DNA-encoded Libraries Research Articles

ABPP-CoDEL: Activity-Based Proteome Profiling-Guided Discovery of Tyrosine-Targeting Covalent Inhibitors from DNA-Encoded Libraries.

DNA-encoded chemical library (DEL) has been extensively used for lead compound discovery for decades in academia and industry. Incorporating an electrophile warhead into DNA-encoded compounds recently permitted the discovery of covalent ligands that selectively react with a particular cysteine residue. However, noncysteine residues remain underexplored as modification sites of covalent DELs. Herein, we report the design and utility of tyrosine-targeting DELs of 67 million compounds. Proteome-wide reactivity analysis of tyrosine-reactive sulfonyl fluoride (SF) covalent probes suggested three enzymes (phosphoglycerate mutase 1, glutathione s-transferase 1, and dipeptidyl peptidase 3) as models of tyrosine-targetable proteins. Enrichment with SF-functionalized DELs led to the identification of a series of tyrosine-targeting covalent inhibitors of the model enzymes. In-depth mechanistic investigation revealed their novel modes of action and reactive ligand-accessible hotspots of the enzymes. Our strategy of combining activity-based proteome profiling and covalent DEL enrichment (ABPP-CoDEL), which generated selective covalent binders against a variety of target proteins, illustrates the potential use of this methodology in further covalent drug discovery.

Rational Screening for Cooperativity in Small-Molecule Inducers of Protein-Protein Associations.

The hallmark of a molecular glue is its ability to induce cooperative protein-protein interactions, leading to the formation of a ternary complex, despite weaker binding toward one or both individual proteins. Notably, the extent of cooperativity distinguishes molecular glues from bifunctional compounds, which constitute a second class of inducers of protein-protein interactions. However, apart from serendipitous discovery, there have been limited rational screening strategies for the high cooperativity exhibited by molecular glues. Here, we propose a binding-based screen of DNA-barcoded compounds on a target protein in the presence or absence of a presenter protein, using the "presenter ratio", the ratio of ternary enrichment to binary enrichment, as a predictive measure of cooperativity. Through this approach, we identified a range of cooperative, noncooperative, and uncooperative compounds in a single DNA-encoded library screen with bromodomain containing protein (BRD)9 and the VHL-elongin C-elongin B (VCB) complex. Our most cooperative hit compound, 13-7, exhibits micromolar binding affinity to BRD9 but nanomolar affinity for the ternary complex with BRD9 and VCB, with cooperativity comparable to classical molecular glues. This approach may enable the rational discovery of molecular glues for preselected proteins and thus facilitate the transition to a new paradigm of small-molecule therapeutics.

Covalent Capture and Selection of DNA-Encoded Chemical Libraries via Photo-Activated Lysine-Selective Crosslinkers.

Covalent crosslinking probes have arisen as efficient toolkits to capture and elucidate biomolecular interaction networks. Exploiting the potential of crosslinking in DNA-encoded chemical library (DEL) selection methods significantly boosted bioactive ligand discovery in complex physiological contexts. Herein, we incorporated o-nitrobenzyl alcohol (o-NBA) as a photo-activated lysine-selective crosslinker into divergent DEL formats and achieved covalent capture of ligand-target interactions featuring improved crosslinking efficiency and site specificity. In addition, covalent DEL selection was realized with the modularly designed o-NBA-functionalized mock libraries.

Control of the antitumour activity and specificity of CAR T cells via organic adapters covalently tethering the CAR to tumour cells.

On-target off-tumour toxicity limits the anticancer applicability of chimaeric antigen receptor (CAR) T cells. Here we show that the tumour-targeting specificity and activity of T cells with a CAR consisting of an antibody with a lysine residue that catalytically forms a reversible covalent bond with a 1,3-diketone hapten can be regulated by the concentration of a small-molecule adapter. This adapter selectively binds to the hapten and to a chosen tumour antigen via a small-molecule binder identified via a DNA-encoded library. The adapter therefore controls the formation of a covalent bond between the catalytic antibody and the hapten, as well as the tethering of the CAR T cells to the tumour cells, and hence the cytotoxicity and specificity of the cytotoxic T cells, as we show in vitro and in mice with prostate cancer xenografts. Such small-molecule switches of T-cell cytotoxicity and specificity via an antigen-independent 'universal' CAR may enhance the control and safety profile of CAR-based cellular immunotherapies.

Discovery of a 53BP1 Small Molecule Antagonist Using a Focused DNA-Encoded Library Screen.

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC50 of 0.47 ± 0.09 μM in a TR-FRET assay and Kd values of 0.85 ± 0.17 and 0.79 ± 0.52 μM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.

