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Ligand Affinity Measurements for the Community Structure Activity Resource
Center(CSAR)
www.CSARDock.org
Jim Delproposto
Drug Docking and Screening Data ResourceRFA‐GM‐08‐008
• Funded by the NIH• Solicit structures and affinity data for target complexes from the industrial
and academic communities • Curate data and refine as needed• Enhance collection by creation of new data
• Standardized assay conditions• Specifically designed to test aspects of docking and scoring algorithms
• Make data publically available
“The goal is to increase the amount of high quality data publicly available for development, validation, and benchmarking of liganddocking and screening software.”
•High quality structures both apo and with small molecules bound
•Affinity data that can be directly related to the structure
•Physicochemical properties of the small molecules
•Thermodynamic properties of ligand binding
•Standardized conditions
What data is useful for structure based drug design?
Incomplete and fragmented data
Lack of confidence • no redundancy in data sets
Attention to detail• structures data may not completely match affinity data• low quality mixed with high quality data• No standardized conditions
Lack of specific data –•data that addresses problem areas of SBDD
What’s wrong with the current data?
• Better data – confidence in the data
• More data – complete data and new data
• Standardized data– identical conditions
• Specific data – data that addresses problem areas of SBDD
It’s All About the Data
We don’t make the docking and scoring products. We provide the data to make them better.
Generating New Experimental Data
3 methods are used to collect affinity data
Crystal structures
Physicochemical properties of ligands
Small Volume ITC
∆H
KD
Thermofluor®
Protein vs Protein‐Ligand complex
Thermofluor from Johnson & Johnson
4 replicates
Octet RED
Octet RED Data
Time0 50 100 150 200
0.90
0.95
1.00
1.05
1.10
1.153.91E-087.81E-081.56E-073.13E-076.25E-071.25E-062.5E-06
CSAR Projects
Complete• CDK2 and CDK2 with Cyclin A• LpxC• Hsp90
In progress• Chk1• Urokinase
CDK2‐CyclinA
PDB:1FIN
Melting of CDK2‐CyclinA complex on the Thermofluor
Reference
CS247 CS245
Affinity of 2.1nMAffinity of 1.07µM
CDK2 Cyclin A Crosslinking
Compound CS248 AffinitiesCDK2 vs. CDK2 CyclinA Complex
KD= 5.07E-07KD= 3.68E-07
Compound CS11 AffinitiesCDK2 vs. CDK2 CyclinA Complex
KD= 1.71E-08KD= 9.21E-08
Data confirming this ~6 fold increase using ITC
CDK2 vs CDK2 with CyclinA
LpxC
Real world LpxC data
Data Transforms
Slope Subtraction
100 200 300 400 500-0.1
0.0
0.1
0.2
0.3
0.4
0.5
Slope Subtracted
3-7run3100 200 300 400 500-0.1
0.0
0.1
0.2
0.3
0.4
0.5
Not Slope Subtracted
3-7run3
100 200 300 400 500-0.1
0.0
0.1
0.2
0.3
0.4
0.5
Not Slope Subtracted
3-7run3100 200 300 400 500
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
Slope Subtracted
3-7run3
Kd from curvefit Kd = 1.65E-07Steady State Dissociation Kd = 1.53E-07Steady State Association Kd = 1.74E-07
Current Curve fitting
Real binding
No detectable disassociation
Association phase Disassociation phase
Competitive binding for slow disassociation compounds
Competitive binding experiment 20uM of reference compound vs increasing concentrations of tight binding compound.
Disassociation signal is only from the reference compound.
0%
100%
What is the affinity of a compound?
What is the affinity of a compound?
WWW.CSARDOCK.ORG• CSAR data set release 2012: ligands, affinities, complexes, and docking decoys.
Dunbar JB Jr, Smith RD, Damm‐Ganamet KL, Ahmed A, Esposito EX, Delproposto J, Chinnaswamy K, Kang YN, Kubish G, Gestwicki JE, Stuckey JA, Carlson HA.J Chem Inf Model. 2013 Aug 26;53(8):1842‐52. doi: 10.1021/ci4000486. Epub 2013 May 10.
CSAR (Community Structure‐Activity Resource)
• Who are we :• Principal Investigators
• Heather A. Carlson• Jeanne A. Stuckey
•Researchers• Aqeel Ahmed• William Clay Brown• Krishnapriya Chinnaswamy• Kelly Damm‐Ganamet• James Delproposto• James B. Dunbar Jr.• Richard D. Smith
•Who are we (cont):• Consultants
• Charles Brooks III• Bruce Palfey• Janet Smith
•Web Programming• Shelly Yang
• System Administration• Allen Bailey
• Advisory Board• Michael Gilson• Steven Muchmore• Paul Labute• Anthony Nicholls• Tudor Oprea• Catherine Peishoff• Deborah Loughney• Peter Preusch• Janna Wehrle
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