High-throughput Screening Campaign Research Articles

Synergizing Chemical Structures and Bioassay Descriptions for Enhanced Molecular Property Prediction in Drug Discovery.

The precise prediction of molecular properties can greatly accelerate the development of new drugs. However, in silico molecular property prediction approaches have been limited so far to assays for which large amounts of data are available. In this study, we develop a new computational approach leveraging both the textual description of the assay of interest and the chemical structure of target compounds. By combining these two sources of information via self-supervised learning, our tool can provide accurate predictions for assays where no measurements are available. Remarkably, our approach achieves state-of-the-art performance on the FS-Mol benchmark for zero-shot prediction, outperforming a wide variety of deep learning approaches. Additionally, we demonstrate how our tool can be used for tailoring screening libraries for the assay of interest, showing promising performance in a retrospective case study on a high-throughput screening campaign. By accelerating the early identification of active molecules in drug discovery and development, this method has the potential to streamline the identification of novel therapeutics.

Creating a more strategic small molecule biophysical hit characterization workflow

To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.

Design and Synthesis of 1,3-Diarylpyrazoles and Investigation of Their Cytotoxicity and Antiparasitic Profile.

Herein, we report a series of 1,3-diarylpyrazoles that are analogues of compound 26/HIT 8. We previously identified this molecule as a 'hit' during a high-throughput screening campaign for autophagy inducers. A variety of synthetic strategies were utilized to modify the 1,3-diarylpyrazole core at its 1-, 3-, and 4-position. Compounds were assessed in vitro to identify their cytotoxicity properties. Of note, several compounds in the series displayed relevant cytotoxicity, which warrants scrutiny while interpreting biological activities that have been reported for structurally related molecules. In addition, antiparasitic activities were recorded against a range of human-infective protozoa, including Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania infantum. The most interesting compounds displayed low micromolar whole-cell potencies against individual or several parasitic species, while lacking cytotoxicity against human cells.

Discovery of Novel Binders to Sterol Regulatory Element-Binding Protein-1 by High-Throughput Screening.

Sterol regulatory element-binding protein-1 (SREBP-1) is a transcription factor that regulates the expression of genes related to fatty acid biosynthesis. Its high expression and activation in obesity and associated metabolic diseases make it a potential therapeutic target. However, the role of SREBP-1 in the development and exacerbation of these diseases remains unclear, partly because of the impossibility of inhibiting its function because of the lack of specific inhibitors. Here, we aimed to identify small-molecule compounds that directly bind to SREBP-1 using the recombinant N-terminal region of SREBP-1a, which is required for its transcriptional activity. A high-throughput screening campaign was conducted using a thermal shift assay and surface plasmon resonance assay to evaluate the compound affinity and specificity, which resulted in the identification of two compounds. Future analysis of their structure-activity relationships may lead to the development of specific SREBP-1 inhibitors, thereby potentially validating SREBP-1 as a therapeutic target for obesity and resultant atherosclerotic diseases.

Discovery of Strong 3-Nitro-2-Phenyl-2H-Chromene Analogues as Antitrypanosomal Agents and Inhibitors of Trypanosoma cruzi Glucokinase.

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.

Discovery of CVN293, a Brain Permeable KCNK13 (THIK-1) Inhibitor Suitable for Clinical Assessment.

The potassium (K+) ion channel KCNK13 is specifically expressed in human microglia with elevated expression observed in post-mortem human brain tissue from patients with Alzheimer's disease. Modulation of KCNK13 activity by a small-molecule inhibitor is proposed as a potential treatment for neurodegenerative diseases. Herein, we describe the evolution of a series of KCNK13 inhibitors derived from a high-throughput screening campaign, resulting in CVN293, a potent, selective, and brain permeable clinical candidate molecule. CVN293 demonstrated a concentration-dependent inhibition of the NLRP3-inflammasome mediated production of IL-1β from LPS-primed murine microglia. Cross-species pharmacokinetic data of CVN293 are also disclosed. These findings support the advancement of CVN293 in clinical trials.

AI is a viable alternative to high throughput screening: a 318-target study

High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery.

Machine Learning Assisted Hit Prioritization for High Throughput Screening in Drug Discovery.

