a zymogen and when converted to proteolytically active plasmin dissolves preformed fibrin clots and extracellular matrix components. Here, we characterize the role of plasmin(ogen) in the complement cascade. Plasminogen binds the central complement protein C3, the C3 cleavage products C3b and C3d, and C5. Plasminogen binds to C3, C3b, C3d, and C5 via lysine residues, and the interaction is ionic strength-dependent. Plasminogen and Factor H bind C3b; however, the two proteins bind to different sites and do not compete for binding. Plasminogen affects complement action in multiple ways. Plasminogen enhanced Factor I-mediated C3b degradation in the presence of the cofactor Factor H. Plasminogen when activated to plasmin inhibited complement as demonstrated by hemolytic assays using either rabbit or sheep erythrocytes. Similarly, plasmin either in the fluid phase or attached to surfaces inhibited complement that was activated via the alternative and classical pathways and cleaved C3b to fragments of 68, 40, 30, and 17 kDa. The C3b fragments generated by plasmin differ in size from those generated by the complement protease Factor I, suggesting that plasmin-mediated C3b cleavage fragments lack effector function. Plasmin also cleaved C5 to products of 65, 50, 30, and 25 kDa. Thus, plasmin(ogen) regulates both complement and coagulation, the two central cascade systems of a vertebrate organism. This complement-inhibitory activity of plasmin provides a new explanation why pathogenic microbes utilize plasmin(ogen) for immune evasion and tissue penetration.     

Comparison of structure-activity relations of corazonin using two different bioassay systems

The structure-activity relations of [His(7)]-corazonin were studied using two different bioassay systems; i.e. inhibitory effect on spinning rate in the silkworm, Bombyx mori, and darkening response in albino nymphs of the migratory locust, Locusta migratoria. Deletion of the N-terminus, shortening of the peptide and single amino acid substitutions reduced activity in a similar manner except for the minimum effective dose in the two insects. The results also revealed that the residues at position 1, 3 and 5 were particularly important for biological activity. Despite the different physiological affects, the two insect species exhibited similar structure-activity relationships, suggesting that they might have similar receptor systems.     

Studies of structure-activity relationships of human interleukin-2

Human interleukin-2 (IL-2) has 3 cysteine residues; cysteines 58 and 105 form an intramolecular disulfide bridge, whereas cysteine 125 has a free sulfhydryl group. In this study, site-specific mutagenesis has been used to modify the cysteine residues of recombinant Escherichia coli-derived IL-2 (rIL-2) to evaluate the functional structure of IL-2. Substitution or deletion of cysteine 105 disrupted the disulfide bridge and yielded a mutant protein which was 8-10 times less active than wild type rIL-2. A similar modification at position 58, however, reduced the activity of rIL-2 by more than 250-fold. Although substitution of serine for cysteine 125 did not affect IL-2 activity, deletion of cysteine 125 or deletion of amino acids in the vicinity of this cysteine yielded mutant proteins with little, if any, activity. These results indicate that the protein structure in the vicinity of both positions 58 and 125 is more critical than that close to position 105. These findings may provide a clue to the understanding of the functional structure of human IL-2.     

Anti-inflammatory effects of 7-methoxycryptopleurine and structure-activity relations of phenanthroindolizidines and phenanthroquinolizidines

A cryptopleurine analogue, 7-methoxycryptopleurine, a phenanthroquinolizidine, was first found to exert potent anti-inflammatory activity in vitro and in vivo as well as have remarkable cytotoxic activity against cancer cells. The non-planar structure between the two major moieties, phenanthrene and indolizidine/quinolizidine, played a crucial role in the activity of phenanthroindolizidines or phenanthroquinolizidines in terms of cytotoxic effects on cancer cells and anti-inflammatory activity. We also showed that increase in planarity and rigidity of the indolizidine/quinolizidine moiety and change of the amine group into an amide by introducing a keto group to phenanthroindolizidines or phenanthroquinolizidines at the equivalent position 9 of tylophorine significantly reduced their activities. Moreover, in general, phenanthroquinolizidines are more potent than their respective phenanthroindolizines.     

Synthesis and structure-activity relationships of elafin, an elastase-specific inhibitor.

Elafin, an elastase-specific inhibitor isolated from human skin, and its related peptides were synthesized by the solution procedure, and their inhibitory activities were measured against various enzymes. During the oxidative folding reactions of the reduced peptides, the ratio of the active product to the inactive product was varied by changing the concentration of guanidine HCl and the amount of redox reagents. The disulfide structures of fully active synthetic elafin and the inactive product were determined by amino acid analysis, gas-phase sequencing and mass spectrometry of their proteolytic fragments. The relationship between structure and inhibitory activities and/or the folding reaction was examined and the amino terminal part of the elafin molecule was found to have a great influence on the folding reactions, but not on the inhibitory activities.     

Staphylococcal complement inhibitor: structure and active sites

The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 A resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31-Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.     

Primate terminal complement inhibitor homologues of human CD59

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L22862 (African green monkey CD59) and L22863 (baboon CD59)     

Understanding the structural characteristics of compstatin by conformational space annealing

The structural characteristics of the 13-residue compstatin molecule are investigated using the conformational space annealing (CSA) method with CHARMM force field and the GBSA continuum solvent model. In order to sample conformations in the energy range of the minimized NMR structures, we have used the stopping criterion to the CSA search when a conformation whose energy is less than -490 kcal/mol is found. With this stopping criterion, a great variety of conformations are generated around experimentally known structures. Twenty independent CSA runs starting from random states find 1000 representative conformations in the energy landscape of the compstatin, which are classified into thirty-one structural families. The majority of the conformations (94.4%) are in the coil state. Other conformers containing a 3(10)-helix, a pi-helix, a beta-hairpin, and an alpha-helix are also found.