DNA-encoded Library Screening Research Articles

An Analysis of Successful Hit-to-Clinical Candidate Pairs.

An analysis of 156 published clinical candidates from the Journal of Medicinal Chemistry between 2018 and 2021 was conducted to identify lead generation strategies most frequently employed leading to drug candidates. As in a previous publication, the most frequent lead generation strategies resulting in clinical candidates were from known compounds (59%) followed by random screening approaches (21%). The remainder of the approaches included directed screening, fragment screening, DNA-encoded library screening (DEL), and virtual screening. An analysis of similarity was also conducted based on Tanimoto-MCS and revealed most clinical candidates were distant from their original hits; however, most shared a key pharmacophore that translated from hit-to-clinical candidate. An examination of frequency of oxygen, nitrogen, fluorine, chlorine, and sulfur incorporation in clinical candidates was also conducted. The three most similar and least similar hit-to-clinical pairs from random screening were examined to provide perspective on changes that occur that lead to successful clinical candidates.

Streamlined DNA-encoded small molecule library screening and validation for the discovery of novel chemotypes targeting BET proteins

Targeting aberrant epigenetic programs that drive tumorigenesis is a promising approach to cancer therapy. DNA-encoded library (DEL) screening is a core platform technology increasingly used to identify drugs that bind to protein targets. Here, we use DEL screening against bromodomain and extra-terminal motif (BET) proteins to identify inhibitors with new chemotypes, and successfully identified BBC1115 as a selective BET inhibitor. While BBC1115 does not structurally resemble OTX-015, a clinically active pan-BET inhibitor, our intensive biological characterization revealed that BBC1115 binds to BET proteins, including BRD4, and suppresses aberrant cell fate programs. Phenotypically, BBC1115-mediated BET inhibition impaired proliferation in acute myeloid leukemia, pancreatic, colorectal, and ovarian cancer cells invitro. Moreover, intravenous administration of BBC1115 inhibited subcutaneous tumor xenograft growth with minimal toxicity and favorable pharmacokinetic properties invivo. Since epigenetic regulations are ubiquitously distributed across normal and malignant cells, it will be critical to evaluate if BBC1115 affects normal cell function. Nonetheless, our study shows integrating DEL-based small-molecule compound screening and multi-step biological validation represents a reliable strategy to discover new chemotypes with selectivity, efficacy, and safety profiles for targeting proteins involved in epigenetic regulation in human malignancies.

DEL-Dock: Molecular Docking-Enabled Modeling of DNA-Encoded Libraries.

DNA-encoded library (DEL) technology has enabled significant advances in hit identification by enabling efficient testing of combinatorially generated molecular libraries. DEL screens measure protein binding affinity though sequencing reads of molecules tagged with unique DNA barcodes that survive a series of selection experiments. Computational models have been deployed to learn the latent binding affinities that are correlated to the sequenced count data; however, this correlation is often obfuscated by various sources of noise introduced in its complicated data-generation process. In order to denoise DEL count data and screen for molecules with good binding affinity, computational models require the correct assumptions in their modeling structure to capture the correct signals underlying the data. Recent advances in DEL models have focused on probabilistic formulations of count data, but existing approaches have thus far been limited to only utilizing 2-D molecule-level representations. We introduce a new paradigm, DEL-Dock, that combines ligand-based descriptors with 3-D spatial information from docked protein-ligand complexes. 3-D spatial information allows our model to learn over the actual binding modality rather than using only structure-based information of the ligand. We show that our model is capable of effectively denoising DEL count data to predict molecule enrichment scores that are better correlated with experimental binding affinity measurements compared to prior works. Moreover, by learning over a collection of docked poses we demonstrate that our model, trained only on DEL data, implicitly learns to perform good docking pose selection without requiring external supervision from expensive-to-source protein crystal structures.

