Ligand Library Research Articles

TOLEDO: enhancing Maestro GUI for non-expert users to perform massive MD simulations

Classical Molecular Dynamics (MD) simulates the dynamical evolution of biological systems at the atomic level. Using MD in conjunction with high-performance computing (HPC) architectures, we can evaluate the possible interactions between a ligand library against one protein target to find a drug that can influence a protein target to cure a disease. Simultaneously, we can also obtain information about their dynamic evolution. One of the primary software packages for MD simulations is Desmond, which employs Maestro for the setup, execution, and analysis of MD through a graphical user interface (GUI), which is suitable even for non-expert users. However, using the GUI, users can typically run only one short (less than 1000 ns) MD each time. Our work aims to create a method/protocol to run several MD simulations simultaneously on a remote HPC cluster within Maestro-Desmond. In this work, we provide TOLEDO (Throughput Optimization of Ligand-Protein Systems Exploration through Dynamics simulation in Optimized HPC systems) to overcome such limitations and run several MD simulations simultaneously. The best feature of TOLEDO is its independence from the usual time constraints of many clusters, with storage space being the only limitation. To run TOLEDO, we prepare/set up the protein-ligand complex before running MD via Maestro GUI. Next, we run the main TOLEDO script for several MD simulations on a supercomputer. When TOLEDO finishes, users obtain reports and graphics. The obtained results are easily interpretable. In essence, TOLEDO significantly enhances MD throughput beyond the capabilities of the Maestro GUI.

Synthesis of a Small Library of Glycoderivative Putative Ligands of SGLT1 and Preliminary Biological Evaluation.

Sodium-glucose co-transporter 1 (SGLT1) is primarily expressed on the membrane of enterocytes, a type of epithelial cell found in the intestines, where it mediates the unidirectional absorption of glucose and galactose. Beyond its well-established role in nutrient absorption, SGLT1 also plays a protective role in maintaining the integrity of the intestinal barrier. Specifically, the natural ligand of SGLT1 (d-glucose) and a synthetic C-glucoside developed by our group can induce a protective anti-inflammatory effect on the intestinal epithelium. In this paper, we report the creation of a small library of C-glycoside, putative ligands for SGLT1, to gain further insights into its unclear mechanism of action. Preliminary biological experiments performed on an in vitro model of doxorubicin-induced mucositis, a severe intestinal inflammatory condition, indicate that the aromatic moiety present in all the compounds of the library is crucial for biological activity, while the sugar component appears to have less influence. These findings will be exploited to develop new, more potent anti-inflammatory compounds and to better understand and rationalize the protective mechanism of action.

Impact of MTHFD2 Expression in Bladder/Breast Cancer and Screening of Its Potential Inhibitor.

Genes of folate-mediated 1 carbon metabolism are found to be highly upregulated in tumor cells and promote cancer cell proliferation. The current study aimed to determine the expression of the MTHFD2 gene in bladder and breast cancers. Furthermore, the determination of potential ligand-based inhibitors against MTHFD2 was performed in comparison with those of chemotherapeutic drugs and natural plant-based compounds. Semiquantitative expression analysis along with structure-based virtual ligand library screening was done to find plausible inhibitors. MTHFD2 expression was significantly increased with tumor stage progression both in low- and high-grade bladder cancer and especially in triple-negative breast cancer. Virtual ligand-based library screening against the three-dimensional MTHFD2 protein structure led to the identification of plausible inhibitors like MCULE-8109969891-0 and MCULE-9715677418-0-1 that displayed lower binding free energy as compared to that of already documented LY345899. Similar scaffold commercial drugs leucal (LEU), epirubicin (EPI), and lometrexol also displayed strong binding to the active site of MTHFD2. EPI and LEU in combinatorial therapy were also tested in vitro on MDA-MB-231 cells. The high doses of LEU in combination with EPI showed a significant reduction in cell viability at 2 and 3 μM concentrations. The interaction of breast cancer serum with high expression of MTHFD2 also showed an increase in binding of epirubicin in the presence of leucovorin. The decrease in the absorbance spectra of epirubicin at 37 and 53 °C displayed the stability induced by LEU on the interaction of EPI with the MTHFD2 binding pocket. Leucovorin tends to stabilize the interaction as the binding affinity is high even at 53 °C. Thus, MTHFD2 might be used as a cancer biomarker since its expression level changes drastically with tumor progression. Further experimental studies are required to establish the potential mode of inhibition of the novel small ligands. Future in vivo trials may validate the effectiveness of the combinatorial therapy.

