Highly biased agonism for GPCR ligands via nanobody tethering.

Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present an approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.

Backbone Modification Provides a Long-Acting Inverse Agonist of Pathogenic, Constitutively Active PTH1R Variants.

Parathyroid hormone 1 receptor (PTH1R) plays a key role in mediating calcium homeostasis and bone development, and aberrant PTH1R activity underlies several human diseases. Peptidic PTH1R antagonists and inverse agonists have therapeutic potential in treating these diseases, but their poor pharmacokinetics and pharmacodynamics undermine their in vivo efficacy. Herein, we report the use of a backbone-modification strategy to design a peptidic PTH1R inhibitor that displays prolonged activity as an antagonist of wild-type PTH1R and an inverse agonist of the constitutively active PTH1R-H223R mutant both in vitro and in vivo. This peptide may be of interest for the future development of therapeutic agents that ameliorate PTH1R malfunction.

Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome.

The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor that plays key roles in regulating calcium homeostasis and skeletal development via binding the ligands, PTH and PTH-related protein (PTHrP), respectively. Eiken syndrome is a rare disease of delayed bone mineralization caused by homozygous PTH1R mutations. Of the three mutations identified so far, R485X, truncates the PTH1R C-terminal tail, while E35K and Y134S alter residues in the receptor's amino-terminal extracellular domain. Here, using a variety of cell-based assays, we show that R485X increases the receptor's basal rate of cAMP signaling and decreases its capacity to recruit ß-arrestin2 upon ligand stimulation. The E35K and Y134S mutations each weaken the binding of PTHrP leading to impaired ß-arrestin2 recruitment and desensitization of cAMP signaling response to PTHrP but not PTH. Our findings support a critical role for interaction with ß-arrestin in the mechanism by which the PTH1R regulates bone formation.

Altered signaling at the PTH receptor via modified agonist contacts with the extracellular domain provides a path to prolonged agonism in vivo.

The parathyroid hormone type 1 receptor (PTHR1), a Class B GPCR, is activated by long polypeptides, including drugs for osteoporosis and hypoparathyroidism. The PTHR1 engages peptide agonists via a two-step mechanism. Initial contact involves the extracellular domain (ECD), which has been thought to contribute primarily to receptor-peptide binding, and then the N terminus of the peptide engages the receptor transmembrane domain (TMD), which is thought to control the message conveyed to intracellular partners. This mechanism has been suggested to apply to other Class B GPCRs as well. Here, we show that modification of a PTHR1 agonist at ECD-contact sites can alter the signaling profile, an outcome that is not accommodated by the current two-step binding model. Our data support a modified two-step binding model in which agonist orientation on the ECD surface can influence the geometry of agonist-TMD engagement. This expanded binding model offers a mechanism by which altering ECD-contact residues can affect signaling profile. Our discoveries provide a rationale for exploring agonist modifications distal from the TMD-contact region in future efforts to optimize therapeutic performance of peptide hormone analogs.

Kinetic and Thermodynamic Insights into Agonist Interactions with the Parathyroid Hormone Receptor-1 from a New NanoBRET Assay.

Polypeptides that activate the parathyroid hormone receptor-1 (PTHR1) are important in human physiology and medicine. Most previous studies of peptide binding to this receptor have involved the displacement of a radiolabeled ligand. We report a new assay format based on bioluminescence resonance energy transfer (BRET). Fusion of a NanoLuc luciferase (nLuc) unit to the N-terminus of the PTHR1 allows the direct detection of binding by an agonist peptide bearing a tetramethylrhodamine (TMR) unit. Affinity measurements from the BRET assay align well with results previously obtained via radioligand displacement. The BRET assay offers substantial operational benefits relative to affinity measurements involving radioactive compounds. The convenience of the new assay allowed us to explore several questions raised by earlier reports. For example, we show that although the first two residues of PTH(1-34) (the drug teriparatide) are critical for PTHR1 activation, these two residues contribute little or nothing to affinity. Comparisons among the well-studied agonists PTH(1-34), PTHrP(1-34), and "long-acting PTH" (LA-PTH) reveal that the high affinity of LA-PTH arises largely from a diminished rate constant for dissociation relative to the other two. A D-peptide recently reported to be comparable to PTH(1-34) as an agonist of the PTHR1 was found not to bind detectably to the receptor and to be a very weak agonist.

