Brian J. Arey

Characterization of Bone Structure in Leptin Receptor-Deficient Zucker (fa/fa) Rats

To investigate the role of leptin in bone formation, the skeleton of the obese female leptin receptor‐deficient Zucker rat was examined using pQCT, μCT, and histomorphometry. A trend toward decreasing structural and bone formation parameters in these rats as they age suggest that leptin has a small positive effect on bone.

Synthesis of (bis)Sulfonic acid, (bis)Benzamides as follicle-Stimulating hormone (FSH) antagonists

Screening efforts identified (bis)sulfonic acid, (bis)benzamides (1-3) as compounds that interact with the follicle stimulating-hormone receptor (FSHR) and inhibit FSH-stimulated cAMP accumulation with IC(50) values in the low micromolar range. Structure-activity relationship studies using novel analogues of 1-3 revealed that two phenylsulfonic acid moieties were necessary for activity and that the carbon-carbon double bond of the stilbene sub-series was the optimum spacer connecting these groups. Selected analogues (2, 14, and 50) were also able to block FSHR-dependent estradiol production in rat primary ovarian granulosa cells and progesterone secretion in a clonal mouse adrenal Y1 cell line. IC(50) values for these compounds in these assays were in the low micromolar range. Optimization of the benzoic acid side chains of 1-3 led to gains in selectivity versus activity at the thyroid stimulating hormone (TSH) receptor (TSHR). For instance, while stilbene (bis)sulfonic acid congener 2 was only 10-fold selective for FSHR over TSHR, analogue 50 with an IC(50) value of 0.9 microM in the FSHR-cAMP assay was essentially inactive at 30 microM in the TSHR-cAMP assay.

Discovery and Structure−Activity Relationships of Trisubstituted Pyrimidines/Pyridines as Novel Calcium-Sensing Receptor Antagonists

The trisubstituted pyrimidine 1 was identified through high-throughput screening as a novel calcium-sensing receptor (CaSR) antagonist. Small molecule CaSR antagonists and/or negative allosteric modulators have the potential to act as an anabolic agent for the treatment of osteoporosis. The investigation of structure-activity relationships around 1 resulted in the identification of 18c and 18d, which showed efficacy at promoting PTH release in vivo and exhibited improved potency and solubility over the original lead 1.

Are circulating gonadotropin isoforms naturally occurring biased agonists? Basic and therapeutic implications

The gonadotropins, luteinizing hormone, human chorionic gonadotropin and follicle-stimulating hormone, are key regulators of reproduction. As a result of this function, they have been the focus of research for many years. Isolated or recombinant proteins have been successfully used therapeutically for the treatment of infertility; and, in the case of compounds that block gonadotropin activity, for their potential utility in contraception. Until recently, selective small molecules modulating gonadotropin receptor activity have proven difficult to identify. The gonadotropins are glycoproteins that are released into the plasma as differently glycosylated isoforms and bind to specific G protein-coupled receptors. The degree of glycosylation on the gonadotropins has been shown to be important for the biological activities of these hormones and is differentially regulated depending on the steroidal status. Recent data from the study of glycosylated variants of LH, hCG and FSH have revealed that these isoforms have distinct signaling properties that allow for gonadotropin pleiotropic signals to be transduced effectively at the level of the receptor. Thus, glycosylated variants of the gonadotropins behave as biased agonists. Recently, newly developed, small molecule, synthetic allosteric compounds have been identified that are capable of mimicking this biased signaling. This opens the door to development of orally available, drug-like therapies for reproductive disorders that offer similar pleiotropic richness as that offered by the complex, endogenous hormones.

