Human complement component C3 (specifically the β-chain)
Compstatin binds to and inhibits complement component C3. It shows species specificity, being active against primate C3 (including human and baboon) but not against non-primate C3. Binding affinity studies using surface plasmon resonance indicate it binds to native C3, C3b, and C3c, but not to C3d. The affinity for C3b and C3c is reported to be 22-fold and 74-fold lower, respectively, than for native C3 .
Compstatin TFA targets complement component C3. It binds to native C3 and inhibits its cleavage by C3 convertase. Binding of Compstatin to native C3 is a multistep reaction that is preferred over its binding to the C3 fragments C3b and C3c. It shows species specificity, being active against primate C3 (human and baboon) but not non-primate C3. It blocks C3 convertase-mediated C3 cleavage, thereby blocking complement activation and amplification.

In vitro, compstatin has a half-life of roughly two hours in human blood[2]. Compstatin creates a hydrophobic cluster in solution by producing a β-turn at residues Gln-5–Gly-8, Ile–1–Val–4, and Thr–13, along with the disulfide bridge Cys-2–Cys12[3].

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Compstatin inhibits complement activation in human serum. In an ex vivo pig kidney xenograft model perfused with fresh human blood, Compstatin (administered at a concentration in the blood of 60 µM) completely inhibited the generation of C3 activation products and terminal complement complex (TCC/C5b-9) over a 60-minute observation period, whereas in the control group these products increased fivefold and eightfold, respectively .
Structural studies using 2D NMR determined that Compstatin contains a type I β-turn (Gln5-Asp6-Trp7-Gly8), which is critical for its conformational stability and functional activity .
Alanine scanning mutagenesis studies revealed that residues Val3, Gln5, Asp6, Trp7, and Gly8 are crucial for inhibitory activity, with substitution of Gly8 causing a 100-fold decrease in potency. In contrast, substitution of Val4, His9, His10, and Arg11 resulted in minimal change in activity .

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In vitro, Compstatin binds to native C3 and inhibits its cleavage by C3 convertase. The IC50 for complement activation inhibition is approximately 12 microM (as measured by inhibition of complement-mediated hemolysis). Compstatin binds to baboon C3 and is resistant to proteolytic cleavage in baboon blood (similar to humans). It inhibits the activation of the classical and alternative complement pathways.

When administered as a bolus injection plus infusion, compstatin (21 mg/kg) results in total suppression. Compstatin completely prevents complement activation generated by heparin and prostamine in vivo, however without changing heart rate or pulmonary, central venous, or systemic artery pressures [1]. In baboon plasma, compstatin is stable for more than 24 hours[1]. The group that received Compstatin perfusion had a considerably longer survival rate for pig xenografts than the control group [2].

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In a baboon model of heparin/protamine complex-induced complement activation (a simulation of cardiac surgery inflammation), intravenous Compstatin (total dose of 21 mg/kg administered as a combination of bolus and infusion) completely inhibited in vivo complement activation. This inhibition occurred without adversely affecting heart rate, systemic arterial pressure, central venous pressure, or pulmonary arterial pressure, indicating a favorable safety profile in this acute setting .

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Compstatin TFA is a research tool for studying complement in primate models. Newer analogs of compstatin have improved inhibitory potency (subnanomolar affinity) and enhanced pharmacokinetic properties, showing efficacy in clinically relevant primate models of disease. The compstatin family of C3 inhibitors is currently being evaluated in clinical trials. Compstatin is reaching its prime for bedside intervention.

The binding kinetics of Compstatin to C3 and its fragments were analyzed using surface plasmon resonance (SPR). Purified human complement proteins (native C3, C3b, C3c) were immobilized on a sensor chip. Compstatin in solution was flowed over the chip at various concentrations to measure association and dissociation rates. The data showed that binding to native C3 was biphasic, while binding to C3b and C3c followed a 1:1 Langmuir binding model, allowing for the calculation of comparative binding affinities .
A photo-crosslinking study was performed to identify the binding site on C3. A Compstatin analog containing a photoactivatable amino acid (Bpa) was synthesized. This analog was incubated with purified C3 protein and then exposed to UV light to form a covalent bond between the peptide and its binding site on C3. Subsequent analysis (SDS-PAGE, immunoblotting) identified a 40-kDa region on the β-chain of C3 as the binding site .