Influence of Pnictogen and Ligand Framework on the Lewis Acidity and Steric Environment in Pnictogen Pincer Complexes.

Pnictogen pincer complexes are a fascinating class of compounds due to their dynamic molecular and electronic structures, and valuable stoichiometric or catalytic reactivity. As recognition of their unique chemistry has grown, so too has the library of pincer ligands employed and pnictogen centres engaged to prepare them. Here we computationally study how the choice of pincer ligand framework and pnictogen influence the electronic and steric outcomes of the complexes obtained. The most relevant electronic parameter is the pnictogen-centred electrophilicity, which has been quantified by fluoride ion affinities and LUMO energies, while the most relevant steric parameter is the crowding around the central pnictogen, which has been quantified by the %Vbur values and visualized using steric maps. The resulting trends are analyzed with reference to binding pocket size, acceptor orbital type, electronic delocalization, π-donor strengths, and heteroatom incorporation. Thus, considering 16 ligand frameworks and 4 heavy pnictogen centres, this study provides a broad-spectrum view of stereo-electronic variation in pnictogen pincer complexes, which, together with a recent study on geometric variation in the same family, provides a substantial dataset to guide future molecular design and reactivity studies.

Scalable, compressed phenotypic screening using pooled perturbations.

High-throughput phenotypic screens using biochemical perturbations and high-content readouts are constrained by limitations of scale. To address this, we establish a method of pooling exogenous perturbations followed by computational deconvolution to reduce required sample size, labor and cost. We demonstrate the increased efficiency of compressed experimental designs compared to conventional approaches through benchmarking with a bioactive small-molecule library and a high-content imaging readout. We then apply compressed screening in two biological discovery campaigns. In the first, we use early-passage pancreatic cancer organoids to map transcriptional responses to a library of recombinant tumor microenvironment protein ligands, uncovering reproducible phenotypic shifts induced by specific ligands distinct from canonical reference signatures and correlated with clinical outcome. In the second, we identify the pleotropic modulatory effects of a chemical compound library with known mechanisms of action on primary human peripheral blood mononuclear cell immune responses. In sum, our approach empowers phenotypic screens with information-rich readouts to advance drug discovery efforts and basic biological inquiry.

New insight into the molecular mechanism of TCM Bufei Huoxue formula for chronic obstructive pulmonary disease based on network pharmacology and experimental verification.

To unveil the mechanism of the Bufei Huoxue formula (BHF) for chronic obstructive pulmonary disease (COPD) through integrated network pharmacology (NP) and experimental verification. LC-MS was first applied to the analysis of both in vitro and in vivo samples from BHF for chemical profiling. Then a ligand library was prepared for NP to reveal the mechanism of BHF against COPD. Finally, verification was performed using an animal model related to the results from the NP. A ligand library containing 170 compounds from BHF was obtained, while 357 targets related to COPD were filtered to construct a PPI network. GO and KEGG analysis demonstrated that bavachin, paeoniflorin, and demethylation of formononetin were the major compounds for BHF against COPD, which mainly by regulating the PI3K/Akt pathway. The experiments proved that BHF could alleviate lung injury and attenuate the release of TNF-α and IL-6 in the lung and BALF in a dose-dependent manner. Western blot further demonstrated the down-regulated effect of BHF on p-PI3K. BHF provides a potent alternative for the treatment of COPD, and the mechanism is probably associated with regulating the PI3K/AKT pathway to alleviate inflammatory injury by bavachin, paeoniflorin, and demethylation of formononetin.

