Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
-
Upload
rguha -
Category
Technology
-
view
414 -
download
0
Transcript of Prioritizing Scaffolds for Hit Selection in High Throughput Screening Programs
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
Biology Chemistry
Informa%cs ACOM
NCGC
Assay development and op2miza2on
Compound Op2miza2on
Automa2on, Compound management
SAR analysis, method & tool development
Small Molecules
Genome wide RNAi
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
Target Iden%fica%on
Lead Discovery
Lead Op%miza%on
Clinical Development
Hun%ng for Leads
• Sensi%vity • Scaling
Assay Op%miza%on
• Fluorescence • High Content
Primary Screening • Select subset
to follow up • Diversity
Cherry Picking
• Counter screen
• Explore SAR
Confirma%on
HTS
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
!
!!!!
!
!!!!!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!!!!
!
!
!
!
!
!
!!
!
!!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!!!!!
!
!!!!!!
!!
!
!
!
!!
!
!!!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!!!!
!!!!!!!
!!
!
!
!
!
!!
!
!
!!!!!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!
!
!!
!!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!!!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!!
!!!!!
!!!!!!
!
!
!
!!!!
!
!
!!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!!
!
!
!!
!!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!!!!!!!!!!!!!
!!
!!
!
!!
!
!
!!!
!!!!!
!!
!
!
!
!
!!
!
!!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!!!
!
!!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!!
!!!
!
!!!
!
!
!
!
!
!
!!!
!
!
!!!!!!!!!
!!
!
!
!
!!
!!!!!!!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!!
!!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!!
!!
!
!
!
!
!
!
!!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!!!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!!
!
!!
!
!
!!
!
!
!
!
!!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!!!
!
!
!
!
!!
!
!!
!!!
!!!
!!!!!!!
!
!
!
!!!!!!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!
!!
!
!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!!!
!!!
!
!!!!!
!
!
!
!!!!!
!
!
!!
!!!!
!!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!!
!!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!
!!!!!!!
!!
!!!!!!!
!
!
!!
!!!!!!
!
!!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!!!!!!!!!!!!!
!
!!!
!
!!!!
!
!!
!
!
!!
!
!
!!
!
!
!
!
!
!!
!!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!!!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!!
!
!!!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!!!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!!!!!!!!!!!!!!!
05
01
00
15
0
Plate Number
Sig
nal
Negative
Positive
Sample
The qHTS Paradigm
• Tradi%onal single point screens can miss useful hits
• qHTS involves concentra%on response assays on a high‐throughput scale
• The CRC allows us to categorize hits in a more fine‐grained manner
Inglese, J et al, Proc. Natl. Acad. Sci., 2006, 103, 11473‐11478
!
!!!!
!
!!!!!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!!!!
!
!
!
!
!
!
!!
!
!!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!!!!!
!
!!!!!!
!!
!
!
!
!!
!
!!!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!!!!
!!!!!!!
!!
!
!
!
!
!!
!
!
!!!!!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!
!
!!
!!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!!!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!!
!!!!!
!!!!!!
!
!
!
!!!!
!
!
!!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!!
!
!
!!
!!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!!!!!!!!!!!!!
!!
!!
!
!!
!
!
!!!
!!!!!
!!
!
!
!
!
!!
!
!!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!!!
!
!!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!!
!!!
!
!!!
!
!
!
!
!
!
!!!
!
!
!!!!!!!!!
!!
!
!
!
!!
!!!!!!!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!!
!!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!!
!!
!
!
!
!
!
!
!!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!!!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!!
!
!!
!
!
!!
!
!
!
!
!!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!!!
!
!
!
!
!!
!
!!
!!!
!!!
!!!!!!!
!
!
!
!!!!!!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!
!!
!
!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!!!
!!!
!
!!!!!
!
!
!
!!!!!
!
!
!!
!!!!
!!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!!
!!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!
!!!!!!!
!!
!!!!!!!
!
!
!!
!!!!!!
!
!!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!!!!!!!!!!!!!
!
!!!
!
!!!!
!
!!
!
!
!!
!
!
!!
!
!
!
!
!
!!
!!
!
!
!!
!
!
!
!
!
!!
!
!
!
!
!!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!!!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!!
!
!!!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!!!
!
!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!!!!!!!!!!!!!!!
