Journal: PLoS ONE

Article Title: Effects of Stimulation of Soluble Guanylate Cyclase on Diabetic Nephropathy in Diabetic eNOS Knockout Mice on Top of Angiotensin II Receptor Blockade

doi: 10.1371/journal.pone.0042623

Figure Lengend Snippet: Experimental progress of blood pressure and blood glucose in diabetic eNOS knockout mice treated with riociguat (3 mg/kg/d), telmisartan (1 mg/kg/d), both (3 mg/kg/d and 1 mg/kg/d) or vehicle.

Article Snippet: Riociguat [BAY 63-2521; Methyl 2-[1-2(-fluorobenzyl)-1 H -pyrazolo[3,4- b ]pyridin-3-yl]-4,6—diaminopyrimidin-5-ylmethylcarbamat] was synthesized by Bayer Pharma AG as described and dissolved in Transcutol®/Cremophor®/water (10/20/70).

Techniques: Knock-Out

Journal: PLoS ONE

Article Title: Effects of Stimulation of Soluble Guanylate Cyclase on Diabetic Nephropathy in Diabetic eNOS Knockout Mice on Top of Angiotensin II Receptor Blockade

doi: 10.1371/journal.pone.0042623

Figure Lengend Snippet: Final urinary albumin as well as organ weights and histology of diabetic mice after 11 weeks of treatment with riociguat (3 mg/kg/d), telmisartan (1 mg/kg/d), riociguat and telmisartan (3 mg/kg/d and 1 mg/kg/d) or vehicle and of non-diabetic control mice, respectively.

Article Snippet: Riociguat [BAY 63-2521; Methyl 2-[1-2(-fluorobenzyl)-1 H -pyrazolo[3,4- b ]pyridin-3-yl]-4,6—diaminopyrimidin-5-ylmethylcarbamat] was synthesized by Bayer Pharma AG as described and dissolved in Transcutol®/Cremophor®/water (10/20/70).

Techniques: Control

Journal: Pharmacy and Therapeutics

Article Title: Riociguat (Adempas): a Novel Agent For the Treatment of Pulmonary Arterial Hypertension and Chronic Thromboembolic Pulmonary Hypertension

doi:

Figure Lengend Snippet: Adverse Events Reported in at Least 5% of Riociguat-Treated Patients And at a Frequency Greater Than Placebo in Phase 3 Clinical Trials 24 , 25

Article Snippet: 3 In October 2013, the Food and Drug Administration approved riociguat (Adempas, Bayer Healthcare Pharmaceuticals), the first agent in a novel therapeutic class called soluble guanylate cyclase (sGC) stimulators.

Techniques: Clinical Proteomics, Reflux, Infection

Journal: bioRxiv

Article Title: The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

doi: 10.1101/2023.05.03.537252

Figure Lengend Snippet: Note that all error bars represent ± SEM and * p< 0.05, ** p≤ 0.01, *** p≤ 0.001, **** p≤ 0.0001. A. The mRNA levels of GUCY1A1 and GUCY1B1 were measured by qPCR in our panel of isogenically matched cell variants of progressive castration-resistance: LNCaP SB0 (parental CSPC), and the counterpart emergent CRPC lines, SB5, SB5X, and SB5XX under the indicated culture conditions. ActinB was used as the normalization control. The p-values are from an unpaired two-tailed Student’s t test. B. Western blotting of total protein lysates (15 µg) from the lines in (A) indicate loss of sGCβ1 protein in emergent CRPC lines vs. their CSPC SB0 counterpart. Immunoblotting against both sGC subunits as well as AR expression, with β actin as the loading control is shown. To assess effects of acute androgen deprivation, cells were cultured for 30 hours in CSS media. Molecular weights of the markers from the original immunoblot are indicated on the right. C. Emergent CRPC cells show significantly dampened sGC activity. The cell lines indicated in (A) were cultured in FBS or CSS media for 30 hours and then treated with 20 µM riociguat for 30 minutes, to stimulate sGC signaling, following which cGMP levels were measured. The p-values are from an unpaired two-tailed Student’s t test.

Article Snippet: Twenty-four hours after plating, cells were changed into either media supplemented with 5% FBS or 5% CSS was added to the cells, and were treated with either 50 μM, 100 μM or 200 μM riociguat (Cayman chemical, 9000554) or DMSO vehicle control (Sigma, D8418).