Computational quest, synthesis and anticancer profiling of 3-methyl quinoxaline-2-one-based active hits against the tyrosine kinase

BackgroundCancer is the predominant cause of mortality and a remarkable obstacle to elevating life anticipation in every nation on globe. Hepatocellular carcinoma (HCC), a hyper-vascular tumour, develops and progresses due to angiogenesis, a key feature of malignancy. HCC exhibits high neoangiogenic activity because of the need to generate new blood vessels for tumour growth.MethodsThe present work includes the construction of virtual library of ligands, virtual screening using the Dockthor-VS server, ADMET study using the SwissADME and Osiris property explorer. All the synthesized compounds were characterized by UV, IR, NMR and mass spectroscopic techniques. MTT assay was done to find the IC50 of the synthesized compounds against HepG2 cell line. The more active compound found is subjected to the molecular dynamics simulation study.ResultsThe ligands exhibited good docking scores, ADMET profile compared to the reference drugs. The target compounds were obtained with the satisfactory yields of 66–82%. The best activity against the HepG2 cancer cell line is observed with the compound SA-4 with IUPAC name (2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)-N-(5-(3-nitrophenyl)-5H-thiazolo[4,3-b] [1,3,4] thiadiazol-2-yl) acetamide). The experimental results obtained show correlation with the in silico results. MD simulation of the compound SA-4 indicates the moderate stability of the protein-ligand complex in real time environment.ConclusionThe results obtained suggest that the compound SA-4 has the potential to be a promising anticancer agent effective against the VEGFR-2 and FGFR-4.Graphical abstract

Fluorine-Thiol Displacement Stapling on the Disordered αB of pKID Domain Increases Its Helicity and Affinity to KIX

AbstractThe development of high-affinity ligands specifically targeting intrinsically disordered protein interactions has remained challenging due to the lack of well-defined binding pockets and shallow binding surfaces commonly found at their interfaces. Here, we employed our fluorine-thiol displacement reaction (FTDR) peptide-stapling platform to synthesize a library of peptide-based ligands derived from the αB-helix of the disordered pKID to target its binding partner KIX. Our library revealed that helical formation and affinity to KIX is highly favored when the αB peptide was stapled at sites corresponding to Arg135 and Ser142, further supporting the hypothesis that stabilization of αB significantly influences the overall binding affinity of pKID to KIX. We also found that the highest binding peptide, αB-RSpS, may form secondary contacts at the MLL site on KIX in addition to binding at the primary pKID site. Lastly, no binding to KIX was observed for any αB-stapled peptide that lacked the conserved helix-flanking prolines Pro132 and Pro146. Conserved helix-flanking prolines have previously been shown to modulate the binding affinities of other disordered domains in other proteins including MLL and p53. However, to our knowledge this is the first evidence within αB of pKID.

Mathematical consideration of massive estimation of dissociation rate constant for genotype–phenotype linking molecules bound to targets through washing/selection and next-generation sequencing

As one of methods for in vitro selection, a flow reactor type washing/selection system seems to be effective, where a ligand library is composed of “genotype–phenotype linking molecules”. In this system, high affinity ligands are selected by their respective “residual ratio” given by exp(−koff×t), where koff is the dissociation rate constant and t is the washing time. In this paper, we mathematically considered the following possibility. When the washing/selection dynamics obeys the residual ratio exp(−koff×t) deterministically and mole fraction measurement for sampled sequences by next-generation sequencing (NGS) is performed ideally, the “relative value” of koff for each of high-ranking sequences can be estimated simultaneously. In addition to these, when the residual ratio for the whole ligand population is measured correctly, the “absolute value” for each sequence can be estimated. We deduced formulas to present the relative and absolute estimates, and mathematically analyzed the effect of fluctuations in the number of NGS reads on the estimates in details. These were confirmed by numerical simulations.

Targeted Protein Localization by Covalent 14-3-3 Recruitment.

14-3-3 proteins have a unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that interactions of 14-3-3 with specific phosphorylated substrate proteins can be enhanced through small-molecule natural products or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates and potential neo-substrates to sequester and inhibit their function remains elusive. One of the 14-3-3 proteins, 14-3-3σ or SFN, has cysteine C38 at the substrate-binding interface, near the sites where previous 14-3-3 molecular glues have been found to bind. In this study, we screen a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance the interaction of 14-3-3σ with not only druggable transcription factors such as estrogen receptor (ERα) but also challenging oncogenic transcription factors such as YAP and TAZ, which are part of the Hippo transducer pathway. We identify a hit EN171 that covalently targets both C38 and C96 on 14-3-3 to enhance 14-3-3 interactions with ERα, YAP, and TAZ, leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions but could also be used as a covalent 14-3-3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 and BLC6 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14-3-3 molecular glue for challenging transcription factors such as YAP and TAZ and demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

MDFit: automated molecular simulations workflow enables high throughput assessment of ligands-protein dynamics.

Molecular dynamics (MD) simulation is a powerful tool for characterizing ligand-protein conformational dynamics and offers significant advantages over docking and other rigid structure-based computational methods. However, setting up, running, and analyzing MD simulations continues to be a multi-step process making it cumbersome to assess a library of ligands in a protein binding pocket using MD. We present an automated workflow that streamlines setting up, running, and analyzing Desmond MD simulations for protein-ligand complexes using machine learning (ML) models. The workflow takes a library of pre-docked ligands and a prepared protein structure as input, sets up and runs MD with each protein-ligand complex, and generates simulation fingerprints for each ligand. Simulation fingerprints (SimFP) capture protein-ligand compatibility, including stability of different ligand-pocket interactions and other useful metrics that enable easy rank-ordering of the ligand library for pocket optimization. SimFPs from a ligand library are used to build & deploy ML models that predict binding assay outcomes and automatically infer important interactions. Unlike relative free-energy methods that are constrained to assess ligands with high chemical similarity, ML models based on SimFPs can accommodate diverse ligand sets. We present two case studies on how SimFP helps delineate structure-activity relationship (SAR) trends and explain potency differences across matched-molecular pairs of (1) cyclic peptides targeting PD-L1 and (2) small molecule inhibitors targeting CDK9.

Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation.

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.

Chemical space exploration with Molpher: Generating and assessing a glucocorticoid receptor ligand library.

Computational exploration of chemical space is crucial in modern cheminformatics research for accelerating the discovery of new biologically active compounds. In this study, we present a detailed analysis of the chemical library of potential glucocorticoid receptor (GR) ligands generated by the molecular generator, Molpher. To generate the targeted GR library and construct the classification models, structures from the ChEMBL database as well as from the internal IMG library, which was experimentally screened for biological activity in the primary luciferase reporter cell assay, were utilized. The composition of the targeted GR ligand library was compared with a reference library that randomly samples chemical space. A random forest model was used to determine the biological activity of ligands, incorporating its applicability domain using conformal prediction. It was demonstrated that the GR library is significantly enriched with GR ligands compared to the random library. Furthermore, a prospective analysis demonstrated that Molpher successfully designed compounds, which were subsequently experimentally confirmed to be active on the GR. A collection of 34 potential new GR ligands was also identified. Moreover, an important contribution of this study is the establishment of a comprehensive workflow for evaluating computationally generated ligands, particularly those with potential activity against targets that are challenging to dock.

Structure-Activity Study on Substituted, Core-Extended, and Dyad Naphthalene Diimide G-Quadruplex Ligands Leading to Potent Antitrypanosomal Agents.

Several G-quadruplex nucleic acid (G4s) ligands have been developed seeking target selectivity in the past decade. Naphthalene diimide (NDI)-based compounds are particularly promising due to their biological activity and red-fluorescence emission. Previously, we demonstrated the existence of G4s in the promoter region of parasite genomes, assessing the effectiveness of NDI-derivatives against them. Here, we explored the biological activity of a small library of G4-DNA ligands, exploiting the NDI pharmacophore, against both Trypanosoma brucei and Leishmania major parasites. Biophysical and biological assays were conducted. Among the various families analyzed, core-extended NDIs exhibited the most promising results concerning the selectivity and antiparasitic effects. NDI 16 emerged as the most potent, with an IC50 of 0.011 nM against T. brucei and remarkable selectivity vs MRC-5 cells (3454-fold). Fascinating, 16 is 480-fold more potent than the standard drug pentamidine (IC50 = 5.3 nM). Cellular uptake and parasite localization were verified by exploiting core-extended NDI red-fluorescent emission.