05
01
00
15
0
Plate NumberS
ignal
NegativePositiveSample
1 2 3 4 5 6 7 8 9 10
Concentra%on Response Curves
• Each compound is characterized by 4 Hill parameters
• Also evaluate p‐values and R2
• Finally we classify each curve
Concentration
Activity
S0
50%
SInf
AC50
Curve Class Usage • Heuris%c assessment of the significance of a
concentra%on response curve
• We aggregate certain curve classes into “ac%ve”, “inconclusive” and “inac%ve” categories
• Inconclusive is a “catch all” category (i.e., if it not clearly ‘ac%ve’ or ‘inac%ve’)
9
Inac2ve
Ac2ve
Inconclusive
Hit Selec%on
• Easy to iden%fy ini%al set of ac%ves • Ranges from 50 to 5000 molecules
• How do we select a subset that will likely retest as ac%ve in secondary assays? – Avoid false nega%ves as far as possible
• We’d also like to be able to explore SAR’s
300K
1000
300
Nu
mb
er o
f M
ole
cu
les
Cherry
Picks
HTS
Hit Selec%on – Prior Work
• The simplest method is purely sta%s%cal – Select compounds above/below an ac%vity threshold
• Many methods are based on similarity to known ac%ves or neighborhoods – Hit‐directed nearest neighbor – Predic%ve models – Local hit rate analysis
Shanmugasundaram, V. et al, J. Med. Chem., 2005, 48, 240‐248 Selzer P. et al, J. Chem. Inf. Model., 2006, 46, 2319‐2323 Posner, B. et al, J. Chem. Inf. Model., 2009, 49, 2202‐2210
Activity
Frequency
0200
400
600
800
1000
1200
Why Use Fragments?
• Conceptually equivalent to clustering • Very fast with pre‐computed fragments compared to clustering – No similarity matrix calcula%ons
• Simple to implement – Iden%fy fragments associated with ac%ve compounds – For a given fragment iden%fy the ac%ve, inac%ve, inconclusive members
– Rank these fragment “series” for follow up
The Fragment Analysis Workflow
Identify Scaffolds
Calculate Scaffold Statistics
1. Min/Max potency and efficacy2. Ratio of actives to size of member set3. Complexity of the scaffold4. Promiscuity (based on active members)
Flourescence Flagging
Scaffold Reduction
Priority Series
Non-Priority Series
SingletonsFlourescence Flagging
Promiscuity & PotencyFilter
Promiscuity & PotencyFilter
Promiscuity & PotencyFilter
Scaffold Genera%on
• We employ Murcko fragments • Pre‐generate fragments on compound registra%on and store them in the database
• For a given screen, we iden%fy fragments based on the ac%ve compounds and then proceed with the analysis – Ignore “common” fragments (benzene, furan, …)
Scaffold Genera%on
Scaffold Reduc%on
• The reduc%on process tries to iden%fy a subset of core‐like fragments
• What is core‐like? – Somewhat subjec%ve
• Ini%ally, we iden%fy fragments that are substructures of another fragment – Keep the smaller fragment – Not always the best choice
• What makes a good scaffold? – Size, complexity, … – Do the members represent an SAR or not?