Efficient prioritization of bioactive compounds from high throughput screening campaigns is a fundamental challenge for accelerating drug development efforts. In this study, we present the first data-driven approach to simultaneously detect assay interferents and prioritize true bioactive compounds. By analyzing the learning dynamics during training of a gradient boosting model on noisy high throughput screening data using a novel formulation of sample influence, we are able to distinguish between compounds exhibiting the desired biological response and those producing assay artifacts. Therefore, our method enables false positive and true positive detection without relying on prior screens or assay interference mechanisms, making it applicable to any high throughput screening campaign. We demonstrate that our approach consistently excludes assay interferents with different mechanisms and prioritizes biologically relevant compounds more efficiently than all tested baselines, including a retrospective case study simulating its use in a real drug discovery campaign. Finally, our tool is extremely computationally efficient, requiring less than 30 s per assay on low-resource hardware. As such, our findings show that our method is an ideal addition to existing false positive detection tools and can be used to guide further pharmacological optimization after high throughput screening campaigns.

High-throughput nephelometry methodology for qualitative determination of aqueous solubility of chemical libraries

The purpose of the protocol reported in this work is the solubility profiling of large chemical libraries using nephelometry. This technique allows the qualitative classification of compounds as highly, moderately, or poorly water-soluble. The described methodology is not intended to yield quantitative solubility values of the studied compounds but can be used as a primary solubility assessment of large chemical libraries, to guide hit prioritization after High Throughput Screening (HTS) campaigns.

Discovery of a Novel Orally Active, Selective LPA Receptor Type 1 Antagonist, 4-(4-(2-Isopropylphenyl)-4-((2-methoxy-4-methylphenyl)carbamoyl)piperidin-1-yl)-4-oxobutanoic Acid, with a Distinct Molecular Scaffold.

Lysophosphatidic acid receptor 1 (LPAR1) antagonists show promise as potentially novel antifibrotic treatments. In a human LPAR1 β-arrestin recruitment-based high-throughput screening campaign, we identified urea 19 as a hit with a LPAR1 IC50 value of 5.0 μM. Hit-to-lead activities revealed that one of the urea nitrogen atoms can be replaced by carbon and establish the corresponding phenylacetic amide as a lead structure for further optimization. Medicinal chemistry efforts led to the discovery of piperidine 18 as a potent and selective LPAR1 antagonist with oral activity in a mouse model of LPA-induced skin vascular leakage. The molecular scaffold of 18 shares no obvious structural similarity with any other LPAR1 antagonist disclosed so far.

Development of a high-throughput screening platform to identify new therapeutic agents for Medulloblastoma Group 3

Pediatric brain tumors (PBTs) represent about 25 % of all pediatric cancers and are the most common solid tumors in children and adolescents. Medulloblastoma (MB) is the most frequently occurring malignant PBT, accounting for almost 10 % of all pediatric cancer deaths. MB Group 3 (MB G3) accounts for 25–30 % of all MB cases and has the worst outcome, particularly when associated with MYC amplification. However, no targeted treatments for this group have been developed so far.Here we describe a unique high throughput screening (HTS) platform specifically designed to identify new therapies for MB G3. The platform incorporates optimized and validated 2D and 3D efficacy and toxicity models, that account for tumor heterogenicity, limited efficacy and unacceptable toxicity from the very early stage of drug discovery. The platform has been validated by conducting a pilot HTS campaign with a 1280 lead-like compound library. Results showed 8 active compounds, targeting MB reported targets and several are currently approved or in clinical trials for pediatric patients with PBTs, including MB. Moreover, hits were combined to avoid tumor resistance, identifying 3 synergistic pairs, one of which is currently under clinical study for recurrent MB and other PBTs.

A digital image colorimetry system based on smart devices for immediate and simultaneous determination of enzyme-linked immunosorbent assays

Standard enzyme-linked immunosorbent assays based on microplates are frequently utilized for various molecular sensing, disease screening, and nanomedicine applications. Comparing this multi-well plate batched analysis to non-batched or non-standard testing, the diagnosis expenses per patient are drastically reduced. However, the requirement for rather big and pricey readout instruments prevents their application in environments with limited resources, especially in the field. In this work, a handheld cellphone-based colorimetric microplate reader for quick, credible, and novel analysis of digital images of human cancer cell lines at a reasonable price was developed. Using our in-house-developed app, images of the plates are captured and sent to our servers, where they are processed using a machine learning algorithm to produce diagnostic results. Using FDA-approved human epididymis protein of ovary IgG (HE4), prostate cancer cell line (PC3), and bladder cancer cell line (5637) ELISA tests, we successfully examined this mobile platform. The accuracies for the HE4, PC3, and 5637 tests were 93%, 97.5%, and 97.2%, respectively. By contrasting the findings with the measurements made using optical absorption EPOCH microplate readers and optical absorption Tecan microplate readers, this approach was found to be accurate and effective. As a result, digital image colorimetry on smart devices offered a practical, user-friendly, affordable, precise, and effective method for quickly identifying human cancer cell lines. Thus, healthcare providers might use this portable device to carry out high-throughput illness screening, epidemiological investigations or monitor vaccination campaigns.

