Pharmacological Evaluation of Different Compound Dilution and Transfer Paradigms on an Enzyme Assay

1
Pharmacological Evaluation of Different Compound
Dilution and Transfer Paradigms on an Enzyme
Assay in Low Volume 384-well Format.

• Tim Spicer, Yvonne Fitzgerald, Neil Burford,
  Sandra Matson, Moneesh Chatterjee, Mark
  Gilchrist, Jim Myslik and Jonathan OConnell.
• Bristol-Myers Squibb Company, 5 Research Parkway,
  Wallingford, CT 06492

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Introduction

• Miniaturization of assays to low volume 384-well
  and 1536-well formats has dramatically reduced
  HTS cost and improved throughput.
• However, miniaturization has posed some
  challenges.
• The difficulty in accurately transferring
  nanoliter volumes of compound in 100 DMSO into
  assay plates
• The low DMSO tolerance of assays (particularly
  cell-based assays)
• This has necessitated an aqueous intermediate
  dilution of compound prior to addition into assay
  plates.
• The effect of this aqueous intermediate dilution
  on compound solubility has often been questioned.
• Recent advances in liquid handling technology has
  achieved reliable, cost-effective and efficient
  nanoliter transfer of compound in 100 DMSO into
  assay plates, improving solubility whilst working
  within the boundary of DMSO tolerance for the
  assay.

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Aim

• To study the effect of different compound
  transfer paradigms on IC50 determinations for
  1090 compounds in a low volume 384-well format
  enzyme assay

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Nanoliter Dispensing Technology Echo 550

• Non-contact acoustic dispensing in the 5-50nL
  range
• Throughput 9 mins / 1536-well plate
• Cherry picking
• Dead volume 6.5mL for LV 384 COC plate source
• No washing, no tips, no carryover
• Small footprint

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How it Works
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Droplet Ejection
droplet
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1536-well Print
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Example of Echo 550 Performance DATA
ECHO 550 dispense into 1536-well plate
Plate CV in 384-well plate lt1 Best in Class
for tested nanoliter dispensers
Combined Echo, Flexdrop and Viewlux data
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Low Volume 384-well Enzyme Assay

• Initial hit identification was conducted using a
  100nl dispense of 100 DMSO )(1 DMSO in final
  assay) using a Velocity 11 VPrep fixed tip head
  (part number 06134.001)
• Target active compounds have tendency to be
  hydrophobic
• Concentration response curve (CRC) source plates
  were produced (10 point, 3-fold dilution of
  compounds from 3 mM in 100 DMSO) for 1090
  compounds.
• Assays were performed using three compound
  transfer paradigms in a 10 ml final assay volume
  in triplicate plates
• intermediate aqueous dilution ( a 110 dilution
  of compound in 100 DMSO with aqueous buffer)
  followed by a 1 ml addition to assay
• 100nl transfer of compound in 100 DMSO via VPrep
  
• 100nl transfer of compound in 100 DMSO via Echo
  550

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Results

• Compound transfer of 100nl in 100 DMSO resulted
  in a trend toward detecting compounds with higher
  potency compared with compound transfer using
  intermediate aqueous dilution (Figure 1)
• 110 compounds (10 of total) that were initially
  too weak (selection criteria of lt20 mM IC50)
  using aqueous intermediate dilution, were
  identified using the 100 nl of 100 DMSO transfer
  paradigm (Figure 1)

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Comparison of Compound Dispense Paradigm on
IC50 (mM)
Figure 1
Compounds below the line appear less potent using
aqueous intermediate dilution than using ECHO
dispense
Initial selection criteria was lt20mM IC50
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Results

• Compound IC50s using the Echo transfer paradigm
  were triaged based on the quality of the curves
  produced to reduce error in IC50 values due to
  poor curve fitting.
• i.e. curves selected had defined Ymin and Ymax
  plateau with a Hill slope ranging between 0.3 and
  3.
• This reduced the data set to 343 compounds (as
  many compounds were too weak in potency to
  achieve a Ymax plateau)
• Triaged compounds were categorized based on the
  fold difference in IC50 between the various
  transfer paradigms (Table 1, Figure 2)

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IC50 Correlation for Triaged Data Set
Less than 3-fold difference in IC50
Figure 2
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IC50 Fold DifferenceBetween Transfer Paradigms
Table 1
24 of compounds were gt 3-fold weaker in potency
and 8 produced No IC50 value in the aqueous
intermediate dilution paradigm compared with the
Echo transfer. This suggests that transfer of
compound in nanoliter volumes in 100 DMSO in
this enzyme assay led to an increased
sensitivity in detection of compounds known to be
hydrophobic.
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Conclusions

• Miniaturized low volume 384-well and 1536-well
  assays currently use the Echo 550 integrated onto
  automated platforms for just in time compound
  transfer for HTS
• Improvement in data quality has resulted from
• Transferring compound in 100 DMSO into the assay
  plate
• Accurate and reproducible transfer of compound
• More information on transfer integrity and source
  composition