Techniques: Control, Two Tailed Test, Western Blot, Expressing, Cell Culture, Activity Assay

Journal: bioRxiv

Article Title: The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

doi: 10.1101/2023.05.03.537252

Figure Lengend Snippet: Note that all immunoblots show the molecular weight of the markers run on the original membrane on the right. All error bars represent ± SEM and * p< 0.05, ** p≤ 0.01, *** p≤ 0.001, **** p≤ 0.0001. A. Emergent CRPC and fully CRPC panel of cell lines show progressively lower p16INK4a induction under AD culture compared to parental SB0 cells, indicating their ability to evade ADIS. Total protein lysates (15 µg) were immunoblotted against the indicated proteins. B. ADIS is associated with increasing levels of sGCβ1 protein. Total LNCaP SB0 protein lysates (15 µg) harvested at the specified time points and culture conditions were immunoblotted against the indicated proteins. C. The mRNA levels of GUCY1A1 and GUCY1B1 were measured via qPCR in LNCaP cells deleted for sGCβ1 by CRISPR-Cas9 modification (KO sGCβ1). ActinB was used for normalization. The p-values were determined by an unpaired two-tailed Student’s t test. D. Knockout of sGCβ1 in LNCaP cells was validated by immunoblotting for loss of protein. Total protein lysates (10 µg) for sGCβ1 levels from counterpart control and KO sGCβ1 LNCaP cell lines were immunoblotted. E. Knockout of sGCβ1 abrogates sGC activity. Baseline and riociguat-stimulated cGMP levels from control and counterpart KO sGCβ1 LNCaP lines are shown. The p-values were determined by an unpaired two-tailed Student’s t test. F. Knockout of sGCβ1 in LNCaP CSPC cells enables their proliferation under AD culture. Cell proliferation curves were established by plating 50,000 cells for KO sGCβ1 or matched control LNCaP cells and monitoring growth in FBS-supplemented (left) or CSS- supplemented (right) media over 9 days. The p values were determined by comparing WT sGCβ1 vs KO sGCβ1 cell numbers at day 9 using unpaired two-tailed Student’s t tests. G. Knockout of sGCβ1 decreases number of CSPC cells that undergo ADIS. WT and counterpart KO sGCβ1 LNCaP cells were cultured under AD conditions for 13 days and then assayed for the senescence-associated beta-galactosidase (SA-beta-gal) activity (left). A minimum of 150 cells across 4-5 high powered fields per sample was evaluated for positively stained cells. Percentage positivity of staining was determined by the number of SA-beta-gal-stained cells normalized to the total number of cells per field (right). Unpaired two-tailed t-test with Welch’s correction was used to determine p-values, and outliers were excluded via the ROUT method (Q=1%). H. KO sGCβ1 LNCaP cells show lower p16 INK4a protein levels relative to its wt counterpart cells following prolonged culture in CSS- supplemented culture to induce ADIS. Western blotting of total protein lysates (10µg) is shown for the indicated cell lines, following culture in FBS or CSS media for 13 days. I. Loss of sGCβ1 promotes castration-resistant tumor growth by LNCaP CSPC cells. The KO sGCβ1 or matched WT LNCaP cells were subcutaneously injected at a single site per 5-week old, Nu/Nu, castrated male mice. Tumor formation kinetics are shown. Wilcoxon two-sample test was used to determine the p-value. J. Endpoint tumor volumes (mm 3 ) for the KO sGCβ1 and matched WT groups (from I) are shown. Each point represents the tumor from an individual mouse. Final tumor incidence: WT 1/5; KO sGCβ1 4/7. Wilcoxon two-sample test was used to determine the p-value. K. Endpoint tumor weights (mg) in the KO sGCβ1 and matched WT sGCβ1 groups were measured. Each point represents an individual tumor. Wilcoxon two-sample test was used to determine the p value. Note that absence of a tumor mass upon necropsy is indicated as zero. L. Intratumoral cGMP levels are lower in KO sGCβ1 tumors relative to wt counterparts. The p-values were established via an unpaired two-tailed Student’s t test. M. Immunoblotting for sGCβ1 levels in tumor lysates (30µg) corresponding to the tumors evaluated for cGMP in (L) is shown. Endpoint tumor weights are indicated for each individual tumor. N. Analysis of GUCY1A1 and GUCY1B1 mRNA levels from four distinct epithelial clusters (two basal, two luminal) derived from human prostate tissue. Expression levels from publicly available scRNA-seq data from the Single Cell Portal at Broad Institute

Article Snippet: Twenty-four hours after plating, cells were changed into either media supplemented with 5% FBS or 5% CSS was added to the cells, and were treated with either 50 μM, 100 μM or 200 μM riociguat (Cayman chemical, 9000554) or DMSO vehicle control (Sigma, D8418).

