Inflammation, regardless of whether it is provoked by infection or by tissue damage, starts with the activation of macrophages which initiate a cascade of inflammatory responses by producing the cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (ref. 1). Three naturally occurring ligands for the IL-1 receptor (IL1R) exist: the agonists IL-1alpha and IL-1beta and the IL-1-receptor antagonist IL1RA (ref. 2). IL-1 is the only cytokine for which a naturally occurring antagonist is known. Here we describe the crystal structure at 2.7 A resolution of the soluble extracellular part of type-I IL1R complexed with IL1RA. The receptor consists of three immunoglobulin-like domains. Domains 1 and 2 are tightly linked, but domain three is completely separate and connected by a flexible linker. Residues of all three domains contact the antagonist and include the five critical IL1RA residues which were identified by site-directed mutagenesis. A region that is important for biological function in IL-1beta, the 'receptor trigger site' is not in direct contact with the receptor in the IL1RA complex. Modelling studies suggest that this IL-1beta trigger site might induce a movement of domain 3.

Interleukin-I (IL-1) molecules are cytokines involved in the acute-phase response against infection and injury. Three naturally occurring IL-1 molecules are known, two agonists: IL-la and IL-1P, and one antagonist, the 1L-1 receptor antagonist (IL-lra). Although IL-1 action protects the organism by enhancing the response to pathogens, its overproduction can lead to pathology and has been implicated in disease states that include septic shock, rheumatoid arthritis, graft versus host disease and certain leukemias. The crystal structure of IL-lra has been solved at 0.21-nm resolution by molecular replacement using the ILlp structure as a search model. The crystals contain two independent IL-lra molecules which are very similar, IL-lra has the same fold as IL-la and IL-ID. The fold consists of twelve P-strands which form a six-stranded P-barrel, closed on one side by three P-hairpin loops. Cys69 and Cysll6 are linked via a disulfide bond and Pro53 has been built in the cis-conformation. Comparison of the IL-lra structure with the IL-la and IL-1P structures present in the Protein Data Bank shows that a putative receptor interaction region, involving the N-terminus up to the beginning of strand pl and the loops D and G, is very different in the three IL-1 molecules. Other putative interaction regions, as identified with mutagenesis studies, are structurally conserved and rigid, allowing precise and specific interactions with the IL-1 receptor.

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC 50's ‫؍‬ 20 M) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC 50 ‫؍‬ 2 nM) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC 50 ‫؍‬ 980 nM) and estradiol production from primary rat ovarian granulosa cells (EC 50 ‫؍‬ 10.5 nM). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy. Follicle-stimulating hormone (FSH)2 is a glycoprotein hormone produced by the anterior pituitary that plays a key role in stimulating ovulation and spermatogenesis. Like other members of the glycoprotein hormone family (luteinizing hormone, chorionic gonadotropin, and thyroid-stimulating hormone (TSH)), FSH is a heterodimeric protein of ϳ30,000 Da that consists of a common ␣-subunit joined noncovalently to a hormone-specific ␤-subunit. FSH activity is mediated through binding to the FSH receptor (FSH-R), which belongs to family I of the large 7-transmembrane-spanning, G protein-coupled receptor (GPCR) superfamily. The glycoprotein hormone receptors are unique among the members of the GPCR family, because they recognize protein hormones and are dominated by a large N-terminal extracellular region (366 amino acids in the case of the FSH-R) that is the predominant site of hormone binding (1, 2) and is required for signal transduction.The binding of FSH to its receptor results in the activation of adenylyl cyclase through heterotrimeric G proteins. Interaction of the activated FSH-R with G s initiates the cAMP signaling cascade (3). The ability of the FSH-R to activate adenylyl cyclase was exploited to generate a Chinese hamster ovary (CHO) FSH-R reporter cell line (4 -6). Screening of a collection...

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Two families of peptides that specifically bind the extracellular domain of the human type I interleukin 1 (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-la to the type I IL-1 receptor with IC5o values of 45-140 ,uM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.