DNA-encoded Library Technology Research Articles

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.

Visible-Light Photocatalysis as an Enabling Technology for Drug Discovery: A Paradigm Shift for Chemical Reactivity.

Visible light-mediated photocatalysis, which relies on the ability of photocatalysts to absorb low-energy visible light and engage in single-electron transfer (SET) or energy transfer (ET) processes with organic substrates, has emerged as one of the fastest growing fields in organic synthesis. This catalytic platform enables a highly selective approach to promote radical-based organic transformations which unlocks unique reaction pathways. Due to the extremely mild conditions of these transformations and compatibility in aqueous environments, photocatalysis has emerged as an enabling technology in drug discovery. Photocatalysis is uniquely positioned for application in pharmaceutical development because of its demonstrated potential for broad functional group tolerance, biocompatibility, site-specific selectivity, and operational simplicity. This review will highlight the recent advances of visible-light photocatalysis through its application in peptide functionalization, protein bioconjugation, Csp 3-Csp 2 cross-coupling, late-stage functionalization, isotopic labeling, DNA-encoded library technology (DELT), and microenvironment mapping (μMap).

Navigating the DNA encoded libraries chemical space

DNA-encoded library (DEL) technology is a novel ligand identification strategy that allows the synthesis and screening of unprecedented chemical diversity more efficiently than conventional methods. However, no reports have been published to systematically study how to increase the diversity and improve the molecular property space that can be covered with DEL. This report describes the development and application of eDESIGNER, an algorithm that comprehensively generates all possible library designs, enumerates and profiles samples from each library and evaluates them to select the libraries to be synthesized. This tool utilizes suitable on-DNA chemistries and available building blocks to design and identify libraries with a pre-defined molecular weight distribution and maximal diversity compared with compound collections from other sources.

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.

On-DNA Cross-Dehydrogenative Coupling Reaction toward the Synthesis of Focused DNA-Encoded Tetrahydroisoquinoline Libraries.

To expand the chemical space of DNA-encoded library (DEL) technology, we have developed the DNA-compatible cross-dehydrogenative coupling reaction between tetrahydroisoquinolines (THIQs) and a series of nucleophiles such as nitromethane, terminal alkynes, and so on. Both photo- and metal-promoted conditions have been successfully established for this C-1 derivation of THIQs, as exemplified by the addition of nitromethane. All of these new DNA-compatible transformations have paved the way for the divergent oriented synthesis of THIQ-focused DELs.

Solution-Phase DNA-Compatible Pictet-Spengler Reaction Aided by Machine Learning Building Block Filtering.

SummaryThe application of machine learning toward DNA encoded library (DEL) technology is lacking despite obvious synergy between these two advancing technologies. Herein, a machine learning algorithm has been developed that predicts the conversion rate for the DNA-compatible reaction of a building block with a model DNA-conjugate. We exemplify the value of this technique with a challenging reaction, the Pictet-Spengler, where acidic conditions are normally required to achieve the desired cyclization between tryptophan and aldehydes to provide tryptolines. This is the first demonstration of using a machine learning algorithm to cull potential building blocks prior to their purchase and testing for DNA-encoded library synthesis. Importantly, this allows for a challenging reaction, with an otherwise very low building block pass rate in the test reaction, to still be used in DEL synthesis. Furthermore, because our protocol is solution phase it is directly applicable to standard plate-based DEL synthesis.

The Impact of Variable Selection Coverage on Detection of Ligands from a DNA-Encoded Library Screen.

DNA-encoded library (DEL) technology has become a prominent screening platform in drug discovery owing to the capacity to screen billions or trillions of compounds in a single experiment. Although numerous successes with DEL technology have been reported, we are unaware of a rigorous examination of the many different variables that can influence a screen's success. Herein, we explore the impact of variable sample sequencing depth on the detection of tool compounds with known affinities toward a given target while simultaneously probing the effect of initial compound input. Our sequencing data confirm reports that high-affinity compounds can be discovered directly from a DEL screen, but we demonstrate that a mismatch between selection output and sequencing quantity can obscure useful ligands. Our results highlight the importance of selection coverage in grasping the entire picture of a DEL screen where the signal of a weak or underrepresented ligand may be suppressed by the inherent noise of a selection. These potential missed ligands may be critical to the success or failure of a drug discovery program.

DNA-Encoded Library Screening as Core Platform Technology in Drug Discovery: Its Synthetic Method Development and Applications in DEL Synthesis.

DNA-encoded library technology (DELT) was introduced to our medicinal chemistry society more than 20 years ago. The application of DELT in the development of clinical candidates has been actively reported in the literature recently. A few representative examples include RIP1K inhibitors for inflammatory diseases and sEH inhibitors for endothelial dysfunction or abnormal tissue repair, among many others. Here, the authors would like to recall the recent developments in on-DNA synthetic methodologies for DEL construction and to analyze recent examples in the literature of DELT-based drug development efforts pursued in both the academic and industrial sectors. With this perspective, we hope to provide a useful summary of recent DELT-based drug discovery research and to discuss the future scope of DELT in medicinal chemistry.

Multifunctional Building Blocks Compatible with Photoredox-Mediated Alkylation for DNA-Encoded Library Synthesis.

DNA-encoded library (DEL) technology has emerged as a novel interrogation modality for ligand discovery in the pharmaceutical industry. Given the increasing demand for a higher proportion of C(sp3)-hybridized centers in DEL platforms, a photoredox-mediated cross-coupling and defluorinative alkylation process is introduced using commercially available alkyl bromides and structurally diverse α-silylamines. Notably, no protecting group strategies for amines are necessary for the incorporation of a variety of amino-acid-based organosilanes, providing crucial branching points for further derivatization.

