Gaining confidence in high-throughput screening.

T he search for novel organic molecules with biological activity on human, animal, and plant physiological systems has passed through many phases over the centuries. From first steps testing single molecules on whole living systems to fully automated high-throughput screening (HTS) testing tens or hundreds of thousands of molecules per day on purified protein targets, the search has become ever more complex (1, 2). However, the increase in success has not been proportional to the effort and expense entailed. In particular, when considering the screening of “small molecules” (molecular mass <1,000 Da), the results of contemporary HTS have been plagued with problems of false positives, false negatives (3), and the abnormal behavior of certain molecules resulting from their physicochemical properties rather than their biological activity (4). In PNAS, Miller et al. (5) describe a significant evolution of current HTS technology that increases the confidence in the detection of truly active molecules by an order of magnitude. High-potency, highly specific molecular ligands are of great importance to modern medicine and agriculture and can also be valuable research tools that significantly aid the elucidation of metabolic pathways and control mechanisms. In its infancy, searching—or “screening” as it is now called—for active molecules relied on the analysis of plant and animal extracts and was a laborious, slow, and time-consuming process. As technology progressed and more rational methods were elaborated in the 1980s, screening evolved to a “process” whereby series of novel synthetic molecules were tested systematically for activity on one or even several different “targets” or target systems. In the 1990s highthroughput robotic screening methods based on microtiter plates were developed that took advantage of industrial-scale automation and large-scale data processing. This has allowed modern-day drug screening laboratories to “process” several tens or even hundreds of thousands of molecules per day (1, 2). The early promise of this technology has not, however, been fully realized (6), as is clearly evidenced by the ongoing efforts of (particularly) the pharmaceutical industry to identify novel molecules that are specific and potent ligands for novel targets. Indeed, the number of new drugs (termed new medical entities, or NMEs) is decreasing: in the period 2005–2010, 50% fewer NMEs were approved compared with the previous 5 y (7). In 2007, for example, only 19 NMEs were approved by the US Food and Drug Administration, the lowest number approved since 1983. Although the introduction and application of large-scale combinatorial chemistry methods, in silico virtual screening, X-ray crystallography, and sophisticated molecular modeling have helped to understand how small molecules bind to large proteins, such as enzymes and G proteincoupled receptors, HTS is still mostly based on random searching and often resembles a highly developed and very expensive search for the proverbial needle in a haystack. There are several reasons for this limited success, and one of the most important is that modern HTS technologies usually only test molecules for activity at a single concentration and thus (at least initially) completely ignore the all-important “dose–response” relationship that underlies the basis of most molecular interactions in biological systems. This results in many false-positive and falsenegative findings (3). Although HTS certainly reveals many “active” molecules, it also reveals numerous false positives and many molecules with bizarre dose–response relationships that on further examination render them useless as potential medicines or research tools (8). Indeed, the time, energy, and costs of the analysis of these false positives is one factor that currently restricts the discovery potential of HTS methods. The loss in economic value due to false negatives (molecules that are in reality active but are measured as inactive in HTS) is impossible to assess but has always been a recognized, omnipresent difficulty. Miller et al. (5) describe a screening technique and strategy that represents a clear advance toward identifying in the first-pass screening campaign only those molecules that are truly acting in a reproducible and dose-dependent fashion. This system was used to screen a library of marketed drugs for inhibition of the enzyme protein tyrosine phosphatase 1B, a target for type 2 diabetes mellitus, obesity, and cancer, and the authors identified a number of unique inhibitors. Their screening system is based on the use of droplets in a microfluidic system as independent microreactors, which play the same role as the wells of a microtiter plate. However, the reaction volume, which in conventional HTS microplate wells is a few microliters, is reduced to a few picolitres, a reduction of ≈1 millionfold. Droplet-based microfluidics is a rapidly developing technology (for reviews see refs. 9 and 10) that is already commercialized for targeted sequencing and digital In hi bi tio n (% )