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For in vitro binding and functional assays, the complement-mediated hemolytic assay (CH50 assay) is used. Guinea pig erythrocytes (for classical pathway) or rabbit erythrocytes (for alternative pathway) are sensitized. Varying concentrations of Compstatin TFA (0-100 microM) are pre-incubated with human serum (diluted in GVB++ buffer). Erythrocytes are added, and after 30-60 minutes at 37degC, the reaction is stopped by cold buffer. The supernatant is analyzed for hemoglobin release by spectrophotometry (OD 405 nm). Percent inhibition is calculated relative to control. IC50 is determined from the dose-response curve. Binding affinity to purified C3 can be measured by SPR or ITC.Compstatin TFA is not typically used in direct cell-based assays for cytotoxicity or proliferation. It is used in complement deposition assays on cells. For example, human endothelial cells or target cells are incubated with human serum (a source of complement) in the presence or absence of Compstatin TFA (0-100 microM). After washing, deposition of C3b/iC3b on cell surfaces is detected by flow cytometry using an anti-C3 antibody. Inhibition of deposition is measured. To test cell viability, standard MTT assays can be performed to confirm no direct cytotoxicity of compstatin at tested concentrations.For ex vivo animal studies, compstatin derivatives are evaluated in non-human primate models. For baboon studies, compstatin is administered intravenously at doses of 1-50 mg/kg. Plasma samples are collected at baseline and at various time points after administration. Complement activity is assessed by CH50 assay or by measuring plasma C3a or sC5b-9 levels by ELISA. Inhibition of complement activation is calculated. For efficacy studies, primate models of disease (e.g., myocardial infarction, xenotransplantation) are used. Typical endpoints include tissue complement deposition, organ function, and survival.Compstatin TFA (molecular formula for compstatin: ~MW 1550.8 Da; for TFA salt, MW slightly higher). The TFA salt ensures optimal solubility (10 mg/mL in DMSO/PBS). Compstatin is a 13-residue cyclic peptide with a disulfide bridge. Newer analogs have enhanced pharmacokinetic properties with improved half-life and bioavailability. Compstatin is stored as a lyophilized powder at -20degC. It is soluble in water (1 mg/mL) and DMSO.Compstatin TFA is a research-grade peptide not for human use. At research concentrations (e.g., 1-50 microM in vitro), compstatin is well-tolerated. In animal studies, compstatin has shown a favorable safety profile with no significant adverse effects at therapeutic doses. As a specific C3 inhibitor, it does not interfere with other immune functions upstream of C3. For human therapeutic use, compstatin derivatives are in clinical development.Standard safety precautions for handling peptides should be followed.Compstatin TFA is a cyclic 13-residue peptide (sequence: H-ICVVQDWGHHRCT-NH2 with disulfide bridge between Cys2-Cys12). It is the parent compound of a family of C3-targeted complement inhibitors that are being developed for clinical use for complement-mediated diseases, including age-related macular degeneration (AMD), paroxysmal nocturnal hemoglobinuria (PNH), and various inflammatory conditions. It is for research use only and not approved for human use. Purity ≥95-98% by HPLC.

Not explicitly detailed in the provided search results for the specified articles. However, related work (referenced in ) mentions the use of hemolysis assays (complement-mediated lysis of red blood cells) to evaluate the inhibitory activity of Compstatin and its analogs, confirming its function in preventing membrane attack complex formation.

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Animal/Disease Models: Juvenile baboons (P. Anubis) weighing 10.5-28.8 kg[1].
Doses: 50, 25 mg/kg 60 min after heparin and 2 min before protamine.
Route of Administration: A bolus injection.
Experimental Results: Completely inhibited complement activation induced by heparin–protamine complexes.

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Baboon Model of Heparin/Protamine-Induced Inflammation: Adult baboons were anesthetized and instrumented for hemodynamic monitoring. Compstatin was administered intravenously. The dosing regimen that achieved complete inhibition consisted of an initial intravenous bolus, followed by a continuous infusion, totaling 21 mg/kg. Heparin and protamine were subsequently administered to induce complement activation, and blood samples were taken to measure complement activation products .
Ex Vivo Pig Xenograft Perfusion Model: Kidneys from Norwegian Landrace pigs were surgically removed. An ex vivo perfusion circuit was established using fresh, heparinized human AB blood from healthy volunteers. Compstatin was directly added to the human blood reservoir at a final concentration of 60 µM before the start of kidney perfusion. Perfusion conditions (temperature, oxygenation, pH) were carefully controlled to mimic physiological conditions. Graft survival was defined as the time until a sharp rise in vascular resistance rendered perfusion impossible .