Techniques: Western Blot, Molecular Weight, Membrane, CRISPR, Modification, Two Tailed Test, Knock-Out, Control, Activity Assay, Cell Culture, Staining, Injection, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

doi: 10.1101/2023.05.03.537252

Figure Lengend Snippet: Note that all error bars represent ± SEM and * p< 0.05, ** p≤ 0.01, *** p≤ 0.001, **** p≤ 0.0001. A. sGC activity is dampened in established CRPC relative to CSPC, but elevated relative to emergent CRPC variants. LNCaP SB0, SB5, SB5X, SB5XX and LNAI cells were cultured in FBS- or CSS-supplemented media for 5 days, then treated with 20 µM riociguat for 30 minutes before measuring cGMP levels. p-values were determined by an unpaired two-tailed Student’s t-test. B. Comparison of relative sGC activity in androgen-replete (FBS) vs. androgen-deprived (CSS) LNAI CRPC cells shows AD enhances sGC activity. Values for cGMP levels from and are plotted together. p-values were determined by an unpaired two-tailed Student’s t-test. C. Androgen deprivation sensitizes CRPC cells to riociguat. LNAI cells were plated in triplicate in 96-well plates and treated with vehicle (DMSO) or riociguat in FBS or CSS media for 72 hours, with daily redosing. The IC 50 s for each curve were modeled using a four-parameter log-logistic function and are shown in the table beside each graph. Wilcoxon two-sample test was used to determine p-values. D. LNAI cells were plated in FBS media as in C, then incubated with 10 µM enzalutamide for 24 hours before dosing with the indicated various concentrations of riociguat alone or riociguat with enzalutamide for 72 hours, with daily redosing. Viability and IC 50 s as well as p-values were established as in . Wilcoxon two-sample test was used to determine p-values. E. LNAI cells were plated in 10 cm dishes and treated with the indicated riociguat doses in either FBS or CSS media for 72 hours, with daily redosing. Following treatment, cells were harvested and immunoblotting of 26 µg total protein lysates was carried out against the specified antibodies. Molecular weights of the markers run on the original membrane are shown on the right. F. LNAI cells were plated in triplicate in 96-well plates and treated with vehicle (DMSO) or riociguat in FBS or CSS media for 20 hours. Intracellular superoxide levels were assessed by PI fluorescence, following incubation with 10 µM HEt. Unpaired two-tailed Student’s t-test were used to determine p-values. G. LNAI cells were plated in triplicate in 96-well plates and treated with vehicle (DMSO) or riociguat in FBS or CSS media for 20 hours. Total cellular peroxide levels were assessed by FITC fluorescence, following incubation with 10 µM CM- H 2 DCF-DA. Unpaired two-tailed Student’s t-test were used to determine p-values. H. LNAI cells were plated in triplicate in 96-well plates and treated with vehicle (DMSO) or riociguat in FBS or CSS media for 20 hours. Cells levels of cellular hydroxyl and peroxynitrite radicals were assessed by FITC fluorescence, following incubation with 10 µM HPF. Unpaired two-tailed Student’s t-test were used to determine p-values.

Article Snippet: Twenty-four hours after plating, cells were changed into either media supplemented with 5% FBS or 5% CSS was added to the cells, and were treated with either 50 μM, 100 μM or 200 μM riociguat (Cayman chemical, 9000554) or DMSO vehicle control (Sigma, D8418).

