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Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs

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Rajarshi Guha
Rajarshi Guha
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Priori%zing Scaffolds for Hit Selec%on  in High Throughput Screening  Programs   Rajarshi Guha, Dac‐Trung Nguyen  NIH Chemical Genomics Center   6th Indo‐US Workshop on Mathema%cal Chemistry  8th – 10th January, 2010 
Outline  •  HTS and qHTS at the NCGC  •  Hit selec%on & cherry picks  •  The fragment based approach 
NIH Chemical Genomics Center  Assay development  Compound  and op2miza2on  Op2miza2on  Small Molecules  Biology  Chemistry  NCGC  Informa%cs  ACOM  Genome wide RNAi  SAR analysis, method &  Automa2on, Compound  tool development  management 
High Throughput Screening  •  Assay thousands to  hundreds of thousands  of compounds  –  Biochemical, gene%c,  pharmacological assays  •  Rapidly iden%fy novel  modulators of biological  systems  •  Lots of raw data –  automated analy%cs are  essen%al 
Hun%ng for Leads  Target  Lead  Lead  Clinical  Iden%fica%on  Discovery  Op%miza%on  Development  HTS  Primary  Confirma%on  •  Sensi%vity  Screening  •  Select subset  •  Scaling  •  Fluorescence  to follow up  •  Counter  •  High Content  •  Diversity  screen  •  Explore SAR  Assay  Cherry Picking  Op%miza%on 
High Throughput Screening  •  Given a screen, we  must iden%fy ac%ve  compounds and  confirm their ac%vity  •  Most HTS campaigns  are run at a single  concentra%on  Negative 150 Positive ! ! ! ! ! ! ! Sample ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! !! !! ! ! ! ! ! !! ! ! !! ! !! ! !! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! !! ! ! ! ! ! ! ! !! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! !! ! !! ! ! !! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! !! ! !! ! !! ! ! ! !! !! ! !! ! !! ! !! ! !! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! !! ! !! ! !! ! ! ! !! !! !! ! !! !! ! ! ! !! !! ! ! ! ! ! ! ! !! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! !! ! ! !!!! !! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! Signal ! ! ! !! ! ! ! ! ! !! !! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! !!! ! 100 ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! !!!! ! ! !! ! ! !! !! !! !! ! ! ! ! ! ! ! ! !! ! ! ! !! !!!! ! ! ! ! ! ! ! ! ! ! !! ! ! !! !!! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! !! !! ! ! ! ! ! !!! !!! ! !! ! !! !! ! ! ! ! ! ! !! ! !! ! ! ! ! !! !!! ! !! !! !! ! ! ! ! ! !! !! ! ! !! ! ! ! ! !! ! ! ! !! ! ! ! !! !! ! ! ! !! ! ! ! ! ! !! ! ! !! ! ! ! ! !! ! ! !! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! !!! ! ! ! ! ! ! ! ! ! !! ! