Modulation of virus-induced NF-κB signaling by NEMO coiled coil mimics

Abstract

Protein-protein interactions featuring intricate binding epitopes remain challenging targets for synthetic inhibitors. Interactions of NEMO, a scaffolding protein central to NF-κB signaling, exemplify this challenge. Various regulators are known to interact with different coiled coil regions of NEMO, but the topological complexity of this protein has limited inhibitor design. We undertook a comprehensive effort to block the interaction between vFLIP, a Kaposi's sarcoma herpesviral oncoprotein, and NEMO using small molecule screening and rational design. Our efforts reveal that a tertiary protein structure mimic of NEMO is necessary for potent inhibition. The rationally designed mimic engages vFLIP directly causing complex disruption, protein degradation and suppression of NF-κB signaling in primary effusion lymphoma (PEL). NEMO mimic treatment induces cell death and delays tumor growth in a PEL xenograft model. Our studies with this inhibitor reveal the critical nexus of signaling complex stability in the regulation of NF-κB by a viral oncoprotein.

Protein Macrocyclization for Tertiary Structure Stabilization

“…The presented cyclic proteins have mainly found application as biocatalysts or ligands for cellular receptors. There is growing evidence that stabilized tertiary structures can also allow the inhibition of protein‐protein interactions[ 90 , 91 , 92 , 93 ] that are not addressable with classic peptidomimetic approaches. [ 94 , 95 ] Therefore, it can be expected that cyclic proteins will find more fields of application in the future.…”

Recent Advances in Developing Treatments of Kaposi’s Sarcoma Herpesvirus-Related Diseases

“…A conformationally constrained, stapled IKKγ peptide derived from the IKKγ-vFLIP interaction site interferes with the binding of IKKγ to vFLIP and enhances apoptosis in PEL cell lines [91]. Also, a tertiary protein structure mimic of the vFLIPinteraction site in the IKKγ/NEMO helix was able to induce cell death in PEL cell lines and to delay tumor growth in a PEL xenograft mouse model [88]. These findings indicate that it may be feasible to develop small molecule inhibitors targeting the vFLIP-IKKγ/NEMO interaction and showing a therapeutic effect against some KSHV-associated diseases.…”

“…vFLIP is a potent activator of the NF-kB pathway and counteracts Fas-induced apoptosis [83,84]. Silencing vFLIP using siRNA [85] or using NF-kB inhibitors such as Bay 11-7082 [83,[86][87][88] induces PEL cell apoptosis, suggesting that vFLIP may also represent an attractive therapeutic target. In order to activate the NF-kB pathway, vFLIP directly interacts with IKKγ/NEMO, a key player in the canonical NF-kB pathway [85,89].…”

WT1 Tumor Antigen is Overexpressed in Kaposi Sarcoma and is Regulated by KSHV vFLIP

“…Plasmids and antibodies WT vFLIP and vFLIP NFκB dead mutant inducible cell lines were established using previously described doxycycline inducible lentiviral vectors 42 . Stable transduced cell lines were established in HUVEC and HuARLT-1 by puromycin selection at 1µg/ml and doxycycline 1-2µg/ml for inducible expression of WT vFLIP and vFLIP null NFκB.…”