DNA-encoded library versus RNA-encoded library selection enables design of an oncogenic noncoding RNA inhibitor.

Nature evolves molecular interaction networks through persistent perturbation and selection, in stark contrast to drug discovery, which evaluates candidates one at a time by screening. Here, nature's highly parallel ligand-target search paradigm is recapitulated in a screen of a DNA-encoded library (DEL; 73,728 ligands) against a library of RNA structures (4,096 targets). In total, the screen evaluated ∼300 million interactions and identified numerous bona fide ligand-RNA three-dimensional fold target pairs. One of the discovered ligands bound a 5'GAG/3'CCC internal loop that is present in primary microRNA-27a (pri-miR-27a), the oncogenic precursor of microRNA-27a. The DEL-derived pri-miR-27a ligand was cell active, potently and selectively inhibiting pri-miR-27a processing to reprogram gene expression and halt an otherwise invasive phenotype in triple-negative breast cancer cells. By exploiting evolutionary principles at the earliest stages of drug discovery, it is possible to identify high-affinity and selective target-ligand interactions and predict engagements in cells that short circuit disease pathways in preclinical disease models.

Integrated, Continuous Emulsion Creamer.

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.

Dose-Response Activity-Based DNA-Encoded Library Screening.

Dose-response, or "conforming" behavior, increases confidence in a screening hit's authenticity. Here, we demonstrate dose-response solid-phase DNA-encoded library (DEL) screening. Compound dose in microfluidic droplets is modulated via the UV intensity of photocleavage from DEL beads. A 55,296-member DEL was screened at different UV intensities against model enzyme drug targets factor Xa (FXa) and autotaxin (ATX). Both screens yielded photochemical dose-dependent hit rates (FXa hit rates of 0.08/0.05% at 100/30% UV exposure; ATX hit rates of 0.24/0.08% at 100/20% UV exposure). FXa hits contained structures reflective of FXa inhibitors and four hits inhibited FXa (IC50 = 4.2 ± 0.1, 7.4 ± 0.3, 9.0 ± 0.3, and 19 ± 2 µM.) The top ATX hits (two dihydrobenzamidazolones and a tetrahydroisoquinoline) were validated as inhibitors (IC50 = 7 ± 2, 13 ± 2, and 1 ± 0.3 µM). Photochemical dose-response DEL screening data prioritized hits for synthesis, the rate-limiting step in DEL lead identification.

Antibacterial Discovery via Phenotypic DNA-Encoded Library Screening.

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.

Chiral lipid bilayers are enantioselectively permeable.

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, generated at a rate of 480 per minute) and benchmark this system by monitoring fluorescent dye DIB permeation over time. Enantioselective permeation of alkyne-labelled amino acids (Ala, Val, Phe, Pro) and dipeptides through a chiral phospholipid bilayer was demonstrated using DIB transport measurements; the biological L enantiomers permeated faster than the D enantiomers (from 1.2-fold to 6-fold for Ala to 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.

Activity-Based DNA-Encoded Library Screening.

Robotic high-throughput compound screening (HTS) and, increasingly, DNA-encoded library (DEL) screening are driving bioactive chemical matter discovery in the postgenomic 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 nanomole-scale synthesis and validation (20 active), guiding candidate selection for synthesis at scale (5/5 compounds with IC50 values of 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.

Natural variation in the strength and direction of male mating preferences for female pheromones in Drosophila melanogaster.

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.