Abstract 5346: Discovery of new targeted protein degraders using DNA-encoded chemistry

Abstract Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplars exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant that rapidly generated bispecific nanomolar degraders of ERα, with PROTACs inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization. Citation Format: Anthony D. Keefe, Jeremy S. Disch, Jennifer Duffy, Esther C. Lee, Diana Gikunju, Betty Chan, Benjamin D. Levin, Michael I. Monteiro, Sarah A. Talcott, Anthony Lau, Fei Zhou, Anton Kozhushnyan, Neil E. Westlund, Patrick B. Mullins, Yan Yu, Moritz von Rechenberg, Junyi Zhang, Yelena Arnautova, Yanbin Liu, Ying Zhang, Andrew J. McRiner, Anna Kohlmann, Matthew A. Clark, John W. Cuozzo, Christelle Huguet, Shilpi Arora. Discovery of new targeted protein degraders using DNA-encoded chemistry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5346.

Abstract P6-10-12: Discovery and Development of Next-Generation Estrogen Receptor Mutant Inhibitors using DNA-Encoded Chemical Library Screening

Abstract Background: Activating somatic ESR1 mutations Y537S and D538G occur more frequently in endocrine therapy-resistant metastatic breast cancer, which is associated with an aggressive phenotype and poor survival in breast cancer patients. These gain of function mutant receptors are constitutively active and allow resistance to first-line endocrine therapies. Therefore, the development of next-generation small molecule drugs targeting mutant estrogen receptor (ER) is an important priority. Here, we searched the small molecule inhibitors for Y537S and D538D ER mutants using DNA-encoded chemical library screening. Methods: Wild type (WT) and mutant ER ligand binding domain (LBD) proteins were expressed in E. coli. The soluble proteins were purified by Ni-NTA chromatography followed by anion exchange and size exclusion chromatography. Homogeneous time-resolved fluorescence (HTRF) and fluorescent polarization (FP) assays were performed in these purified proteins. We employed a DNA-encoded chemical library affinity selection using our in-house collection of 6 billion compounds. Hit compounds were resynthesized and validated in biochemical assays. Finally, we have performed functional studies in CRISPR-Cas9 knock-in of Y537S and D538G mutant MCF-7 breast cancer cells. Results: We have successfully purified microgram amounts of ERα LBD of WT, Y537S, and D538G proteins. To test whether the purified WT and mutant proteins are active, HTRF and FP assays were performed in the presence and absence of estradiol and 4OH tamoxifen. Steroid receptor coactivator 3 (SRC3) peptide binding to the WT ER protein occurred only in the presence of estradiol. However, Y537S and D538G proteins are recruited by the SRC3 peptide in the absence of estradiol, indicating that these mutants are constitutively active and bind to SRC3. Furthermore, an in vitro biochemical FP assay was also established for WT and mutants in the presence of estradiol and 4OH tamoxifen. The screen of our multibillion small molecule collection of DNA-encoded chemical libraries identified several hits in WT and mutant ER. To confirm the selection output, we synthesized off-DNA compounds and validated these in biochemical and cell-based studies. We have identified that the compounds, CDD-1272 and CDD-1274, are active in HTRF and FP assays. Furthermore, these compounds inhibit WT and mutant cell growth in the presence of estradiol. More importantly, CDD-1274 degrades ER mutant and cyclin D1 proteins. In addition, CDD-1274 induced p21 protein expression in WT and mutant cells. Conclusions: We have identified potent novel ER mutant binders by using our DNA-encoded chemical library platform. Our compounds are active in biochemical and ER mutant cell lines, suggesting these molecules are potential chemical probes to explore in in vivo models of breast cancer. Support: NIH/NCI R03 CA259664 and CPRIT RP220524 to MP. Citation Format: Murugesan Palaniappan, Kurt M. Bohren, Yong Wang, Damian W. Young, Suzanne A. Fuqua, Martin M. Matzuk. Discovery and Development of Next-Generation Estrogen Receptor Mutant Inhibitors using DNA-Encoded Chemical Library Screening [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-12.

