WIN Site Research Articles

The NTE domain of PTENα/β promotes cancer progression by interacting with WDR5 via its SSSRRSS motif

PTENα/β, two variants of PTEN, play a key role in promoting tumor growth by interacting with WDR5 through their N-terminal extensions (NTEs). This interaction facilitates the recruitment of the SET1/MLL methyltransferase complex, resulting in histone H3K4 trimethylation and upregulation of oncogenes such as NOTCH3, which in turn promotes tumor growth. However, the molecular mechanism underlying this interaction has remained elusive. In this study, we determined the first crystal structure of PTENα-NTE in complex with WDR5, which reveals that PTENα utilizes a unique binding motif of a sequence SSSRRSS found in the NTE domain of PTENα/β to specifically bind to the WIN site of WDR5. Disruption of this interaction significantly impedes cell proliferation and tumor growth, highlighting the potential of the WIN site inhibitors of WDR5 as a way of therapeutic intervention of the PTENα/β associated cancers. These findings not only shed light on the important role of the PTENα/β-WDR5 interaction in carcinogenesis, but also present a promising avenue for developing cancer treatments that target this pathway.

PARP1 interacts with WDR5 to enhance target gene recognition and facilitate tumorigenesis

Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear protein that attaches negatively charged poly (ADP-ribose) (PAR) to itself and other target proteins. While its function in DNA damage repair is well established, its role in target chromatin recognition and regulation of gene expression remains to be better understood. This study showed that PARP1 interacts with SET1/MLL complexes by binding directly to WDR5. Notably, although PARP1 does not modulate WDR5 PARylation or the global level of H3K4 methylation, it exerts locus-specific effects on WDR5 binding and H3K4 methylation. Interestingly, PARP1 and WDR5 show extensive co-localization on chromatin, with WDR5 facilitating the recognition and expression of target genes regulated by PARP1. Furthermore, we demonstrated that inhibition of the WDR5 Win site impedes the interaction between PARP1 and WDR5, thereby inhibiting PARP1 from binding to target genes. Finally, the combined inhibition of the WDR5 Win site and PARP shows a profound inhibitory effect on the proliferation of cancer cells. These findings illuminate intricate mechanisms underlying chromatin recognition, gene transcription, and tumorigenesis, shedding light on previously unrecognized roles of PARP1 and WDR5 in these processes.

Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Small molecule WDR5 inhibitors down-regulate lncRNA expression.

WD repeat domain 5 (WDR5) plays an important role as a scaffold protein in both protein-protein and RNA-protein complexes involved in epigenetic gene regulation. In particular, some of these lncRNAs were reported to regulate the expression of genes in cis as well as themselves through binding WDR5. In this report, we investigate the two known binding sites of WDR5 in relation to lncRNA binding and expression. The WBM binding site mediates both protein-protein and lncRNA-protein interactions while the WIN site, which is on the opposite side of the protein, is only known to mediate protein-protein interactions. To dissect the function of different binding sites on WDR5, we characterized them with selective peptide ligands using fluorescence polarization and used these to demonstrate the selectivity of small molecule inhibitors of these two major binding sites. RNA immunoprecipitation experiments were performed to show that lncRNA-WDR5 complex formation could be interrupted using a WBM site inhibitor. Finally, we demonstrated that WDR5 regulated lncRNAs are down regulated with different sensitivity toward the corresponding inhibitors, demonstrating the potential of targeting lncRNA-protein interactions to reduce oncogenic lncRNA expression.

Discovery and Structure-Based Design of Inhibitors of the WD Repeat-Containing Protein 5 (WDR5)-MYC Interaction.

WDR5 is a critical chromatin cofactor of MYC. WDR5 interacts with MYC through the WBM pocket and is hypothesized to anchor MYC to chromatin through its WIN site. Blocking the interaction of WDR5 and MYC impairs the recruitment of MYC to its target genes and disrupts the oncogenic function of MYC in cancer development, thus providing a promising strategy for the treatment of MYC-dysregulated cancers. Here, we describe the discovery of novel WDR5 WBM pocket antagonists containing a 1-phenyl dihydropyridazinone 3-carboxamide core that was identified from high-throughput screening and subsequent structure-based design. The leading compounds showed sub-micromolar inhibition in the biochemical assay. Among them, compound 12 can disrupt WDR5-MYC interaction in cells and reduce MYC target gene expression. Our work provides useful probes to study WDR5-MYC interaction and its function in cancers, which can also be used as the starting point for further optimization toward drug-like small molecules.