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An in vitro human blood biotransformation study showed that the main degradation pathway of Compstatin is the enzymatic removal (cleavage) of the N-terminal isoleucine (Ile1) residue. This degradation can be blocked by chemically modifying the peptide through N-acetylation, resulting in a more stable analog that is less susceptible to enzymatic degradation in the blood.

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In the baboon study, an effective anticomplement dose of Compstatin (21 mg/kg, intravenously) did not cause any adverse hemodynamic effects, such as changes in heart rate or blood pressure, indicating that there was no acute cardiovascular toxicity at this dose in the study model.

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[1]. Inhibition of heparin/protamine complex-induced complement activation by Compstatin in baboons. Clin Immunol. 2000 Sep;96(3):212-21.

[2]. Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused pig xenografts. Xenotransplantation. 1999 Feb;6(1):52-65.

[3]. Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition. J Biol Chem. 2007 Oct 5;282(40):29241-7.

[4]. Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library. J Immunol. 1996 Jul 15;157(2):884-91.

Compstatin is a 13-amino acid cyclic peptide discovered by screening a phage-displayed library of random peptides antagonistic to C3b. Its sequence is Ile-Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys-Thr, cyclized via a disulfide bond between Cys2 and Cys12. It inhibits three complement activation pathways (classical, lectin, and alternative pathways) by binding to C3 and preventing proteolytic cleavage by C3 convertases, thereby blocking the generation of downstream effector molecules such as C3a, C5a, and the membrane attack complex (C5b-9). Due to its species-specificity for primate C3, non-human primate models (such as baboons) are crucial for preclinical in vivo evaluation. Compstatin is considered the prototype for designing complement-targeted therapies, and has been used to develop multiple generations of analogues with stronger efficacy and better drug properties for clinical development in various inflammatory diseases.

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C68H100F3N23O19S2

1664.79

1565.73437

Compstatin;206645-99-0

172873810

White to off-white solid powder

22

23

25

109

3170

14

C.CC[C@H](C)[C@@H](C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC1=O)C(C)C)C(C)C)CCC(=O)N)CC(=O)O)CC2=CNC3=CC=CC=C32)CC4=CN=CN4)CC5=CN=CN5)CCCN=C(N)N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N)N

NNKABSGFRUMXBK-MIGPDYDPSA-N

InChI=1S/C66H99N23O17S2.CH4/c1-8-32(6)50(68)63(104)86-46-27-108-107-26-45(62(103)89-53(33(7)90)54(69)95)85-56(97)39(14-11-17-74-66(70)71)80-59(100)43(20-36-24-73-29-78-36)83-58(99)42(19-35-23-72-28-77-35)79-48(92)25-76-55(96)41(18-34-22-75-38-13-10-9-12-37(34)38)82-60(101)44(21-49(93)94)84-57(98)40(15-16-47(67)91)81-64(105)51(30(2)3)88-65(106)52(31(4)5)87-61(46)102;/h9-10,12-13,22-24,28-33,39-46,50-53,75,90H,8,11,14-21,25-27,68H2,1-7H3,(H2,67,91)(H2,69,95)(H,72,77)(H,73,78)(H,76,96)(H,79,92)(H,80,100)(H,81,105)(H,82,101)(H,83,99)(H,84,98)(H,85,97)(H,86,104)(H,87,102)(H,88,106)(H,89,103)(H,93,94)(H4,70,71,74);1H4/t32-,33+,39-,40-,41-,42-,43-,44-,45-,46-,50-,51-,52-,53-;/m0./s1

2-[(4R,7S,10S,13S,19S,22S,25S,28S,31S,34R)-4-[[(2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl]carbamoyl]-34-[[(2S,3S)-2-amino-3-methylpentanoyl]amino]-25-(3-amino-3-oxopropyl)-7-[3-(diaminomethylideneamino)propyl]-10,13-bis(1H-imidazol-5-ylmethyl)-19-(1H-indol-3-ylmethyl)-6,9,12,15,18,21,24,27,30,33-decaoxo-28,31-di(propan-2-yl)-1,2-dithia-5,8,11,14,17,20,23,26,29,32-decazacyclopentatriacont-22-yl]acetic acid;methane

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~100 mg/mL (~60.07 mM)
H2O :~50 mg/mL (~30.03 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (1.65 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (1.65 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (1.65 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)