One Reaction Served Three Ways: The On-DNA Ugi 4C-3C Reaction for the Formation of Lactams.

Developing new on-DNA reactions is paramount to the development of new encoded libraries in the pursuit of novel pharmaceutical lead compounds. Lactam-containing molecules have been shown to be effective in a wide range of therapeutic areas and therefore represent a promising target for further investigation by DNA-encoded library screening. In pursuit of this motif, we report a novel method for the introduction of lactam-containing structures onto a DNA headpiece through the Ugi four-center three-component reaction (4C-3CR). This novel method is successful in three different approaches to give unique on-DNA lactam structures: on-DNA aldehyde coupled with isonitriles and amino acids; on-DNA isonitrile coupled with aldehydes and amino acids; and on-DNA isonitrile coupled with amines and acid aldehydes.

Discovery of Highly Potent and BMPR2-Selective Kinase Inhibitors Using DNA-Encoded Chemical Library Screening.

The discovery of monokinase-selective inhibitors for patients is challenging because the 500+ kinases encoded by the human genome share highly conserved catalytic domains. Until now, no selective inhibitors unique for a single transforming growth factor β (TGFβ) family transmembrane receptor kinase, including bone morphogenetic protein receptor type 2 (BMPR2), have been reported. This dearth of receptor-specific kinase inhibitors hinders therapeutic options for skeletal defects and cancer as a result of an overactivated BMP signaling pathway. By screening 4.17 billion "unbiased" and "kinase-biased" DNA-encoded chemical library molecules, we identified hits CDD-1115 and CDD-1431, respectively, that were low-nanomolar selective kinase inhibitors of BMPR2. Structure-activity relationship studies addressed metabolic lability and high-molecular-weight issues, resulting in potent and BMPR2-selective inhibitor analogs CDD-1281 (IC50 = 1.2 nM) and CDD-1653 (IC50 = 2.8 nM), respectively. Our work demonstrates that DNA-encoded chemistry technology (DEC-Tec) is reliable for identifying novel first-in-class, highly potent, and selective kinase inhibitors.

Dearomative intermolecular [2 + 2] photocycloaddition for construction of C(sp3)-rich heterospirocycles on-DNA.

DNA-encoded library (DEL) screens have significantly impacted new lead compound identification efforts within drug discovery. An advantage of DELs compared to traditional screening methods is that an exponentially broader chemical space can be effectively screened using only nmol quantities of billions of DNA-tagged, drug-like molecules. The synthesis of DELs containing diverse, sp3-rich spirocycles, an important class of molecules in drug discovery, has not been previously reported. Herein, we demonstrate the synthesis of complex and novel spirocyclic cores via an on-DNA, visible light-mediated intermolecular [2 + 2] cycloaddition of olefins with heterocycles, including indoles, azaindoles, benzofurans, and coumarins. The DNA-tagged exo-methylenecyclobutane substrates were prepared from easily accessible alkyl iodides and styrene derivatives. Broad reactivity with many other DNA-conjugated alkene substrates was observed, including unactivated and activated alkenes, and the process is tolerant of various heterocycles. The cycloaddition was successfully scaled from 10 to 100 nmol without diminished yield, indicative of this reaction's suitability for DNA-encoded library production. Evaluation of DNA compatibility with the developed reaction in a mock-library format showed that the DNA barcode was maintained with high fidelity, with <1% mutated sequences and >99% amplifiable DNA from quantitative polymerase chain reaction (PCR) and next generation sequencing (NGS).

Photoredox-Mediated Deoxygenative Alkylation of DNA-Tagged Alkenes with Activated Alcohols.

