Robotic high-throughput compound screening (HTS) and, increasingly, DNA-encoded library (DEL) screening are driving bioactive chemical matter discovery in the post-genome era. HTS enables activity-based investigation of highly complex targets using static compound libraries. Conversely, DEL grants efficient access to novel chemical diversity, although screening is limited to affinity-based selections. Here, we describe an integrated droplet-based microfluidic circuit that directly screens solid-phase DELs for activity. An example screen of a 67,100-member library for inhibitors of the phosphodiesterase autotaxin yielded 35 high-priority structures for nanomolescale synthesis and validation (20 active), guiding candidate selection for synthesis at scale (5/5 compounds with IC50s 4-10 μM). We further compared activity-based hits with those of an analogous affinity-based DEL selection. This miniaturized screening platform paves the way toward applying DELs to more complex targets (signaling pathways, cellular response), and represents a distributable approach to small molecule discovery.

Significance Drug discovery generally investigates one target at a time, in sharp contrast to living organisms, which mold ligands and targets by evolution of highly complex molecular interaction networks. We recapitulate this modality of discovery by encoding drug structures in DNA, allowing the entire DNA-encoded library to interact with thousands of RNA fold targets, and then decoding both drug and target by sequencing. This information serves as a filter to identify human RNAs aberrantly produced in cancer that are also binding partners of the discovered ligand, leading to a precision medicine candidate that selectively ablates an oncogenic noncoding RNA, reversing a disease-associated phenotype in cells.

Homochiral membrane bilayers organize biological functions in all domains of life. The membrane’s permeability–its key property–correlates with a molecule’s lipophilicity, but the role of the membrane’s rich and uniform stereochemistry as a permeability determinant is largely ignored in empirical and computational measurements. Here, we describe a new approach to measuring permeation using continuously generated microfluidic droplet interface bilayers (DIBs, 480/min) and benchmark this system by monitoring fluorescent dye DIB permeation over time. Permeation of non-fluorescent, alkyne-labeled molecules was measured using a fluorogenic click reaction. DIB transport measurements revealed enantioselective permeation of alkyne-labeled amino acids (Ala, Val, Phe, Pro) and dipeptides through a chiral phospholipid bilayer; the biological L enantiomers permeated faster than D (1.2–6-fold; Ala–Pro). Enantioselective permeation both poses a potentially unanticipated criterion for drug design and offers a kinetic mechanism for the abiotic emergence of homochirality via chiral transfer between sugars, amino acids, and lipids.

Automated and reproducible sample handling is a key requirement for high-throughput compound screening and currently demands heavy reliance on expensive robotics in screening centers. Integrated droplet microfluidic screening processors are poised to replace robotic automation by miniaturizing biochemical reactions to the droplet scale. These processors must generate, incubate, and sort droplets for continuous droplet screening, passively handling millions of droplets with complete uniformity, especially during the key step of sample incubation. Here, we disclose an integrated microfluidic emulsion creamer that packs ("creams") assay droplets by draining away excess oil through microfabricated drain channels. The drained oil coflows with creamed emulsion and then reintroduces the oil to disperse the droplets at the circuit terminus for analysis. Creamed emulsion assay incubation time dispersion was 1.7%, 3-fold less than other reported incubators. The integrated, continuous emulsion creamer (ICEcreamer) was used to miniaturize and optimize measurements of various enzymatic activities (phosphodiesterase, kinase, bacterial translation) under multiple- and single-turnover conditions. Combining the ICEcreamer with current integrated microfluidic DNA-encoded library bead processors eliminates potentially cumbersome instrumentation engineering challenges and is compatible with assays of diverse target class activities commonly investigated in drug discovery.