Allosteric modulators of glycoprotein hormone receptors: discovery and therapeutic potential

The glycoprotein hormones, luteinizing hormone, follicle-stimulating hormone and thyroid stimulating hormone, are important regulators of reproductive and metabolic processes. However, because of the nature of their ligand-receptor interactions that contain multiple contact sites, classical small molecule drug discovery strategies have not been successful. However, recent advances in screening and combinatorial chemistry strategies have identified chemical series that act allosterically as positive, negative or mixed modulators of the glycoprotein hormone receptors. This review will discuss the discovery and highlight the currently known series of allosteric modulators to this therapeutically important family of G-protein coupled receptors. Lastly, we will present potential mechanisms whereby the different series could modulate receptor function in the context of currently held theory and known structure of G protein-coupled receptors.

A global model to define the behavior of partial agonists (bell-shaped dose-response inducers) in pharmacological evaluation of activity in the presence of the full agonist

The dose-response models for full agonists and for a particular type of partial agonist can be described by sigmoidal curves and bell-shaped curves, respectively. The methods currently used to evaluate the interaction of a full agonist and a partial agonist require a large number of experimental units and base their analysis on nonlinear regression analysis, which may not be statistically appropriate. We propose an appropriate design and a global nonlinear model to evaluate such interactions. The new model allows us to estimate the interaction parameters and the parameters that characterize the individual partial agonist curve and the full agonist curve.

Editorial: The Physiology and Pharmacology of Leucine-rich Repeat GPCRs.

