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.

Analysis of host gene expression changes reveals distinct roles for the cytoplasmic domain of the Epstein-Barr virus receptor/CD21 in B-cell maturation, activation, and initiation of virus infection.

UNLABELLED: Epstein-Barr virus (EBV) attachment to human CD21 on the B-cell surface initiates infection. Whether CD21 is a simple tether or conveys vital information to the cell interior for production of host factors that promote infection of primary B cells is controversial, as the cytoplasmic fragment of CD21 is short, though highly conserved. The ubiquity of CD21 on normal B cells, the diversity of this population, and the well-known resistance of primary B cells to gene transfer technologies have all impeded resolution of this question. To uncover the role(s) of the CD21 cytoplasmic domain during infection initiation, the full-length receptor (CD21=CR), a mutant lacking the entire cytoplasmic tail (CT), and a control vector (NEO) were stably expressed in two pre-B-cell lines that lack endogenous receptor. Genome-wide transcriptional analysis demonstrated that stable CD21 surface expression alone (either CR or CT) produced multiple independent changes in gene expression, though both dramatically decreased class I melanoma-associated antigen (MAGE) family RNAs and upregulated genes associated with B-cell differentiation (e.g., C2TA, HLA-II, IL21R, MIC2, CD48, and PTPRCAP/CD45-associated protein). Temporal analysis spanning 72 h revealed that not only CR- but also CT-expressing lines initiated latency. In spite of this, the number and spectrum of transcripts altered in CR- compared with CT-bearing lines at 1 h after infection further diverged. Differential modulation of immediate early cellular transcripts (e.g., c-Jun and multiple histones), both novel and previously linked to CD21-initiated signaling, as well as distinct results from pathway analyses support a separate role for the cytoplasmic domain in initiation of intracellular signals. IMPORTANCE: Membrane proteins that mediate virus attachment tether virus particles to the cell surface, initiating infection. In addition, upon virus interaction such proteins may transmit signals to the interior of the cell that support subsequent steps in the infection process. Here we show that expression of the Epstein-Barr virus B-cell attachment receptor, CD21, in B cells that lack this receptor results in significant changes in gene expression, both before and rapidly following EBV-CD21 interaction. These changes translate into major signaling pathway alterations that are predicted to support stable infection.

Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus.

Kaposi sarcoma is considered a neoplasm of lymphatic endothelium infected with Kaposi sarcoma-associated herpesvirus. It is characterized by the expression of lymphatic lineage-specific genes by Kaposi sarcoma tumor cells. Here we show that infection of differentiated blood vascular endothelial cells with Kaposi sarcoma-associated herpesvirus leads to their lymphatic reprogramming; induction of approximately 70% of the main lymphatic lineage-specific genes, including PROX1, a master regulator of lymphatic development; and downregulation of blood vascular genes.

EBV attachment stimulates FHOS/FHOD1 redistribution and co-aggregation with CD21: formin interactions with the cytoplasmic domain of human CD21.

CD21 is a multifunctional receptor for Epstein-Barr virus (EBV), for C3dg and for CD23. Upon engagement of immune complexes CD21 modulates immunoreceptor signaling, linking innate and adaptive immune responses. The mechanisms enabling CD21 to independently relay information between the exterior and interior of the cell, however, remain unresolved. We show that formin homologue overexpressed in spleen (FHOS/FHOD1) binds the cytoplasmic domain of human CD21 through its C terminus. When expressed in cells, EGFP-FHOS localizes to the cytoplasm and accumulates with actin in membrane protrusions. Plasma membrane aggregation, redistribution and co-localization of both proteins are stimulated when EBV (ligand) binds CD21. Though widely expressed, FHOS RNA is most abundant in the littoral cell, a major constituent of the red pulp of human spleen believed to function in antigen filtration. Formins are molecular scaffolds that nucleate actin by a pathway distinct from Arp2/3 complex, linking signal transduction to actin reorganization and gene transcription. Thus, ligand stimulation of FHOS-CD21 interaction may transmit signals through promotion of cytoskeletal rearrangement. Moreover, formin recruitment to sites of actin assembly initiated by immunoreceptors could be a general mechanism whereby co-receptors such as CD21 modulate intracellular signaling.

Characterization of Kaposi sarcoma-associated herpesvirus/human herpesvirus-8 infection of human vascular endothelial cells: early events.

Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) is causally associated with Kaposi sarcoma (KS). The absence of a cell culture system that effectively reproduces the composite mechanisms governing initiation and maintenance of HHV-8 infection (lytic and latent) in KS endothelial cells, however, has left important questions unanswered. Here, we report a culture system in which the earliest events that accompany HHV-8 infection could be surveyed in primary endothelial cells. Binding of HHV-8 to microvascular dermal endothelial cells (MVDECs) was directly compared with other primary target cells implicated in HHV-8-associated diseases. Virus attachment, fusion, internalization and transport within MVDECs was monitored by electron microscopy. Studies of genome configuration revealed that rapid circularization of the viral DNA occurred on entry, though by 72 hours after infection linear DNAs accumulated and early as well as late lytic RNAs (T1.1, K8.1) could be detected. The latency transcripts (LT1/LT2) were first detected on day 8, demonstrating that both lytic and latent infection were initiated. Although most lytic transcripts accrued until passage, open-reading frame-74 RNAs fluctuated with a fixed periodicity, suggesting that early replication after infection of MVDECs was synchronous.

The crystal structure of human CD21: Implications for Epstein-Barr virus and C3d binding.

Human complement receptor type 2 (CD21) is the cellular receptor for Epstein-Barr virus (EBV), a human tumor virus. The N-terminal two short consensus repeats (SCR1-SCR2) of the receptor interact with the EBV glycoprotein gp350/220 and also with the natural CD21 ligand C3d. Here we present the crystal structure of the CD21 SCR1-SCR2 fragment in the absence of ligand and demonstrate that it is able to bind EBV. Based on a functional analysis of wild-type and mutant CD21 and molecular modeling, we identify a likely region for EBV attachment and demonstrate that this region is not involved in the interaction with C3d. A comparison with the previously determined structure of CD21 SCR1-SCR2 in complex with C3d shows that, in both cases, CD21 assumes compact V-shaped conformations. However, our analysis reveals a surprising degree of flexibility at the SCR1-SCR2 interface, suggesting interactions between the two domains are not specific. We present evidence that the V-shaped conformation is induced by deglycosylation of the protein, and that physiologic glycosylation of CD21 would result in a more extended conformation, perhaps with additional epitopes for C3d binding.