Building Block-Centric Approach to DNA-Encoded Library Design.

DNA-encoded library technology grants access to nearly infinite opportunities to explore the chemical structure space for drug discovery. Successful navigation depends on the design and synthesis of libraries with appropriate physicochemical properties (PCPs) and structural diversity while aligning with practical considerations. To this end, we analyze combinatorial library design constraints including the number of chemistry cycles, bond construction strategies, and building block (BB) class selection in pursuit of ideal library designs. We compare two-cycle library designs (amino acid + carboxylic acid, primary amine + carboxylic acid) in the context of PCPs and chemical space coverage, given different BB selection strategies and constraints. We find that broad availability of amines and acids is essential for enabling the widest exploration of chemical space. Surprisingly, cost is not a driving factor, and virtually, the same chemical space can be explored with "budget" BBs.

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.

Hydrogel-Encapsulated Beads Enable Proximity-Driven Encoded Library Synthesis and Screening.

Encoded combinatorial library technologies have dramatically expanded the chemical space for screening but are usually only analyzed by affinity selection binding. It would be highly advantageous to reformat selection outputs to "one-bead-one-compound" solid-phase libraries, unlocking activity-based and cellular screening capabilities. Here, we describe hydrogel-encapsulated magnetic beads that enable such a transformation. Bulk emulsion polymerization of polyacrylamide hydrogel shells around magnetic microbeads yielded uniform particles (7 ± 2 µm diameter) that are compatible with diverse in-gel functionalization (amine, alkyne, oligonucleotides) and transformations associated with DNA-encoded library synthesis (acylation, enzymatic DNA ligation). In a case study of reformatting mRNA display libraries, transcription from DNA-templated magnetic beads encapsulated in gel particles colocalized both RNA synthesis via hybridization with copolymerized complementary DNA and translation via puromycin labeling. Two control epitope templates (V5, HA) were successfully enriched (50- and 99-fold, respectively) from an NNK5 library bead screen via FACS. Proximity-driven library synthesis in concert with magnetic sample manipulation provides a plausible means for reformatting encoded combinatorial libraries at scale.

Liposomal Permeation Assay for Droplet-Scale Pharmacokinetic Screening.

Combinatorial library screening increasingly explores chemical space beyond the Ro5 (bRo5), which is useful for investigating "undruggable" targets but suffers compromised cellular permeability and therefore bioavailability. Moreover, structure-permeation relationships for bRo5 molecules are unclear partially because high-throughput permeation measurement technology for encoded combinatorial libraries is still nascent. Here, we present a permeation assay that is scalable to combinatorial library screening. A liposomal fluorogenic azide probe transduces permeation of alkyne-labeled molecules into small unilamellar vesicles via copper-catalyzed azide-alkyne cycloaddition. Control alkynes (e.g., propargylamine, various alkyne-labeled PEGs) benchmarked the assay. Cell-permeable macrocyclic peptides, exemplary bRo5 molecules, were alkyne labeled and shown to retain permeability. The assay was miniaturized to microfluidic droplets with high assay quality (Z' ≥ 0.5), demonstrating excellent discrimination of photocleaved known membrane-permeable and -impermeable model library beads. Droplet-scale permeation screening will enable pharmacokinetic mapping of bRo5 libraries to build predictive models.

Translating the Genome into Drugs.