– Intui%on and experience also play a role
Scoring Scaffolds
Scaffolds, R‐groups & SAR
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!8 !6 !4 !2 0
!8
!6
!4
!2
0
ObservedPredicted
Evaluate topological and physicochemical descriptors for the R‐groups
Fit PLS or ridge regression model
Characterize the SAR landscape
Guha, R et al. J. Chem. Inf. Model., 2008,48, 646‐658
Scaffold QSAR ‐ Drawbacks
• Many scaffolds have few (5 to 10) members • Invariably, more features than observa%ons • If the number of R‐groups is large, the feature matrix can be very sparse – Less of a problem for combinatorial libraries
• A linear fit may not be the best approach to correla%ng R‐groups to the ac%vi%es – Difficult to choose a model type a priori
• SBll working on it …
Measuring Promiscuity • Promiscuity is measured on a compound‐wise basis
• Defined in terms of how many %mes it is ac%ve in a pre‐defined set of assays versus how many %mes it’s not ac%ve
• Promiscuity ranges from 1 to 10 – 1 indicates it was not ac%ve in any assay – 10 indicates it was ac%ve in all assays – 0 indicates it wasn’t tested
• Scaffold promiscuity is the mean promiscuity of its ac%ve members
Measuring Promiscuity
Promiscuity Score
Fre
qu
en
cy
1 2 3 4 5 6 7
05
00
10
00
15
00
20
00
1 2 3 4 5 6 7
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Promiscuity Score
Wid
th o
f C
onfidence Inte
rval
€
Promiscuity Score =10Nactive + Ninactive
Nactive + Ninactive
Fluorescence Flagging
• Depends on the nature of the screen • For a single compound, flag if ac%ve in spectral profiling assay or LDR fluorescence counterscreen
• For a series – Flag if a single ac%ve compound in the series is ac%ve in spectral profiling assay or LDR fluorescence counterscreen
– Or if > 50% of the ac%ve series members are ac%ve in LDR/Lamin‐A/Betaglobin
qHTS for Tau Inhibitors
• Fibrils of the microtubule associated protein tau are observed in Alzheimers pa%ents
• Goal was to iden%fy small molecule inhibitors of tau oligomeriza%on
• Screened 292K compounds, iden%fied 4788 ac%ves
• Selected a novel aminothienopyridazine series
Crowe, A. et al, Biochemistry, 2009, 48, 7732‐7745
Fragment Analysis on Tau‐2 Identify Scaffolds
1. Consider the 4788 actives2. Ignore non-specific scaffolds
Calculate Scaffold Statistics
1. Min/Max potency and efficacy2. Ratio of actives to size of member set3. Complexity of the scaffold4. Promiscuity (based on active members)
Flourescence Flagging
1. If a single active member is active in cho-gfp, the cluster is flagged fluorescent2. If a single active member is active in LDR retest, the cluster is flagged fluorescent2. If 50% of the actives are active in LDR/ Lamin-A/Betaglobin, the cluster is flagged
Non flourescent other scaffolds 514
Scaffold Reduction
Use a substructure method to collapse scaffolds into more "core-like" scaffolds.
Priority series (scaffolds with > 10% actives) 46
Potency & Promiscuity Filter
1. Keep series whose minimum potency < X uM2. Keep series with promiscuity < Y
Priority series (scaffolds with > 10% actives) ?
Non-priority series (scaffolds with < 10% actives)
459
Potency & Promiscuity Filter
1. Keep series whose minimum potency < X uM2. Keep series with promiscuity < Y
Non-priority series (scaffolds with < 10% actives)
?
Identify singleton compounds (i.e., compounds with no precomputed scaffold)
866
Potency & Promiscuity Filter
1. Keep cmpds whose minimum potency < X uM2. Keep cmpds with promiscuity < Y
Identify singleton compounds (i.e., compounds with no precomputed scaffold)
?
Flourescence Flagging
1. If compound is active in cho-gfp/spec-profile/ LDR retest, the cluster is flagged fluorescent
• All singletons
• 2 ac%ves, 1 inac%ve from priority series
• 1 ac%ve from non‐priority series
Selec2on Strategy
Effects of Potency Filters on Selec%ons
!7.5 !7.0 !6.5 !6.0 !5.5 !5.0 !4.5
!8
0!
60
!4
0!
20
0
log Concentration (M)
Activity
!!
!
!
!!
!
! !
!
!
!
!
!
!!
! !
!! !
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
! !
!
The Top Priority Series
Scaffold MCS
The Top Priority Series
Scaffold
MCS
!7.5 !7.0 !6.5 !6.0 !5.5 !5.0 !4.5
!3
0!
20
!1
00
log Concentration (M)
Activity
!
!
!
!
!
!
!
!
!
!
! !
!
!
!
!
!
!
The Scaffold Explorer
• Currently %ed to NCGC database infrastructure
Summary
• Fragment based hit selec%on is a cheaper alterna%ve to clustering & neighborhood methods
• Easily integrated with other metrics such as promiscuity
• What makes a “good” fragment is s%ll tricky to encode algorithmically – Encoding the “SARability” of a fragment series is key – But, this is one component of a comprehensive fragment score
• Allows us to rapidly drill into series of interest in qHTS projects
Acknowledgements
• Ruili Huang • Wenwei Huang
• Ajit Jadhav