Biased GPCR signaling by the native parathyroid hormone-related protein 1 to 141 relative to its N-terminal fragment 1 to 36.

The parathyroid hormone (PTH)-related protein (PTHrP) is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling, and causes hypercalcemia in patients with malignancy. Although mature forms of PTHrP in the body consist of splice variants of 139, 141, and 173 amino acids, our current understanding on how endogenous PTHrP transduces signals through its cognate G-protein coupled receptor (GPCR), the PTH type 1 receptor (PTHR), is largely derived from studies done with its N-terminal fragment, PTHrP1-36. Here, we demonstrate using various fluorescence imaging approaches at the single cell level to measure kinetics of (i) receptor activation, (ii) receptor signaling via Gs and Gq, and (iii) receptor internalization and recycling that the native PTHrP1-141 displays biased agonist signaling properties that are not mimicked by PTHrP1-36. Although PTHrP1-36 induces transient cAMP production, acute intracellular Ca2+ (iCa2+) release and ß-arrestin recruitment mediated by ligand-PTHR interactions at the plasma membrane, PTHrP1-141 triggers sustained cAMP signaling from the plasma membrane and fails to stimulate iCa2+ release and recruit ß-arrestin. Furthermore, we show that the molecular basis for biased signaling differences between PTHrP1-36 and properties of native PTHrP1-141 are caused by the stabilization of a singular PTHR conformation and PTHrP1-141 sensitivity to heparin, a sulfated glycosaminoglycan. Taken together, our results contribute to a better understanding of the biased signaling process of a native protein hormone acting in conjunction with a GPCR.

Functional Properties of Two Distinct PTH1R Mutants Associated With Either Skeletal Defects or Pseudohypoparathyroidism.

Consistent with a vital role of parathyroid hormone (PTH) receptor type 1 (PTH1R) in skeletal development, homozygous loss-of-function PTH1R mutations in humans results in neonatal lethality (Blomstrand chondrodysplasia), whereas such heterozygous mutations cause a primary failure of tooth eruption (PFE). Despite a key role of PTH1R in calcium and phosphate homeostasis, blood mineral ion levels are not altered in such cases of PFE. Recently, two nonlethal homozygous PTH1R mutations were identified in two unrelated families in which affected members exhibit either dental and skeletal abnormalities (PTH1R-V204E) or hypocalcemia and hyperphosphatemia (PTH1R-R186H). Arg186 and Val204 map to the first transmembrane helix of the PTH1R, and thus to a critical region of this class B G protein-coupled receptor. We used cell-based assays and PTH and PTH-related protein (PTHrP) ligand analogs to assess the impact of the R186H and V204E mutations on PTH1R function in vitro. In transiently transfected HEK293 cells, PTH1R-R186H mediated cyclic adenosine monophosphate (cAMP) responses to PTH(1-34) and PTHrP(1-36) that were of comparable potency to those observed on wild-type PTH1R (PTH1R-WT) (half maximal effective concentrations [EC50s] = 0.4nM to 1.2nM), whereas the response-maxima were significantly reduced for the PTH1R-V204E mutant (maximum effect [Emax] = 81%-77% of PTH1R-WT, p ≤ 0.004). Antibody binding to an extracellular hemagglutinin (HA) tag was comparable for PTH1R-R186H and PTH1R-WT, but was significantly reduced for PTH1R-V204E (maximum binding level [Bmax] = 44% ± 11% of PTH1R-WT, p = 0.002). The potency of cAMP signaling induced by a PTH(1-11) analog was reduced by ninefold and threefold, respectively, for PTH1R-R186H and PTH1R-V204E, relative to PTH1R-WT, and a PTH(1-15) radioligand analog that bound adequately to PTH1R-WT exhibited little or no specific binding to either mutant receptor. The data support a general decrease in PTH1R surface expression and/or function as a mechanism for PFE and a selective impairment in PTH ligand affinity as a potential PTH1R-mutation-based mechanism for pseudohypoparathyroidism. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein­coupled receptor (GPCR) that regulates mineral ion, vitamin D, and bone homeostasis. Activation of the PTHR by PTH induces both transient cell surface and sustained endosomal cAMP production. To address whether the spatial (location) or temporal (duration) dimension of PTHR-induced cAMP encodes distinct biological outcomes, we engineered a biased PTHR ligand (PTH7d) that elicits cAMP production at the plasma membrane but not at endosomes. PTH7d stabilized a unique active PTHR conformation that mediated sustained cAMP signaling at the plasma membrane due to impaired ß-arrestin coupling to the receptor. Experiments in cells and mice revealed that sustained cAMP production by cell surface PTHR failed to mimic the pharmacological effects of sustained endosomal cAMP production on the abundance of the rate-limiting hydroxylase catalyzing the formation of active vitamin D, as well as increases in circulating active vitamin D and Ca2+ and in bone formation in mice. Thus, similar amounts of cAMP generated by PTHR for similar lengths of time in different cellular locations, plasma membrane and endosomes, mediate distinct physiological responses. These results unveil subcellular signaling location as a means to achieve specificity in PTHR-mediated biological outcomes and raise the prospect of rational drug design based upon spatiotemporal manipulation of GPCR signaling.

