June 1, 2010 (Vol. 30, No. 11)
Ultrasonic Fluid Processor Re-Solubilizes Fragment Precipitates and Can Increase Yields
Fragment-based drug discovery (FBDD) is gaining recognition for its many advantages over high-throughput screening. Key benefits include better hit-to-lead rates and broad chemical space for possible compound scaffolds. Due to the low affinity of fragments to the biological target, fragment libraries may contain weak hits and therefore scientists screen fragment libraries at high concentrations, sometimes as high as 200 mM.
Even though many fragments have high solubility in DMSO, environmental shocks introduced by DMSO hydration or repeat freeze/thaw cycles can cause compounds to crash out of solution and consequently affect the accuracy of FBDD screening results.
To resolve this issue, Microsonic Systems developed the Hendrix SM100 Ultrasonic Fluid Processor. At the core of the Hendrix SM100 is lateral ultrasonic thrust™ (LUT™) technology, a form of nonfocused ultrasonic energy generated using Fresnel lens elements.
LUT technology works by using a MEMS-based transducer, which when excited with RF power generates ultrasonic waves. These ultrasonic waves pass into the sample as broad beams of acoustic energy. As shown in Figure 1, the waves create regions of strong LUT energy that in turn create strong fluid motion in the form of a rapid vortex.
LUT technology allows for a very dynamic range of power control and, unlike other ultrasonic methods, does not cause cavitation. At high power, LUT technology can be used for solubilization and for thawing applications; at low power, the same technology can be used for assay mixing or for bead suspension.
The lack of cavitation helps preserve the integrity of samples when the higher power settings are utilized. By adding the Hendrix SM100 to the FBDD process, researchers are able to re-solubilize precipitated fragment libraries and consequently recover samples that are often unavailable using conventional methods. This secondary recovery process both increases sample concentrations and improves the accuracy of screening results. See Figure 2 for secndary recovery process map.
Primary Screening Activity
With the assistance of Michael Jobling, head of compound management at Elan Pharmaceuticals, we conducted experiments to analyze how secondary recovery could positively impact drug screening results. We re-solubilized precipitated fragment samples back into solution in DMSO, and then sent 24 samples to primary screening.
The percent inhibition results were compared to the control group, which had visible precipitates at the bottom of the tubes and did not go through the ultrasonic secondary recovery process.
In the control experiment, 29% of the screened fragments showed less than 20% inhibition, 17% of the group showed 20–40% inhibition, 21% of the group showed 40–60% inhibition, 4% showed 60-80% inhibition, and 29% showed high activity with greater than 80% inhibition.
In the secondary recovery experiment, with precipitated samples recovered back into solution, the primary screening accuracy increased: 38% of the screened fragments showed high activity with greater than 80% inhibition, 12% showed 60–80% inhibition, and 17% showed 40–60% inhibition. The differences between the control and the secondary recovery experiments are summarized in Figure 3.
Fragments put through the Hendrix SM100 for secondary recovery increased activity by an average of 68% with a maximum increase in inhibition of 384%.
Reduced IC50 Values
We compared the IC50 values of 12 samples before and after the secondary recovery ultrasonic process. The IC50 values of all the test fragments left-shifted to lower concentrations; 9 out of the 12 samples showed more than two digits in percent reduction after fragment library secondary recovery, and four showed over 80% reduction.
In particular, the IC50 value of one sample shifted from 56 nM to 0.56 nM—a two logs reduction in IC50 value. This indicates that the potencies of these fragment samples were much stronger than originally thought.
The Hendrix SM100 supports a wide range of common labware: 96-, 384-, and 1,536-well plates; flat bottom, U-bottom, V-bottom labware; 2-D barcoded tubes; and scintillation vials. Because the Hendrix SM100 has 16×24 arrays of piezoelectric acoustic elements, it enables rapid fluid processing and reduces the hours-long solubilization process to minutes.
Recovering a rack of 96-fragment samples took five to ten minutes depending on sample volume and the amount of precipitates; given the small amount of additional time required, the quality improvements observed in both primary and secondary screening were significant.
Fragment libraries are screened at much higher concentrations than small molecule libraries due to their low affinity to biological targets. This makes fragment libraries more susceptible to precipitation and consequently affects the accuracy of screening results.
The data presented here shows that the Hendrix SM100 ultrasonic fluid processor can be used to recover the precipitated fragment samples back into solution, and thus increase both the sample concentration and the accuracy of screening results. After putting the fragment library through secondary recovery using the Hendrix SM100 system, we observed higher percent inhibition in primary screening and lower IC50 values in secondary screening.
By adding secondary recovery into the drug discovery process, we observed improvements in the quantity and the quality of primary screening leads as well as in the quality of secondary screening results.