Discovery of Potent Small-Molecule Inhibitors of WDR5-MYC Interaction.

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple processes. It is also a prominent target for pharmacological inhibition in diseases such as cancer, aging, and neurodegenerative disorders. Interactions between WDR5 and various partners are essential for sustaining its function. Most drug discovery efforts center on the WIN (WDR5 interaction motif) site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe the discovery of novel WDR5 inhibitors for the other WBM (WDR5 binding motif) pocket on this scaffold protein, to disrupt WDR5 interaction with its binding partner MYC by high-throughput biochemical screening, subsequent molecule optimization, and biological assessment. These new WDR5 inhibitors provide useful probes for future investigations of WDR5 and an avenue for targeting WDR5 as a therapeutic strategy.

Discovery, evaluation and mechanism study of WDR5-targeted small molecular inhibitors for neuroblastoma.

Neuroblastoma is the most common and deadliest tumor in infancy. WDR5 (WD Repeat Domain 5), a critical factor supporting an N-myc transcriptional complex via its WBM site and interacting with chromosome via its WIN site, promotes the progression of neuroblastoma, thus making it a potential anti-neuroblastoma drug target. So far, a few WIN site inhibitors have been reported, and the WBM site disruptors are rare to see. In this study we conducted virtual screening to identify candidate hit compounds targeting the WBM site of WDR5. As a result, 60 compounds were selected as candidate WBM site inhibitors. Cell proliferation assay demonstrated 6 structurally distinct WBM site inhibitors, numbering as compounds 4, 7, 11, 13, 19 and 22, which potently suppressed 3 neuroblastoma cell lines (MYCN-amplified IMR32 and LAN5 cell lines, and MYCN-unamplified SK-N-AS cell line). Among them, compound 19 suppressed the proliferation of IMR32 and LAN5 cells with EC50 values of 12.34 and 14.89 μM, respectively, and exerted a moderate inhibition on SK-N-AS cells, without affecting HEK293T cells at 20 μM. Analysis of high-resolution crystal complex structure of compound 19 against WDR5 revealed that it competitively occupied the hydrophobic pocket where V264 was located, which might disrupt the interaction of MYC with WDR5 and further MYC-medicated gene transcription. By performing RNA-seq analysis we demonstrated the differences in molecular action mechanisms of the compound 19 and a WIN site inhibitor OICR-9429. Most interestingly, we established the particularly high synergy rate by combining WBM site inhibitor 19 and the WIN site inhibitor OICR-9429, providing a novel therapeutic avenue for neuroblastoma.

Discovery of Potent Orally Bioavailable WD Repeat Domain 5 (WDR5) Inhibitors Using a Pharmacophore-Based Optimization.

WD repeat domain 5 (WDR5) is a nuclear scaffolding protein that forms many biologically important multiprotein complexes. The WIN site of WDR5 represents a promising pharmacological target in a variety of human cancers. Here, we describe the optimization of our initial WDR5 WIN-site inhibitor using a structure-guided pharmacophore-based convergent strategy to improve its druglike properties and pharmacokinetic profile. The core of the previous lead remained constant while a focused SAR effort on the three pharmacophore units was combined to generate a new in vivo lead series. Importantly, this new series of compounds has picomolar binding affinity, improved cellular antiproliferative activity and selectivity, and increased kinetic aqueous solubility. They also exhibit a desirable oral pharmacokinetic profile with manageable intravenous clearance and high oral bioavailability. Thus, these new leads are useful probes toward studying the effects of WDR5 inhibition.