Exploring the Lower Limit of Individual DNA-Encoded Library Molecules in Selection.

DNA-encoded library (DEL) technology has been used as an ultra-high-throughput screening approach for hit identification of drug targets. This process is an affinity-based selection and requires incubation of DEL molecules with the target. Currently, in most reported cases, the input (i.e., the copy number) of individual DEL molecules varies from 105 to 107. With the ever-increasing DEL size and screening cost, lowering the input of DEL molecules while maintaining an appropriate signal-to-noise ratio in a selection is of paramount importance. In this article, we varied the input of DEL ranging from 103 to 105 in selections with two different protein targets to explore the lower limit of DEL molecule input. The results could facilitate the optimization of the DEL selection process and reduce costs related to library consumption.

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.

Abstract 981: Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design

Abstract Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, ‘fit-for-purpose’ PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable. Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.

Open-Air Alkylation Reactions in Photoredox-Catalyzed DNA-Encoded Library Synthesis

DNA-encoded library (DEL) technology is a powerful tool commonly used by the pharmaceutical industry for the identification of compounds with affinity to biomolecular targets. Success in this endeavor lies in sampling diverse chemical libraries. However, current DELs tend to be deficient in C(sp3) carbon counts. We report unique solutions to the challenge of increasing both the chemical diversity of these libraries and their C(sp3) carbon counts by merging Ni/photoredox dual catalytic C(sp2)-C(sp3) cross-coupling as well as photoredox-catalyzed radical/polar crossover alkylation protocols with DELs. The successful integration of multiple classes of radical sources enables the rapid incorporation of a diverse set of alkyl fragments.

Mild and Efficient Palladium-Mediated C-N Cross-Coupling Reaction between DNA-Conjugated Aryl Bromides and Aromatic Amines.

DNA-encoded library technology (ELT) has emerged in the pharmaceutical industry as a powerful tool for hit and lead generation. Over the last 10 years, a number of DNA-compatible chemical reactions have been published and used to synthesize libraries. Among the most commonly used reactions in medicinal chemistry is the C-N bond formation, and its application to DNA-encoded library technology affords an alternative approach to identify high-affinity binders for biologically relevant protein targets. Herein we report a newly developed Pd-promoted C-N cross coupling reaction between DNA-conjugated aryl bromides and a wide scope of arylamines in good to excellent yields. The mild reaction conditions should facilitate the synthesis of novel DNA-encoded combinatorial libraries.

DNA-Encoded Macrocyclic Peptide Library.

DNA-encoded library technology (ELT) is a cutting-edge enabling technology platform for drug discovery. Here we describe how to design and synthesize a macrocyclic DNA-encoded library; how to perform selection, sequencing, and data analysis to identify potential active peptides; and how to synthesize off-DNA peptides to confirm activity. This approach provides an effective tool for pharmaceutical research based on peptides.

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

Ruthenium Promoted On-DNA Ring-Closing Metathesis and Cross-Metathesis.

DNA-encoded library technology (ELT) is now widely used in pharmaceutical, biotechnological, and academic research for hit identification and target validation. New on-DNA reactions are keys to exploring greater chemical space and accessing challenging chemotypes such as configurationally constrained macrocycles. Herein, we describe the first on-DNA ring-closing metathesis (RCM) and cross-metathesis (CM) reactions promoted by fast initiating Grubbs Ru reagents. Under the optimized conditions, MgCl2 was used to protect the DNA from Ru-induced decomposition. The substrate scope for on-DNA RCM was established and the same conditions were applied to a CM reaction with good conversion.

Discovering Drugs with DNA-Encoded Library Technology: From Concept to Clinic with an Inhibitor of Soluble Epoxide Hydrolase.

DNA-encoded chemical library technology was developed with the vision of its becoming a transformational platform for drug discovery. The hope was that a new paradigm for the discovery of low-molecular-weight drugs would be enabled by combining the vast molecular diversity achievable with combinatorial chemistry, the information-encoding attributes of DNA, the power of molecular biology, and a streamlined selection-based discovery process. Here, we describe the discovery and early clinical development of GSK2256294, an inhibitor of soluble epoxide hydrolase (sEH, EPHX2), by using encoded-library technology (ELT). GSK2256294 is an orally bioavailable, potent and selective inhibitor of sEH that has a long half life and produced no serious adverse events in a first-time-in-human clinical study. To our knowledge, GSK2256294 is the first molecule discovered from this technology to enter human clinical testing and represents a realization of the vision that DNA-encoded chemical library technology can efficiently yield molecules with favorable properties that can be readily progressed into high-quality drugs.

Leitmolekülsuche mit DNA-codierten Bibliotheken

Structure-based drug design requires lead molecules, which traditionally are found by tedious and expensive screening campaigns. By contrast, screening of DNA-encoded small molecule libraries can be much more efficient. Here, the principle and application of the recently developed DNA-encoded library technology (DELT) are discussed.

Zirconium(IV)-Catalyzed Ring Opening of on-DNA Epoxides in Water.

DNA-encoded library technology (ELT) has spurred wide interest in the pharmaceutical industry as a powerful tool for hit and lead generation. In recent years a number of "DNA-compatible" chemical modifications have been published and used to synthesize vastly diverse screening libraries. Herein we report a newly developed, zirconium tetrakis(dodecyl sulfate) [Zr(DS)4 ] catalyzed ring-opening of on-DNA epoxides in water with amines, including anilines. Subsequent cyclization of the resulting on-DNA β-amino alcohols leads to a variety of biologically interesting, nonaromatic heterocycles. Under these conditions, a library of 137 million on-DNA β-amino alcohols and their cyclization products was assembled.