Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays.

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds.

Derivatization of inhibitor of apoptosis protein (IAP) ligands yields improved inducers of estrogen receptor α degradation.

Aberrant expression of proteins often underlies many diseases, including cancer. A recently developed approach in drug development is small molecule-mediated, selective degradation of dysregulated proteins. We have devised a protein-knockdown system that utilizes chimeric molecules termed specific and nongenetic IAP-dependent protein erasers (SNIPERs) to induce ubiquitylation and proteasomal degradation of various target proteins. SNIPER(ER)-87 consists of an inhibitor of apoptosis protein (IAP) ligand LCL161 derivative that is conjugated to the estrogen receptor α (ERα) ligand 4-hydroxytamoxifen by a PEG linker, and we have previously reported that this SNIPER efficiently degrades the ERα protein. Here, we report that derivatization of the IAP ligand module yields SNIPER(ER)s with superior protein-knockdown activity. These improved SNIPER(ER)s exhibited higher binding affinities to IAPs and induced more potent degradation of ERα than does SNIPER(ER)-87. Further, they induced simultaneous degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) and delayed degradation of X-linked IAP (XIAP). Notably, these reengineered SNIPER(ER)s efficiently induced apoptosis in MCF-7 human breast cancer cells that require IAPs for continued cellular survival. We found that one of these molecules, SNIPER(ER)-110, inhibits the growth of MCF-7 tumor xenografts in mice more potently than the previously characterized SNIPER(ER)-87. Mechanistic analysis revealed that our novel SNIPER(ER)s preferentially recruit XIAP, rather than cIAP1, to degrade ERα. Our results suggest that derivatized IAP ligands could facilitate further development of SNIPERs with potent protein-knockdown and cytocidal activities against cancer cells requiring IAPs for survival.

Discovery of an Irreversible and Cell-Active BCL6 Inhibitor Selectively Targeting Cys53 Located at the Protein-Protein Interaction Interface.

B-cell lymphoma 6 (BCL6) is the most frequently involved oncogene in diffuse large B-cell lymphomas (DLBCLs). BCL6 shows potent transcriptional repressor activity through interactions with its corepressors, such as BCL6 corepressor (BCOR). The inhibition of the protein-protein interaction (PPI) between BCL6 and its corepressors suppresses the growth of BCL6-dependent DLBCLs, thus making BCL6 an attractive drug target for lymphoma treatment. However, potent small-molecule PPI inhibitor identification remains challenging because of the lack of deep cavities at PPI interfaces. This article reports the discovery of a potent, cell-active small-molecule BCL6 inhibitor, BCL6-i (8), that operates through irreversible inhibition. First, we synthesized irreversible lead compound 4, which targets Cys53 in a cavity on the BCL6-BTB domain dimer by introducing an irreversible warhead to high-throughput screening hit compound 1. Further chemical optimization of 4 based on kinact/KI evaluation produced BCL6-i with a kinact/KI value of 1.9 × 104 M-1 s-1, corresponding to a 670-fold improvement in potency compared to that of 4. By exploiting the property of irreversible inhibition, engagement of BCL6-i to intracellular BCL6 was confirmed. BCL6-i showed intracellular PPI inhibitory activity between BCL6 and its corepressors, thus resulting in BCL6-dependent DLBCL cell growth inhibition. BCL6-i is a cell-active chemical probe with the most potent BCL6 inhibitory activity reported to date. The discovery process of BCL6-i illustrates the utility of irreversible inhibition for identifying potent chemical probes for intractable target proteins.

In Vivo Knockdown of Pathogenic Proteins via Specific and Nongenetic Inhibitor of Apoptosis Protein (IAP)-dependent Protein Erasers (SNIPERs).

Many diseases, especially cancers, result from aberrant or overexpression of pathogenic proteins. Specific inhibitors against these proteins have shown remarkable therapeutic effects, but these are limited mainly to enzymes. An alternative approach that may have utility in drug development relies on selective degradation of pathogenic proteins via small chimeric molecules linking an E3 ubiquitin ligase to the targeted protein for proteasomal degradation. To this end, we recently developed a protein knockdown system based on hybrid small molecule SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers) that recruit inhibitor of the apoptosis protein (IAP) ubiquitin ligases to specifically degrade targeted proteins. Here, we extend our previous study to show a proof of concept of the SNIPER technology in vivo By incorporating a high affinity IAP ligand, we developed a novel SNIPER against estrogen receptor α (ERα), SNIPER(ER)-87, that has a potent protein knockdown activity. The SNIPER(ER) reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice. Mechanistically, it preferentially recruits X-linked IAP (XIAP) rather than cellular IAP1, to degrade ERα via the ubiquitin-proteasome pathway. With this IAP ligand, potent SNIPERs against other pathogenic proteins, BCR-ABL, bromodomain-containing protein 4 (BRD4), and phosphodiesterase-4 (PDE4) could also be developed. These results indicate that forced ubiquitylation by SNIPERs is a useful method to achieve efficient protein knockdown with potential therapeutic activities and could also be applied to study the role of ubiquitylation in many cellular processes.