Development of an automated flow chemistry affinity-based purification process for DNA-encoded chemistry

An automated flow chemistry platform for DNA-encoded library (DEL) technologies requires the integration of a purification process for DNA-tagged substrates. It facilitates the development of further DEL reactions, building block rehearsal, and library synthesis. Therefore, a recently developed, manual affinity-based batch purification process for DNA-tagged substrates based on dispersive solid-phase extraction (DSPE) was transferred to automated flow chemistry using tailored 3D-printed microfluidic devices and open-source lab automation equipment. The immobilization and purification steps use Watson–Crick base pairing for a compound-encoding single-stranded DNA, which allows for the thorough removal of impurities and contaminations by washing steps and operationally simple recovery of the purified DNA-encoded compounds. This work optimized the annealing step for flow incubation and DNA purification was accomplished by flow DSPE washing/elution steps. The manually performed batch affinity-based purification process was compared with the microfluidic process by determining qualitative and quantitative DNA recovery parameters. It aimed at comparing batch and flow purification processes with regard to DNA recovery and purity to benefit from the high potential for automation, precise process control, and higher information density of the microfluidic purification process for DNA-tagged substrates. Manual operations were minimized by applying an automation strategy to demonstrate the potential for integrating the microfluidic affinity-based purification process for DNA-tagged substrates into an automated DNA-encoded flow chemistry platform.Graphical

Recording Binding Information Directly into DNA-Encoded Libraries Using Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT) is an unusual DNA polymerase that adds untemplated dNTPs to 3'-ends of DNA. If a target protein is expressed as a TdT fusion and incubated with a DNA-encoded library (DEL) in the presence of dATP, the binders of the target induce proximity between TdT and the DNA, promoting the synthesis of a poly-adenine (polyA) tail. The polyA tail length is proportional to the binding affinity, effectively serving as a stable molecular record of binding events. The polyA tail is also a convenient handle to enrich binders with magnetic poly(dT)25 beads before sequencing. In a benchmarking system, we show that ligands spanning nanomolar to double-digit micromolar binding can be cleanly identified by TdT extension, whereas only the tightest binding ligands are identified by classical affinity selection. The method is simple to implement and can function on any DEL that bears a free 3'-end.

DNA-Compatible Cyanomethylation of (Hetero)aryl Halides or Triflates under a Tandem Reaction for DNA-Encoded Library Synthesis.

A DNA-compatible reaction has been developed for the cyanomethylation of (hetero)aryl halides or triflates via a tandem process involving palladium-mediated Suzuki-Miyaura coupling and base-promoted isoxazole fragmentation. This one-pot protocol employs easily accessible starting materials, exhibits a wide substrate scope, and results in no significant DNA damage. Additionally, the resulting (hetero)arylacetonitriles can be converted into the corresponding carboxylic acids, which may be utilized for the synthesis of DNA-encoded chemical libraries.

Novel molecule BBC0901 inhibits BRD4 and acts as a catabolic regulator in the pathogenesis of osteoarthritis

Osteoarthritis (OA) is induced by matrix degradation and inflammation mediated by bromo-domain-containing protein 4 (BRD4)-dependent catabolic factors. BRD4 acts as both a transcriptional regulator and an epigenetic reader. BBC0901 was identified as an inhibitor of BRD4 using a DNA-encoded library screening system. We aimed to demonstrate the effects of BBC0901 on OA pathogenesis by in vitro, ex vivo, and in vivo analyses. BBC0901 inhibited the expression of catabolic factors that degrade cartilage without significantly affecting the viability of mouse articular chondrocytes. Additionally, ex vivo experiments under conditions mimicking OA showed that BBC0901 suppressed extracellular matrix degradation. RNA sequencing analysis of gene expression patterns showed that BBC0901 inhibited the expression of catabolic factors, such as matrix metalloproteinases (MMPs) and cyclooxygenase (COX)2, along with reactive oxygen species (ROS) production. Furthermore, intra-articular (IA) injection of BBC0901 into the knee joint blocked osteoarthritic cartilage destruction by inhibition of MMP3, MMP13, COX2, interleukin (IL)6, and ROS production, thereby obstructing the nuclear factor kappa-light-chain-enhancer of activated B cell and mitogen activated protein kinase signaling. In conclusion, BBC0901-mediated BRD4 inhibition prevented OA development by attenuating catabolic signaling and hence, can be considered a promising IA therapeutic for OA.

End-to-End Semi-automated Mid-scale Protein Screening Platform for Drug Discovery Research.

The drug discovery landscape is ever-evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have built a higher-throughput mid-scale semi-automated protein expression and screening platform to accelerate drug discovery research. The workflow described here enables comprehensive expression and purification screening assessment of challenging or difficult-to-express recombinant proteins in a fast and efficient manner by delivering small but sufficient amounts of high-quality proteins. The platform has been implemented for a wide range of applications that include identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co-expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for insoluble or aggregation-prone proteins. The approach allows parallel expression, purification, and characterization of 24 different samples using co-infection or a polycistronic approach in insect cells and enables parallel testing of multiple parameters to improve protein yields. The strategy has been successfully adopted for screening intracellular and secreted proteins in Escherichia coli, mammalian transient expression, and baculovirus expression vector systems. Proteins purified from this platform are used for several structural and functional screens, such as negative staining, biochemical activity assays, mass spectrometry, surface plasmon resonance, and DNA-encoded chemical library screens. In this article, for simplicity, we have focused on detailed expression and purification screening of intracellular protein complexes from insect cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Baculovirus generation via homologous recombination Support Protocol 1: Anti-glycoprotein 64 antibody assay Basic Protocol 2: Generation of insect cell biomass expressing target protein(s) Basic Protocol 3: Mid-scale affinity purification Support Protocol 2: Automated method for affinity purification on Hamilton STAR Basic Protocol 4: Size exclusion chromatography Support Protocol 3: Chromeleon 7 operation on Vanquish Duo.

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.

Meta-GTM: Visualization and Analysis of the Chemical Library Space.

In chemical library analysis, it may be useful to describe libraries as individual items rather than collections of compounds. This is particularly true for ultra-large noncherry-pickable compound mixtures, such as DNA-encoded libraries (DELs). In this sense, the chemical library space (CLS) is useful for the management of a portfolio of libraries, just like chemical space (CS) helps manage a portfolio of molecules. Several possible CLSs were previously defined using vectorial library representations obtained from generative topographic mapping (GTM). Given the steadily growing number of DEL designs, the CLS becomes "crowded" and requires analysis tools beyond pairwise library comparison. Therefore, herein, we investigate the cartography of CLS on meta-(μ)GTMs─"meta" to remind that these are maps of the CLS, itself based on responsibility vectors issued by regular CS GTMs. 2,5 K DELs and ChEMBL (reference) were projected on the μGTM, producing landscapes of library-specific properties. These describe both interlibrary similarity and intrinsic library characteristics in the same view, herewith facilitating the selection of the best project-specific libraries.

DNA-Conjugated Cyclopropane Derivatives Constructed from Sulfonium Ylides with α,β-Unsaturated Ketones.

Here, we report a DNA-compatible reaction for the generation of cyclopropane derivatives using thiolides with α,β-unsaturated ketones in the absence of transition metal and N2 protection, which is convenient for DNA encoded library (DEL) construction. This approach allows the rapid and efficient production of a series of DEL libraries of potentially biologically active cyclopropanes and spirocyclopropyl oxindole derivatives.

Diversity-oriented synthesis encoded by deoxyoligonucleotides

Diversity-oriented synthesis (DOS) is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors and derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We also show screening results for three diverse protein targets. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Building Block-Based Binding Predictions for DNA-Encoded Libraries.

DNA-encoded libraries (DELs) provide the means to make and screen millions of diverse compounds against a target of interest in a single experiment. However, despite producing large volumes of binding data at a relatively low cost, the DEL selection process is susceptible to noise, necessitating computational follow-up to increase signal-to-noise ratios. In this work, we present a set of informatics tools to employ data from prior DEL screen(s) to gain information about which building blocks are most likely to be productive when designing new DELs for the same target. We demonstrate that similar building blocks have similar probabilities of forming compounds that bind. We then build a model from the inference that the combined behavior of individual building blocks is predictive of whether an overall compound binds. We illustrate our approach on a set of three-cycle OpenDEL libraries screened against soluble epoxide hydrolase (sEH) and report performance of more than an order of magnitude greater than random guessing on a holdout set, demonstrating that our model can serve as a baseline for comparison against other machine learning models on DEL data. Lastly, we provide a discussion on how we believe this informatics workflow could be applied to benefit researchers in their specific DEL campaigns.

Impact of organic chemistry conditions on DNA durability in the context of DNA-encoded library technology

High-power screening (HPS) technologies, such as DNA-encoded library (DEL) technology, could exponentially increase the dimensions of the chemical space accessible for drug discovery. The intrinsic fragile nature of DNA is associated with cumbersome limitations and DNA durability (e.g., depurination, loss of phosphate groups, adduct formation) is compromised in numerous organic chemistry conditions that require empirical testing. An atlas of reaction conditions (temperature, pH, solvent/buffer, ligands, oxidizing reagents, catalysts, scavengers in function of time) that have been systematically tested in multiple combinations, indicates precisely limits useful for DEL construction. More importantly, this approach could be used broadly to effectively evaluate DNA-compatibility of any novel on-DNA chemical reaction, and it is compatible with different molecular methodologies. This atlas and the general approach presented, by allowing novel reaction conditions to be performed in presence of DNA, should greatly help in expanding the DEL chemical space as well as any field involving DNA durability.

Discovery, SAR Study of GST Inhibitors from a Novel Quinazolin-4(1H)-one Focused DNA-Encoded Library.