A novel and selective fluorescent ligand for the study of adenosine A2B receptors.

Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.

Investigating natural plant products as potential inhibitors to disrupt NS1 β-roll domain polymerisation in DENV2: a detailed computational chemistry approach

ABSTRACT The aim of this investigation is to design a novel drug, screened through various computational methods, that harnesses the historical evidence of plant products inhibiting viral infections. The beta-roll domain of Flavivirus NS1, crucial for its stability, membrane interaction and viral RNA replication, underscores its significance in disease pathogenesis, being the sole non-structural protein detected in the sera of DENV-infected patients. Our goal is to develop optimised compounds that disrupt NS1 β-roll domain polymerisation, mitigating hydrophobic core formation, to effectively inhibit DENV2 cytopathic infection by leveraging natural interference with viral replication. We created 10 plant-based ligand libraries and applied scaffold hopping and bioisosteric replacement to improve them. Computational methodologies, including virtual screening, molecular dynamics simulation, contact matrix and dynamic cross correlation matrix, were employed for analysing the dynamics of the protein with the association of ligand. ADMET analysis of ligand 126 revealed a favourable ADME profile with no observed toxicity properties. Calculations of pKd, pKi and pIC50 values indicated a heightened affinity of the ligand for the receptor protein, suggesting enhanced inhibitory action and a reduced dissociation rate. These encouraging findings suggest a greater potency of the drug, warranting further in vitro testing for validation.

Comprehensive analysis of resorcinyl-imidazole Hsp90 inhibitor design

Human Hsp90 chaperones are implicated in various aspects of cancer. Due to this, Hsp90 has been explored as potential target in cancer treatment. Initial attempts to use Hsp90 inhibitors in drug trials failed due to toxicity and inefficacy. The next generation of drugs were less toxic but still insufficiently effective in a clinical setting. Recently, a lot of effort is being put into understanding the consequences of Hsp90 isoform selective inhibition, expecting that this might hold the key in targeting Hsp90 for disease treatment. Here we investigate a series of compounds containing the aryl-resorcinol scaffold with a 5-membered ring as a promising class of new human Hsp90 inhibitors, reaching nanomolar affinity. We compare how the replacement of 5-membered ring, from thiadiazole to imidazole, as well as a variety of their substituents, influences the potency of these inhibitors for Hsp90 alpha and beta isoforms. To further elucidate the dissimilarity in ligand selectivity between the isoforms, a mutant protein was constructed and tested against the ligand library. In addition, we performed a series of molecular dynamics (MD) and docking simulations to further explain our experimental findings as well as evaluated key compounds in cell assays. Our results deepen the understanding of Hsp90 isoform ligand selectivity and serve as an informative base for further Hsp90 inhibitor optimization.

Structural insights into the unexpected agonism of tetracyclic antidepressants at serotonin receptors 5-HT1eR and 5-HT1FR.

Serotonin [5-hydroxytryptamine (5-HT)] acts via 13 different receptors in humans. Of these receptor subtypes, all but 5-HT1eR have confirmed roles in native tissue and are validated drug targets. Despite 5-HT1eR's therapeutic potential and plausible druggability, the mechanisms of its activation remain elusive. To illuminate 5-HT1eR's pharmacology in relation to the highly homologous 5-HT1FR, we screened a library of aminergic receptor ligands at both receptors and observe 5-HT1eR/5-HT1FR agonism by multicyclic drugs described as pan-antagonists at 5-HT receptors. Potent agonism by tetracyclic antidepressants mianserin, setiptiline, and mirtazapine suggests a mechanism for their clinically observed antimigraine properties. Using cryo-EM and mutagenesis studies, we uncover and characterize unique agonist-like binding poses of mianserin and setiptiline at 5-HT1eR distinct from similar drug scaffolds in inactive-state 5-HTR structures. Together with computational studies, our data suggest that these binding poses alongside receptor-specific allosteric coupling in 5-HT1eR and 5-HT1FR contribute to the agonist activity of these antidepressants.

Directional multiobjective optimization of metal complexes at the billion-system scale.

The discovery of transition metal complexes (TMCs) with optimal properties requires large ligand libraries and efficient multiobjective optimization algorithms. Here we provide the tmQMg-L library, containing 30k diverse and synthesizable ligands with robustly assigned charges and metal coordination modes. tmQMg-L enabled the generation of 1.37 million palladium TMCs, which were used to develop and benchmark the Pareto-Lighthouse multiobjective genetic algorithm (PL-MOGA). With fine control over aim and scope, this algorithm maximized both the polarizability and highest occupied molecular orbital-lowest unoccupied molecular orbital gap of the TMCs within selected regions of the Pareto front, without requiring prior knowledge on the objective limits. Instead of genetic operations on small ligand fragments, the PL-MOGA did whole-ligand mutation and crossover operations, which in chemical spaces containing billions of systems, yielded thousands of highly diverse TMCs in an interpretable manner.

Abstract 5814: EPriL macrocycles as a platform for the rapid generation of effective kinase degrader antibody conjugates (DACs)

Abstract The therapeutic success of antibody drug conjugates (ADCs) drives a continuous search for novel payloads that can increase therapeutic window and thereby widen the applications for ADCs. Recently, heterobifunctional degraders have gained great interest as payloads, and degrader antibody conjugates (DACs) are seen as a novel therapeutic modality. Heterobifunctional degraders consist of a small molecule ligand that binds a target (protein of interest or POI), a spacer and an E3 ligase ligand, which can catalyze target degradation. Owing to their catalytic activity, degraders can have better potency than the equivalent inhibitors, making them suitable as ADC payload [1]. Degrader payloads can target a wider variety of mechanisms than classic payloads, which use the same antitumor strategies as chemotherapy, such as tubulin binding or topoisomerase inhibition. Since many FDA-approved targeted therapies are based on the inhibition of protein kinases, we investigated degraders of these kinases as DAC payloads. Kinase DACs could bring enhanced targeting and therefore better therapeutic window to a field where classic ADC payloads have often shown substantial toxicities. As many heterobifunctional degraders show poor cell membrane penetration, their inhibitory potential could be increased as part of a DAC, where endocytic uptake is followed by intracellular release of the degrader payload. To identify kinase degrader payloads, we present a workflow based on a platform called Energetically Privileged Ligands (EPriLs). EPriLs are macrocycle scaffolds that bind non-covalently in the kinase ATP pocket. Their unique binding mode avoids contacts with amino acid positions where resistance to kinase inhibitors frequently occurs. EPriL macrocycles can be decorated appropriately to rationally design specific inhibitors for many therapeutically relevant kinases, and provide synthetic handles to couple them to VHL or CRBN ligands to generate effective kinase degraders. Here we describe how EPriL kinase degraders can be developed into effective DACs, using consecutive libraries of EPriL ligands, spacers, E3 ligase ligands and linkers. First, suitable degraders are identified, based on rapid and deep target degradation and potent antiproliferative activity on target cell lines. Degraders are then transformed into maleimide-linked degraders using convenient attachment of enzymatically cleavable linkers. In a medium throughput fashion, these maleimides are coupled to antibodies to generate DACs, which are tested for stability and biological potency. Applying this workflow to various well-validated kinase targets in oncology resulted in a promising kinase targeting DAC with favorable ADME properties, clear potentiation compared to the parent degrader, and increased selectivity for tumor cell lines. 1] Dragovich et al., Chem. Soc. Rev. (2022) 51, 3886-3897. Citation Format: Joost C. Uitdehaag, Jos e Man, Michelle Muller, Freek an Cauter, Sander an Gemert, Milan Hoffmann, Yvonne G. an Mil, Winfried R. Mulder, Martine B. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri e Wit, Erik Ensing, Rogier C. Buijsman. EPriL macrocycles as a platform for the rapid generation of effective kinase degrader antibody conjugates (DACs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5814.