DNA-encoded library (DEL) screens have become a key technology to find small molecule binders to biological targets for drug discovery applications. The development of new DNA-compatible chemistries to expand the accessible DEL chemical space is imperative to enhance screen success across broad target classes and modalities. Additionally, reactions that use commonly available building blocks as well as those that enable the fsp3 of library members to be increased would have high impact for accessing diverse drug-like structures. Herein, we report a DNA-compatible Giese-type addition of nonstabilized C-centered radicals generated by the deoxygenation of preactivated alcohols into on-DNA olefins. Although alcohols have been historically underused as a building block class within DEL synthesis, their activation to a xanthate enables Csp3-Csp3 coupling to furnish sp3-rich products. This reaction is compatible with multiple classes of functional groups, does not damage the DNA tag, and is suitable for use in DEL productions.

DNA-Encoded Library Screening To Inform Design of a Ribonuclease Targeting Chimera (RiboTAC).

Ribonuclease targeting chimeras (RiboTACs) induce degradation of an RNA target by facilitating an interaction between an RNA and a ribonuclease (RNase). We describe the screening of a DNA-encoded library (DEL) to identify binders of monomeric RNase L to provide a compound that induced dimerization of RNase L, activating its ribonuclease activity. This compound was incorporated into the design of a next-generation RiboTAC that targeted the microRNA-21 (miR-21) precursor and alleviated a miR-21-associated cellular phenotype in triple-negative breast cancer cells. The RNA-binding module in the RiboTAC is Dovitinib, a known receptor tyrosine kinase (RTK) inhibitor, which was previously identified to bind miR-21 as an off-target. Conversion of Dovitinib into this RiboTAC reprograms the known drug to selectively affect the RNA target. This work demonstrates that DEL can be used to identify compounds that bind and recruit proteins with effector functions in heterobifunctional compounds.

Advancing ASMS with LC-MS/MS for the discovery of novel PDCL2 ligands from DNA-encoded chemical library selections.

A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due to globozoospermia caused by impaired sperm head formation, indicating that PDCL2 is a potential target for male contraception. Herein, our study for the first time developed a biophysical assay for PDCL2 allowing us to screen a series of small molecules, to study structure-activity relationships, and to discover two PDCL2 binders with novel chemical structure. To identify a PDCL2 ligand for therapeutic male contraception, we performed DNA-encoded chemical library (DECL) screening and off-DNA hit validation using a unique affinity selection mass spectrometry (ASMS) biophysical profiling strategy. We employed the screening process of DECL, which contains billions of chemically unique DNA-barcoded compounds generated through individual sequences of reactions and different combinations of functionalized building blocks. The structures of the PDCL2 binders are proposed based on the sequencing analysis of the DNA barcode attached to each individual DECL compound. The proposed structure is synthesized through multistep reactions. To confirm and determine binding affinity between the DECL identified molecules and PDCL2, we developed an ASMS assay that incorporates liquid chromatography with tandem mass spectrometry (LC-MS/MS). After a screening process of PDCL2 with DECLs containing>440 billion compounds, we identified a series of hits. The selected compounds were synthesized as off-DNA small molecules, characterized by spectroscopy data, and subjected to our ASMS/LC-MS/MS binding assay. By this assay, we discovered two novel compounds, which showed good binding affinity for PDCL2 in comparison to other molecules generated in our laboratory andwhich were further confirmed by a thermal shift assay. With the ASMS/LC-MS/MS assay developed in this paper, we successfully discovered a PDCL2 ligand that warrants further development as a male contraceptive.

DNA-encoded library screening on two validated enzymes of the peptidoglycan biosynthetic pathway

Screening of DNA-encoded libraries is an emerging technology for discovering hits against protein targets. With the recent launch of the DELopen platform, a facile screening of 4.4 billion compounds is available to accelerate the drug discovery process. Here we report an affinity-based screening of the DELopen library for the first time. The screening was performed against two bacterial enzymes of the peptidoglycan biosynthetic pathway, N-acetylglucosamine-enolpyruvyl transferase (MurA) and d-alanine:d-alanine ligase (DdlB). Several binders were obtained and selected for off-DNA synthesis. Hits with confirmed inhibitory potency were deconstructed into smaller fragments. In this way, two new MurA inhibitors with antibacterial activity were obtained and are available for further optimization.

