RESUMEN
HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.
Asunto(s)
Linfocitos B/fisiología , Reparación del ADN por Unión de Extremidades/genética , Proteínas de Unión al ADN/genética , ADN/genética , Cambio de Clase de Inmunoglobulina/genética , Animales , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Ratones , Ratones Endogámicos C57BL , Translocación Genética/genéticaRESUMEN
5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers". We show that 5'-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5'-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5'-Pme.
Asunto(s)
Proteínas de Neoplasias , Biosíntesis de Proteínas , Proteínas de Neoplasias/genética , Canadá , Metilación , ARN Mensajero/genética , ARN/metabolismoRESUMEN
The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-µM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.
Asunto(s)
Proteínas de Unión al ADN , Péptidos , Humanos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Polarización de Fluorescencia/métodos , Péptidos/química , Péptidos/farmacología , Unión Proteica , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-CoV-2. Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) protein, enabling virus-host membrane fusion and infection of the airways. We present here a recombinant production strategy for enzymatically active TMPRSS2 and characterization of its matured proteolytic activity, as well as its 1.95 Å X-ray cocrystal structure with the synthetic protease inhibitor nafamostat. Our study provides a structural basis for the potent but nonspecific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that explain specificity. TMPRSS2 cleaved SARS-CoV-2 S protein at multiple sites, including the canonical S1/S2 cleavage site. We ranked the potency of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.4 nM to 120 µM and determined inhibitor mechanisms of action, providing the groundwork for drug development efforts to selectively inhibit TMPRSS2.
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COVID-19 , SARS-CoV-2 , Serina Endopeptidasas/metabolismo , Humanos , Péptido Hidrolasas , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del VirusRESUMEN
We herewith applied a priori a generic hit identification method (POEM) for difficult targets of known three-dimensional structure, relying on the simple knowledge of physicochemical and topological properties of a user-selected cavity. Searching for local similarity to a set of fragment-bound protein microenvironments of known structure, a point cloud registration algorithm is first applied to align known subpockets to the target cavity. The resulting alignment then permits us to directly pose the corresponding seed fragments in a target cavity space not typically amenable to classical docking approaches. Last, linking potentially connectable atoms by a deep generative linker enables full ligand enumeration. When applied to the WD40 repeat (WDR) central cavity of leucine-rich repeat kinase 2 (LRRK2), an unprecedented binding site, POEM was able to quickly propose 94 potential hits, five of which were subsequently confirmed to bind in vitro to LRRK2-WDR.
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Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Simulación del Acoplamiento Molecular , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Sitios de Unión , Dominios Proteicos , Humanos , Ligandos , Unión Proteica , Repeticiones WD40 , AlgoritmosRESUMEN
DCAF1 functions as a substrate recruitment subunit for the RING-type CRL4DCAF1 and the HECT family EDVPDCAF1 E3 ubiquitin ligases. The WDR domain of DCAF1 serves as a binding platform for substrate proteins and is also targeted by HIV and SIV lentiviral adaptors to induce the ubiquitination and proteasomal degradation of antiviral host factors. It is therefore attractive both as a potential therapeutic target for the development of chemical inhibitors and as an E3 ligase that could be recruited by novel PROTACs for targeted protein degradation. In this study, we used a proteome-scale drug-target interaction prediction model, MatchMaker, combined with cheminformatics filtering and docking to identify ligands for the DCAF1 WDR domain. Biophysical screening and X-ray crystallographic studies of the predicted binders confirmed a selective ligand occupying the central cavity of the WDR domain. This study shows that artificial intelligence-enabled virtual screening methods can successfully be applied in the absence of previously known ligands.
Asunto(s)
Inteligencia Artificial , Proteínas Portadoras , Ligandos , Proteínas Portadoras/química , Ubiquitina-Proteína Ligasas/metabolismo , Aprendizaje AutomáticoRESUMEN
The human pseudouridine synthase PUS7 is a versatile RNA modification enzyme targeting many RNAs thereby playing a critical role in development and brain function. Whereas all target RNAs of PUS7 share a consensus sequence, additional recognition elements are likely required, and the structural basis for RNA binding by PUS7 is unknown. Here, we characterize the structure-function relationship of human PUS7 reporting its X-ray crystal structure at 2.26 Å resolution. Compared to its bacterial homolog, human PUS7 possesses two additional subdomains, and structural modeling studies suggest that these subdomains contribute to tRNA recognition through increased interactions along the tRNA substrate. Consistent with our modeling, we find that all structural elements of tRNA are required for productive interaction with PUS7 as the consensus sequence of target RNA alone is not sufficient for pseudouridylation by human PUS7. Moreover, PUS7 binds several, non-modifiable RNAs with medium affinity which likely enables PUS7 to screen for productive RNA substrates. Following tRNA modification, the product tRNA has a significantly lower affinity for PUS7 facilitating its dissociation. Taken together our studies suggest a combination of structure-specific and sequence-specific RNA recognition by PUS7 and provide mechanistic insight into its function.