The Editorial on the Research Topic The Physiology and Pharmacology of Leucine-rich Repeat GPCRs G protein-coupled receptors represent a large family of proteins that act as receptors for many types of physiological ligands, including peptides, metabolites, and lipids. These receptors are important for understanding physiology since they contribute to the regulation of all major organ systems. Additionally, they are also a key focus for the development of therapeutics for the treatment of pathophysiology and are still recognized as the most druggable class of macromolecules today. GPCRs are classified into separate subfamilies (Classes A, B, and C) based on protein sequence homology in their transmembrane domains. Within the Class A family of GPCRs, these receptors can be further placed into sub-groups based on other structural features and similarities in function. In this Special Topic for Frontiers in Endocrinology: Molecular and Structural Endocrinology, we have focused on a subfamily of Class A GPCRs, the leucine-rich repeat family of receptors (LGR). The Physiology and Pharmacology of Leucine-rich Repeat GPCRs captures the continuum of structure to function, agonist to effector, and reproduction to metabolism that provides an overview of this important family of receptors. The LGRs are characterized by the leucine-rich repeat structural motif (1) that provides the rigid structure of their large extracellular domains. The predicted heptahelical transmembrane domains and their sequence homology in this region with other receptors classify them as Class A GPCRs (2). Furthermore, there are currently three sub-groups of LGRs recognized. The Type A receptors have been extensively studied and are receptors for the pituitary and placental glycoprotein hormones. The endogenous ligands for the Type B (R-spondins) and C (relaxin) receptors have only recently been identified and rapid progress has been made that has advanced understanding of their structure and function. These receptors are important mediators in the regulation of diverse physiological process, such as reproduction, cardio-renal function, cell growth, and stem cell differentiation. Within this Special Topic, we seek to provide an understanding of this family of receptors while addressing both future opportunities and challenges that lay ahead. Petrie et al. provide an overview of LGRs in terms of structure and organization that places this family of receptors within the larger context. This review provides in depth knowledge of the RXFP1 and RXFP2 receptors whose cognate ligands are two insulin- related peptides, H2 relaxin, and INSL3. Thus, this review while calling out the uniqueness of the Type C LGRs RXFP1 and RXFP2, also introduces us nicely to the three Types of LGRs based on the size of their leucine-rich repeat extracellular domains. Continuing on this theme of structure and function, activation of the most well studied of the LGRs, the gonadotropin receptors, is reviewed. The structural basis of activation of the gonadotropin receptors in the absence of a full crystal structure remains an enigma. However, we do know that the large extracellular domains are the binding site of the large heterodimeric ligands for these receptors. A remaining question is the potential role of the other parts of the receptor structure in determining function. Banerjee and Mahale provide evidence using site-directed mutagenesis that signaling of the LH receptor is dependent on specific residues of the extracellular loops. Furthermore, Grzesik et al. demonstrate that differences in signaling by the two physiological ligands of the LH receptor, are at least in part, mediated by the hinge region of the extracellular domain. While both receptors coexist in the same cell during folliculogenesis, it is unclear how their agonist-induced signals are parsed out. Further refining this thought, this paper addresses how one receptor can bind two nearly identical ligands and produce two different signaling profiles. It turns out that both hormone and extracellular loops coordinate to produce the breadth of nuance seen in signaling of these receptors. This seems a critical point if small molecules are to be developed which mimic some or all of these signaling patterns. The physiological and therapeutic importance of the Type B and C receptors is discussed as well. The importance of RXFP1 in cancers is exemplified by Thanasupawat et al. in a review of new ligands for RXFP1 other than the canonical relaxin and INSL3; specifically C1q/TNF-related proteins and the role of RXFP1 as a brain cancer promoter. The theme is further explored with an in-depth look by Li et al. of the Type B LGR4 where we learn that this receptor that is critical for developmental signaling and tissue homeostasis has as its ligand, R-spondins. We learn that R-spondins are the sole secreted potentiators of Wnt signaling and stem cell maintenance and appreciate that the Type B LGRs do not signal via G-proteins but do interact with the FZD–LRP complex to stimulate unique signaling pathways. The LGR receptor family has been the subject of drug development for decades. Focusing on improving the natural ligand for therapeutics based on these receptors, a perspective is provided by Szkudlinski on a journey toward the successful development of superagonists of the thyroid-stimulating hormone receptor, a Type A LGR. This review shines light on the potential of utilizing the naturally occurring ligands as a scaffold for engineering by structure-based drug design to develop “super biosimilars.” This is contrasted with the development of small molecule agonists and antagonists that act at this same class of LGR Type A as described by Nataraja et al. Here, we learn that it is possible to bypass the complicated interactions of heterodimeric ligands and the large extracellular domains of the LGR Type A receptors, to effect activation or inhibition with molecules a fraction of the size of the natural ligands. Although this represents a clear step forward in our ability to develop small molecule agonists and antagonists to these receptors, it is not without its challenges. In what would seem to be a reasonable assumption that cAMP as readout would predict efficacy in vivo, this is shown to not necessarily be the case. In the end, primary cells and iterative testing reveal the true candidate. This concept is further exemplified in the article by Huang et al. An overwhelming majority of the preclinical animal testing for relaxin treatment includes rodent models and, thus, the inability of small molecule agonists to activate the mouse receptor has hampered preclinical studies. In a search for animal models to study RXFP1 small molecule agonists as potential acute heart failure therapeutics, it was determined that non-human primate and porcine species could be used but the standard laboratory mouse model was unable to respond to the lead compound! These examples illustrate how development of small molecule therapeutics is fraught with potential pitfalls and how appropriate models are needed for screening and selection of leads. Finally, in an era of optogenetics and real-time inquiry, the use of transgenic methods may yield some recourse. Narayan describes how, for the gonadotropin receptors, a combination of knockout and knock-in approaches can yield novel mouse models that either simulates human disease or tests whether genomic variants can explain disease. In this regard, the luteinizing hormone receptor has been very well studied using transgenic animals to better understand the effect of mutations causing constitutive activation as a model for Familial Male Precocious Puberty. Advanced methods in cellular imaging are also available which will aid in the study of LGR signaling. Certainly in the area of the gonadotropin receptors, these methods have contributed to an understanding that, although the canonical cAMP pathway is operative in the gonadotropin receptors, additional pathways are likely at play. Thus, Ayoub et al. describes the use of bioluminescence resonance energy transfer (BRET) to study activation of gonadotropin receptors in living cells. Using this method, they confirm that these receptors exhibit biased agonism. It has been nearly a century since Smith discovered the relationship between pituitary extracts and follicular development and 60 years since Hisaw discovered relaxin as a hormone that could cause a loosening of the pubic symphysis prior to parturition. These seminal findings have led to the identification of the importance of these peptides on the physiology of the reproductive system but have also ultimately revealed a more complex endocrine role of these hormones and the identification of a unique family of receptors. Research over the ensuing decades has revealed that the LGRs exert varied and comprehensive controls on processes that include but are certainly not limited to reproduction and point to their potential as therapeutic targets to treat disease.

