title: Small molecule inhibition of the TNF family cytokine CD40 ligand through a subunit fracture mechanism.
abstract: BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC(50) = 25 μM and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein's 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.
authors.affiliation: Department of Drug Discovery, Biogen Idec, 12 Cambridge Center, Cambridge, MA 02142, United States. laura.silvian@biogenidec.com.
authors.forename: Laura F
authors.initials: LF
authors.lastname: Silvian
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Friedman
authors.forename: Kathy
authors.initials: K
authors.lastname: Strauch
authors.forename: Teresa G
authors.initials: TG
authors.lastname: Cachero
authors.forename: Eric S
authors.initials: ES
authors.lastname: Day
authors.forename: Fang
authors.initials: F
authors.lastname: Qian
authors.forename: Brian
authors.initials: B
authors.lastname: Cunningham
authors.forename: Amy
authors.initials: A
authors.lastname: Fung
authors.forename: Lihong
authors.initials: L
authors.lastname: Sun
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Shipps
authors.forename: Lihe
authors.initials: L
authors.lastname: Su
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Zheng
authors.forename: Gnanasambandam
authors.initials: G
authors.lastname: Kumaravel
authors.forename: Adrian
authors.initials: A
authors.lastname: Whitty
mesh_terms: D000818:Animals; D019013:CD40 Antigens / Q000276:immunology / Q000302:isolation & purification; D023201:CD40 Ligand / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000302:isolation & purification; D002460:Cell Line; D006224:Cricetinae; D018360:Crystallography, X-Ray; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology / Q000302:isolation & purification; D008958:Models, Molecular; D015394:Molecular Structure; D008970:Molecular Weight; D011485:Protein Binding / Q000187:drug effects; D011725:Pyridines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011759:Pyrrolidines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C570596:2-(2'-(biphenyl-3-ylmethylcarbamoyl)-6,6'-bis(2-(pyrrolidin-1-ylmethyl)pyrrolidine-1-carbonyl)-4,4'-bipyridine-2-carboxamido)-2-cyclohexylacetic acid; D019013:CD40 Antigens; D007141:Immunoglobulin Fc Fragments; D011725:Pyridines; D011759:Pyrrolidines; D023201:CD40 Ligand