Discovery of TIGIT inhibitors based on DEL and machine learning.

Drug discovery has entered a new period of vigorous development with advanced technologies such as DNA-encoded library (DEL) and artificial intelligence (AI). The previous DEL-AI combination has been successfully applied in the drug discovery of classical kinase and receptor targets mainly based on the known scaffold. So far, there is no report of the DEL-AI combination on inhibitors targeting protein-protein interaction, including those undruggable targets with few or unknown active scaffolds. Here, we applied DEL technology on the T cell immunoglobulin and ITIM domain (TIGIT) target, resulting in the unique hit compound 1 (IC50 = 20.7 μM). Based on the screening data from DEL and hit derivatives a1-a34, a machine learning (ML) modeling process was established to address the challenge of poor sample distribution uniformity, which is also frequently encountered in DEL screening on new targets. In the end, the established ML model achieved a satisfactory hit rate of about 75% for derivatives in a high-scored area.

Palladium-Mediated Carbonylative Suzuki Coupling for DNA-Encoded Library Synthesis.

Developing new DNA-compatible reactions is key to expanding the accessible chemical space of DNA-encoded library (DEL) technology. Here we disclose the first report of a DNA-compatible carbonylative Suzuki coupling of DNA-conjugated (hetero)aryl iodides with (hetero)aryl boronic acids to access di(hetero)aryl ketones, a valuable structural motif present within several approved or clinically advanced small molecules. The reported DNA-compatible, Pd(OAc)2-mediated system is mild, uses a robust protocol, has a wide substrate scope for both coupling partners, is suitable for large-scale DEL productions, and provides a source of previously unexplored chemical matter for DEL screens.

RNA-templated chemical synthesis of proapoptotic L- and d-peptides

Nucleic acid-programmed reactions find application in drug screening and nucleic acid diagnosis, and offer prospects for a RNA-sensitive prodrug approach. We aim for the development of a nucleic acid-templated reaction providing nucleic acid-linked molecules that can act on intracellular protein targets. Such reactions would be useful for in situ drug synthesis and activity-based DNA-encoded library screening. In this report, we show native chemical ligation-like chemical peptidyl transfer reactions between peptide-PNA conjugates. The reaction proceeds on RNA templates. As a chemical alternative to ribosomal peptide synthesis access to both L- and d-peptides is provided. In reactions affording 9 to 14 amino acid long pro-apoptotic L- and d-peptides, we found that certain PNA sequence motifs and combinations of cell penetrating peptides (CPPs) cause surprisingly high reactivity in absence of a template. Viability measurements demonstrate that the products of templated peptidyl transfer act on HeLa cells and HEK293 cells. Of note, the presence of cysteine, which is required for NCL chemistry, can enhance the bioactivity. The study provides guidelines for the application of peptide-PNA conjugates in templated synthesis and is of interest for in situ drug synthesis and activity-based DNA-encoded library screening.

Machine Learning on DNA-Encoded Library Count Data Using an Uncertainty-Aware Probabilistic Loss Function.