Biasing Receptor Tyrosine Kinase Signaling Pathways

Receptor tyrosine kinases (RTK) are a relatively small family of integral membrane receptors. However, RTKs comprise nodes at the center of vastly complex signaling networks involving hundreds of signaling proteins. These signaling networks have essential functions in virtually all aspects of animal cell growth, development, and differentiation. Dysregulation of these networks has been implicated in neoplastic and other diseases. The very complexity of RTK-mediated signaling creates opportunities for the identification of pathway-biased synthetic peptide or small molecule ligands that can selectively modulate a portion of an RTK signaling network. Such pathway-biased molecules may afford exquisite selectivity and utility in the treatment of diseases associated with disorders in RTK signaling.

An Historical Introduction to Biased Signaling

Organisms have evolved the ability to sense and respond to external chemical and physical cues through specific receptors. Within cells, receptors interact with internal and external partners in a cell-specific fashion in order to develop tissue specificity of function. The recognition of ligands and the ultimate activation of signal transduction pathways by receptors are dependent upon the conformational fluidity of the receptor. Nature has evolved several different types of receptors that we categorize based upon their structure and signaling activity; however, the general principles of activation of intracellular signals are similar. Ligands to nuclear receptors and G protein coupled receptors have been found to induce ligand-specific signal-transduction pathways, most recently termed biased signaling. Given the importance of receptor conformation in signal transduction activation, it is likely that biased signaling also occurs with cytokine receptor/receptor tyrosine kinases and ligand-gated ion channels. Indeed, recent observations have found that these receptor classes also exhibit ligand-dependent biased signaling properties. In this chapter, we will briefly discuss the evolution of pharmacology and the receptor theory up to elucidation of biased signaling, and provide a general overview of how biased signaling can impact physiology, pharmacology and the development of new therapeutics. Subsequent chapters will provide more in depth information on how biased signaling affects each receptor class, how biased signaling is impacted by the intracellular milieu, and how elucidation of this phenomenon can be harnessed to better understand physiology and design improved therapeutics.

Conformational Mechanisms of Signaling Bias of Ion Channels

Ion channels represent some of the most ancient mechanisms for cells to sense and respond to their external environment. The physiological importance of these proteins is suggested by their evolutionary stability and through numerous naturally occurring disease states derived from channel dysfunction. The primary role of ion channels is to regulate the passage of ions across the cell membrane, and the alteration of intracellular ion concentration can be seen as the primary signaling mechanism of ion channels. Ion channels display characteristic ion selectivity that is provided by the very structure and juxtaposition of the ion channel subunits within the cell membrane. However, recent observations have found that some ion channels demonstrate altered ion selectivity that is time- and conformationally dependent. Additionally, some classes of ion channels have been shown to activate signaling enzymes more often considered as part of the signal transduction pathways of other receptors. Thus, these observations suggest that ion channels have the capability to display functional selectivity (biased signaling) recently attributed to nuclear receptors or G protein-coupled receptors. Moreover, some channels have been found to have altered ion selectivity or activation of associated signaling enzymes in a ligand-dependent fashion. These revelations not only provide a new perspective on the function of ion channels but also expand the potential roles of ion channels in modulating cell activity. Perhaps more importantly, biased signaling in ion channels may provide another opportunity to more efficiently exploit this class of receptor for therapeutic benefit.