Ligand-Dependent Effects of Methionine-8 Oxidation in Parathyroid Hormone Peptide Analogues.

LA-PTH is a long-acting parathyroid hormone (PTH) peptide analogue in preclinical development for hypoparathyroidism (HP). Like native PTH, LA-PTH contains a methionine at position 8 (Met8) that is predicted to be critical for function. We assessed the impact of Met oxidation on the functional properties of LA-PTH and control PTH ligands. Oxidation of PTH(1-34) resulted in marked (~20-fold) reductions in binding affinity on the PTH receptor-1 (PTHR1) in cell membranes, similarly diminished potency for 3',5'-cyclic AMP signaling in osteoblastic cell lines (SaOS-2 and UMR106), and impaired efficacy for raising blood calcium in mice. Surprisingly, oxidation of LA-PTH resulted in little or no change in these functional responses. The signaling potency of oxidized-LA-PTH was, however, reduced approximately 40-fold compared to LA-PTH in cells expressing a PTHR1 construct that lacks the N-terminal extracellular domain (ECD). Molecular modeling revealed that while Met8 of both LA-PTH and PTH(1-34) is situated within the orthosteric ligand-binding pocket of the receptor's transmembrane domain bundle (TMD), the Met8 sidechain position is shifted for the 2 ligands so that on Met8 oxidation of PTH(1-34), steric clashes occur that are not seen with oxidized LA-PTH. The findings suggest that LA-PTH and PTH(1-34) engage the receptor differently in the Met8-interaction environment of the TMD bundle, and that this interaction environment can be allosterically influenced by the ECD component of the ligand-receptor complex. The findings should be useful for the future development of novel PTH-based peptide therapeutics for diseases of bone and mineral ion metabolism.

Allosteric interactions in the parathyroid hormone GPCR-arrestin complex formation.