Discovery of benzimidazole derivatives as orally active renin inhibitors: Optimization of 3,5-disubstituted piperidine to improve pharmacokinetic profile.

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio.

Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands.

Chromosomal translocation occurs in some cancer cells, which results in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate the BCR-ABL protein. We have devised a protein knockdown system by hybrid molecules named Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER), which is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins, and a couple of SNIPER(ABL) against BCR-ABL protein have been developed recently. In this study, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL). The resulting SNIPER(ABL)-39, in which dasatinib is conjugated to an IAP ligand LCL161 derivative by polyethylene glycol (PEG) × 3 linker, shows a potent activity to degrade the BCR-ABL protein. Mechanistic analysis suggested that both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) play a role in the degradation of BCR-ABL protein. Consistent with the degradation of BCR-ABL protein, the SNIPER(ABL)-39 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and Crk like proto-oncogene (CrkL), and suppressed the growth of BCR-ABL-positive CML cells. These results suggest that SNIPER(ABL)-39 could be a candidate for a degradation-based novel anti-cancer drug against BCR-ABL-positive CML.

Discovery of high-affinity BCL6-binding peptide and its structure-activity relationship.

B cell lymphoma 6 (BCL6) is a transcriptional repressor that interacts with its corepressors BcoR and SMRT. Since this protein-protein interaction (PPI) induces activation and differentiation of B lymphocytes, BCL6 has been an attractive drug target for potential autoimmune disease treatments. Here we report a novel BCL6 inhibitory peptide, F1324 (Ac-LWYTDIRMSWRVP-OH), which we discovered using phage display technology; we also discuss this peptide's structure-activity relationship (SAR). For BCL6(5-129) binding, KD and IC50 values of F1324 were 0.57 nM and 1 nM according to the results of an SPR analysis and cell-free ELISA assay, respectively. In contrast, BcoR(Arg498-514Pro) and SMRT(Leu1422-Arg1438) exhibited relatively weak micromole-order binding to BCL6. Furthermore, Fusion protein AcGFP-F1324 transiently expressed in HEK293T cells inhibited intracellular PPI in cell-based M2H assay. By examination of the truncation and fragmentation of F1324, the C-terminal sequence WRVP, which is similar to the BcoR(509-512) sequence WVVP, was identified as being critical for BCL6 binding. In addition, subsequent single-crystal X-ray diffraction analysis of F1324/BCL6(5-129) complex revealed that the high affinity of F1324 was caused by effective interaction of its side chains while its main chain structure was similar to that of BcoR(Arg498-514Pro). To our knowledge, F1324 is the strongest BCL6-binding peptide yet reported.

Discovery of selective ATP-competitive eIF4A3 inhibitors.

Eukaryotic initiation factor 4A3 (eIF4A3), an ATP-dependent RNA helicase, is a core component of exon junction complex (EJC). EJC has a variety of roles in RNA metabolism such as translation, surveillance, and localization of spliced RNA. It is worthwhile to identify selective eIF4A3 inhibitors with a view to investigating the functions of eIF4A3 and EJC further to clarify the roles of the ATPase and helicase activities in cells. Our chemical optimization of hit compound 2 culminated in the discovery of ATP-competitive eIF4A3 inhibitor 18 with submicromolar ATPase inhibitory activity and excellent selectivity over other helicases. Hence, compound 18 could be a valuable chemical probe to elucidate the detailed functions of eIF4A3 and EJC.

Discovery of a novel B-cell lymphoma 6 (BCL6)-corepressor interaction inhibitor by utilizing structure-based drug design.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor that can form complexes with corepressors via protein-protein interactions (PPIs). The complexes of BCL6 and corepressors play an important role in the formation of germinal centers (GCs), and differentiation and proliferation of lymphocytes. Therefore, BCL6-corepressor interaction inhibitors would be drug candidates for managing autoimmune diseases and cancer. Starting from high-throughput screening hits 1a and 2a, we identified a novel BCL6-corepressor interaction inhibitor 8c (cell-free enzyme-linked immunosorbent assay [ELISA] IC50=0.10µM, cell-based mammalian two-hybrid [M2H] assay IC50=0.72µM) by utilizing structure-based drug design (SBDD) based on an X-ray crystal structure of 1a bound to BCL6. Compound 8c also showed a good pharmacokinetic profile, which was acceptable for both in vitro and in vivo studies.

