ラン藻ゲノム交流会 2006 年 7 月 6 日

1. HIGH QUALITY MODELING DATABASE OF HUMAN, RAT AND MOUSE GENES FOR THREE DIMENSIONAL PROTEIN STRUCTURE AND THE DRUG DESIGN (Department of Biomolecular Design, School of Pharmaceutical Sciences, Kitasato University) Mitsuo Iwadate, Kazuhiko Kanou, Daisuke Takaya, Genki Terashi, Mayuko Takeda-Shitakaand Hideaki Umeyama,. ラン藻ゲノム交流会2006年7月6日

2. Contents • The 3-dmensional structure database, “human-rat-mouse FAMSBASE”, modeled by the “famd” method proposed by 　“famd” team in CASP6 of 2004. • GDT-TSestimatimation is used for the model accuracy. In an international contest "CASP6", GDT-TS is an important index showing how correct the modeled protein structure is. • In silico screening of Oncogene “Brk”.

3. Human Rat Mouse Human Rat Mouse Sequence Database • Homo sapiens (ENSEMBLE) hsap0 28063 in 2004 (36272 in 2005)- Metazoa Chordata ENSEMBL • Homo sapiens(H-Invitational) hsap1 39067 in 2004 (39091 in 2005) - Metazoa Chordata JBIRC • Homo sapiens cDNA huge0 2037 - Metazoa Chordata Kazusa • Rattus norvegicus28682 in rnor0 (27122 in 2005) Metazoa Chordata ENSEMBL • Mus musculus mmus0 24928 in 2004 (30364 in 2005) Metazoa Chordata ENSEMBL Total: 122777

4. BLAST from NCBI original profile 7 homology search and alignment methods are used in this research • FASTA • BLAST • PSI-BLAST • IMPALA • RPS-BLAST • PSF-BLAST • PFAM-BLAST

5. Our scoring functionwas made by our “famd” team in CASP6. • SCORE=f(program,Len, Hom, SS) Program=7 kinds Len=(Model length/ORF length) Hom=Homology % SS= Matching Ratio of Secondary Structure between PSI-PRED 1) and STRIDE2). The higher value of this function obtained from protein sequence alignment is made as the GDT-TS score shows a higher value. Top 5 score alignments are selected and used to model the 3-dimensional structure. • McGuffin LJ etc “The PSIPRED protein structure prediction server.” Bioinformatics. 16, 404-405. (2000) • Frishman,D & Argos,P. “Knowledge-based secondary structure assignment.”Proteins, 23, 566-579. (1995)

6. 2 types of functions were used in this paper. • SCORE=f(program, Len, Hom, SS) for alignment selection • estimated GDT_TS= KNN(program, e-value, Homology %, Target Amino Acid Length, Model Amino Acid Length) for model quality estimation

7. 5.7Å 2.2Å 2.2Å 5.3Å 3.5Å 3.5Å 0.8Å 0.8Å 0.8Å 6.5Å 1.9Å 1.9Å 1.9Å 4.2Å GDT_TS(Global Distance Test Total Score) GDT_TS=(GDT_P1+ GDT_P2+ GDT_P4+ GDT_P8)/4 Where GDT_Pn Percent of residues under Ca distance cutoff <=n.0 Å. example GDT_P1=(1/9)*100 GDT_P2=(2/9)*100 GDT_P4=(4/9)*100 GDT_P8=(8/9)*100 GDT_TS=41.7 Chain A 8.1Å 5.7Å 2.2Å 5.3Å 3.5Å 0.8Å 0.8Å 1.9Å 6.5Å 4.2Å Chain B

8. Number of Modeled Genes Most (99%=121191/12277) of Genes are modeled 99% of genes are modeled 36% of genes are (gene length base=raw) GDT_TS ≧40 92% of genes are model length base GDT_TS ≧40

9. 1e-50 1e-30 1e-10 1e-3 Multi Regions are Modeled Top 5 models of each amino acid region are modeled. Target Amino Acid length

10. ⑤ σ=10.92 Real GDT_TS=80~100 ④ σ=11.88 Real GDT_TS=60~80 ⑤ Real GDT_TS=40~60 ③ σ=10.86 ④ ② σ=10.53 Real GDT_TS=20~40 Real GDT_TS=0~20 ① σ=4.83 ③ ② ① Results of GDT_TS Estimation 10-fold Jack-Knife Cross Validation test Corr. Coeff =0.95 Estimated DGT_TS Plotted points of 0.28 million Real GDT_TS

11. GDT_TS=70 GDT_TS=100 GDT_TS=80 GDT_TS=40 GDT_TS=60 GDT_TS=30 GDT_TS=50 GDT_TS=90 GDT_TS=20 GDT_TS examples

12. 70 100 80 40 60 30 50 90 20 GDT_TS and model quality

13. Target Amino Acid length Estimated GDT_TS histgram of human Raw GDT_TS Model Length Base GDT_TS =(Raw GDT_TS)*(target length / model length)

14. Estimated GDT_TS histgram of human-rat-mouse Raw GDT_TS Model Length Base GDT_TS =(Raw GDT_TS)*(target length / model length)