Fragment library screening by X-ray crystallography and binding site analysis on thioredoxin glutathione reductase of Schistosoma mansoni.

Schistosomiasis is caused by parasites of the genus Schistosoma, which infect more than 200 million people. Praziquantel (PZQ) has been the main drug for controlling schistosomiasis for over four decades, butdespite that it is ineffective against juvenile worms and size and taste issues with its pharmaceutical forms impose challenges for treating school-aged children. It is also important to note that PZQ resistant strains can be generated in laboratory conditions and observed in the field, hence its extensive use in mass drug administration programs raises concerns about resistance, highlighting the need to search for new schistosomicidal drugs. Schistosomes survival relies on the redox enzyme thioredoxin glutathione reductase (TGR), a validated target for the development of new anti-schistosomal drugs. Here we report a high-throughput fragment screening campaign of 768 compounds against S. mansoni TGR (SmTGR) using X-ray crystallography. We observed 49 binding events involving 35 distinct molecular fragments which were found to be distributed across 16 binding sites. Most sites are described for the first time within SmTGR, a noteworthy exception being the "doorstop pocket" near the NADPH binding site. We have compared results from hotspots and pocket druggability analysis of SmTGR with the experimental binding sites found in this work, with our results indicating only limited coincidence between experimental and computational results. Finally, we discuss that binding sites at the doorstop/NADPH binding site and in the SmTGR dimer interface, should be prioritized for developing SmTGR inhibitors as new antischistosomal drugs.

Carbohydrate-active enzyme (CAZyme) discovery and engineering via (Ultra)high-throughput screening.

Carbohydrate-active enzymes (CAZymes) constitute a diverse set of enzymes that catalyze the assembly, degradation, and modification of carbohydrates. These enzymes have been fashioned into potent, selective catalysts by millennia of evolution, and yet are also highly adaptable and readily evolved in the laboratory. To identify and engineer CAZymes for different purposes, (ultra)high-throughput screening campaigns have been frequently utilized with great success. This review provides an overview of the different approaches taken in screening for CAZymes and how mechanistic understandings of CAZymes can enable new approaches to screening. Within, we also cover how cutting-edge techniques such as microfluidics, advances in computational approaches and synthetic biology, as well as novel assay designs are leading the field towards more informative and effective screening approaches.

Optimization of Selectivity and Pharmacokinetic Properties of Salt-Inducible Kinase Inhibitors that Led to the Discovery of Pan-SIK Inhibitor GLPG3312.

Salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 are serine/threonine kinases and form a subfamily of the protein kinase AMP-activated protein kinase (AMPK) family. Inhibition of SIKs in stimulated innate immune cells and mouse models has been associated with a dual mechanism of action consisting of a reduction of pro-inflammatory cytokines and an increase of immunoregulatory cytokine production, suggesting a therapeutic potential for inflammatory diseases. Following a high-throughput screening campaign, subsequent hit to lead optimization through synthesis, structure-activity relationship, kinome selectivity, and pharmacokinetic investigations led to the discovery of clinical candidate GLPG3312 (compound 28), a potent and selective pan-SIK inhibitor (IC50: 2.0 nM for SIK1, 0.7 nM for SIK2, and 0.6 nM for SIK3). Characterization of the first human SIK3 crystal structure provided an understanding of the binding mode and kinome selectivity of the chemical series. GLPG3312 demonstrated both anti-inflammatory and immunoregulatory activities in vitro in human primary myeloid cells and in vivo in mouse models.

Experimental structure based drug design (SBDD) applications for anti-leishmanial drugs: A paradigm shift?