DNA-encoded library (DEL) screening and quantitative structure-activity relationship (QSAR) modeling are two techniques used in drug discovery to find novel small molecules that bind a protein target. Applying QSAR modeling to DEL selection data can facilitate the selection of compounds for off-DNA synthesis and evaluation. Such a combined approach has been done recently by training binary classifiers to learn DEL enrichments of aggregated "disynthons" in order to accommodate the sparse and noisy nature of DEL data. However, a binary classification model cannot distinguish between different levels of enrichment, and information is potentially lost during disynthon aggregation. Here, we demonstrate a regression approach to learning DEL enrichments of individual molecules, using a custom negative-log-likelihood loss function that effectively denoises DEL data and introduces opportunities for visualization of learned structure-activity relationships. Our approach explicitly models the Poisson statistics of the sequencing process used in the DEL experimental workflow under a frequentist view. We illustrate this approach on a DEL dataset of 108,528 compounds screened against carbonic anhydrase (CAIX), and a dataset of 5,655,000 compounds screened against soluble epoxide hydrolase (sEH) and SIRT2. Due to the treatment of uncertainty in the data through the negative-log-likelihood loss used during training, the models can ignore low-confidence outliers. While our approach does not demonstrate a benefit for extrapolation to novel structures, we expect our denoising and visualization pipeline to be useful in identifying structure-activity trends and highly enriched pharmacophores in DEL data. Further, this approach to uncertainty-aware regression modeling is applicable to other sparse or noisy datasets where the nature of stochasticity is known or can be modeled; in particular, the Poisson enrichment ratio metric we use can apply to other settings that compare sequencing count data between two experimental conditions.

Palladium-Mediated C-N Coupling of DNA-Conjugated (Hetero)aryl Halides with Aliphatic and (Hetero)aromatic Amines.

DNA-encoded chemical library (DEL) screens are a powerful hit generation tool in drug discovery, but the diversity of DEL chemical matter is dependent on developing robust reaction conditions that may be used on hundreds to millions of substrate combinations and that are compatible with the platform. Here, we disclose the first report of a general, aqueous, DNA-compatible C-N coupling condition that can now couple aliphatic amines, in addition to (hetero)aromatic amines, with a variety of (hetero)aryl iodides, bromides, and chlorides. The reported BippyPhos-Pd(OAc)2 catalyst system has a wide substrate scope for both coupling partners, is operationally feasible for large scale DEL productions, uses common DEL building block solution stocks, and enables an expansion of DEL-accessible, drug-like chemical space.

N-Alkyl Linkers for DNA-Encoded Chemical Libraries.

A series of novel N-alkyl linkers that connect small-molecule library members with their encoding DNA oligonucleotides has been developed. In comparison with the standard amide linker (usually constructed with oligo-AOP-NH2 ), the N-alkyl linker is not only more chemically stable, but also provides better structural diversity at the linkage point. Chemical variety in the vicinity of the polyglycol terminus, in particular, could affect binding interactions with the target protein. It could have been neglected in previous DNA-encoded chemical library (DEL) synthesis and screening studies due to the limited linkage alternatives. With these linkers, one can produce versatile key intermediates as Cycle 1 products directly amenable to Cycle 2 chemistry without the use of protecting groups. As a result, a DEL synthesis process that uses the fewest chemical conversions, such as 3-step, 3-cycle DELs, can achieve higher synthetic efficiency while creating less DNA tag degradation, resulting in higher quality DELs.

Discovery of SARS-CoV-2 main protease covalent inhibitors from a DNA-encoded library selection

Covalent inhibitors targeting the main protease (Mpro, or 3CLpro) of SARS-CoV-2 have shown promise in preclinical investigations. Herein, we report the discovery of two new series of molecules that irreversibly bind to SARS-CoV-2 Mpro. These acrylamide containing molecules were discovered using our covalent DNA-encoded library (DEL) screening platform. Following selection against SARS-CoV-2 Mpro, off-DNA compounds were synthesized and investigated to determine their inhibitory effects, the nature of their binding, and to generate preliminary structure-activity relationships. LC-MS analysis indicates a 1:1 (covalent) binding stoichiometry between our hit molecules and SARS-CoV-2 Mpro. Fluorescent staining assay for covalent binding in the presence of cell lysate suggests reasonable selectivity for SARS-CoV-2 Mpro. And lastly, inhibition of enzymatic activity was also observed against a panel of 3CLpro enzymes from different coronavirus strains, with IC50 values ranging from inactive to single digit micromolar. Our results indicate that DEL selection is a useful approach for identifying covalent inhibitors of cysteine proteases.

A Multifaceted Hit-Finding Approach Reveals Novel LC3 Family Ligands.

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.