Peptide ligands of class B G-protein-coupled receptors act via a two-step binding process, but the essential mechanisms that link their extracellular binding to intracellular receptor-arrestin interactions are not fully understood. Using NMR, crosslinking coupled to mass spectrometry, signaling experiments and computational approaches on the parathyroid hormone (PTH) type 1 receptor (PTHR), we show that initial binding of the PTH C-terminal part constrains the conformation of the flexible PTH N-terminal signaling epitope before a second binding event occurs. A 'hot-spot' PTH residue, His9, that inserts into the PTHR transmembrane domain at this second step allosterically engages receptor-arrestin coupling. A conformational change in PTHR intracellular loop 3 permits favorable interactions with ß-arrestin's finger loop. These results unveil structural determinants for PTHR-arrestin complex formation and reveal that the two-step binding mechanism proceeds via cooperative fluctuations between ligand and receptor, which extend to other class B G-protein-coupled receptors.

Improved GPCR ligands from nanobody tethering.

Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody's specificity. Here we develop a new type of conjugate that consists of a nanobody and a peptidic ligand for a G protein-coupled receptor (GPCR), fused via their C-termini. We address activation of parathyroid hormone receptor-1 (PTHR1) and improve the signaling activity and specificity of otherwise poorly active N-terminal peptide fragments of PTH by conjugating them to nanobodies (VHHs) that recognize PTHR1. These C-to-C conjugates show biological activity superior to that of the parent fragment peptide in vitro. In an exploratory experiment in mice, a VHH-PTH peptide conjugate showed biological activity, whereas the corresponding free peptide did not. The lead conjugate also possesses selectivity for PTHR1 superior to that of PTH(1-34). This design approach, dubbed "conjugation of ligands and antibodies for membrane proteins" (CLAMP), can yield ligands with high potency and specificity.

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia.

Jansen's metaphyseal chondrodysplasia (JMC) is a rare disease of bone and mineral ion physiology that is caused by activating mutations in PTHR1. Ligand-independent signaling by the mutant receptors in cells of bone and kidney results in abnormal skeletal growth, excessive bone turnover, and chronic hypercalcemia and hyperphosphaturia. Clinical features further include short stature, limb deformities, nephrocalcinosis, and progressive losses in kidney function. There is no effective treatment option available for JMC. In previous cell-based assays, we found that certain N-terminally truncated PTH and PTHrP antagonist peptides function as inverse agonists and thus can reduce the high rates of basal cAMP signaling exhibited by the mutant PTHR1s of JMC in vitro. Here we explored whether one such inverse agonist ligand, [Leu11 ,dTrp12 ,Trp23 ,Tyr36 ]-PTHrP(7-36)NH2 (IA), can be effective in vivo and thus ameliorate the skeletal abnormalities that occur in transgenic mice expressing the PTHR1-H223R allele of JMC in osteoblastic cells via the collagen-1α1 promoter (C1HR mice). We observed that after 2 weeks of twice-daily injection and relative to vehicle controls, the IA analog resulted in significant improvements in key skeletal parameters that characterize the C1HR mice, because it reduced the excess trabecular bone mass, bone marrow fibrosis, and levels of bone turnover markers in blood and urine. The overall findings provide proof-of-concept support for the notion that inverse agonist ligands targeted to the mutant PTHR1 variants of JMC can have efficacy in vivo. Further studies of such PTHR1 ligand analogs could help open paths toward the first treatment option for this debilitating skeletal disorder. © 2019 American Society for Bone and Mineral Research.

Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism.

The type-1 parathyroid hormone receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH forms a complex with the PTHR1 that is rapidly internalized and induces prolonged cAMP production from endosomes. In contrast, PTHrP induces only transient cAMP production, which primarily arises from receptors on the cell surface. We show that backbone modification of PTH(1-34)-NH2 and abaloparatide (a PTHrP derivative) with a single homologous ß-amino acid residue can generate biased agonists that induce prolonged cAMP production from receptors at the cell surface. This unique spatiotemporal profile could be useful for distinguishing effects associated with the duration of cAMP production from effects associated with the site of cAMP production.

Parathyroid hormone(1-34) and its analogs differentially modulate osteoblastic Rankl expression via PKA/SIK2/SIK3 and PP1/PP2A-CRTC3 signaling.

