Discovery of a dual action first-in-class peptide that mimics and enhances CNS-mediated actions of thyrotropin-releasing hormone

Abstract

Thyrotropin-releasing hormone (TRH) displays multiple CNS-mediated actions that have long been recognized to have therapeutic potential in treating a wide range of neurological disorders. Investigations of CNS functions and clinical use of TRH are hindered, however, due to its rapid degradation by TRH-degrading ectoenzyme (TRH-DE). We now report the discovery of a set of first-in-class compounds that display unique ability to both potently inhibit TRH-DE and bind to central TRH receptors with unparalleled affinity. This dual pharmacological activity within one molecular entity was found through selective manipulation of peptide stereochemistry. Notably, the lead compound of this set, l-pyroglutamyl-l-asparaginyl-l-prolyl-d-tyrosyl-d-tryptophan amide (Glp-Asn-Pro-d-Tyr-d-TrpNH₂), is effective in vivo at producing and potentiating central actions of TRH without evoking release of thyroid-stimulating hormone (TSH). Specifically, this peptide displayed high plasma stability and combined potent inhibition of TRH-DE (K_i 151 nM) with high affinity binding to central TRH receptors (K_i 6.8 nM). Moreover, intraperitoneal injection of this peptide mimicked and augmented the effects of TRH on behavioural activity in rat. Analogous to TRH, it also antagonized pentobarbital-induced narcosis when administered intravenously. This discovery provides new opportunities for probing the role of TRH actions in the CNS and a basis for development of novel TRH-based neurotherapeutics.

Introduction

Thyrotropin-releasing hormone (TRH) is an important multifunctional signaling molecule in the CNS (Bauer et al., 1999). It is recognized that this naturally occurring tripeptide (Glp-His-ProNH₂) has considerable influence on the activity of a number of neurobiological systems through CNS-mediated actions that are independent of the hypothalamic-pituitary-thyroid (HPT) axis (Gary et al., 2003). These central effects of TRH have attracted widespread attention particularly because of their potential therapeutic application across a broad platform of neurological conditions, such as CNS trauma, epilepsy, depression, spinocerebellar degeneration (SCD), Alzheimer's Disease and feeding disorders, for which there exists a pressing need for new and effective therapies (for review see Faden and Salzman, 1992, Kubek and Garg, 2002, Vetulani and Nalepa, 2000, Luo et al., 2002, Bennett et al., 1997, Nillni and Sevarino, 1999, Horita, 1998, Steward et al., 2003).

Critically, the rapid inactivation of TRH by the neuropeptide-specific ectopeptidase TRH-degrading ectoenzyme (TRH-DE, EC 3.4.19.6), also known as pyroglutamyl aminopeptidase II (PPII), impedes both therapeutic use of TRH (Kelly, 1995) and research into the biological functions of TRH in the CNS. In attempts to overcome this constraint much emphasis has been placed on the development of degradation-stabilized agonist analogs of TRH (Gary et al., 2003, Kelly, 1995, Horita, 1998, Metcalf, 1982). To accentuate CNS-mediated actions of TRH, a major goal of agonist analog design has been to separate central from endocrine actions; accordingly, such analogs would ideally bind with high affinity to central TRH receptors but have minimal effect on TSH release (Gary et al., 2003, Kelly, 1995, Horita, 1998, Metcalf, 1982). To date, all reported degradation-stabilized TRH analogs have reduced affinity compared to TRH for central TRH receptors and most retain some TSH-releasing activity. Interestingly, several TRH-like peptides, in which the central histidyl residue is replaced by another amino acid, have been shown to display central TRH effects without evoking TSH release (Vonhof et al., 1990, Lloyd et al., 2001, Pekary et al., 2005). Such peptides, however, have been shown to display poor affinity for central TRH receptors in competitive radioligand binding studies (Kelly et al., 2002, Hinkle et al., 2002). Further, it has been reported that their properties limit CNS access and hence neuropharmacological application (Prokai et al., 1999). As a consequence of their low affinity for known TRH receptors, it has been suggested that the central actions of these TRH-like peptides may be mediated by hitherto unidentified receptors (Hinkle et al., 2002, Colson and Gershengorn, 2006). Two TRH receptor subtypes have been identified in rat thus far: TRHR1 and TRHR2 (Cao et al., 1998, Itadani et al., 1998). A third TRH receptor subtype has recently been cloned in Xenopus laevis (xTRHR3) (Bidaud et al., 2002) and possibly a mammalian homologue of this receptor, or some other orphan receptor, may mediate CNS effects of TRH-related peptides (Lu et al., 2003). TRHR1 and TRHR2 have a distinct amino acid sequence and distribution pattern, but both display a similar high-affinity for [³H][3-Me-His²]TRH. Hence, [³H][3-Me-His²]TRH cannot be used to discriminate between these two known TRH receptor subtypes. TRHR1 is believed to be responsible for mediating the endocrine actions of TRH and TRHR2 appears to be strategically located to mediate central TRH actions, though it has not been detected in humans (Colson and Gershengorn, 2006). Subtype-selective analogues would be useful for probing the functions of these two receptors. Recently, analogues with reduced binding affinity in comparison to TRH have been identified that display a degree of selectivity for TRHR2 (Kaur et al., 2007).

The use of TRH-DE inhibitors represents an alternative approach to enhance the neuropharmacological actions of TRH (Charli et al., 1989, Kelly et al., 2000). TRH-DE is distributed primarily in serum and CNS tissues; peripheral organs, with the exception of lung and liver, are almost devoid of activity (Bauer et al., 1999). This enzyme is a particularly attractive therapeutic target because it displays extraordinary functional selectivity for TRH. Moreover, TRH is not degraded by any other enzyme in a position to affect TRH signaling (Turner, 1997). Consequently, TRH-DE inhibitors should enhance the biological actions of TRH exclusively.

We have previously shown that replacement of the central His residue in TRH with Asn confers resistance to proteolysis by TRH-DE and efficient inhibition of this enzyme (Kelly et al., 2000). Further, we have demonstrated that addition of hydrophobic l-amino acids to the C-terminus of Glp-Asn-ProNH₂ substantially improves inhibitory potency (Kelly et al., 2005). Although Glp-Asn-ProNH₂ is not susceptible to hydrolysis by general amino- and carboxy-peptidases because of the presence of an N-terminal Glp residue and an amidated C-terminus (Turner, 1997), inclusion of l-amino acids in the extension may be expected to render such peptides susceptible to degradation. Replacement of l-amino acids by d-amino acids is a common method used to confer stability to proteolysis, as well as to stabilize bioactive conformations (Hruby, 2002). Indeed, replacement of the hydrophobic l-amino acid residues with their d-isomers in C-terminally extended analogs of Glp-Asn-ProNH₂ was a key development leading to the discovery of the set of compounds reported herein, which display unique capability not only to inhibit TRH-DE with high potency, but also bind to central native TRH receptors with an affinity greater than that of all other previously described TRH analogs. Compounds with such potent dual-action have not been described before and are of both experimental and therapeutic interest. Moreover, we demonstrate that one member of this new class of compound, Glp-Asn-Pro-d-Tyr-d-TrpNH₂, is effective in producing central TRH actions in vivo while not evoking TSH release, thereby obviating any changes in thyroid status. Thus, this peptide not only contains the dual functionalities of an ectopeptidase inhibitor and receptor binding ligand within one molecular structure, but also displays properties in vivo that are highly desirable for a TRH analog with investigative and therapeutic application.