1. Module 4: High Throughput Screen Presentation BENG 162 Group A6 December 11, 2014 Alex Boone, Derrick Buntin, Frank Fleming, Keith Hoffman

2. Overview 1.Goals for Module 4 2.Background for Osteogenic Differentiation 3.Methodology and Rationale for Growth and Differentiation Screens 4.Data Results and Analysis Z' values, normalization, discarding data, hit picking, pathways 5.Discussion 6.Conclusion

3. Goals 1.Develop an understanding for the essential principles of chemical genomics 2.Screen for ~600 small molecules that promote/ inhibit osteogenic differentiation 3.Use the screened hits to determine which signaling pathway modulators influence osteogenic differentiation

4. Osteogenic Differentiation Background In Vitro Adult Mesenchymal Stem Cells combined with Differentiaion Media to form osteoblasts and eventually osteoclasts In Vivo Two Forms: Intramembranous and Endochondral Ossification Bone Alkaline Phosphatase Specific form of alkaline phosphatase produced by osteoblasts

5. Osteogenic Differentiation in Diverse Pathologies Osteoporosis - abnormally low bone density Osteopetrosis - abnornally high bone density Paget's disease - bone overgrowth and weakness Osteogenesis imperfecta - brittle bone disease Osteomalacia - softening of bone Bone Spurs - abnormal bone growth protrusions Rickets - bone tenderness

6. Osteogenesis as a Therapeutic Target Positive Hits from Growth Data Used to Promote Osteogenic Differentiation and Bone Formation e.g. Osteoporosis, Osteogenesis Imperfecta Positive Hits from Differentiation Data Used to Reduce the Severity of Abnormal Bone Growth e.g. Bone Spurs, Paget's Disease

7. Tissue Engineering Applications Regeneration of Bone Tissue Increasing Bone Density New Bone Formation Bone Marrow Replacement for Patients Suffering from Blood Diseases Increase Vascularization of Bone Graft Using Hydrogels or 3-D Printed Scaffolds

8. Methodology for Growth & Differentiation Screens 96-well Plates - MC3T3-E1 osteoblast -Positive controls in Growth Plate promote differentiation -Positive controls in Diff. Plate inhibit differentiation Alkaline Phosphatase Assay -para nitrophenyl phosphate (pNPP) -non-specific substrate cleaved via hydrolyis by alkaline phosphatase to form p-nitrophenol (yellow in color) Plate Reader -absorbance measured at 405 nm

9. Methodology for Growth & Differentiation Screens Overview of 96-Well Plate Negative Control Positive Control Experimental Compounds

10. Rationale for Growth Screen Used to Determine Promoters of Osteogenic Differentiation The Presence of Yellow Color in Growth Screen is Indicative of a Hit In this case, Hit Signifies Compound is an Promoter of Differentiation and Differentiation has Occurred

11. Rationale for Differentiation Screen Used to Determine Inhibitors of Osteogenic Differentiation The Absence of Yellow Color in Differentiation Screen Indicates a Hit In this case, Hit Signifies Compound is an Inhibitor of Differentiation and Differentiation has not Occurred

12. Group A6 Individual Data

13. Normalization of Activity in Growth Plates

14. Growth Z' Values

15. Normalization of Activity in Differentiation Plates

16. Differentiation Z' Values

17. Discarding Data

18. Growth Volcano Plot Hit Requirements: -Log(p-value) > 1 Activity > 0.2

19. Growth Screen Hits

20. Differentiation Volcano Plot Hit Requirements: -Log(p-value) > 20 Activity > 0.8

21. Differentiation Screen Hits

22. Module 3 Hit 2 (Trichostatin) - ss d G11 Hit 3 (Thapsigargin) - ss d G7 Hit 4 (Wnt Agonist) - ss a H10 Hit 5 (VEGF Inducer) - ss a H7 This data illustrates which molecules are effective at inhibiting osteogenic differentiation at varying concentrations.

23. p38 MAPK1 Pathway Hits Target in Pathway - Product Affecting Target MAPK 11 - Anisomycin, Streptomyces griseolus MAPK 12 - Anisomycin, Streptomyces griseolus MAPK 13 - Anisomycin, Streptomyces griseolus MAPK 14 - Anisomycin, Streptomyces griseolus

24. p38 MAPK1 Signaling

25. p38 MAPK1 Pathway Differentiation Hits Resulting in the Downregulation of: - Regulation of Mitotic Activity - Transcription - Dendritic Cell Endocytosis - Translation

26. FGF Signaling Pathway

27. RAR Activation Pathway

28. Wnt/Beta-Catenin Signaling Pathway

29. Wnt/Ca+ pathway

30. TGF-Beta Signaling for Positive Growth Hit

31. Limitations of a High Throughput Assay In vivo vs. in vitro Coverage of the full biological response landscape Accounting for chemical metabolism Ability to account for diversity in the population Accounting for chemical interactions between multiple compounds Determining a significant and adverse level of perturbation Insufficient accounting for epigenetic effects Controlling false negatives and false positives

32. Comparison to High Throughput Screening in Industry Large scale for industry production with the use of 384 or 1536 well screens Accuracy and speed of automated systems Computational speed and data storage Miniaturization reducing the cost of individual screens Higher throughput with larger compound libraries

33. Next Steps Individual assays on each hit to eliminate the chance of false positives and to establish an accurate activity level specific to each compound Run assays on other chemicals throughout the targeted pathways to determine their effect on differentiation Analyze adjoining pathways to observe the upstream/downstream effect of each compound

34. Problems & Uncertainties Determining which data to discard -method other than standard error of the mean Hit Picking -adjusting the p-value for the large sample size lead to no definitive hits -more quantitative calculation Target Pathway Selection -familiarity with alternative software

35. Conclusion Conducted a High Throughput Screening to Determine Promoters and Inhibitors of Osteogenic Differentiation Growth Screen Contained 4 Hits Differentiation Screen Contained 33 Hits Hits affected Differentiation Pathways for p38 MAPK1, FGF Signaling, RAR Activation, Wnt/Beta-Catenin Signaling, Wnt/Ca+ pathway Hit for Growth Pathway influences TGF-Beta Signaling

36. References file:///Users/farf4/Downloads/9783642023996-c1.pdf http://www.drkaslow.com/html/alkaline_phosphatase. html http://www.drkaslow.com/html/alkaline_phosphatase. html http://www.usbjd.org/projects/RBDPN_op.cfm?dirID=25 2 http://www.usbjd.org/projects/RBDPN_op.cfm?dirID=25 2 http://www.usbjd.org/projects/RBDPN_op.cfm?dirID=25 2 http://www.usbjd.org/projects/RBDPN_op.cfm?dirID=25 2 http://www.mayoclinic.org/diseases- conditions/osteomalacia/basics/definition/con- 20029393 http://www.mayoclinic.org/diseases- conditions/osteomalacia/basics/definition/con- 20029393

37. References http://en.wikipedia.org/wiki/Dendritic_cell http://www.isu.edu/~galisusa/alp_sop.html http://www.ijbs.com/v08p0272.htm