A Novel Telomerase Activator Suppresses Lung Damage in a Murine Model of Idiopathic Pulmonary Fibrosis

<div><p>The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity <i>ex vivo</i> and <i>in vivo</i>. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in <i>mTERT</i> heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2–4 fold both in hematopoietic progenitors <i>ex vivo</i> and in bone marrow and lung tissue <i>in vivo,</i> respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L), a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC) or lung fibroblasts <i>ex vivo</i> with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.</p> </div

Treatment with GRN510 enhances telomerase function in the lung using a bleomycin-induced model of pulmonary fibrosis. A.

<p>Analysis of telomerase activity levels in lung tissue for Tert Het mice dosed with GRN510 at 10 mg/kg/d for 21 days post-Bleomycin treatment (23 days total). Intratracheal injection of Bleomycin was performed on day 0. The mean telomerase activity level for the GRN510 treated and vehicle control mice are shown (n = 4–10 per group). The average activity level for the vehicle control group was arbitrarily assigned a value of 1.0. Asterisk, * denotes significant difference relative to the vehicle control mice (Student's t Test). <b>B.</b> Analysis of the effect of telomerase inhibitor GRN163L on GRN510-mediated telomerase activation in the lung. Mice were dosed with GRN510 (10 mg/kg/d) and/or the telomerase inhibitor GRN163L (13 mg/kg/d) as indicated for 21 days (n = 5–10 for all groups). The average activity level for the vehicle control group was arbitrarily assigned a value of 1.0. Asterisks, (*, **) denotes significance amongst the indicated groups (pvalue≤0.03, 1-way ANOVA; P≤0.01 comparing GRN510+GRN163L treated group to GRN510 treated group Student’s t Test). <b>C.</b> Sample southern blot for analysis of terminal restriction fragment length of lung tissue after GRN510 (TA-telomerase activator) treatment. A control sample for untreated Tert Het mice is represented by 'θ'. Size of molecular weight standards are shown on the left (Kb). <b>D.</b> Quantitative analysis of mean TRF length lung tissue after 3 weeks of GRN510 treatment. For each group, n = 5–10. Asterisk * denotes significance amongst indicated groups; P = 0.03, 1-way ANOVA). <b>E.</b> Mean TRF length analysis of PBS control mice. Analysis was performed after 3 weeks of dosing (all mice dosed as described above). N = 4 per group. Asterisk, * denotes significance amongst groups (P = 0.02, 1-way ANOVA). For all plots, mean ± SEM is shown.</p

GRN510 and GRN163L treatments did not affect the structure and the function of the lungs.

<p>Mice were treated with GRN510, GRN163L, and GRN510+ GRN163L for 3 weeks. Expression of α-smooth muscle actin (αSMA) and collagen deposition was evaluated as well as the baseline lung mechanics. <b>A.</b> Representative picorsirius red staining (upper panel) and immunostaining of α−SMA (lower panel) are shown; X200. <b>B.</b> Collagen deposition was quantified using ImagePro software by blinded personnel to the treatment groups. <b>C.</b> Dynamic compliance (ml/cm H2O) was measured. Treatment with the 2 components, alone or in combination indicated no difference in compliance among groups. Statistical analysis was performed by 1-way ANOVA (Prism, GraphPad Software, Inc). N = 4 per group (same animals as assessed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058423#pone.0058423.s001" target="_blank">Fig. 1</a>E); For all plots, mean ± SEM is shown.</p

Reduced cellular senescence in bleomycin-injured lung tissue with GRN510 treatment.

<p>All analyses were performed on the same mice as in Fig. 4. <b>A.</b> Analysis of SA-β galactosidase activity in the lung tissue. Shown are representative paraffin-embedded lung sections after staining for SA-β gal activity (blue staining). Increased SA-β gal activity was detected in the mice challenged with bleomycin and treated with vehicle or GRN510 and GRN163L. In contrast, reduced SA-β gal activity was detected in the mice challenged with bleomycin and treated with GRN510. Positive stained cells are encircled. Magnification 40X. Representative paraffin embedded lung sections immunohistochemically stained for MH2A using enhance blank staining solution (KPL, Gaithersburg, MD); n = 3 different specimens with 3 fields of view analyzed per section; Hematoxylin was used as a negative counter-stain. Magnification of 40X. Circles denote positive staining. <b>B.</b> Analysis of <i>SIRT6</i> and <i>p21</i> expression in the lung. Targeted genes and β-actin expression at the mRNA level was determined by qRT-PCR. Analysis was performed in triplicate for each specimen. N = 5–10 animals per group. Asterisk, * indicates significance amongst indicated groups (P = 0.002, 1-way ANOVA). <b>C.</b> Western analysis of p53 expression. A trend in increase expression was associated with bleomycin treatment that was partially reverted by GRN510 treatment. For all plots mean ± SEM is shown.</p

The GRN510-mediated protection from bleomycin-induced fibrosis is dependent on telomerase activation.

<p>Tert Het mice subjected to Bleomycin-induced fibrosis were treated with GRN510 (10 mg/kg/d) and the telomerase inhibitor GRN163L as indicated. <b>A.</b> Representative histological sections were obtained from the whole left pulmonary lobe taken from each animal 11 days after bleomycin-induced injury and stained with H&E (Top panel) or with picrosirius red (Lower panel). To minimize variation, samples were processed under identical conditions. <b>B.</b> Levels of picrosirius red staining were quantified with ImagePro software. Asterisk, * indicates significance amongst indicated groups (P = 0.02, 1-way ANOVA). <b>C.</b> RT-PCR expression levels of procollagen1α mRNA in the lung. Analysis was performed in triplicate for each specimen. Asterisk, * denotes significance amongst indicated groups (P = 0.04, 1-way ANOVA). N = 5–10 animals per group; for all plots, mean ± SEM is shown.</p

Optimization of the Potency and Pharmacokinetic Properties of a Macrocyclic Ghrelin Receptor Agonist (Part I): Development of Ulimorelin (TZP-101) from Hit to Clinic

High-throughput screening of Tranzyme Pharma’s proprietary macrocycle library using the aequorin Ca²⁺-bioluminescence assay against the human ghrelin receptor (GRLN) led to the discovery of novel agonists against this G-protein coupled receptor. Early hits such as <b>1</b> (<i>K</i>_i = 86 nM, EC₅₀ = 134 nM) though potent in vitro displayed poor pharmacokinetic properties that required optimization. While such macrocycles are not fully rule-of-five compliant, principally due to their molecular weight and clogP, optimization of their pharmacokinetic properties proved feasible largely through conformational rigidification. Extensive SAR led to the identification of <b>2</b> (<i>K</i>_i = 16 nM, EC₅₀ = 29 nM), also known as ulimorelin or TZP-101, which has progressed to phase III human clinical trials for the treatment of postoperative ileus. X-ray structure and detailed NMR studies indicated a rigid peptidomimetic portion in <b>2</b> that is best defined as a nonideal type-I′ β-turn. Compound <b>2</b> is 24% orally bioavailable in both rats and monkeys. Despite its potency, in vitro and in gastric emptying studies, <b>2</b> did not induce growth hormone (GH) release in rats, thus demarcating the GH versus GI pharmacology of GRLN

Discovery of Potent and Orally Bioavailable Macrocyclic Peptide–Peptoid Hybrid CXCR7 Modulators

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide–peptoid hybrids with high CXCR7 binding affinities (<i>K</i>_i < 100 nM) and measurable passive permeability (<i>P</i>_app > 5 × 10^–6 cm/s). Moreover, bioactive peptide <b>25</b> (<i>K</i>_i = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration