Broad Institute Epstein-Barr LMP-1 Inhibitor Probe Project
The aim of this study is to elucidate the molecular mechanisms used by LMP1 during EBV oncogenesis. We have adapted a suite of siRNA assays for use with small molecules and derived an LMP1 TES2-inducible HEK293 cell line with a stably integrated NF-kB luciferase reporter, which was validated through RNAi screening and whole-genome transcription profiling. This inducible system allows us to focus on primary LMP1 effects on a NF-kB-dependent reporter in the presence of each compound and to study small molecule inhibitor effects prior to the onset of secondary effects. ..more
Elliot Kieff,Brigham & Women's Hospital,firstname.lastname@example.org,617-525-4252
Benjamin Guwurz,Brigham & Women's Hospital,BGEWURZ@PARTNERS.ORG
The aim of this study is to elucidate the molecular mechanisms used by LMP1 during EBV oncogenesis. We have adapted a suite of siRNA assays for use with small molecules and derived an LMP1 TES2-inducible HEK293 cell line with a stably integrated NF-kB luciferase reporter, which was validated through RNAi screening and whole-genome transcription profiling. This inducible system allows us to focus on primary LMP1 effects on a NF-kB-dependent reporter in the presence of each compound and to study small molecule inhibitor effects prior to the onset of secondary effects.
There is significant therapeutic potential for a highly LMP1-selective compound. An LMP1-specific inhibitor could spare pitfalls that have limited NF-kB inhibitor development by sparing canonical NF-kB pathways integral to innate and adaptive immune responses. An LMP1/NF-kB selective probe might be used as a novel chemotherapeutic agent to treat established LMP1+ malignancies. Alternatively, an LMP1-selective probe could be used prophylactically to prevent EBV-associated lymphoproliferative disorders during periods of profound immunosuppression.
There are three major applications for molecular probes discovered through this screening process:
1) identification of LMP1/NFkB pathway components that have thus far not been elucidated;
2) development of probes that inhibit NF-kB components for which antagonists are currently unavailable; and
3) development of therapeutic agents for human malignancies that depend on LMP1/NF-kB for survival.
Target activity in primary screen: IC 50 < 1 uM
Selectivity: IC 50 > 20 uM in TNF-a and Il1-b stimulated cells
Biological Mode of Action: cellular
Cellular Toxicity: Selectively toxic to LCL's at IC50 < 1 uM
Functional Groups to be avoided:
1. Chemically reactive groups
2.Metabolically labile groups
3. pH sensitive or hydrolytically unstable groups
Chemical Solubility Criteria: > 20 uM in PBS
Epstein-Barr Virus, LMP1, Latent Membrane Protein 1, NF-kappaB, NF-kB, lymphoproliferative disorder
Nuclear Factor kappaB (NF-kappaB) is a family of transcription factors implicated in the development and progression of multiple cancers. NF-kappaB drives both growth, anti-apoptotic and metabolic programs important for cell proliferation, differentiation and survival. Multiple receptors can activate NF-kappaB through 'canonical' or 'non-canonical' signal transduction pathways. Multiple immune cytokines activate NF-kappaB, including the pro-inflammatory cytokines TNF-a and IL1-b. NF-kappaB activation by oncogenic viruses or as a consequence of mutations in human cancers frequently underlies malignant cell growth. NF-kappaB hyperactivation is a salient feature of numerous lymphoproliferative disorders, including diffuse large B cell lymphoma (DLBCL), Hodgkin disease, and multiple myeloma. The genetic basis for elevated NF-kappaB activity in these cancers remains largely unknown, though appears to arise at least in part from mutation of key NF-kappaB pathway regulators. NF-kappaB pathway components are therefore promising targets for the development of rational chemotherapeutic agents.
Epstein-Barr Virus (EBV) is a ubiquitous Herpesvirus that is an important cause of Hodgkin's Disease, other Lymphoproliferative Diseases, and Nasopharyngeal Carcinoma. EBV infection mimics NF-kappaB hyperactivation states present in many malignancies. The EBV oncoprotein LMP1 (Latent Membrane Protein 1) constitutively activates both canonical and noncanonical NF-kB pathways in a ligand-independent fashion. LMP1 is expressed in most EBV-associated lymphoproliferative and epithelial malignancies. LMP1 activates NF-kappaB via two cytoplasmic signaling domains. The membrane proximal "TES1" domain activates a non-canonical NF-kappaB pathway, while the membrane distal "TES2" domain activates canonical NF-kappaB. EBV-transformed B cells require persistent NF-kappaB activation for growth and survival, and rapidly undergo apoptosis upon expression of a dominant-negative NF-kappaB super-repressor. Cellular factors that mediate LMP1 effects therefore constitute potential targets for rational therapeutic intervention.
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