A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy

Marcinkowski, Patrick; Hoyer, Inna; Specker, Edgar; Furkert, Jens; Rutz, Claudia; Neuenschwander, Martin; Sobottka, Sebastian; Sun, Han; Nazaré, Marc; Berchner‐Pfannschmidt, Utta; Kries, Jens Peter von; Eckstein, Anja; Schülein, Ralf; Krause, Gerd

doi:10.1089/thy.2018.0349

Cited by 56 publications

(48 citation statements)

References 67 publications

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“…For each structure, the grid box was built in accordance with coordinates of the allosteric ligand-binding sites, as described by other authors [ 101 , 102 ]. The grid dimension is a 12 Å buffer zone in accordance to reference ligand size.…”

Section: Methodsmentioning

confidence: 99%

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats

Bakhtyukov

Derkach

Dar’in

et al. 2020

IJMS

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Low-molecular-weight agonists of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LHCGR), which interact with LHCGR transmembrane allosteric site and, in comparison with gonadotropins, more selectively activate intracellular effectors, are currently being developed. Meanwhile, their effects on testicular steroidogenesis have not been studied. The purpose of this work is to perform a comparative study of the effects of 5-amino-N-tert-butyl-4-(3-(1-methylpyrazole-4-carboxamido)phenyl)-2-(methylthio)thieno[2,3-d] pyrimidine-6-carboxamide (TP4/2), a LHCGR allosteric agonist developed by us, and hCG on adenylyl cyclase activity in rat testicular membranes, testosterone levels, testicular steroidogenesis and spermatogenesis in young (four-month-old), aging (18-month-old) and diabetic male Wistar rats. Type 1 diabetes was caused by a single streptozotocin (50 mg/kg) injection. TP4/2 (20 mg/kg/day) and hCG (20 IU/rat/day) were administered for 5 days. TP4/2 was less effective in adenylyl cyclase stimulation and ability to activate steroidogenesis when administered once into rats. On the 3rd–5th day, TP4/2 and hCG steroidogenic effects in young adult, aging and diabetic rats were comparable. Unlike hCG, TP4/2 did not inhibit LHCGR gene expression and did not hyperstimulate the testicular steroidogenesis system, moderately increasing steroidogenic proteins gene expression and testosterone production. In aging and diabetic testes, TP4/2 improved spermatogenesis. Thus, during five-day administration, TP4/2 steadily stimulates testicular steroidogenesis, and can be used to prevent androgen deficiency in aging and diabetes.

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Section: Methodsmentioning

confidence: 99%

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats

Bakhtyukov

Derkach

Dar’in

et al. 2020

IJMS

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“…The racemate S37 had been separated into its enantiomers S37a (eluted first) and S37b (eluted second) by chiral high-performance liquid chromatography, as described previously (Marcinkowski et al, 2019).…”

Section: Crystal Structure Determination Of S37amentioning

confidence: 99%

“…On the basis of the mentioned previous findings and by combining mutagenesis, modeling and small ligand modulators, we aim to shed light on these critical points. We have studied the effect of our recently discovered highly TSHR selective small-molecule NAM S37 as racemate and its active enantiomer S37a (Marcinkowski et al, 2019): 1) on stepwise N-terminally truncated TSHR constructs, 2) on the TMD alone and 3) on point mutations distributed across the hinge region, all three ECL and the TMD. The TSHR constructs were activated either by TSH and/or by a smallmolecule PAM-C2 (Neumann et al, 2009(Neumann et al, , 2016.…”

Section: Introductionmentioning

confidence: 99%

Thyrotropin Receptor: Allosteric Modulators Illuminate Intramolecular Signaling Mechanisms at the Interface of Ecto- and Transmembrane Domain

