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.

Full antagonism of the estrogen receptor without a prototypical ligand side chain.

Resistance to endocrine therapies remains a major clinical problem for the treatment of estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor, and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.

Evolution of a mass spectrometry-grade protease with PTM-directed specificity.

Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (kcat/KM = 6.9 × 105 M-1⋅s-1). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.

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.

Ligand-binding dynamics rewire cellular signaling via estrogen receptor-α.

Ligand-binding dynamics control allosteric signaling through the estrogen receptor-α (ERα), but the biological consequences of such dynamic binding orientations are unknown. Here, we compare a set of ER ligands having dynamic binding orientation (dynamic ligands) with a control set of isomers that are constrained to bind in a single orientation (constrained ligands). Proliferation of breast cancer cells directed by constrained ligands is associated with DNA binding, coactivator recruitment and activation of the estrogen-induced gene GREB1, reflecting a highly interconnected signaling network. In contrast, proliferation driven by dynamic ligands is associated with induction of ERα-mediated transcription in a DNA-binding domain (DBD)-dependent manner. Further, dynamic ligands showed enhanced anti-inflammatory activity. The DBD-dependent profile was predictive of these signaling patterns in a larger diverse set of natural and synthetic ligands. Thus, ligand dynamics directs unique signaling pathways and reveals a new role of the DBD in allosteric control of ERα-mediated signaling.

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.

Bicyclic core estrogens as full antagonists: synthesis, biological evaluation and structure-activity relationships of estrogen receptor ligands based on bridged oxabicyclic core arylsulfonamides.

Compounds that block estrogen action through the estrogen receptor (ER) or downregulate ER levels are useful for the treatment of breast cancer and endocrine disorders. In our search for structurally novel estrogens having three-dimensional core scaffolds, we found some compounds with a 7-oxabicyclo[2.2.1]heptene core that bound well to the ERs. The best of these compounds, a phenyl sulfonate ester (termed OBHS for oxabicycloheptene sulfonate), was a partial antagonist on both ERα and ERß. Although OBHS bears no structural resemblance to other estrogen antagonists, it appears to achieve its partial antagonist character by stabilizing a novel conformation of the ER that involves a significant distortion of helix-11. To enhance the antagonist properties of these oxabicyclo[2.2.1]heptane core ligands, we expanded the functional diversity of OBHS by replacing the sulfonate with secondary or tertiary sulfonamides (-SO(2)NR-), isoelectronic and potentially isostructural molecular replacements. An array of 16 OBHS sulfonamide analogues were prepared through a Diels-Alder reaction of a 3,4-diarylfuran using various N-aryl vinyl sulfonamide dienophiles. While the more polar secondary sulphonamides were weak ligands, certain of the tertiary sulfonamides had very good ER binding affinity. In HepG2 cell reporter gene assays, the sulphonamides had moderate potency, but they showed lower intrinsic transcriptional activity on ERα than the selective estrogen receptor modulator (SERM) hydroxytamoxifen or OBHS, and they were inverse agonists on ERß. Thus, the behaviour of these OBH-sulfonamides more closely mirrors the activity of full antagonists like the drug fulvestrant (ICI 182 780), and their greater antagonist biocharacter appears to arise from an accentuated distortion of helix-11.

Activation domain-dependent degradation of somatic Wee1 kinase.

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G(2) and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.

A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway.

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the "coactivator trap," to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1-3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54(nrb)) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.

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.

Mapping the phosphorylation sites of Ulk1.