The Human Genome Project ultimately aimed to translate DNA sequence into drugs. With the draft in hand, the Molecular Libraries Program set out to prosecute all genome-encoded proteins for drug discovery with automated high-throughput screening (HTS). This ambitious vision remains unfulfilled, even while innovations in sequencing technology have fully democratized access to genome-scale sequencing. Why? While the central dogma of biology allows us to chart the entirety of cellular metabolism through sequencing, there is no direct coding for chemistry. The rules of base pairing that relate DNA gene to RNA transcript and amino acid sequence do not exist for relating small-molecule structure with macromolecular binding partners and subsequently cellular function. Obtaining such relationships genome-wide is unapproachable via state-of-the-art HTS, akin to attempting genome-wide association studies using turn-of-the-millennium Sanger DNA sequencing.Our laboratory has been engaged in a multipronged technology development campaign to revolutionize molecular screening through miniaturization in pursuit of genome-scale drug discovery capabilities. The compound library was ripe for miniaturization: it clearly needed to become a consumable. We employed DNA-encoded library (DEL) synthesis principles in the development of solid-phase DELs prepared on microscopic beads, each harboring 100 fmol of a single library member and a DNA tag whose sequence describes the structure of the library member. Loading these DEL beads into 100 pL microfluidic droplets followed by online photocleavage, incubation, fluorescence-activated droplet sorting, and DNA sequencing of the sorted DEL beads reveals the chemical structures of bioactive compounds. This scalable library synthesis and screening platform has proven useful in several proof-of-concept projects involving current clinical targets.Moving forward, we face the problem of druggability and proteome-scale assay development. Developing biochemical or cellular assays for all genome-encoded targets is not scalable and likely impossible as most proteins have ill-defined or unknown activity and may not function outside of their native contexts. These are the dark undruggable expanses, and charting them will require advanced synthesis and analytical technologies that can generalize probe discovery, irrespective of mature protein function, to fulfill the Genome Project's vision of proteome-wide control of cellular pharmacology.

Study of an RNA-Focused DNA-Encoded Library Informs Design of a Degrader of a r(CUG) Repeat Expansion.

A solid-phase DNA-encoded library (DEL) was studied for binding the RNA repeat expansion r(CUG)exp, the causative agent of the most common form of adult-onset muscular dystrophy, myotonic dystrophy type 1 (DM1). A variety of uncharged and novel RNA binders were identified to selectively bind r(CUG)exp by using a two-color flow cytometry screen. The cellular activity of one binder was augmented by attaching it with a module that directly cleaves r(CUG)exp. In DM1 patient-derived muscle cells, the compound specifically bound r(CUG)exp and allele-specifically eliminated r(CUG)exp, improving disease-associated defects. The approaches herein can be used to identify and optimize ligands and bind RNA that can be further augmented for functionality including degradation.

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.

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.

DNA-Encoded Chemistry: Drug Discovery from a Few Good Reactions.

Click chemistry, proposed nearly 20 years ago, promised access to novel chemical space by empowering combinatorial library synthesis with a "few good reactions". These click reactions fulfilled key criteria (broad scope, quantitative yield, abundant starting material, mild reaction conditions, and high chemoselectivity), keeping the focus on molecules that would be easy to make, yet structurally diverse. This philosophy bears a striking resemblance to DNA-encoded library (DEL) technology, the now-dominant combinatorial chemistry paradigm. This review highlights the similarities between click and DEL reaction design and deployment in combinatorial library settings, providing a framework for the design of new DEL synthesis technologies to enable next-generation drug discovery.

Multiplexed Enzyme Activity-Based Probe Display via Hybridization.

Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼106 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼103 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types.

Considerations for Achieving Maximized DNA Recovery in Solid-Phase DNA-Encoded Library Synthesis.

DNA-encoded library (DEL) technology enables rapid, economical synthesis, and exploration of novel chemical space. Reaction development for DEL synthesis has recently accelerated in pace with a specific emphasis on ensuring that the reaction does not compromise the integrity of the encoding DNA. However, the factors that contribute to a reaction's "DNA compatibility" remain relatively unknown. We investigated several solid-phase reactions and encoding conditions and determined their impact on DNA compatibility. Conditions that minimized the accessibility of reactive groups on the DNA encoding tag (switching solvent, low temperature, double-stranded encoding tag) significantly improved compatibility. We showcased this approach in the multistep synthesis of an acyldepsipeptide (ADEP1) fragment, which preserved 73% of DNA for a >100-fold improvement over canonical conditions. These results are particularly encouraging in the context of multistep reaction sequences to access natural product-like scaffolds and more broadly underscore the importance of reconciling the biophysical properties and reactivity of DNA with chemistry development to yield high-quality libraries of those scaffolds.

Cycloisomerization of Olefins in Water.