et al. 2019

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The large TSH-bound ectodomain of the thyrotropin receptor (TSHR) activates the transmembrane domain (TMD) indirectly via an internal agonist (IA). The ectodomain/TMD interface consists of a converging helix, a Cys-Cys-bridge-linked IA, and extracellular loops (ECL). To investigate the intramolecular course of molecular activation, especially details of the indirect activation, we narrowed down allosteric inhibition sites of negative allosteric modulator (NAM) by mutagenesis, homology modeling, and competition studies with positive allosteric modulator (PAM). From the inhibitory effects of NAM S37a on: 1) chimeras with swapped ectodomain, 2) stepwise N-terminal truncations, 3) distinct constitutively active mutations distributed across the hinge region and ECL, but not across the TMD, we conclude that S37a binds at the ectodomain/TMD interface, between the converging helix, ECL1, and the IA. This is also supported by the noncompetitive inhibition of PAM-C2-activation by S37a in the TSHR-TMD construct lacking the ectodomain. Mutagenesis studies on the IA and ECL were guided by our refined model of the ectodomain/TMD interface and indicate an interaction with the TSHR-specific residues E404 (preceding IA) and H478 (ECL1). At this new allosteric interaction site, NAM S37a blocks both TSH-and PAM-induced activation of the TSHR. Our refined models, mutations, and new allosteric binding pocket helped us to gain more detailed insights into the intramolecular course of TSHR activation at the ectodomain/TMD interface, including the delocalization of the converging helix and rearrangement of the conformation of IA. These changes are embedded between the ECL and cooperatively trigger active conformations of TMD. SIGNIFICANCE STATEMENT The intramolecular activation mechanisms of the TSHR appear to be distinct from those of other G protein-coupled receptors, as the TSHR has a uniquely large N-terminal ectodomain that includes the hormone binding site and an internal agonist sequence. We present new molecular and structural insights into the interface between ectodomain and transmembrane domain in the TSHR, as well as the transfer of activation to the transmembrane domain. This knowledge is critical for understanding activation or inhibition of the receptor by allosteric ligands. We have identified a new allosteric antagonist binding pocket that is located exactly at this interface and possesses specific features that may allow the generation of potent highly TSHR-selective drugs, of potential value for the treatment of Graves' orbitopathy.

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“…S37a exhibits also a slight partial agonistic effect, with the advantage of not totally blocking the TSHR but retaining a low cAMP level. Initial pharmacokinetic PK in vivo studies showed good bioavailability and tolerance in mice [122].…”

Section: Negative Allosteric Modulatorsmentioning

confidence: 99%

Intervention Strategies into Glycoprotein Hormone Receptors for Modulating (Mal–)function, with Special Emphasis on the TSH Receptor

Krause

Marcinkowski

2018

Horm Metab Res

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The thyrotropin receptor (TSHR), the lutropin- (LHR), and the follicotropin receptor (FSHR) belong to glycoprotein hormone receptors (GPHR), a subgroup of the class A G-protein coupled receptors. In this review, the unique features of GPHR have been taken into account for their pharmacological interventions: i) The respective hormone and stimulating or blocking antibodies are binding on the large ectodomain that is ii) via a hinge region, containing iii) an internal tethered agonist linked to the transmembrane domain. iv) Multimerization and mechanisms for negative or positive cooperativity of GPHR upon ligand binding and v) dimer- and oligomeric arrangements enabling trans-activation on GPHR signaling are considered. Available knowledge concerning the modulation of the GPHR (mal)-function and associated structural aspects by diverse entities such as antibodies, chaperones, peptides, small molecule agonists, inverse agonists, and antagonists is summarized. The TSHR is important with respect to autoimmune [Graves’ disease (GD), Graves’ orbitopathy (GO)] or non-autoimmune thyroid dysfunctions and cancer-development. To date there is neither an agonist nor antagonist modulator of pathogenic such as TSHR signaling in the clinics. However, several different ligands monoclonal stimulating and inhibiting antibodies and small molecule drug-like ligands have been reported in the last decade. In special focus are the most recent findings regarding the development and use of small molecule TSHR ligands. Finally, limitations of current knowledge and lack of information are discussed highlighting the need for intensified efforts towards understanding the interplay of TSHR multimers, especially their interaction with drug-like ligands. Important in this context is the biased ligand development.

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A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy

Cited by 56 publications

References 67 publications

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats

Thyrotropin Receptor: Allosteric Modulators Illuminate Intramolecular Signaling Mechanisms at the Interface of Ecto- and Transmembrane Domain

Intervention Strategies into Glycoprotein Hormone Receptors for Modulating (Mal–)function, with Special Emphasis on the TSH Receptor

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