The DNA-encoded library (DEL) is a powerful hit-generation tool in drug discovery. This study describes a new DEL with a privileged scaffold quinazolin-4(3H)-one developed by a robust DNA-compatible multicomponent reaction and a series of novel glutathione S-transferase (GST) inhibitors that were identified through affinity-mediated DEL selection. A novel inhibitor 16 was subsequently verified with an inhibitory potency value of 1.55 ± 0.02 μM against SjGST and 2.02 ± 0.20 μM against hGSTM2. Further optimization was carried out via various structure-activity relationship studies. And especially, the co-crystal structure of the compound 16 with the SjGST was unveiled, which clearly demonstrated its binding mode was quite different from the known GSH-like compounds. This new type of probe is likely to play a different role compared with the GSH, which may provide new opportunities to discover more potent GST inhibitors.

Development of a microfluidic photochemical flow reactor concept by rapid prototyping.

The transfer from batch to flow chemistry is often based on commercial microfluidic equipment, such as costly complete reactor systems, which cannot be easily tailored to specific requirements of technologies such as DNA-encoded library technology (DELT), in particular for increasingly important photochemical reactions. Customized photoreactor concepts using rapid prototyping technology offer a modular, flexible, and affordable design that allows for adaptation to various applications. In order to validate the prototype reactors, a photochemical pinacol coupling reaction at 368nm was conducted to demonstrate the transfer from batch to flow chemistry. The conversion rates were optimized by adapting the design parameters of the microfluidic flow photoreactor module. Subsequently, the photoreactor module has been extended to an application with DNA-tagged substrates by switching to LEDs with a wavelength of 454nm. The successful recovery of DNA confirmed the feasibility of the modular-designed flow photo reactor. This collaborative approach holds enormous potential to drive the development of DELT and flow equipment design.

DNA-Compatible Copper/TEMPO Oxidation for DNA-Encoded Libraries.

Aldehydes are important synthons for DNA-encoded library (DEL) construction, but the development of a DNA-compatible method for the oxidation of alcohols to aldehydes remains a significant challenge in the field of DEL chemistry. We report that a copper/TEMPO catalyst system enables the solution-phase DNA-compatible oxidation of DNA-linked primary activated alcohols to aldehydes. The semiaqueous, room-temperature reaction conditions afford oxidation of benzylic, heterobenzylic, and allylic alcohols in high yield, with DNA compatibility verified by mass spectrometry, qPCR, Sanger sequencing, and ligation assays. Subsequent transformations of the resulting aldehydes demonstrate the potential of this method for robust library diversification.

Abstract P2154: A Novel Allosteric Modulator Of The β 1 AR Identified By DNA-encoded Small Molecule Library Screening Demonstrates Unique Pharmacology And Function

While traditional β-blockers (i.e. competitive orthosteric antagonists of β-adrenergic receptors; βARs) are widely used as cardiovascular therapeutics, adverse effects such as fatigue and the nonselective inhibition of multiple receptor subtypes often limit maximal effectiveness. An emerging approach to enhance therapeutic targeting is to identify allosteric modulators that act cooperatively with orthosteric ligands. In contrast to orthosteric ligands which bind the endogenous ligand binding site, allosteric modulators bind to regions that are topographically distinct from the orthosteric pocket and can enhance (positive allosteric modulator; PAM) or reduce (negative allosteric modulator; NAM) the activities of orthosteric agonists/antagonists. Since allosteric regions exhibit greater sequence and structural diversity among receptor subtypes relative to the more highly conserved orthosteric pocket, allosteric modulators are more likely to be subtype specific and/or generate less adverse effects. We therefore embarked on a DNA-encoded small molecule library screen to identify novel allosteric modulators of the β 1 AR. Following multiple rounds of affinity selection using purified, functional, β 1 ARs reconstituted in lipid nanodiscs and HitGen’s OpenDEL® small molecule library containing more than 1 billion unique compounds, we identified Compound 11 (C11) as an allosteric modulator with unique pharmacological properties. Notably, C11 binds to the β 1 AR with micromolar affinity and enhances the binding affinity of orthosteric agonists and certain antagonists to the β 1 AR. In contrast to its positive cooperative effect on ligand binding, cell signaling assays showed C11 potently inhibits G protein and β-arrestin signaling downstream of the β 1 AR. Importantly, C11 showed high β 1 AR specificity with no effect on β 2 AR or AT 1 R signaling. These results suggest that C11 is a β 1 AR-specific PAM in terms of ligand binding but a NAM in terms of agonist efficacy, belonging to a largely under-characterized class of allosteric modulators termed PAM-antagonists. With an extremely unique pharmacological profile, C11 is a promising potential therapeutic and experiments evaluating its ability to modulate β 1 AR signaling in vivo are ongoing.