15. 70 100 80 40 60 30 50 90 20 Model Length Base GDT_TS and model quality

16. Search for Gene name Search Screen Search for Genome name Search for Reference PDB Search for Expected Values Search for Homology Values Search for Annotation keywords Search for Sequence

17. example (text keyword: kinase) Input of “kinase”

18. Estimated GDT_TS histgram example (text keyword: kinase) Human Raw GDT_TS Model Length Base GDT_TS

19. Estimated GDT_TS histgram example (text keyword: kinase) Human-Rat-Mouse Raw GDT_TS Model Length Base GDT_TS

20. HIT000012587.3 in jbic0 Search Result Example GDTTS=90 GDTTS=48

21. 70 100 80 40 60 30 50 90 20 Model Length Base GDT_TS and model quality 48 90

22. Brk is possible to be a therapeutic target in cancer Breast tumor kinase (Brk) … • is a soluble Src-like protein-tyrosine kinase. • is overexpressed in the majority of breast cancers, but absent from normal mammary tissues. Barker KT et al. (1997) Oncogene, 15:799-805. • RNA interference of Brk significantly suppressed proliferation of breast carcinoma cells. Harvey AJ and Crompton MR. (2003) Oncogene, 22 5006-5010. • activates rac1 and promotes cell migration and invasion by phosphorylating paxillin. • is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. Chen HY et al. (2004) Mol Cell Biol. 24, 10558-72. • Brk interacts with protein kinase B/Akt, a serine/threonine kinase involved in cell growth and survival. Zhang P et al. (2005) JBC. 280, 1982-1991. • associate with insulin receptor substate-4 (IRS-4) and effects cell proliferation, growth, survival … Qie H et al. (2005) Oncogene 24, 5656-5664.

23. 90 80 GDT_TS is estimated 81.90± 11.39 In the FAMSBASE, Human Brk GDT_TS is 82.04±11.39 or Breast tumor kinase (Brk) This oncogene Human Brk increases in the breast cancer. >Brk HOMOLOGY MATCH MISMATCH INSERTION DELETION >1Y57_A 443 44.4% 196 2 0 7 5 GPKYVGLWDFKSRTDEELSFRAGDVFHVARKEEQWWWATLLDEAGGAVAQGYVPHNYLAERETVESEPWFFGCISRSEAV VTTFVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLAHSLSTG---QTGYIPSNYVAPSDSIQAEEWYFGKITRRESE RRLQAEGNATGAFLIRVSEKPSADYVLSVRDTQA-----VRHYKIWRRAGGRLHLNEAVSFLSLPELVNYHRAQSLSHGL RLLLNAENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCH RLAAPCRKHEPEPLP-HWDDWERPREEFTLCRKLGSGYFGEVFEGLWKDRVQVAIKVISRDNLLHQQMLQSEIQAMKKLR RLTTVCPTSKPQTQGLAKDAWEIPRESLRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEA-FLQEAQVMKKLR HKHILALYAVVSVGDPVYIITELMAKGSLLELLRDSDEKVLPVSELLDIAWQVAEGMCYLESQNYIHRDLAARNILVGEN HEKLVQLYAVVS-EEPIYIVTEYMSKGSLLDFLKGETGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAANILVGEN TLCKVGDFGLARLIKEDVYL-SHDHNIPYKWTAPEALSRGHYSTKSDVWSFGILLHEMFSRGQVPYPGMSNHEAFLRVDA LVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVER GYRMPCPLECPPSVHKLMLTCWCRDPEQRPCFKALRERLSSFTSYENP GYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYFTSTEP Homology 44.44% E-value under 1e-200 Length of Brk 451a.a. Model lenth 441a.a. Two model examples are shown above.

24. Ligand: MPZ a des-methyl analog of the antileukemia drug imatinib (STI571; Gleevec). 1Y57_A (Proto-Oncogene Tyrosine-Protein Kinase Src) Human Brk model

25. Human Rat Mouse Brk models of Human-Rat-Mouse

26. ５Å １０Å Ligand binding site

27. Homology around the binding site Mouse vs Rat Human vs Rat Human vs Mouse

28. In silico screening system chart Target protein selection and modeling Database of ligand molecules Superinposed ligand atoms called A group by Dali algorism ① ② Ligand atoms are superimposed on A group Using the interacting function with some structures including some protein conformations, ligand docking. ③ Ligand conformation determination after changing the ligand conformation largely ④ Calculation of interaction energy between protein and ligand Ligand ranking for binding to protein This system is composed of the pathway from 1 to 4.

29. In silico ligand screening, the smaller area of the binding site in the protein are used. Brk model In silico ligand screening, the smaller area of the binding site in the protein are used. 綺麗な絵

30. Results of ligand screening 8685 compounds in MDL ComprehensiveMedicinalChemistry database MPZ Gleevec analog Hits (Top 10)

31. Conclusions • Protein modeling database “human-rat-mouse FAMSBASE” was constructed using the “famd” method, and, additionally, algorism of this “famd” method has been validated especially for side-chain torsion angle correctness. • GDT_TS of all models in “human-rat-mouse FAMSBASE” were estimated and written on the database. • The models having highly estimated GDT_TS for oncogene Brk tyrosine kinases were made and applicable in silico screening.