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

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

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

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

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

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

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

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

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

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

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

Group A6 Individual Data

Normalization of Activity in Growth Plates

Growth Z' Values

Normalization of Activity in Differentiation Plates

Differentiation Z' Values

Discarding Data

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

Growth Screen Hits

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

Differentiation Screen Hits

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.

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

p38 MAPK1 Signaling

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

FGF Signaling Pathway

RAR Activation Pathway

Wnt/Beta-Catenin Signaling Pathway

Wnt/Ca+ pathway

TGF-Beta Signaling for Positive Growth Hit

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

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

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

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

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

References file:///Users/farf4/Downloads/ c1.pdf html html conditions/osteomalacia/basics/definition/con conditions/osteomalacia/basics/definition/con

References