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! !! !! ! !! ! ! !!!! ! !!!! ! !! ! !! !! !! ! ! !!!! ! ! ! ! !!! !!! !! ! ! ! ! ! !! ! !!! !!!! !! ! ! ! ! ! ! ! ! !!! !! !! ! ! ! ! ! ! !! ! ! ! ! !!! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! !!!! ! ! ! ! ! ! ! !! ! ! !! ! !! ! ! !!! !! !! !! ! !!! ! ! ! ! ! ! !!! !!! ! ! ! ! !! ! ! ! !! ! !! !! ! !! ! !! ! !!! !! !! ! ! ! ! !! ! ! ! ! !! ! !! !! ! ! ! !! !!! !! !!!!! !! !! !!! ! ! ! !! !! ! ! !!! !!! !! !!! ! ! ! ! !!! ! ! ! !! ! ! ! ! ! ! !!!! ! !! ! !! ! !! ! ! ! ! ! ! !! ! ! ! ! !! !! ! ! !! ! ! ! ! ! ! !! !! ! !! ! ! !! ! ! ! ! !! !! ! ! !! !! ! ! ! ! ! ! ! !! ! ! !! !!! ! ! !! ! ! !!! !!!! ! ! !! ! ! !!! ! ! ! ! ! ! !! ! ! !! !!! ! !! ! ! ! ! !! ! ! ! !! ! !! ! ! !! ! ! ! ! !!! ! ! ! !! !!! !! ! ! !!!!! ! !!! !! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !!! ! ! ! ! !!!! ! ! ! ! !! ! ! ! !!!! ! ! ! ! ! ! ! !!! ! ! !! ! ! ! ! !! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! !!! ! ! ! ! ! ! !!!! !! ! ! ! ! ! !!! !! ! ! ! ! ! ! ! ! !!! !! ! ! ! ! !! ! !! ! ! ! !!! ! !! ! ! ! ! !!! !! ! ! !!! ! ! ! ! !!! ! ! ! ! !!! ! ! !! ! ! ! ! ! ! !! ! ! ! ! !!! ! ! ! ! ! !! ! ! ! ! ! !!! !!! !!! ! ! ! ! !!!! ! ! !! !! ! ! !!!! !!! ! !!! ! ! ! ! !! !! !!! ! !!!! !! ! !! ! !! ! ! !!! ! !!! ! ! ! !! ! ! ! !! ! !! ! ! ! !! ! ! ! !! !!! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! !!! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !!! ! !! ! !! !!!! ! ! !!!!! !! ! !! ! ! ! !! ! ! ! !! !!! ! ! ! ! ! !! !! ! ! ! ! !!! !! !!!! !! ! ! !! ! ! !! !! !! ! ! ! ! !!! ! !! ! ! ! ! ! ! ! !! ! !! ! !! ! ! ! ! ! !! ! ! ! !! !!! !! ! !!! ! !!! ! ! ! ! ! !! !!!!! ! !!! ! !! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !!!! !! !! ! ! ! !! ! ! ! !! ! ! ! ! ! ! !! ! !!! ! !!!! ! ! !! ! ! ! ! !!! ! !! ! ! ! ! ! !! ! !! ! !!! ! !!!!!! !!! !! ! !! ! !!! ! ! ! ! ! !! ! !! ! ! ! !! !! ! ! ! ! !! ! ! !! ! ! ! ! !! ! ! ! !! ! !! ! ! ! !! ! !!! ! !! !! ! ! ! ! ! !! ! !!!!!!! ! ! !! ! ! ! ! !! ! !! ! !! ! !! !!! !! !! ! !! !! ! ! ! ! ! ! !!!!!! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! !! ! !! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! !! !!! ! ! !! ! !! ! ! ! !!! ! ! ! ! !!! ! ! ! !! ! ! ! ! !! ! !!!! !! ! ! ! !! ! ! ! ! !! ! ! !! ! ! ! ! !! ! !!!! !! !! ! !! ! ! !! ! ! ! ! !! ! !! ! ! ! !! !!! ! ! ! ! ! ! ! !!! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! !! ! ! ! !! ! !! !!! !! ! ! ! !! ! !! !! !! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! !! ! !! ! ! ! !! !!! ! !!! !! !! ! ! ! !! ! ! ! ! !!!! ! ! !! ! ! ! ! !! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! !! ! ! !! ! ! !! ! ! ! !! ! !! ! ! ! ! !! ! 50 ! !!! ! ! ! ! ! ! !! ! ! !! !! !!! ! ! ! ! ! ! ! ! !! !!!! ! ! ! ! ! ! ! ! !!! ! ! ! !! !! ! ! ! ! ! !! !! ! ! ! ! !! ! !! ! ! !! !! ! ! ! !! ! ! !! !! ! ! ! ! ! ! ! ! ! ! !! !! !! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! !!! !! ! ! ! ! !! !! ! ! ! ! ! !! ! ! ! !! ! ! ! !! !! ! ! ! ! ! ! ! ! !! ! ! !! ! ! !! !! ! ! ! ! ! !! !!!! ! ! !! ! !! ! ! ! ! ! !! ! !! !! ! !! ! !! ! !! ! !!! !!!! !! !! ! !!!!! ! ! ! !! ! ! ! ! !! ! !!!! ! ! ! ! ! ! ! ! ! ! ! ! !! !!! ! ! ! ! !! !! !!! ! ! ! !! ! ! ! ! !!!!! ! ! ! ! ! ! !! ! !! !! ! ! ! !!! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! !! ! ! ! ! !! !! ! ! ! ! !! !!! ! !! ! !!! ! ! !! ! ! ! !! ! ! ! ! !! ! !!! ! ! ! !! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! !!! ! ! ! !! ! ! ! ! ! !! ! !! !! !! ! ! ! ! ! !! ! !! ! ! !!! ! ! ! ! ! ! ! ! ! !!! ! !!! ! ! ! ! !! ! ! ! ! ! ! !! ! ! !!! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! !! ! !! ! ! !! ! ! ! !! ! !! ! ! ! ! !! ! !! !! !!! ! ! !! ! ! !! !! !! ! ! ! ! ! !! ! ! !! ! !! !! !! ! ! ! ! !! ! !! ! ! ! ! ! ! ! !!! ! ! ! !! ! ! !! ! ! !! !! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! !! ! !! ! ! ! ! ! !! ! !! ! ! ! ! !! !! ! ! ! ! ! ! ! !! !!! !! ! !! !! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! !! !! ! ! !! ! !! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! !! !!! ! ! !! ! ! ! ! ! ! !!! !! ! ! ! !! ! ! ! ! ! ! ! ! !! !! ! ! ! ! !!! ! ! ! !!! !! ! !! ! ! ! !! ! ! ! ! ! ! ! !!!! !! !! !! ! ! ! ! ! ! ! !!! !! ! ! ! ! ! ! ! ! ! !! ! !!! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! !! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! !! ! ! ! ! ! ! ! 0 Plate Number
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs

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Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs

  • 1. Priori%zing Scaffolds for Hit Selec%on  in High Throughput Screening  Programs   Rajarshi Guha, Dac‐Trung Nguyen  NIH Chemical Genomics Center   6th Indo‐US Workshop on Mathema%cal Chemistry  8th – 10th January, 2010 
  • 3. NIH Chemical Genomics Center  Assay development  Compound  and op2miza2on  Op2miza2on  Small Molecules  Biology  Chemistry  NCGC  Informa%cs  ACOM  Genome wide RNAi  SAR analysis, method &  Automa2on, Compound  tool development  management 
  • 4. High Throughput Screening  •  Assay thousands to  hundreds of thousands  of compounds  –  Biochemical, gene%c,  pharmacological assays  •  Rapidly iden%fy novel  modulators of biological  systems  •  Lots of raw data –  automated analy%cs are  essen%al 
  • 5. Hun%ng for Leads  Target  Lead  Lead  Clinical  Iden%fica%on  Discovery  Op%miza%on  Development  HTS  Primary  Confirma%on  •  Sensi%vity  Screening  •  Select subset  •  Scaling  •  Fluorescence  to follow up  •  Counter  •  High Content  •  Diversity  screen  •  Explore SAR  Assay  Cherry Picking  Op%miza%on 
