Transcript Document

Use of High Throughput
Screening to Determine Lead
Crystallization Conditions and
as a Tool for Basic Research
HT Infrastructure:
The Wet Lab
HT Infrastructure:
Computer Hardware and Software
BACKUP
BIG BROTHER
Reader Tables
RAID1
Data to Primary fileserver
1 TB RAID5 subsystem
Failover fileserver
Sample Database
2 TB RAID5 snapshots
Tape
Optical Disc
Offsite
How can we improve our output?
Improved image quality = Improved lead detection
New
Old
SGPP Lmaj004655AAASeMet
Outcome Verification
Identify the outcome as crystalline
Images courtesy of
Greiner Bio-One
PS
Glass
LBR
Identify the composition
140
120
100
80
60
40
Number of Samples
Samples Screened – All Users
Number of Plates per Month
180
160
20
Jan-04
Nov-03
Sep-03
Jul-03
May-03
Mar-03
Jan-03
Nov-02
Month - Yr
Sep-02
Jul-02
May-02
Mar-02
Jan-02
Nov-01
Sep-01
Jul-01
May-01
Mar-01
Jan-01
Nov-00
Sep-00
Jul-00
May-00
Mar-00
0
HTS: Success Rate
SGPP Samples July 2003 - Jan 2004
30
25
total # samples
no leads
questionable leads
definite leads
20
15
10
5
0
Ju
l-0
Au 3
g0
Se 3
p0
O 3
ct
-0
N 3
ov
-0
D 3
ec
-0
Ja 3
n04
Number of Samples
35
Month-Year
Co-Crystallants
• The SGPP co-crystallants were designed to
promote 2 types of macromolecular
interactions:
– intra-molecular interactions (freezers) and
– inter-molecular interactions (glues)
• The library uses “privileged structural motifs”,
fused-ring aromatics (triazoliums or
guanidiniums) “decorated with bromines”*.
*Hovey,
B., Verlinde, C. L. M. J., Merritt, E. A. & Hol, W. G. J. (1999).
Structure-based discovery of a pore-binding ligand: Towards assembly
inhibitors for cholera and related AB5 toxins. J. Mol. Biol. 285, 1169-1178.
Co-Crystallants Used in This Study
01
02
04
05
09
10
11
12
13
Co-Crystallant Experiments
•
Crystallization in HTS laboratory
–
Compared the crystallization behavior of:
1. native protein
2. protein-DMSO
3. protein-DMSO-10mM Co-Crystallant
•
DLS
–
Look for evidence of aggregation in the presence
of the co-crystallants.
DLS Results
•
Sample aggregation occurred in the presence
of the co-crystallants.
DLS Results and Crystallization Outcomes:
Ldon1686AAA (22)
Venn Diagram of Crystallization Results:
Ldon1686AAA (22)
Venn Diagram of Crystallization Results:
Hen Egg White Lysozyme
Acknowledgements
• Robert J. Collins, Nancy A. Fehrman, Stacey
M. Gulde, Angela M. Lauricella, Carrie A.
Mancuso, Christina K. Veatch
and George T. DeTitta
• Jizhen Li, Brian Carroll, Erkang Fan, Mark
Robien and Wim G.J. Hol
Acknowledgements
• Work supported in part by the John R. Oishei
Foundation, the Cummings Foundation, the
Western New York Foundation, NASA NAG81594, NASA NAG8-1839, NASA NCC8-232,
NIH RR016924 NIH P50 GM-62413 and NIH
P50 GM-64655.
• Special thanks to Greiner Bio-One