Leishmaniasis is a group of neglected tropical diseases caused by at least 20 species of Leishmania protozoa, which are spread by the bite of infected sandflies. There are three main forms of the disease: cutaneous leishmaniasis (CL, the most common), visceral leishmaniasis (VL, also known as kala-azar, the most serious), and mucocutaneous leishmaniasis. One billion people live in areas endemic to leishmaniasis, with an annual estimation of 30,000 new cases of VL and more than 1 million of CL. New treatments for leishmaniasis are an urgent need, as the existing ones are inefficient, toxic, and/or expensive. We have revised the experimental structure-based drug design (SBDD) efforts applied to the discovery of new drugs against leishmaniasis. We have grouped the explored targets according to the metabolic pathways they belong to, and the key achieved advances are highlighted and evaluated. In most cases, SBDD studies follow high-throughput screening campaigns and are secondary to pharmacokinetic optimization, due to the majoritarian belief that there are few validated targets for SBDD in leishmaniasis. However, some SBDD strategies have significantly contributed to new drug candidates against leishmaniasis and a bigger number holds promise for future development.

Abstract IA013: Integrating natural product samples back into drug screening programs

Abstract Natural products have a proven track record in use as anti-cancer agents. As of 2020 natural products (NPs), NP pharmacophores, and NP mimics represent a significant percentage of small molecule-based anti-cancer drugs. However, accessing the potential of NPs in high-throughput screening (HTS) campaigns has been challenging due to the complexity of the crude NP extracts, the time necessary for follow-up isolation efforts, and indefinite capabilities for resupply. This has resulted in NP-based libraries being significantly underrepresented in most recent large-scale HTS programs. The US National Cancer Institute’s Natural Product Extract Repository is one of the world’s largest, most diverse collections of natural products containing >200,000 unique extracts derived from plant, marine and microbial organisms. To address the challenges in screening NPs and encourage increased assay of NP chemical diversity, the NCI created a Program for Natural Product Discovery (NPNPD) which has developed an automated, high-throughput robotics platform that has created 500,000 pre-fractionated NP samples and released them to the public for screening. In addition, automated secondary purification and structure elucidation systems were created that significantly decrease costs and timelines. This presentation will discuss the reasons for, challenges to, and mechanisms by which the NCI is working to increase the pace and efficiency of NP-based drug discovery. Citation Format: Barry R. O'Keefe. Integrating natural product samples back into drug screening programs [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA013.

Discovery of a Novel Benzodiazepine Series of Cbl-b Inhibitors for the Enhancement of Antitumor Immunity.

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) is a RING finger E3 ligase that is responsible for repressing T-cell, natural killer (NK) cell, and B-cell activation. The robust antitumor activity observed in Cbl-b deficient mice arising from elevated T-cell and NK-cell activity justified our discovery effort toward Cbl-b inhibitors that might show therapeutic promise in immuno-oncology, where activation of the immune system can drive the recognition and killing of cancer cells. We undertook a high-throughput screening campaign followed by structure-enabled optimization to develop a novel benzodiazepine series of potent Cbl-b inhibitors. This series displayed nanomolar levels of biochemical potency, as well as potent T-cell activation. The functional activity of this class of Cbl-b inhibitors was further corroborated with ubiquitin-based cellular assays.

A high-throughput screening strategy identifies adenosine 5'-monophosphate as a novel enhancing molecule of the cardio-protective enzyme 5-oxoprolinase (OPLAH)

Abstract Background/Introduction Oxidative stress plays an essential role in the pathophysiology of heart failure (HF), and reducing it might improve outcomes in patients suffering from it. We recently identified 5-oxoproline to be elevated in patients with HF and that the increased levels are associated with a poor prognosis. 5-oxoprolinase (OPLAH) is an enzyme that converts toxic 5-oxoproline to harmless glutamate. We previously showed that the overexpression of OPLAH prevented cardiac damage in experimental models. Purpose To identify small molecules that hold the potential to increase the activity of the antioxidant enzyme OPLAH. Methods and results OPLAH expression vectors were established containing a 6xHis-tag in the N-terminal region and were used to efficiently overexpress enzymatically active recombinant OPLAH protein (rOPLAH) in human embryonic kidney cells (HEK293A) and Spodoptera frugiperda cells (SF9). rOPLAH expressing His-tag was purified under native conditions using an affinity chromatography matrix, and enzymatic activity was proven by measuring the conversion of 5-oxoproline to glutamate using LC-MS/MS. After miniaturization and optimization of a luminescence-based technique that measures OPLAH's ATPase activity, a high-throughput screening campaign of 1280 FDA-approved compounds was performed. Adenosine 5′-monophosphate (5'-AMP) was identified to enhance the ATPase activity of OPLAH by >50%. In addition, an LC-MS/MS dose-response curve exhibited that increasing 5'-AMP concentration resulted in a higher glutamate production using isolated rOPLAH. A thermal shift assay confirmed the interaction of 5'-AMP and OPLAH. Since 5’-AMP is unable to cross the cellular plasma membrane, acetalated dextran spermine-modified nanoparticles loaded with 5'-AMP were produced (AcDXSp@AMP NPs) to target cells. NPs were prepared by the water-in-oil-in-water double emulsion technique. Particle size, polydispersity index and z–potential of the NPs were characterized by dynamic and electrophoretic light scattering. HPLC tested the quantification of encapsulated 5'-AMP and its release over time. The efficacy of OPLAH activation by 5'-AMP-loaded NPs was tested in HeLa cells cultured in the presence of 10 mM 13C-5-oxoproline. Employing AcDXSp@AMP NPs for 12h to cells resulted in about 50% elevation of 13C-glutamate production, showing successful activation of OPLAH by AMP in cells. Conclusion Here, we developed a high-throughput screening for 5-oxprolinase and identified a non-cell permeable enhancer compound, adenosine 5′-monophosphate. Orthogonal studies confirmed 5'-AMP affinity to OPLAH. Finally, we successfully utilized nanoparticles as a drug-delivery system for 5'-AMP and proved its intracellular effect. Our efforts are a blueprint for bringing target discovery to treatment identification. As such, nanoparticle-based delivery systems hold great potential for future trials utilizing impermeable modulators to target heart failure and other diseases.Illustrative workflow