Techniques: Activity Assay, Cell Culture, Two Tailed Test, Comparison, Incubation, Western Blot, Membrane, Fluorescence

Journal: bioRxiv

Article Title: The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

doi: 10.1101/2023.05.03.537252

Figure Lengend Snippet: Note that all error bars represent ± SEM and * p< 0.05, ** p≤ 0.01, *** p≤ 0.001, **** p≤ 0.0001. Note that all immunoblots show the molecular weight of the markers run on the original membrane on the right. A. Cinaciguat decreases the viability of LNAI CRPC cells even under androgen-replete conditions. Cells were plated at 96- well plates in triplicate and treated with riociguat or cinaciguat in FBS media for 72 hours with daily redosing. Cell viabilities, IC 50 s and p-values were established as in . B. Cinaciguat induces apoptosis in LNAI CRPC cells. LNAI cells were plated in 10 cm dishes under the conditions in (A), and harvested following treatment. Total protein lysates (15 µg) were immunoblotted and probed with the indicated antibodies. C. Cinaciguat stimulates sGC activity in LNAI CRPC to a greater extent than in LNCaP CSPC cells. LNCaP SB0, SB5, SB5X, SB5XX and LNAI cells were plated in FBS-supplemented media, and cGMP levels were measured following incubation with either vehicle or 20 µM cinaciguat for 30 minutes. An unpaired two-tailed Student’s t-test was used to determine p-values. D. Western blotting was carried out using total protein lysates (10 µg) from SB0, SB5, SB5X, SB5XX and LNAI cells cultured under FBS- or CSS-supplemented media for 30h. Blots were probed for HSP90 with β actin as the loading control. E. Levels of the redox-protective thiol protein, TRX1, increase with castration resistance. Western blotting was carried out using total protein lysates (15 µg) from SB0, SB5, SB5X, SB5XX and LNAI cells. Blots were probed for TRX1 with β actin as the loading control. F. TRX1 overexpression sensitizes LNAI CRPC cells to riociguat under androgen-replete conditions. Generation of control vector and TRX1-overexpressing LNAI counterpart lines was validated by immunoblotting (inset). The pBL- and pBL.TRX1-expressing LNAI cells were plated as described in A, and treated with riociguat for 72 hours in FBS- supplemented media, with daily drug redosing. Cell viability and IC 50 s were established as in .

Article Snippet: Twenty-four hours after plating, cells were changed into either media supplemented with 5% FBS or 5% CSS was added to the cells, and were treated with either 50 μM, 100 μM or 200 μM riociguat (Cayman chemical, 9000554) or DMSO vehicle control (Sigma, D8418).

Techniques: Western Blot, Molecular Weight, Membrane, Activity Assay, Incubation, Two Tailed Test, Cell Culture, Control, Over Expression, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

doi: 10.1101/2023.05.03.537252

Figure Lengend Snippet: Note that all error bars represent ± SEM and * p< 0.05, ** p≤ 0.01, *** p≤ 0.001, **** p≤ 0.0001. Note that all immunoblots show the molecular weight of the markers run on the original membrane on the right. A. Riociguat reduces castration-resistant tumor growth. LNAI tumor-bearing castrated male Nu/Nu mice were randomized into groups receiving either daily IP injections of riociguat (20 mg/kg/day) or DMSO. Tumor growth was monitored three times weekly. Mean tumor volumes per treatment group across the 24-day experiment are shown. Adjusted areas under the curve (aAUCs) are plotted within the inset for the vehicle group (n=14) and the riociguat-treated group (n=14). Wilcoxon two-sample test was used to determine p-values. B. Endpoint tumor volumes (mm 3 ) in each treatment group (n=14) are shown. Each point represents an individual tumor/mouse. Unpaired two-tailed Student’s t-test was used to determine p-values. C. Representative H&E and Ki67-stained immunohistochemical images for tumors from the vehicle and riociguat-treated groups. The size bar, in white, represents a 50 μm scale. D. TUNEL staining shows riociguat treatment induces apoptosis. The TUNEL assay was carried out on tissue sections from vehicle and riociguat-treated tumors. Representative images of co-localized dUTP/DAPI staining are shown for two separate representative tumors in each indicated group. Images were acquired through identical exposures per channel. The size bar represents a 100 μm scale. E. Western blotting for the apoptotic marker, cleaved-PARP (cl-PARP), in xenograft tumor lysates (15 µg) from vehicle or riociguat- treated mice (left). Actin is used as the loading control. Quantitation of the cl-PARP immunoblot signal from the respective indicated tumor lysates, normalized to actin, is shown (Right). Unpaired two-tailed t-test with Welch’s correction was used to determine p- values. F. Immunoblotting for phospho (Ser239)-VASP in tumor lysates (30 µg) from vehicle or riociguat-treated mice. Total VASP levels are shown as loading control. Quantitation of the p-VASP protein signal, normalized to total VASP, is shown (right). Unpaired two-tailed t-test with Welch’s correction was used to determine p-values. G. Intratumoral cGMP levels were measured in tumor lysates from the vehicle or riociguat tumor groups. Unpaired two-tailed t-test with Welch’s correction was used. H. Intratumoral cGMP levels from riociguat-treated animals correlate inversely with the tumor volumes. Spearman correlation test values are shown. I. Plasma cGMP levels were measured in the vehicle or riociguat groups. Unpaired two-tailed t-test with Welch’s correction was used to determine p-values. J. Plasma cGMP levels from riociguat-treated animals correlate inversely with xenograft tumor volumes. Spearman correlation test values are shown.