WIN site inhibition disrupts a subset of WDR5 function

WDR5 nucleates the assembly of histone-modifying complexes and acts outside this context in a range of chromatin-centric processes. WDR5 is also a prominent target for pharmacological inhibition in cancer. Small-molecule degraders of WDR5 have been described, but most drug discovery efforts center on blocking the WIN site of WDR5, an arginine binding cavity that engages MLL/SET enzymes that deposit histone H3 lysine 4 methylation (H3K4me). Therapeutic application of WIN site inhibitors is complicated by the disparate functions of WDR5, but is generally guided by two assumptions—that WIN site inhibitors disable all functions of WDR5, and that changes in H3K4me drive the transcriptional response of cancer cells to WIN site blockade. Here, we test these assumptions by comparing the impact of WIN site inhibition versus WDR5 degradation on H3K4me and transcriptional processes. We show that WIN site inhibition disables only a specific subset of WDR5 activity, and that H3K4me changes induced by WDR5 depletion do not explain accompanying transcriptional responses. These data recast WIN site inhibitors as selective loss-of-function agents, contradict H3K4me as a relevant mechanism of action for WDR5 inhibitors, and indicate distinct clinical applications of WIN site inhibitors and WDR5 degraders.

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.

Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of SMARCB1, which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of SMARCB1 is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the ‘WIN’ site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the growth response of RT cells to WIN site blockade is independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to induce p53 and block RT cell proliferation in vitro. These data reveal a p53-independent action of WIN site inhibitors and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.

Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders.

Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

Impact of WIN site inhibitor on the WDR5 interactome

SUMMARYThe chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.

WDR5 is a conserved regulator of protein synthesis gene expression.

WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the ‘WIN’ site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks—if any—that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis.

Discovery and Structure-Based Optimization of Potent and Selective WD Repeat Domain 5 (WDR5) Inhibitors Containing a Dihydroisoquinolinone Bicyclic Core.

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple chromatin-centric processes. Overexpression of WDR5 correlates with a poor clinical outcome in many human cancers, and WDR5 itself has emerged as an attractive target for therapy. Most drug-discovery efforts center on the WIN site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe discovery of a novel WDR5 WIN site antagonists containing a dihydroisoquinolinone bicyclic core using a structure-based design. These compounds exhibit picomolar binding affinity and selective concentration-dependent antiproliferative activities in sensitive MLL-fusion cell lines. Furthermore, these WDR5 WIN site binders inhibit proliferation in MYC-driven cancer cells and reduce MYC recruitment to chromatin at MYC/WDR5 co-bound genes. Thus, these molecules are useful probes to study the implication of WDR5 inhibition in cancers and serve as a potential starting point toward the discovery of anti-WDR5 therapeutics.

Fragment screening for a protein-protein interaction inhibitor to WDR5.

The WD40-repeat protein WDR5 scaffolds various epigenetic writers and is a critical component of the mammalian SET/MLL histone methyltransferase complex. Dysregulation of the MLL1 catalytic function is associated with mixed-lineage leukemia, and antagonism of the WDR5-MLL1 interaction by small molecules has been proposed as a therapeutic strategy for MLL-rearranged cancers. Small molecule binders of the “WIN” site of WDR5 that cause displacement from chromatin have been additionally implicated to be of broader use in cancer treatment. In this study, a fragment screen with Surface Plasmon Resonance (SPR) was used to identify a highly ligand-efficient imidazole-containing compound that is bound in the WIN site. The subsequent medicinal chemistry campaign—guided by a suite of high-resolution cocrystal structures with WDR5—progressed the initial hit to a low micromolar binder. One outcome from this study is a moiety that substitutes well for the side chain of arginine; a tripeptide containing one such substitution was resolved in a high resolution structure (1.5 Å) with a binding mode analogous to the native tripeptide. SPR furthermore indicates a similar residence time (kd = ∼0.06 s−1) for these two analogs. This novel scaffold therefore represents a possible means to overcome the potential permeability issues of WDR5 ligands that possess highly basic groups like guanidine. The series reported here furthers the understanding of the WDR5 WIN site and functions as a starting point for the development of more potent WDR5 inhibitors that may serve as cancer therapeutics.

Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity

SUMMARYThe chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the “WIN site” of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.

Discovery of Potent 2-Aryl-6,7-dihydro-5 H-pyrrolo[1,2- a]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design.

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5 H-pyrrolo[1,2- a]imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer.