Dispensing Processes Profoundly Impact Biological, Computational and Statistical Analyses Sean Ekins 1, Joe Olechno 2 Antony J. Williams 3 1 Collaborations in Chemistry, Fuquay Varina, NC. 2 Labcyte Inc, Sunnyvale, CA. 3 Royal Society of Chemistry, Wake Forest, NC. Disclaimer: SE and AJW have no affiliation with Labcyte and have not been engaged as consultants
Where do scientists get chemistry/ biology data? Databases Patents Papers Your own lab Collaborators If I have seen further than others, it is by standing upon the shoulders of giants. Some or all of the above? What is common to all? quality issues Isaac Newton
Data can be found but..drug structure quality is important More groups doing in silico repositioning Target-based or ligand-based Network and systems biology integrating or using sets of FDA drugs..if the structures are incorrect predictions will be too.. Need a definitive set of FDA approved drugs with correct structures Also linkage between in vitro data & clinical data
Structure Quality Issues Database released and within days 100 s of errors found in structures Science Translational Medicine 2011 NPC Browser http://tripod.nih.gov/npc/ DDT, 16: 747-750 (2011) DDT 17: 685-701 (2012)
DDT editorial Dec 2011 Its not just structure quality we need to worry about This editorial led to the current work http://goo.gl/diqhu
Finding structures of Pharma molecules is hard NCATS and MRC made molecule identifiers from pharmas available with no structures Southan et al., DDT, 18: 58-70 (2013)
How do you move a liquid? Plastic leaching Images courtesy of Bing, Tecan McDonald et al., Science 2008, 322, 917. Belaiche et al., Clin Chem 2009, 55, 1883-1884
Moving Liquids with sound: Acoustic Droplet Ejection (ADE) Acoustic energy expels droplets without physical contact Extremely precise Extremely accurate Rapid Auto-calibrating Completely touchless No crosscontamination No leachates No binding %CV 15.0 12.5 10.0 7.5 5.0 2.5 0 0.1 1 10 100 1000 10000 Volume (nl) Comley J, Nanolitre Dispensing, Drug Discovery World, Summer 2004, 43-54 8 Images courtesy of Labcyte Inc. http://goo.gl/k0fjz
Using literature data from different dispensing methods to generate computational models Few molecule structures and corresponding datasets are public Using data from 2 AstraZeneca patents Tyrosine kinase EphB4 pharmacophores (Accelrys Discovery Studio) were developed using data for 14 compounds IC 50 determined using different dispensing methods Analyzed correlation with simple descriptors (SAS JMP) Calculated LogP correlation with log IC 50 data for acoustic dispensing (r 2 = 0.34, p < 0.05, N = 14) Barlaam, B. C.; Ducray, R., WO 2009/010794 A1, 2009 Barlaam, B. C.; Ducray, R.; Kettle, J. G., US 7,718,653 B2, 2010
14 compounds with structures and IC 50 data. Compound # IC 50 Acoustic (µm) IC 50 Tips (µm) Ratio IC 50Tip /IC 50ADE 5 0.002 0.553 4 0.003 0.146 7 0.003 0.778 W7b 0.004 0.152 8 0.004 0.445 W5 0.006 0.087 6 0.007 0.973 W3 0.012 0.049 W1 0.014 0.112 9 0.052 0.170 10 0.064 0.817 W12 0.158 0.250 W11 0.207 14.400 11 0.486 3.030 276.5 48.7 259.3 42.5 111.3 13.7 139.0 4.2 8.2 3.3 12.8 1.6 69.6 6.2 Barlaam, B. C.; Ducray, R., WO 2009/010794 A1, 2009 Barlaam, B. C.; Ducray, R.; Kettle, J. G., US 7,718,653 B2, 2010
A graph of the log IC 50 values for tip-based serial dilution and dispensing versus acoustic dispensing with direct dilution shows a poor correlation between techniques (R 2 = 0.246). 1.5 1 0.5 log IC 50-tips 0-3 -2.5-2 -1.5-1 -0.5 0 0.5 1 1.5-0.5-1 -1.5-2 -2.5 log IC 50-acoustic -3 acoustic technique always gave a more potent IC 50 value
Experimental Process Results Acoustic Model Acoustic Model Acoustic Model 14 Structures with Data Generate pharmacophore models for EphB4 receptor Test models against new data Test models against X-ray crystal structure pharmacophores Tip-based Model Tip-based Model Tip-based Model Results Initial data set of 14 WO2009/010794, US 7,718,653 Independent data set of 12 WO2008/132505 Independent crystallography data Bioorg Med Chem Lett 18:2776; 12 18:5717; 20:6242; 21:2207
Tyrosine kinase EphB4 Pharmacophores Generated with Discovery Studio (Accelrys) Cyan = hydrophobic Green = hydrogen bond acceptor Purple = hydrogen bond donor Acoustic Tip based Each model shows most potent molecule mapping Hydrophobic Hydrogen Hydrogen Observed vs. features (HPF) bond acceptor bond donor predicted IC 50 (HBA) (HBD) r Acoustic mediated process Tip-based process 2 1 1 0.92 0 2 1 0.80 Ekins et al., PLOSONE, In press
Test set evaluation of pharmacophores An additional 12 compounds from AstraZeneca Barlaam, B. C.; Ducray, R., WO 2008/132505 A1, 2008 10 of these compounds had data for tip based dispensing and 2 for acoustic dispensing Calculated LogP and logd showed low but statistically significant correlations with tip based dispensing (r 2 = 0.39 p < 0.05 and 0.24 p < 0.05, N = 36) Used as a test set for pharmacophores The two compounds analyzed with acoustic liquid handling were predicted in the top 3 using the acoustic pharmacophore The Tip-based pharmacophore failed to rank the retrieved compounds correctly
Automated receptor-ligand pharmacophore generation method Pharmacophores for the tyrosine kinase EphB4 generated from crystal structures in the protein data bank PDB using Discovery Studio version 3.5.5 Cyan = hydrophobic Green = hydrogen bond acceptor Purple = hydrogen bond donor Grey = excluded volumes Each model shows most potent molecule mapping Bioorg Med Chem Lett 2010, 20, 6242-6245. Bioorg Med Chem Lett 2008, 18, 5717-5721. Bioorg Med Chem Lett 2008, 18, 2776-2780. Bioorg Med Chem Lett 2011, 21, 2207-2211.
Summary In the absence of structural data, pharmacophores and other computational and statistical models are used to guide medicinal chemistry in early drug discovery. Our findings suggest acoustic dispensing methods could improve HTS results and avoid the development of misleading computational models and statistical relationships. Automated pharmacophores are closer to pharmacophore generated with acoustic data all have hydrophobic features missing from Tipbased pharmacophore model Importance of hydrophobicity seen with logp correlation and crystal structure interactions Public databases should annotate this meta-data alongside biological data points, to create larger datasets for comparing different computational methods.
Serial dilution IC 50 μm Log IC 50 tips 0 10 20 30 40 50 Serial dilution IC 50 μm -40 Acoustic % Inhibition -20 0 20 40 60 80 100 Acoustic vs. Tip-based Transfers Adapted from Spicer et al., Presentation at Drug Discovery Technology, Boston, MA, August 2005 0 10 20 30 40 50 Acoustic IC 50 μm Adapted from Wingfield. Presentation at ELRIG2012, Manchester, UK NOTE DIFFERENT ORIENTATION -40-20 0 20 40 60 80 Aqueous % Inhibition 100 10 4 10 3 10 2 Adapted from Wingfield et al., Amer. Drug Disco. 2007, 3(3):24 10 1 10-1 Data in this presentation 10-2 10-3 10-3 10-2 10-1 1 10 10 2 10 3 10 4 Acoustic IC 50 μm Log IC 50 acoustic No Previous Analysis of molecule properties
Ekins et al., PLOSONE, In press Strengths and Weaknesses Small dataset size focused on one compound series No previous publication describing how data quality can be impacted by dispensing and how this in turn affects computational models and downstream decision making. No comparison of pharmacophores generated from acoustic dispensing and tip-based dispensing. No previous comparison of pharmacophores generated from in vitro data with pharmacophores automatically generated from X-ray crystal conformations of inhibitors. Severely limited by number of structures in public domain with data in both systems Reluctance of many to accept that this could be an issue
The stuff of nightmares? How much of the data in databases is generated by tip based serial dilution methods How much is erroneous Do we have to start again? How does it affect all subsequent science data mining etc Does it impact Pharmas productivity?
Simple Rules for licensing open data Could data open accessibility equal Disruption As we see a future of increased database integration the licensing of the data may be a hurdle that hampers progress and usability. 1: NIH and other international scientific funding bodies should mandate open accessibility for all data generated by publicly funded research immediately Williams, Wilbanks and Ekins. PLoS Comput Biol 8(9): e1002706, 2012 Ekins, Waller, Bradley, Clark and Williams. DDT, 18:265-71, 2013
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