Stephen D. Yanofsky

Allosteric Activation of the Follicle-stimulating Hormone (FSH) Receptor by Selective, Nonpeptide Agonists

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 (EC50s = 20 μm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 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 (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 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.

Analysis of ∅29 and ∅15 genomes by bacterial restriction endonucleases, EcoR1 and HpaI

Abstract The DNAs of the two small Bacillus subtilis phages, ∅29 and ∅15, were analyzed by use of two restriction endonucleases, EcoR1 and HpaI. Five and six EcoR1 generated fragments were obtained from ∅29 and ∅15 genomes, respectively. Eight HpaI generated fragments were obtained from each phage genome, although the sizes of the fragments were considerably different. The molecular weights of each fragment were estimated from the distribution of 32 P-radioactivity in the bands and from the relative electrophoretic mobilities. The five fragments of ∅29 DNA by EcoR1 were isolated in pure form and their sedimentation rates were determined by sucrose gradient centrifugation. The four EcoR1 restriction sites in ∅29 DNA have been mapped by analysis of incomplete digestion products. The ends of the DNA molecules were identified by analysis of EcoR1 digestion fragments of nonprotease-treated ∅29 DNA using agarose gel electrophoresis. From similar experiments, the order of the six fragments of ∅15 DNA produced by EcoR1 digestion is also proposed.

Differing pharmacological activities of thiazolidinone analogs at the FSH receptor

The follicle-stimulating hormone is critical to reproductive success and is an important target for development of novel reproductive therapies. We have recently reported the development of thiazolidinone positive allosteric modulators of the follicle-stimulating hormone receptor. Here, we demonstrate that discrete modifications in the chemical structure of the thiazolidinone agonists produced compounds with different pharmacological properties. Positive allosteric modulators activated adenylate cyclase signaling (Gs). Using an ADP-ribosylation assay we found that both differing glycosylated variants of human FSH (hFSH) and selected thiazolidinone allosteric modulators of the FSHR induce activation of the Gi signaling pathway. Additionally, we observed that some analogs of this class could activate both pathways. These data suggest that the pharmacological activity of thiazolidinone modulators to the FSHR may be due to the ability of these compounds to induce association of the FSHR with either Gs or Gi signaling pathways in an analog-specific manner.

IL-1 antagonist discovery

Interleukin 1 (IL-1) is a pivotal pro-inflammatory cytokine involved in many types of inflammatory and autoimmune diseases [1]. IL-1 production whether initiated by infection or host antigen, initiates an inflammatory cascade which includes the induction of other mediators (e.g. IL-6, IL-8), upregulation of adhesion molecules (e.g. ICAM-1, E-selectin) and potent synergistic activity with other macrophage, proinflammatory cytokines (e.g. tumor necrosis factor) [2,3]. The IL-1 family consists of three structurally related naturally occurring ligands: two agonists, IL-1 α, and IL-1s; and one antagonist, the IL-1 receptor antagonist (IL-1RA) [4-6]. IL-1 is the only cytokine known to have a naturally occurring receptor antagonist. Two distinct cell membrane receptors have been characterized: The type I, IL-1 receptor (IL1RtI) responsible for biological responses; and the type II receptor (IL-1RtII), which is shed, and acts as a decoy to modulate IL-1 activity [7-11]. The existence of a naturally occurring antagonist and a decoy receptor further illustrates the importance and need for the tight regulation of IL-1 activity and its critical role in inflammation.