Discovery of First-in-Class Small Molecule Inhibitors of the IRF4-PU.1/Spi-B Interaction

Introduction Many transcription factors (TF) are promising therapeutic targets in hematological malignancies but have been mislabeled as ‘undruggable’ due to their dynamic structures and lack of feasible paths for bioactive small molecules. Multiple myeloma (MM), the second most common hematological malignancy, is predominantly driven by oncogenic transcription factors such as c-Myc & interferon regulatory factor 4 (IRF4). Attempts at targeting IRF4 have relied on immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, which target upstream TFs of IRF4 - IKZF1 & IKZF3. Unfortunately, IMiDs are highly susceptible to resistance mechanisms in the clinic and fail to provide deep and sustained downregulation of IRF4 due to theirindirect mechanism of action. Currently, no known small molecules that directly bind and inhibit IRF4 exist. Moreover, no probes or screening based assays have been developed for IRF4 or any member of the IRF family.IRF4 is the top ranked dependency of MM and is a validated clinical target given the success of IMiD-based regimens. Thus, orally available small molecules that can directly target IRF4 will have significant benefit for patients with MM. Methods & Results To identify small molecules that inhibit or bind IRF4, we first cloned, expressed, and purified numerous constructs of the interferon accessory domain (IAD - residues 238-420) and full-length IRF4 (residues 1-451). To enable traditional high-throughput screening (HTS) campaigns, we first identified peptide ligands of IRF4 based on the IRF4 - PU.1/Spi-B interaction, an oncogenic signaling node in various hematological malignancies. We profiled peptides derived from PU.1 and Spi-B and confirmed binding to the IAD domain with K ds of 1 - 7 µM by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). Encouraged by the robust signal seen with our FP assay, we further developed a TR-FRET based method using a CoraFluor labeled IAD domain which significantly boosted our signal-to-noise ratio for our HTS efforts (Z' > 0.8). We screened this interaction against the Chembridge2020 drug library, which consists of 50,000 lead-like small molecules. The screen afforded 68 small molecules that had inhibition > 50% by TR-FRET (hit rate of ~0.1%). We prioritized our most potent compound, HIT-1, which has a TR-FRET IC 50 of 2 µM. Further binding validation including ITC, biotin-based pull-downs in human myeloma cell lines (HMCLs), and a BRET based target engagement assay confirm binding to both recombinant and full-length IRF4. Additionally, medicinal chemistry optimization led to the development of 1, with sub-micromolar affinity to IRF4, and an inactive control 2. Biological assessment of compounds 1 and inactive control 2 are underway in HMCLs; initial results show on-target modulation of IRF4 signaling processes. Compound 1 represents the first validated small molecule targeting the ‘undruggable’ IRF family and warrants its use in investigating the IRF4-PU.1/Spi-B interaction in oncology and immunology. Conclusions These small molecules are the first inhibitors of the IRF4-PU.1 interaction. We have developed novel biochemical assays that can be applied to other IRF family members, including IRF8. Additionally, we showcase translatable strategies to build novel biochemical assays for other transcription factors, leveraging known PPIs for probe development. These ligands represent starting points for hetero-bifunctional compounds and chemical probes for modulating IRF4 activity both in vitro and in vivo.