Osteoporosis can result from the loss of sex hormones and/or aging. Abaloparatide (ABL), an analog of parathyroid hormone-related protein (PTHrP(1-36)), is the second osteoanabolic therapy approved by the United States Food and Drug Administration after teriparatide (PTH(1-34)). All three peptides bind PTH/PTHrP receptor type 1 (PTHR1), but the effects of PTHrP(1-36) or ABL in the osteoblast remain unclear. We show that, in primary calvarial osteoblasts, PTH(1-34) promotes a more robust cAMP response than PTHrP(1-36) and ABL and causes a greater activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB). All three peptides similarly inhibited sclerostin (Sost). Interestingly, the three peptides differentially modulated two other PKA target genes, c-Fos and receptor activator of NF-κB ligand (Rankl), and the latter both in vitro and in vivo Knockdown of salt-inducible kinases (SIKs) 2 and 3 and CREB-regulated transcription coactivator 3 (CRTC3), indicated that all three are part of the pathway that regulates osteoblastic Rankl expression. We also show that the peptides differentially regulate the nuclear localization of CRTC2 and CRTC3, and that this correlates with PKA activation. Moreover, inhibition of protein phosphatases 1 and 2A (PP1/PP2A) activity revealed that they play a major role in both PTH-induced Rankl expression and the effects of PTH(1-34) on CRTC3 localization. In summary, in the osteoblast, the effects of PTH(1-34), PTHrP(1-36), and ABL on Rankl are mediated by differential stimulation of cAMP/PKA signaling and by their downstream effects on SIK2 and -3, PP1/PP2A, and CRTC3.

Receptor selectivity from minimal backbone modification of a polypeptide agonist.

Human parathyroid hormone (PTH) and N-terminal fragments thereof activate two receptors, hPTHR1 and hPTHR2, which share ∼51% sequence similarity. A peptide comprising the first 34 residues of PTH is fully active at both receptors and is used to treat osteoporosis. We have used this system to explore the hypothesis that backbone modification of a promiscuous peptidic agonist can provide novel receptor-selective agonists. We tested this hypothesis by preparing a set of variants of PTH(1-34)-NH2 that contained a single ß-amino-acid residue replacement at each of the first eight positions. These homologs, each containing one additional backbone methylene unit relative to PTH(1-34)-NH2 itself, displayed a wide range of potencies in cell-based assays for PTHR1 or PTHR2 activation. The ß-scan series allowed us to identify two homologs, each containing two αâ†’ß replacements, that were highly selective, one for PTHR1 and the other for PTHR2. These findings suggest that backbone modification of peptides may provide a general strategy for achieving activation selectivity among polypeptide-modulated receptors, and that success requires consideration of both ß2- and ß3-residues, which differ in terms of side-chain location.

Progression of Mineral Ion Abnormalities in Patients With Jansen Metaphyseal Chondrodysplasia.

Context: Five different activating PTH/PTH-related peptide (PTHrP) receptor (PTHR1) mutations have been reported as causes of Jansen metaphyseal chondrodysplasia (JMC), a rare disorder characterized by severe growth plate abnormalities and PTH-independent hypercalcemia. Objectives: Assess the natural history of clinical and laboratory findings in 24 patients with JMC and characterize the disease-causing mutant receptors in vitro. Patients and Methods: The H223R mutation occurred in 18 patients. T410P, I458R and I458K each occurred in single cases; T410R was present in a father and his two sons. Laboratory records were analyzed individually and in aggregate. Results: Postnatal calcium levels were normal in most patients, but elevated between 0.15 and 10 years (11.8 ± 1.37 mg/dL) and tended to normalize in adults (10.0 ± 1.03 mg/dL). Mean phosphate levels were at the lower end of the age-specific normal ranges. Urinary calcium/creatinine (mg/mg) were consistently elevated (children, 0.80 ± 0.40; adults, 0.28 ± 0.19). Adult heights were well below the 3rd percentile for all patients, except for those with the T410R mutation. Most patients with JMC had undergone orthopedic surgical procedures, most had nephrocalcinosis, and two had advanced chronic kidney disease. The five PTHR1 mutants showed varying degrees of constitutive and PTH-stimulated cAMP signaling activity when expressed in HEK293 reporter cells. The inverse agonist [L11,dW12,W23,Y36]PTHrP(7-36) reduced basal cAMP signaling for each PTHR1 mutant. Conclusions: Except for T410R, the other PTHR1 mutations were associated with indistinguishable mineral ion abnormalities and cause similarly severe growth impairment. Hypercalciuria persisted into adulthood. An inverse agonist ligand effectively reduced in vitro PTH-independent cAMP formation at all five PTHR1 mutants, suggesting a potential path toward therapy.

Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad ß Residue Distribution.

The parathyroid hormone receptor 1 (PTHR1) is a member of the B-family of GPCRs; these receptors are activated by long polypeptide hormones and constitute targets of drug development efforts. Parathyroid hormone (PTH, 84 residues) and PTH-related protein (PTHrP, 141 residues) are natural agonists of PTHR1, and an N-terminal fragment of PTH, PTH(1-34), is used clinically to treat osteoporosis. Conventional peptides in the 20-40-mer length range are rapidly degraded by proteases, which may limit their biomedical utility. We have used the PTHR1-ligand system to explore the impact of broadly distributed replacement of α-amino acid residues with ß-amino acid residues on susceptibility to proteolysis and agonist activity. This effort led us to identify new PTHR1 agonists that contain α → ß replacements throughout their sequences, manifest potent agonist activity in cellular assays, and display remarkable resistance to proteolysis, in cases remaining active after extended exposure to simulated gastric fluid. The strategy we have employed suggests a path toward identifying protease-resistant agonists of other B-family GPCRs.

Prolonged Pharmacokinetic and Pharmacodynamic Actions of a Pegylated Parathyroid Hormone (1-34) Peptide Fragment.

Polyethylene glycol (PEG) addition can prolong the pharmacokinetic and pharmacodynamic actions of a bioactive peptide in vivo, in part by impeding rates of glomerular filtration. For parathyroid hormone (PTH) peptides, pegylation could help in exploring the actions of the hormone in the kidney; e.g., in dissecting the relative roles that filtered versus blood-borne PTH play in regulating phosphate transport. It could also lead to potential alternate forms of treatment for hypoparathyroidism. We thus synthesized the fluorescent pegylated PTH derivative [Lys13 (tetramethylrhodamine {TMR}), Cys35 (PEG-20,000 Da)]PTH(1-35) (PEG-PTHTMR ) and its non-pegylated counterpart [Lys13 (TMR), Cys35 ]PTH(1-35) (PTHTMR ) and assessed their properties in cells and in mice. In PTHR1-expressing HEK-293 cells, PEG-PTHTMR and PTHTMR exhibited similar potencies for inducing cAMP signaling, whereas when injected into mice, the pegylated analog persisted much longer in the circulation (>24 hours versus ∼ 1 hour) and induced markedly more prolonged calcemic and phosphaturic responses than did the non-pegylated control. Fluorescence microscopy analysis of kidney sections obtained from the injected mice revealed much less PEG-PTHTMR than PTHTMR on the luminal brush-border surfaces of renal proximal tubule cells (PTCs), on which PTH regulates phosphate transporter function, whereas immunostained phosphorylated PKA substrate, a marker of cAMP signaling, was increased to similar extents for the two ligands and for each, was localized to the basolateral portion of the PTCs. Pegylation of a bioactive PTH peptide thus led to prolonged pharmacokinetic/pharmacodynamic properties in vivo, as well as to new in vivo data that support a prominent role for PTH action at basolateral surfaces of renal proximal tubule cells. © 2016 American Society for Bone and Mineral Research.

SIKs control osteocyte responses to parathyroid hormone.

Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.