Novel approach of fragment-based lead discovery applied to renin inhibitors.

A novel approach was conducted for fragment-based lead discovery and applied to renin inhibitors. The biochemical screening of a fragment library against renin provided the hit fragment which showed a characteristic interaction pattern with the target protein. The hit fragment bound only to the S1, S3, and S3SP (S3 subpocket) sites without any interactions with the catalytic aspartate residues (Asp32 and Asp215 (pepsin numbering)). Prior to making chemical modifications to the hit fragment, we first identified its essential binding sites by utilizing the hit fragment's substructures. Second, we created a new and smaller scaffold, which better occupied the identified essential S3 and S3SP sites, by utilizing library synthesis with high-throughput chemistry. We then revisited the S1 site and efficiently explored a good building block attaching to the scaffold with library synthesis. In the library syntheses, the binding modes of each pivotal compound were determined and confirmed by X-ray crystallography and the library was strategically designed by structure-based computational approach not only to obtain a more active compound but also to obtain informative Structure Activity Relationship (SAR). As a result, we obtained a lead compound offering synthetic accessibility as well as the improved in vitro ADMET profiles. The fragments and compounds possessing a characteristic interaction pattern provided new structural insights into renin's active site and the potential to create a new generation of renin inhibitors. In addition, we demonstrated our FBDD strategy integrating highly sensitive biochemical assay, X-ray crystallography, and high-throughput synthesis and in silico library design aimed at fragment morphing at the initial stage was effective to elucidate a pocket profile and a promising lead compound.

Structure-based design of a new series of N-(piperidin-3-yl)pyrimidine-5-carboxamides as renin inhibitors.

The action of the aspartyl protease renin is the rate-limiting initial step of the renin-angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine derivatives 5-14 were designed in an attempt to enhance the renin inhibitory activity of compound 3 identified by our previous fragment-based drug design approach. Introduction of a basic amine essential for interaction with the two aspartic acids in the catalytic site and optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114 ('Leu-in' to 'Leu-out') by a rational structure-based drug design approach led to the discovery of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of compound 14 has been achieved by the overall addition of only seven heavy atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate oral bioavailability in rats.

Antithrombotic and anticoagulant profiles of TAK-442, a novel factor Xa inhibitor, in a rabbit model of venous thrombosis.

The anticoagulant and antithrombotic profiles of TAK-442, a direct factor Xa (FXa) inhibitor, were investigated. TAK-442 showed potent inhibition of human FXa (Ki = 1.8 nM) and high specificity, with a 440-fold greater selectivity than thrombin and negligible effects on trypsin, plasmin, and tissue plasminogen activator (K(i) > 30 microM). [corrected] In human plasma, TAK-442 doubled FXa-induced clotting time, prothrombin time (PT), and activated partial thromboplastin time at 0.19, 0.55, and 0.59 microM, respectively. The relative PT-prolonging potencies of TAK-442, rivaroxaban, and apixaban were 1, 2.0-2.6, and 0.46-1.3, respectively, in 4 different PT reagents. In a rabbit model of venous thrombosis, 50- and 100-micrograms/kg [corrected] TAK-442 (intravenous bolus followed by 1-hour infusion) reduced thrombus formation by 50% and 81%, with plasma anti-FXa activity of 23%-26% and 34%-38%, respectively, and only marginal prolongation of PT and activated partial thromboplastin time. Melagatran, a thrombin inhibitor, showed similar antithrombotic activity to TAK-442. However, 500-micrograms/kg [corrected TAK-442 did not affect bleeding time (BT), whereas the same dose of melagatran significantly prolonged BT by 3.6-fold compared with vehicle control. These findings suggest that TAK-442 has similar antithrombotic effects as melagatran but does not cause BT prolongation, and plasma anti-FXa activity may reliably predict its potency.

Discovery of imidazo[1,5-c]imidazol-3-ones: weakly basic, orally active factor Xa inhibitors.

The coagulation enzyme factor Xa (FXa) has been recognized as a promising target for the development of new antithrombotic agents. We previously found compound 1 to be an orally bioavailable FXa inhibitor in fasted monkeys; however, 1 showed poor bioavailability in rats and fed monkeys. To work out the pharmacokinetic problems, we focused our synthetic efforts on the chemical conversion of the 4-(imidazo[1,2- a]pyridin-5-yl)piperazine moiety of 1 to imidazolylpiperidine derivatives (fused and nonfused), which resulted in the discovery of the weakly basic imidazo[1,5- c]imidazol-3-one 3q as a potent and selective FXa inhibitor. Compound 3q showed favorable oral bioavailability in rats and monkeys under both fasted and fed conditions and antithrombotic efficacy in a rat model of venous thrombosis after oral administration, without a significant increase in bleeding time (unlike warfarin). On the basis of these promising properties, compound 3q was selected for further evaluation.