Preparative reactions that occur efficiently under dilute, buffered, aqueous conditions in the presence of biomolecules find application in ligation, peptide synthesis, and polynucleotide synthesis and sequencing. However, the identification of functional groups or reagents that are mutually reactive with one another, but unreactive with biopolymers and water, is challenging. Shown here are cobalt catalysts that react with alkenes under dilute, aqueous, buffered conditions and promote efficient cycloisomerization and formal Friedel-Crafts reactions. The constraining conditions of bioorthogonal chemistry are beneficial for reaction efficiency as superior conversion at low catalyst concentration is obtained and competent rates in dilute conditions are maintained. Efficiency at high dilution in the presence of buffer and nucleobases suggests that these reaction conditions may find broad application.

Off-DNA DNA-Encoded Library Affinity Screening.

DNA-encoded library (DEL) technology is emerging as a key element of the small molecule discovery toolbox. Conventional DEL screens (i.e., on-DNA screening) interrogate large combinatorial libraries via affinity selection of DNA-tagged library members that are ligands of a purified and immobilized protein target. In these selections, the DNA tags can materially and undesirably influence target binding and, therefore, the experiment outcome. Here, we use a solid-phase DEL and droplet-based microfluidic screening to separate the DEL member from its DNA tag (i.e., off-DNA screening), for subsequent in-droplet laser-induced fluorescence polarization (FP) detection of target binding, obviating DNA tag interference. Using the receptor tyrosine kinase (RTK) discoidin domain receptor 1 (DDR1) as a proof-of-concept target in a droplet-scale competition-binding assay, we screened a 67 100-member solid-phase DEL of drug-like small molecules for competitive ligands of DDR1 and identified several known RTK inhibitor pharmacophores, including azaindole- and quinazolinone-containing monomers. Off-DNA DEL affinity screening with FP detection is potentially amenable to a wide array of target classes, including nucleic acid binding proteins, proteins that are difficult to overexpress and purify, or targets with no known activity assay.

A Solution Phase Platform to Characterize Chemical Reaction Compatibility with DNA-Encoded Chemical Library Synthesis.

DNA-encoded chemical library (DECL) synthesis must occur in aqueous media under conditions that preserve the integrity of the DNA encoding tag. While the identification of "DNA-compatible" reaction conditions is critical for the development of DECL designs that explore previously inaccessible chemical space, reports measuring such compatibility have been largely restricted to methods that do not faithfully capture the impact of reaction conditions on DNA fidelity in solution phase. Here we report a comprehensive methodology that uses soluble DNA substrates that exactly recapitulate DNA's exposure to the chemically reactive species of DECL synthesis. This approach includes the assessment of chemical fidelity (reaction yield and purity), encoding fidelity (ligation efficiency), and readability (DNA compatibility), revealing the fate of the DNA tag during DECL chemistry from a single platform.

Fluorescence-Activated Droplet Sorting for Single-Cell Directed Evolution.

Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ∼2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.

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.

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.

Poisson Statistics of Combinatorial Library Sampling Predict False Discovery Rates of Screening.

Microfluidic droplet-based screening of DNA-encoded one-bead-one-compound combinatorial libraries is a miniaturized, potentially widely distributable approach to small molecule discovery. In these screens, a microfluidic circuit distributes library beads into droplets of activity assay reagent, photochemically cleaves the compound from the bead, then incubates and sorts the droplets based on assay result for subsequent DNA sequencing-based hit compound structure elucidation. Pilot experimental studies revealed that Poisson statistics describe nearly all aspects of such screens, prompting the development of simulations to understand system behavior. Monte Carlo screening simulation data showed that increasing mean library sampling (ε), mean droplet occupancy, or library hit rate all increase the false discovery rate (FDR). Compounds identified as hits on k > 1 beads (the replicate k class) were much more likely to be authentic hits than singletons (k = 1), in agreement with previous findings. Here, we explain this observation by deriving an equation for authenticity, which reduces to the product of a library sampling bias term (exponential in k) and a sampling saturation term (exponential in ε) setting a threshold that the k-dependent bias must overcome. The equation thus quantitatively describes why each hit structure's FDR is based on its k class, and further predicts the feasibility of intentionally populating droplets with multiple library beads, assaying the micromixtures for function, and identifying the active members by statistical deconvolution.

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-µm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.