High-resolution dose–response screening using droplet-based microfluidics

Abstract

A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC 50 values that are highly precise ( 2.40% at 95% confidence) and highly reproducible (CV 2.45%, n 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC 50 of 27 0.83 μM.high-throughput screening | HTS | small molecule library I n the early 16th century the Swiss chemist Paracelsus declared "all substances are poisons, there is none which is not a poison; only the right dose makes a substance non-poisonous." This idea that the biological effects of a chemical compound are dependent upon its concentration was quantified by A. V. Hill in 1910 (1). However, despite the fact that compounds can display complex concentration-dependent relationships, varying in potency, efficacy, and steepness of response, usually just a single measurement at a single concentration (approximately 10 μM) is obtained for each compound in the chemical library during a primary drug screen, even when using state-of-the-art robotic microplate-based screening systems. This results in high numbers of false positives and false negatives (2), as well as the inability to identify subtle, complex pharmacology, such as partial agonism or antagonism. Even when dose-response curves are generated during a quantitative primary screen (3) or, more typically, during the follow-up of a single-point screen, the time and cost limitations mean that the curves typically contain only 7-10 data points each. With ≤10 nonduplicated data points, and as many as four adjustable nonlinear parameters (e.g., in the four-parameter Hill function), the results are highly sensitive to the data quality: For example, the presence of a single outlying data point can substantially alter the fit of the data, unless the outliers are identified and removed (4).We have developed a system that uses droplet-based microfluidics to generate high-quality dose-response data during drug screening. Droplet-based microfluidics is itself a new technology for creating and manipulating picoliter-volume aqueous droplets that function as independent microreactors (for a review see ref. 5). As a result of the miniaturization inherent in this approach, our system (Fig. 1) is capable of generating doseresponse curves at materiall...

Exploring the Existing Drug Space for Novel pTyr Mimetic and SHP2 Inhibitors

“…Cefsulodin is also quite selective for SHP2, displaying, with one exception of SHP1, >10-fold selectivity against all PTPs examined ( Table 1). As reported before, 12 cefsulodin also exhibits inhibitory activity against PTP1B, although with much less potency compared to SHP2 (Table 1).…”

Sensitive and predictable separation of microfluidic droplets by size using in-line passive filter

“…Droplet microfluidics has shown promise in various fields including directed evolution, 1,2 drug screening, 3,4 and droplet-based nucleic acid analysis. 5,6 Compartmentalization of reactions in small droplets using the microfluidic method increases the sensitivity of assays and reduces the cost.…”

Droplet Microfluidic Technique for the Study of Fermentation

“…Earlier, Song et al [17], Damean et al [18], Niu et al [19], Sun et al [20], Wang et al [21] and Agresti et al [22] reported systems to generate sequences of droplets scanning concentrations of one substance (up to four orders of magnitude) and used them to screening of biochemical reactions. Miller et al [23] demonstrated a microfluidic system that exploits Taylor-Aris dispersion to create a concentration gradient within about 10,000 droplets per compound. Guo et al [24] presented an optofluidic device for real-time, high-throughput (2000 droplets per second) quantitative analysis of droplet contents.…”