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Transferasas Intramoleculares/química , ARN de Transferencia/metabolismo , Sitios de Unión , Humanos , Transferasas Intramoleculares/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , ARN de Transferencia/químicaRESUMEN
Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.
Asunto(s)
Código de Histonas , Isobutiratos/metabolismo , Lisina Acetiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Acilcoenzima A/síntesis química , Acilcoenzima A/metabolismo , Acilación , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Células HEK293 , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Isobutiratos/farmacología , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en Tándem , Valina/metabolismo , Factores de Transcripción p300-CBPRESUMEN
Aberrant isoform expression of chromatin-associated proteins can induce epigenetic programs related to disease. The MDS1 and EVI1 complex locus (MECOM) encodes PRDM3, a protein with an N-terminal PR-SET domain, as well as a shorter isoform, EVI1, lacking the N-terminus containing the PR-SET domain (ΔPR). Imbalanced expression of MECOM isoforms is observed in multiple malignancies, implicating EVI1 as an oncogene, while PRDM3 has been suggested to function as a tumor suppressor through an unknown mechanism. To elucidate functional characteristics of these N-terminal residues, we compared the protein interactomes of the full-length and ΔPR isoforms of PRDM3 and its closely related paralog, PRDM16. Unlike the ΔPR isoforms, both full-length isoforms exhibited a significantly enriched association with components of the NuRD chromatin remodeling complex, especially RBBP4. Typically, RBBP4 facilitates chromatin association of the NuRD complex by binding to histone H3 tails. We show that RBBP4 binds to the N-terminal amino acid residues of PRDM3 and PRDM16, with a dissociation constant of 3.0 µM, as measured by isothermal titration calorimetry. Furthermore, high-resolution X-ray crystal structures of PRDM3 and PRDM16 N-terminal peptides in complex with RBBP4 revealed binding to RBBP4 within the conserved histone H3-binding groove. These data support a mechanism of isoform-specific interaction of PRDM3 and PRDM16 with the NuRD chromatin remodeling complex.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteína del Locus del Complejo MDS1 y EV11/química , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Línea Celular , Cristalografía por Rayos X , Humanos , Proteína del Locus del Complejo MDS1 y EV11/genética , Ratones , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína 4 de Unión a Retinoblastoma/química , Proteína 4 de Unión a Retinoblastoma/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
To form extracellular aggregates, amyloidogenic proteins bypass the intracellular quality control, which normally targets unfolded/aggregated polypeptides. Human D76N ß2-microglobulin (ß2m) variant is the prototype of unstable and amyloidogenic protein that forms abundant extracellular fibrillar deposits. Here we focus on the role of the class I major histocompatibility complex (MHCI) in the intracellular stabilization of D76N ß2m. Using biophysical and structural approaches, we show that the MHCI containing D76N ß2m (MHCI76) displays stability, dissociation patterns, and crystal structure comparable with those of the MHCI with wild type ß2m. Conversely, limited proteolysis experiments show a reduced protease susceptibility for D76N ß2m within the MHCI76 as compared with the free variant, suggesting that the MHCI has a chaperone-like activity in preventing D76N ß2m degradation within the cell. Accordingly, D76N ß2m is normally assembled in the MHCI and circulates as free plasma species in a transgenic mouse model.
Asunto(s)
Amiloide/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Mutación Missense , Microglobulina beta-2/metabolismo , Sustitución de Aminoácidos , Amiloide/genética , Animales , Cristalografía por Rayos X , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Ratones , Ratones Transgénicos , Microglobulina beta-2/genéticaRESUMEN
The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15N-NMR-based binding affinity assays and 19F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Humanos , Ligandos , Unión Proteica , Sitios de Unión , Dominios Proteicos , Modelos Moleculares , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Cristalografía por Rayos X , Descubrimiento de Drogas/métodosRESUMEN
Controlling malaria requires new drugs against Plasmodium falciparum. The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the parasite's life cycle. We defined the structure-activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.