Discovery of piperazinylimidazo[1,2-a]pyridines as novel S4 binding elements for orally active factor Xa inhibitors.

We have recently reported the discovery of orally active sulfonylalkylamide Factor Xa (FXa) inhibitors, as typified by compound 1 (FXa IC(50)=0.061 microM). Since the pyridylpiperidine moiety was not investigated in our previous study, we conducted detailed structure-activity relationship studies on this S4 binding element. This investigation led to the discovery of piperazinylimidazo[1,2-a]pyridine 2b as a novel and potent FXa inhibitor (FXa IC(50)=0.021 microM). Further modification resulted in the discovery of 2-hydroxymethylimidazo[1,2-a]pyridine 2e (FXa IC(50)=0.0090 microM), which was found to be a selective and orally bioavailable FXa inhibitor with reduced CYP3A4 inhibition.

Discovery of sulfonylalkylamides: A new class of orally active factor Xa inhibitors.

Factor Xa (FXa) is a trypsin-like serine protease involved in the coagulation cascade and has received great interest as a potential target for the development of new antithrombotic agents. Most of amidine-type FXa inhibitors reported have been found to show extremely poor oral bioavailability. Compound 1 is one of the first reported non-amidine type FXa inhibitors. To discover novel and orally active FXa inhibitors, we investigated flexible linear linkers between the 6-chloronaphthalene ring and the 1-(pyridin-4-yl)piperidine moiety of 1 and found the orally active sulfonylalkylamide 2f with an FXa IC(50) of 0.05 microM, comparable with that of 1. Further modification to reduce the CYP3A4 inhibitory activity of 2f resulted in the potent, selective, and orally active 2-methylpyridine analogue 2s (FXa IC(50) of 0.061 microM), for which the liability of CYP3A4 inhibition was significantly weakened compared to 2f. Compound 2s also showed long lasting anticoagulant activity in cynomolgus monkeys.

Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands.

Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.

Discovery of 3-Benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea Derivatives as Novel and Selective Cyclin-Dependent Kinase 12 (CDK12) Inhibitors.

Cyclin-dependent kinase 12 (CDK12) plays a key role in the coordination of transcription with elongation and mRNA processing. CDK12 mutations found in tumors and CDK12 inhibition sensitize cancer cells to DNA-damaging reagents and DNA-repair inhibitors. This suggests that CDK12 inhibitors are potential therapeutics for cancer that may cause synthetic lethality. Here, we report the discovery of 3-benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea derivatives as novel and selective CDK12 inhibitors. Structure-activity relationship studies of a HTS hit, structure-based drug design, and conformation-oriented design using the Cambridge Structural Database afforded the optimized compound 2, which exhibited not only potent CDK12 (and CDK13) inhibitory activity and excellent selectivity but also good physicochemical properties. Furthermore, 2 inhibited the phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and induced growth inhibition in SK-BR-3 cells. Therefore, 2 represents an excellent chemical probe for functional studies of CDK12 and could be a promising lead compound for drug discovery.

TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.

Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors.

Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor 53a exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of 53a and its analog 52a to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds 52a and 53a represent novel molecular probes for further study of eIF4A3, the EJC, and NMD.

Discovery of a B-Cell Lymphoma 6 Protein-Protein Interaction Inhibitor by a Biophysics-Driven Fragment-Based Approach.

B-cell lymphoma 6 (BCL6) is a transcriptional factor that expresses in lymphocytes and regulates the differentiation and proliferation of lymphocytes. Therefore, BCL6 is a therapeutic target for autoimmune diseases and cancer treatment. This report presents the discovery of BCL6-corepressor interaction inhibitors by using a biophysics-driven fragment-based approach. Using the surface plasmon resonance (SPR)-based fragment screening, we successfully identified fragment 1 (SPR KD = 1200 µM, ligand efficiency (LE) = 0.28), a competitive binder to the natural ligand BCoR peptide. Moreover, we elaborated 1 into the more potent compound 7 (SPR KD = 0.078 µM, LE = 0.37, cell-free protein-protein interaction (PPI) IC50 = 0.48 µM (ELISA), cellular PPI IC50 = 8.6 µM (M2H)) by a structure-based design and structural integration with a second high-throughput screening hit.