Ulk1 is a serine/threonine kinase that controls macroautophagy, an essential homeostatic recycling pathway that degrades bulk cytoplasmic material and directs the turnover of organelles such as peroxisomes and mitochondria. Further, macroautophagy is potently induced by signals that trigger metabolic stress, such as hypoxia and amino acid starvation, where its recycling functions provide macromolecules necessary to maintain catabolic metabolism and cell survival. Substrates for Ulk1 have not been identified, and little is known regarding post-translational control of Ulk1 kinase activity and function. To gain insights into the regulatory mechanisms of Ulk1, we developed a robust purification protocol for Ulk1 and demonstrated that Ulk1 is highly phosphorylated and requires autophosphorylation for stability. Importantly, high-resolution, tandem mass spectrometry identified multiple sites of phosphorylation on Ulk1, including several within domains known to regulate macroautophagy. Differential phosphorylation analyses also identified sites of phosphorylation in the C-terminal domain that depend upon or require Ulk1 autophosphorylation.

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.

Strategies to recover proteins from ocular tissues for proteomics.

We present here the results of protein extraction from different ocular regions using different detergents. Extraction strategies used to determine optimal protein extraction included: pressure cycling and aqueous-organic phase extraction in combination with electrophoretic fractionation for anterior, posterior, and peripapillary sclera. Detergent extraction of proteins from freshly enucleated porcine eyes (n = 8) showed significant differences for different eye regions. Protein yield ranged from 2.3 to 50.7 mug protein/mg for different ocular tissues, with the lens yielding the most protein. ASB-14 and Triton X-100 provided the best protein yields (n = 10) for anterior and posterior sclera. The spectrophotometric measurements for ASB-14 were not consistent with SDS-PAGE densitometry. A combination of 0.5% Triton X-100, 0.5% Tween-20, and 0.1% Genapol C-100 was found optimal for extraction from sclera. Proteins from different regions of the eye are best extracted with different detergents. The pressure cycling technology provided superior extraction compared to the other methods. Additional aqueous-organic phase partitioning enables superior fractionation when compared to SDS-PAGE alone. Organic phase fractionation is compatible with MS and allowed identification of 34, 71, and 77 proteins respectively from anterior, posterior, and peripapillary sclera. The extraction strategy may affect the final outcome in protein profiling by MS or by other methods.

Chemoselective Coupling Preserves the Substrate Integrity of Surface-Immobilized Oligonucleotides for Emulsion PCR-Based Gene Library Construction.

Combinatorial bead libraries figure prominently in next-generation sequencing and are also important tools for in vitro evolution. The most common methodology for generating such bead libraries, emulsion PCR (emPCR), enzymatically extends bead-immobilized oligonucleotide PCR primers in emulsion droplets containing a single progenitor library member. Primers are almost always immobilized on beads via noncovalent biotin-streptavidin binding. Here, we describe covalent bead functionalization with primers (∼106 primers/2.8-µm-diameter bead) via either azide-alkyne click chemistry or Michael addition. The primers are viable polymerase substrates (4-7% bead-immobilized enzymatic extension product yield from one thermal cycle). Carbodiimide-activated carboxylic acid beads only react with oligonucleotides under conditions that promote nonspecific interactions (low salt, low pH, no detergent), comparably immobilizing primers on beads, but yielding no detectable enzymatic extension product. Click-functionalized beads perform satisfactorily in emPCR of a site-saturation mutagenesis library, generating monoclonal templated beads (104-105 copies/bead, 1.4-kb amplicons). This simpler, chemical approach to primer immobilization may spur more economical library preparation for high-throughput sequencing and enable more complex surface elaboration for in vitro evolution.

A cell based screening approach for identifying protein degradation regulators.

Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.

High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium tuberculosis Infections.

The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover "epitope surrogates" (ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 106 beads, 448 000 unique compounds) using fluorescent antihuman IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens.

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-µm quality control beads and 10-µm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Structural mechanism for signal transduction in RXR nuclear receptor heterodimers.

A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA). This SCA network acts as a signalling rheostat to integrate signals between dimer partners, ligands and coregulator-binding sites, thereby affecting signal transmission in RXR heterodimers. These findings define rules guiding how NRs integrate two ligand-dependent signalling pathways into RXR heterodimer-specific responses.