Consortional main: Mechanism of action and physiological role of the complement system: a comprehesive study based on in vitro evolved pathway selective inhibitors

  • Pál, Gábor G. (PI)
  • Boros, Eszter E. (CoPI)
  • Harmat, Veronika V. (CoPI)
  • Hegedüs, Rózsa R. (CoPI)
  • Heja, David (CoPI)
  • Kardos, József J. (CoPI)
  • Mezo, Gábor G. (CoPI)
  • Micsonai, András A. (CoPI)
  • Szakács, Dávid D. (CoPI)
  • Szilágyi, László L. (CoPI)

Project Details

Description

Summary including key goals

The complement system is part of the immune system. Its function is crucial for fighting infections but its abnormal activation leads to serious diseases. It is a complex network of over 30 proteins. It can activate through three different routes, the classical, the lectin and the alternative pathway. Each pathway relies on cascade like activation of specific proteases. The three pathways merge into a common effector cascade. We have recently developed using directed evolution novel peptide inhibitors against MASP-1 and MASP-2, key enzymes of the lectin pathway. The inhibitors totally and selectively block the lectin pathway while the other two pathways remain fully functional. Our key goals are as follows.

1. To develop highly specific substrate-like inhibitors against key proteases of the classical and the alternative pathway as well. For the first C1r and C1s, while for the latter factor D are the targets. These will be produced as recombinant proteins and various peptide- and protein scaffolds will be used in phage display based inhibitor evolution studies.

2. To submit the evolved inhibitors to thorough functional tests and to determine their structure in complex with the inhibited protease. The results should highlight fine molecular details that provide these proteases with extraordinary substrate specificity.

3. To chemically modify inhibitors for affinity chromatography, FRET and other specific studies.

4. To determine the exact protease composition of the lectin-pathway related recognition complexes using fluorescent labeled MASP-1 and MASP-2 inhibitors and FRET analysis as well as with inhibitor based affinity chromatography.

5. To quantitatively assess the selective contribution of individual complement activation pathways to cardiac tissue damage using pathway selective inhibitors and a tissue culture based myocardial infarct model.

Research questions

1. In the serum and in the complement system within highly similar proteases of common evolutionary origin play very different functional roles. Related to this there is a fundamental question: what kind of structural features provide such extraordinary selectivity to these proteases? We aim to answer this question using highly selective substrate-like inhibitors developed against the key proteases using directed evolution.

2. In case of the classical pathway the identity, activation order and specific function of the related proteases are well established. However, it is still debated how the two key enzymes, C1r and C1s are arranged in the C1q complex and exactly what kind of molecular movements take place during the activation process. We aim to answer these questions with using in vitro evolved and chemically labeled protease-selective inhibitors.

3. In the case of the lectin pathway even the protease composition of the related recognition complexes is questioned. It is debated whether MASP-1 and MASP-2 participate in mixed complexes, or only separate MASP-1 and MASP-2 complexes exist. Related to this question the sequence of activation of the two MASP enzymes is unknown. Moreover, it is unknown whether besides auto-activation, cross-activation could also take place, e.g. MASP-1 enzymes could activate MASP-2 zymogens and vice versa. Our selective inhibitors will allow us to reveal the answers.

4. The key initiating protease of the alternative pathway, factor D circulates in the blood in activated form. The genuine activator enzyme of zymogen factor D has been recently questioned. Again, the use of selective inhibitors could provide an answer to this fundamental question.

5. The three pathways contribute to different extent to pathological complement activation. Lack of selective inhibitors frustrated all efforts to decipher the significance of the individual activation pathways in complement related pathological states such as in the massive tissue damage upon cardiac attack. We can quantitatively assess this by using pathway selective inhibitors in a myocardial tissue based model system.

Significance of the research

Normal complement function is indispensable in fighting infections and removing altered (cancerous, apoptotic or necrotic) cells. On the other hand, uncontrolled activation of the complement results in life threatening clinical states. Both physiological as well as pathological significance of the system is very high, thus it would be extremely important to understand the exact activation mechanism of each individual pathway. Selective inhibitors developed against key proteases will be instrumental in deciphering the underlying molecular details. In the various pathologies generally only one of the three pathways is unregulated. Thus, pathway selective inhibitors promise extremely high therapeutic value too. With such inhibitors it becomes possible to block exclusively the unregulated pathway while the other two activation routes remain intact providing protection against pathogens and dangerous altered cells. The existing complement inhibitors block the common effector route. While such inhibitors effectively block the entire complement system, this leads to serious immune suppression. We have recently developed the very first lectin pathway selective inhibitors. With these we can study fundamental questions about the molecular details of the lectin pathway activation and we can also study the extent of the lectin pathway contribution to various physiological and pathological processes. Our long standing experience with in vitro evolution of proteins and the success with the MASP inhibitors make us confident that we will succeed in developing selective inhibitors against the classical and alternative pathway as well. Having selective inhibitors against all three pathways allow for unprecedentedly comprehensive and yet detailed structural, mechanistic and functional studies promising a great theoretical and therapeutic breakthrough in the field.

StatusFinished
Effective start/end date1/02/1031/12/16

Funding

  • Hungarian Scientific Research Fund

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