The global rise of multidrug resistant infections poses an imminent, existential threat. Numerous pipelines have failed to convert biochemically active molecules into bona fide antibacterials, owing to a lack of chemical material with antibacterial-like physical properties in high-throughput screening compound libraries. Here, we demonstrate scalable design and synthesis of an antibacterial-like solid-phase DNA-encoded library (DEL, 7488 members) and facile hit deconvolution from whole-cell Escherichia coli and Bacillus subtilis cytotoxicity screens. The screen output identified two low-micromolar inhibitors of B. subtilis growth and recapitulated known structure–activity relationships of the fluoroquinolone antibacterial class. This phenotypic DEL screening strategy is also potentially applicable to adherent cells and will broadly enable the discovery and optimization of cell-active molecules.

Many animal species communicate using chemical signals. In Drosophila, cuticular hydrocarbons (CHCs) are involved in species and sexual identification, and have long been thought to act as stimulatory pheromones as well. However, a previous study reported that D. melanogaster males were more attracted to females that were lacking CHCs. This surprising result is consistent with several evolutionary hypotheses but is at odds with other work demonstrating that female CHCs are attractive to males. Here, we investigated natural variation in male preferences for female pheromones using transgenic flies that cannot produce CHCs. By perfuming females with CHCs and performing mate choice tests, we found that some male genotypes prefer females with pheromones, some have no apparent preference, and at least one male genotype prefers females without pheromones. This variation provides an excellent opportunity to further investigate the mechanistic causes and evolutionary implications of divergent pheromone preferences in D. melanogaster males.

Connecting genetic variation to trait variation is a grand challenge in biology. Natural 8 populations contain a vast reservoir of fascinating and potentially useful variation, but it 9is unclear if the causal alleles will generally have large enough effects for us to detect. 10Without knowing the effect sizes or allele frequency of typical variants, it is also unclear 11 what methods will be most successful. Here, we use a multi-parent advanced intercross 12 population (the Drosophila Synthetic Population Resource) to map natural variation in 13Drosophila courtship song traits. Most additive genetic variation in this population can be 14 explained by a modest number of highly resolved QTL. Mapped QTL are universally 15 multiallelic, suggesting that individual genes are "hotspots" of natural variation due to a 16 small target size for major mutations and/or filtering of variation by positive or negative 17 selection. Using quantitative complementation in randomized genetic backgrounds, we 18provide evidence that one causal allele is harbored in the gene Fhos, making this one of 19the few genes associated with behavioral variation in any taxon. 20 21 Results 94The Drosophila Synthetic Population Resource (DSPR) was started from 15 95 founder strains collected in Ohio, Georgia, California, and Hawaii in the United States as 96 well as Columbia, South Africa, Spain, Greece, Israel, Malaysia, Taiwan, Peru, Bermuda, 97and Uzbekistan (Table S1). As shown in Figure 2, these strains differ greatly in their 98inter-pulse interval (IPI) and carrier frequency (CF). The DSPR was constructed by 99 mixing strains in two sets of eight: the seven strains that contributed to population A are 100 numbered A1 -A7, the seven strains that contributed to population B are numbered B1 -101 B7, and one strain, AB8, contributed to both. Over 1700 recombinant inbred strains were 102 derived from these populations, and we measured trait values in at least 4 males from 103 1656 of them (N = 4-71, mean N = 16; Figure S1). 104 105Heritability estimates for IPI and CF 106Broad-sense heritability (H 2 ), which includes both additive and non-additive 107 genetic effects, can be estimated in the DSPR as the fraction of trait variation among 108 recombinant inbred strains. Strain effects explained almost half the variation in IPI (46% 109and 45% for A and B populations, respectively) and nearly a third of the variation in CF 110(30% in both populations). We can also estimate the fraction of variation explained by 111additive genetic effects (narrow-sense heritability or h 2 ) using ridge regression [47,48]. 112Rather than estimating the genetic variation explained by strain, this method estimates 113 breeding values using variation among strains in the proportion of shared genomic 114ancestry. It is similar to methods that compare the trait correlation among relatives in a 115 pedigree, but uses direct measurements of shared genomic ancestry rather than a 116 historical pedigree [2,49]. We first estimated the proportion of broad-sense heritability ...