  • 6. High Throughput Screening  •  Given a screen, we  must iden%fy ac%ve  compounds and  confirm their ac%vity  •  Most HTS campaigns  are run at a single  concentra%on  Negative 150 Positive ! ! ! ! ! ! ! Sample ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! !! !! ! ! ! ! ! !! ! ! !! ! !! ! !! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! !! ! ! ! ! ! ! ! !! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! !! ! !! ! ! !! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! !! ! !! ! !! ! ! ! !! !! ! !! ! !! ! !! ! !! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! !! ! !! ! !! ! ! ! !! !! !! ! !! !! ! ! ! !! !! ! ! ! ! ! ! ! !! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! !! ! ! !!!! !! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! Signal ! ! ! !! ! ! ! ! ! !! !! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! !!! ! 100 ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! !!!! ! ! !! ! ! !! !! !! !! ! ! ! ! ! ! ! ! !! ! ! ! !! !!!! ! ! ! ! ! ! ! ! ! ! !! ! ! !! !!! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! !! !! ! ! ! ! ! !!! !!! ! !! ! !! !! ! ! ! ! ! ! !! ! !! ! ! ! ! !! !!! ! !! !! !! ! ! ! ! ! !! !! ! ! !! ! ! ! ! !! ! ! ! !! ! ! ! !! !! ! ! ! !! ! ! ! ! ! !! ! ! !! ! ! ! ! !! ! ! !! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! !!! ! ! ! ! ! ! ! ! ! !! ! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! !! !! ! !! ! ! !!!! ! !!!! ! !! ! !! !! !! ! ! !!!! ! ! ! ! !!! !!! !! ! ! ! ! ! !! ! !!! !!!! !! ! ! ! ! ! ! ! ! !!! !! !! ! ! ! ! ! ! !! ! ! ! ! !!! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! !!!! ! ! ! ! ! ! ! !! ! ! !! ! !! ! ! !!! !! !! !! ! !!! ! ! ! ! ! ! !!! !!! ! ! ! ! !! ! ! ! !! ! !! !! ! !! ! !! ! !!! !! !! ! ! ! ! !! ! ! ! ! !! ! !! !! ! ! ! !! !!! !! !!!!! !! !! !!! ! ! ! !! !! ! ! !!! !!! !! !!! ! ! ! ! !!! ! ! ! !! ! ! ! ! ! ! !!!! ! !! ! !! ! !! ! ! ! ! ! ! !! ! ! ! ! !! !! ! ! !! ! ! ! ! ! ! !! !! ! !! ! ! !! ! ! ! ! !! !! ! ! !! !! ! ! ! ! ! ! ! !! ! ! !! !!! ! ! !! ! ! !!! !!!! ! ! !! ! ! !!! ! ! ! ! ! ! !! ! ! !! !!! ! !! ! ! ! ! !! ! ! ! !! ! !! ! ! !! ! ! ! ! !!! ! ! ! !! !!! !! ! ! !!!!! ! !!! !! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !!! ! ! ! ! !!!! ! ! ! ! !! ! ! ! !!!! ! ! ! ! ! ! ! !!! ! ! !! ! ! ! ! !! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! !!! ! ! ! ! ! ! !!!! !! ! ! ! ! ! !!! !! ! ! ! ! ! ! ! ! !!! !! ! ! ! ! !! ! !! ! ! ! !!! ! !! ! ! ! ! !!! !! ! ! !!! ! ! ! ! !!! ! ! ! ! !!! ! ! !! ! ! ! ! ! ! !! ! ! ! ! !!! ! ! ! ! ! !! ! ! ! ! ! !!! !!! !!! ! ! ! ! !!!! ! ! !! !! ! ! !!!! !!! ! !!! ! ! ! ! !! !! !!! ! !!!! !! ! !! ! !! ! ! !!! ! !!! ! ! ! !! ! ! ! !! ! !! ! ! ! !! ! ! ! !! !!! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! !!! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !!! ! !! ! !! !!!! ! ! !!!!! !! ! !! ! ! ! !! ! ! ! !! !!! ! ! ! ! ! !! !! ! ! ! ! !!! !! !!!! !! ! ! !! ! ! !! !! !! ! ! ! ! !!! ! !! ! ! ! ! ! ! ! !! ! !! ! !! ! ! ! ! ! !! ! ! ! !! !!! !! ! !!! ! !!! ! ! ! ! ! !! !!!!! ! !!! ! !! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !!!! !! !! ! ! ! !! ! ! ! !! ! ! ! ! ! ! !! ! !!! ! !!!! ! ! !! ! ! ! ! !!! ! !! ! ! ! ! ! !! ! !! ! !!! ! !!!!!! !!! !! ! !! ! !!! ! ! ! ! ! !! ! !! ! ! ! !! !! ! ! ! ! !! ! ! !! ! ! ! ! !! ! ! ! !! ! !! ! ! ! !! ! !!! ! !! !! ! ! ! ! ! !! ! !!!!!!! ! ! !! ! ! ! ! !! ! !! ! !! ! !! !!! !! !! ! !! !! ! ! ! ! ! ! !!!!!! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! !! ! !! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! !! !!! ! ! !! ! !! ! ! ! !!! ! ! ! ! !!! ! ! ! !! ! ! ! ! !! ! !!!! !! ! ! ! !! ! ! ! ! !! ! ! !! ! ! ! ! !! ! !!!! !! !! ! !! ! ! !! ! ! ! ! !! ! !! ! ! ! !! !!! ! ! ! ! ! ! ! !!! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! !! ! ! ! !! ! !! !!! !! ! ! ! !! ! !! !! !! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! !! ! !! ! ! ! !! !!! ! !!! !! !! ! ! ! !! ! ! ! ! !!!! ! ! !! ! ! ! ! !! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! !! ! ! !! ! ! !! ! ! ! !! ! !! ! ! ! ! !! ! 50 ! !!! ! ! ! ! ! ! !! ! ! !! !! !!! ! ! ! ! ! ! ! ! !! !!!! ! ! ! ! ! ! ! ! !!! ! ! ! !! !! ! ! ! ! ! !! !! ! ! ! ! !! ! !! ! ! !! !! ! ! ! !! ! ! !! !! ! ! ! ! ! ! ! ! ! ! !! !! !! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! !!! !! ! ! ! ! !! !! ! ! ! ! ! !! ! ! ! !! ! ! ! !! !! ! ! ! ! ! ! ! ! !! ! ! !! ! ! !! !! ! ! ! ! ! !! !!!! ! ! !! ! !! ! ! ! ! ! !! ! !! !! ! !! ! !! ! !! ! !!! !!!! !! !! ! !!!!! ! ! ! !! ! ! ! ! !! ! !!!! ! ! ! ! ! ! ! ! ! ! ! ! !! !!! ! ! ! ! !! !! !!! ! ! ! !! ! ! ! ! !!!!! ! ! ! ! ! ! !! ! !! !! ! ! ! !!! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! !! ! ! ! ! !! !! ! ! ! ! !! !!! ! !! ! !!! ! ! !! ! ! ! !! ! ! ! ! !! ! !!! ! ! ! !! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! !!! ! ! ! !! ! ! ! ! ! !! ! !! !! !! ! ! ! ! ! !! ! !! ! ! !!! ! ! ! ! ! ! ! ! ! !!! ! !!! ! ! ! ! !! ! ! ! ! ! ! !! ! ! !!! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! !! ! !! ! ! !! ! ! ! !! ! !! ! ! ! ! !! ! !! !! !!! ! ! !! ! ! !! !! !! ! ! ! ! ! !! ! ! !! ! !! !! !! ! ! ! ! !! ! !! ! ! ! ! ! ! ! !!! ! ! ! !! ! ! !! ! ! !! !! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! !! ! !! ! ! ! ! ! !! ! !! ! ! ! ! !! !! ! ! ! ! ! ! ! !! !!! !! ! !! !! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! !! !! ! ! !! ! !! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! !! !!! ! ! !! ! ! ! ! ! ! !!! !! ! ! ! !! ! ! ! ! ! ! ! ! !! !! ! ! ! ! !!! ! ! ! !!! !! ! !! ! ! ! !! ! ! ! ! ! ! ! !!!! !! !! !! ! ! ! ! ! ! ! !!! !! ! ! ! ! ! ! ! ! ! !! ! !!! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! !! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! !! ! ! ! ! ! ! ! 0 Plate Number