1. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University CLASSIFICATION AND CHARACTERIZATION OF NATURAL PROTEIN INHIBITORS OF PROTEIN KINASES AGATA MEGLICZ 1, JACEK LELUK 1, BOGDAN LESYNG 1,2 1 Interdisciplinary Centre for Mathematical and Computational Modelling (ICM), Warsaw University, Poland 2 Department of Biophysics, Faculty of Physics, Warsaw University, Poland

2. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Kinase project at ICM Complex comparative studies at the primary structure level Construction of molecular phylogenetic trees Studies on sequence/structure/function relationship Studies on the mechanisms of correlated mutations and variability Genetic principles of differentiation within the kinase and kinase inhibitor families

3. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Kinase protein inhibitors – current knowledge status Although various protein kinase families are relatively well described, there is much less known about natural protein inhibitors that control their activities. Protein kinase inhibitors are not sufficiently well classified into homologous families. There is not much known about their mechanisms of inhibition, and especially about structure-function relationships. The mechanisms of their specific recognition processes is still unclear in many cases. This limits the approaches aiming to select inhibitors of desired structural features and specificity.

4. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University The comparative study of primary structures of natural protein kinase inhibitors includes: -a thorough classification of this group of proteins -selecting the homologous families -describing of each selected family with respect to their mutational variability, structural properties and select regions that are important for their specificity. Our study started by selecting homologous inhibitor sequences. Multiple alignment was carried out and consensus sequences were constructed with the aid of the programs GEISHA (written by Adam Górecki) and Consensus Constructor (both elaborated at ICM).

5. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Multiple alignment of the „cAMP inhibitor family”

6. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Multiple alignment of the Cip inhibitor family

7. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Multiple alignment of the Ink4 inhibitor family

8. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Multiple alignment of the KCIP-1 inhibitor family (part 1/3)

9. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Multiple alignment of the KCIP-1 inhibitor family (part 2/3)

10. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Multiple alignment of the KCIP-1 inhibitor family (part 3/3)

11. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Multiple alignment of the HIT family

12. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University The families of protein kinase inhibitors KCIP-1 - inhibit Ca dependent kinases Ink4 – inhibit cyclin dependent kinases Cip/Kip – inhibit cyclin dependent kinases cAMP – inhibit cAMP dependent kinases The HIT family ??? - supposed to inhibit protein kinase C

13. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Consensus sequences KCIP-1 Cip/Kip Ink4

14. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Homology studies Identity: KCIP – 1 -> 45 - 82% Ink4 -> 37 – 50% Cip/Kip -> 33 – 40% (without the proline rich region) Similarity (genetic relationships): KCIP-1 -> 84 - 90% Ink4 -> 55 - 60% Cip/Kip -> 50 - 62% The most conservative regions: KCIP-1 -> 14.3.3 protein motif: RNLLSVAY (positions: 44-51), YKDSTLIMQLLRDNLTLWTS (positions:211-238) Ink4 -> Ankyrin motifs – a quadruple repeated motif (positions50- 67,81-101,117-134,149-169) Cip/Kip -> A domain reacting with the N-teminal site of the Cdk kinase (31-40,64-68), the NLS motif (255-265,285,289).

15. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Occurrence of six-codon amino acids in KCIP-1 family

16. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Occurrence of six-codon amino acids in Ink4 family

17. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Occurrence of six-codon amino acids in Cip/Kip family

18. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Simplified (planar) diagram of genetic relationships between amino acids In planar diagram the encoding role of the third codon position is ignored. Only first two codon positions are taken into account.

19. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Simplified (planar) diagram of genetic relationships between amino acids The simplified planar diagram emphasizes the special encoding character of six- codon amino acids – Leu, Arg and Ser. The six-codon amino acids may play the role the of „mutational passages” that are not liable to the selection restrictions. These amino acids may influence on the variability range increase. In fact the six-codon amino acids occur unusually frequent at very variable positions. This concerns especially serine, and to lesser extent – arginine. Leucine does not show the correlation between the frequency of occurrence and variability range.

20. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Frequency of six-codon amino acids as a function of position variability in randomly selected proteins of different origin and nature The results for 2686 residues at 606 corresponding positions

21. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Studies on phylogenetic relationships Program SSSS2 (Ela Gajewska and Jacek Leluk) Freely accessible Java application Contact with the authors egajewska@grid.icm.edu.plegajewska@grid.icm.edu.pl, lulu@icm.edu.pllulu@icm.edu.pl Phylogenetic trees generally reveal correlation with observed similarity

22. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Program SSSS2 The basic criteria used for analysis unsignificant significant Length of the sequence Contribution of identities (%) significant unsignificant Distribution of identical positions unsignificant significant

23. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Pairwise similarity estimation by program SSSS2 (Sequence Similarity Significance Statement v. 2)

24. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Pairwise similarity estimation

25. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Phylograms Cip inhibitor familyKCIP inhibitor family

26. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Phylograms Ink4 inhibitor familycAMP inhibitor family

27. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Phylograms HIT inhibitor (?) family

28. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Tertiary structures and correlated mutations within the inhibitor families cAMP (PKA) inhibitor family (PKI 5-24 ) Ink4 inhibitor family KCIP inhibitor family HIT family Cip inhibitor family

29. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Program Corm (written by Adam Górecki) http://tarawa.icm.edu.pl/agorecki/corm Location and characterization of correlated mutations occuring in proteins

30. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Correlated mutations within the Cip/Kip inhibitor family

31. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Correlated mutations within the Ink4 inhibitor family

32. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Correlated mutations within the KCIP-1 inhibitor family

33. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University The results of this comparative analysis can be used in the process of the rational drug design against many pathophysiological states caused by wrong functioning of kinases or their inhibitors. This work was supported by European Centre of Excellence for Multi- scale Biomolecular Modelling, Bioinformatics and Applications (project QLRI-CT-2002-90383) and by Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University.

34. Jacek Leluk, Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University Thank you for your attention ! Bogdan Lesyng Agata Meglicz Jacek Leluk currently at: Leiden University Medical Center The Netherlands