Asunto(s)
Antimaláricos , Malaria Falciparum , Animales , Humanos , Antimaláricos/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum , Animales Modificados Genéticamente , Relación Estructura-ActividadRESUMEN
Damaged DNA-binding protein-1 (DDB1)- and CUL4-associated factor 12 (DCAF12) serves as the substrate recognition component within the Cullin4-RING E3 ligase (CRL4) complex, capable of identifying C-terminal double-glutamic acid degrons to promote the degradation of specific substrates through the ubiquitin proteasome system. Melanoma-associated antigen 3 (MAGEA3) and T-complex protein 1 subunit epsilon (CCT5) proteins have been identified as cellular targets of DCAF12. To further characterize the interactions between DCAF12 and both MAGEA3 and CCT5, we developed a suite of biophysical and proximity-based cellular NanoBRET assays showing that the C-terminal degron peptides of both MAGEA3 and CCT5 form nanomolar affinity interactions with DCAF12 in vitro and in cells. Furthermore, we report here the 3.17â Å cryo-EM structure of DDB1-DCAF12-MAGEA3 complex revealing the key DCAF12 residues responsible for C-terminal degron recognition and binding. Our study provides new insights and tools to enable the discovery of small molecule handles targeting the WD40-repeat domain of DCAF12 for future proteolysis targeting chimera design and development.
RESUMEN
Protein arginine methyltransferase 9 (PRMT9) is a recently identified member of the PRMT family, yet its biological function remains largely unknown. Here, by characterizing an intellectual disability associated PRMT9 mutation (G189R) and establishing a Prmt9 conditional knockout (cKO) mouse model, we uncover an important function of PRMT9 in neuronal development. The G189R mutation abolishes PRMT9 methyltransferase activity and reduces its protein stability. Knockout of Prmt9 in hippocampal neurons causes alternative splicing of ~1900 genes, which likely accounts for the aberrant synapse development and impaired learning and memory in the Prmt9 cKO mice. Mechanistically, we discover a methylation-sensitive protein-RNA interaction between the arginine 508 (R508) of the splicing factor 3B subunit 2 (SF3B2), the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element that is critical for RNA splicing. Additionally, using human and mouse cell lines, as well as an SF3B2 arginine methylation-deficient mouse model, we provide strong evidence that SF3B2 is the primary methylation substrate of PRMT9, thus highlighting the conserved function of the PRMT9/SF3B2 axis in regulating pre-mRNA splicing.
Asunto(s)
Empalme Alternativo , ARN , Animales , Humanos , Ratones , Arginina/metabolismo , Ratones Noqueados , Mutación , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN/genéticaRESUMEN
Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment. A Cbl-b inhibitor, Nx-1607, is currently in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirm potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing the compound's interaction with both the TKBD and LHR, but not the RING domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.
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Neoplasias , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/química , Unión Proteica , Conformación Molecular , FosforilaciónRESUMEN
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.
Asunto(s)
Reparación del ADN , Péptidos y Proteínas de Señalización Intracelular , Humanos , ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/química , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Dominio TudorRESUMEN
WD40 repeat-containing protein 91 (WDR91) regulates early-to-late endosome conversion and plays vital roles in endosome fusion, recycling, and transport. WDR91 was recently identified as a potential host factor for viral infection. We employed DNA-encoded chemical library (DEL) selection against the WDR domain of WDR91, followed by machine learning to predict ligands from the synthetically accessible Enamine REAL database. Screening of predicted compounds identified a WDR91 selective compound 1, with a KD of 6 ± 2 µM by surface plasmon resonance. The co-crystal structure confirmed the binding of 1 to the WDR91 side pocket, in proximity to cysteine 487, which led to the discovery of covalent analogues 18 and 19. The covalent adduct formation for 18 and 19 was confirmed by intact mass liquid chromatography-mass spectrometry. The discovery of 1, 18, and 19, accompanying structure-activity relationship, and the co-crystal structures provide valuable insights for designing potent and selective chemical tools against WDR91 to evaluate its therapeutic potential.
Asunto(s)
ADN , Bibliotecas de Moléculas Pequeñas , ADN/química , Biblioteca de Genes , Ligandos , Aprendizaje Automático , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
DCAF1 is a substrate receptor of two distinct E3 ligases (CRL4DCAF1 and EDVP), plays a critical physiological role in protein degradation, and is considered a drug target for various cancers. Antagonists of DCAF1 could be used toward the development of therapeutics for cancers and viral treatments. We used the WDR domain of DCAF1 to screen a 114-billion-compound DNA encoded library (DEL) and identified candidate compounds using similarity search and machine learning. This led to the discovery of a compound (Z1391232269) with an SPR KD of 11 µM. Structure-guided hit optimization led to the discovery of OICR-8268 (26e) with an SPR KD of 38 nM and cellular target engagement with EC50 of 10 µM as measured by cellular thermal shift assay (CETSA). OICR-8268 is an excellent tool compound to enable the development of next-generation DCAF1 ligands toward cancer therapeutics, further investigation of DCAF1 functions in cells, and the development of DCAF1-based PROTACs.