Betscheider, Dirk;Ray, Somak;Zangen, Dirk;Schultheiss, Eva;Lengauer, Thomas;Jose, Joachim - A new model for an inhibitor of human cathepsin G by bacterial surface display library screening and protein-peptide docking

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Title: A new model for an inhibitor of human cathepsin G by bacterial surface display library screening and protein-peptide docking	P13
Betscheider, Dirk; Ray, Somak; Zangen, Dirk; Schultheiss, Eva; Lengauer, Thomas; Jose, Joachim d.betscheider@mx.uni-saarland.de Pharmaceutical and Medicinal Chemistry, Saarland University, Postfach 15 11 50, D-66041 Saarbrücken, and Max-Planck-Institute for Informatics, Computational Biology and Applied Algorithmics, Stuhlsatzenhausweg 85, 66123 Saarbrücken, Germany

In recent years combinatorial chemistry has become a promising approach to generate new drugs. By this technique a large mixture of compounds is created that can be screened for new potential drug candidates. After a lead has been identified out of a library created by combinatorial chemistry, identifying its structure is a major problem. Solving this problem requires either highly sophisticated logistics during synthesis or subsequent fastidious analytics. If we deal with peptides as leads, such efforts are not necessary, when a library has been created on the surface of a living cell. Here, every variant carries its own label - the gene - which will reveal its sequence and hence its structure by simple and low-cost methods. Cells have the advantage of being self-replicating and can be subjected to high-throughput screening methods. Moreover, the fact that an inhibitor has high affinity to its enzyme can be exploited to specifically label cells that bear an inhibitor on their surface [1].
In the present study we displayed random variations of a peptidic inhibitor of human cathepsin G on the surface of E. coli by the aid of "autodisplay" [2], an efficient surface display system with broad applications [3, 4]. A protocol has been developed by which single cells bearing an active inhibitor can be selected. The protocol is based on fluorescence labeling of whole cells by binding of human cathepsin G and subsequent fluorescence activated cell sorting (FACS) [1]. Random libraries were screened using this protocol and DNA from selected cells was subjected to sequence analysis. Peptides derived from these DNA sequences will be used for inhibition studies and for virtual ligand docking with the crystal structure of the enzyme. For this purpose a protein-peptide docking tool is currently under development. The coupling of both methods, surface display library screening and computational biology is supposed to result in a new model for an optimized inhibitor of human cathepsin G. This model will serve as basis for the synthesis of new potent non-peptidic inhibitors.

[1] Zangen S and Jose J (2002) Pharm. Res. sub.
[2] Maurer J, Jose J, Meyer TF (1997) J Bacteriol, 179:794-804.
[3] Jose J, Bernhardt R, Hannemann F (2001) ChemBioChem, 2:695- 701; Jose J, Bernhardt R, Hannemann F (2002) J Biotechnol, 95: 257-268;
[4] Schultheiss E, Paar C, Schwab H, Jose J (2002) J. Mol. Catal. B: Enz., 18:89-97.