Article Snippet: Twenty-four hours after plating, cells were changed into either media supplemented with 5% FBS or 5% CSS was added to the cells, and were treated with either 50 μM, 100 μM or 200 μM riociguat (Cayman chemical, 9000554) or DMSO vehicle control (Sigma, D8418).

Techniques: Western Blot, Molecular Weight, Membrane, Two Tailed Test, Staining, Immunohistochemical staining, TUNEL Assay, Marker, Control, Quantitation Assay, Clinical Proteomics

Journal: bioRxiv

Article Title: The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

doi: 10.1101/2023.05.03.537252

Figure Lengend Snippet: Note that all error bars represent ± SEM and * p< 0.05, ** p≤ 0.01, *** p≤ 0.001, **** p≤ 0.0001. A. Riociguat treatment oxygenates CRPC tumors. Representative immunofluorescent images of vehicle- or riociguat-treated tumors stained with pimonidazole (Hypoxyprobe) and counterstained with the nuclear dye, DAPI. Endpoint tumor volumes are indicated within each panel. The size bar represents a 50 μm scale. B. Riociguat treatment induces tumor vascular reorganization. Representative immunohistological images of the same tumors from (A), stained for the vascular endothelial marker, CD31. Arrows indicate canalised blood vessels. The size bar represents a 100 μm scale. C. Anti-tumor response to riociguat treatment correlates with loss of the PC stem cell marker, CD44. Representative immunofluorescent images of a vehicle-treated, a riociguat-treated nonresponsive tumor (NR), and two riociguat-responsive (R1, R2) tumors stained for CD44, shown in green, and counterstained with DAPI (blue). D. Cytokine markers of anti-tumor response to riociguat treatment. Sera obtained from vehicle, non-responder, responder 1, and responder 2 animals in (C) were assayed for systemic changes in host (murine) cytokine levels. The indicated cytokines fit the criteria of being the only ones where the non-responder and vehicle-treated values are similar and where the responder 1, responder 2 values trend in the same direction. Each cytokine value is the average of n=2. Results are represented as fold-change relative to the values established from vehicle-treated sera. The p values compare changes in group 1 (vehicle, non-responder) vs those in group 2(responder 1, responder 2), and were established using unpaired two-tailed Student’s tests. E. Riociguat sensitizes CRPC tumors to radiation. LNAI tumor-bearing castrated male Nu/Nu mice were randomized into groups receiving daily IP injections of riociguat (20 mg/kg/day) or vehicle (DMSO) for 96 hours. Following this, animals were further randomized in each group to receive either a single dose of 6 Gy ionizing radiation (IR) or mock irradiation (0 Gy). Daily IP injections of vehicle or riociguat were continued for the duration of the experiment. Mean tumor volumes per treatment group across the 24- day experiment are shown: vehicle/mock IR (n=3), riociguat/mock IR (n=4), vehicle/6Gy IR (n=10), riociguat/6Gy IR (n=11). The p values shown are comparing aAUC of the curves (vehicle/6 Gy IR vs riociguat/6 Gy IR) using a Kruskal-Wallis test. F. Endpoint tumor volumes (mm 3 ) from all treatment groups. Each point represents an individual tumor. Unpaired two-tailed Student’s t tests were used to determine p-values. G. Intratumoral cGMP levels were measured from xenograft tumors across all treatment groups. Unpaired two-tailed t-tests with Welch’s correction were used to determine p-values. H. H&E immunohistology is shown alongside co-localized Hypoxyprobe/DAPI immunofluorescent staining from representative tumors across all treatment groups. I. Overview of the role for sGC signaling in prostate cancer progression, showing the loss of sGC activity in the CSPC-to-CRPC transition through dysregulated heterodimer formation to evade ADIS, and through oxidative inactivation in established CRPC.

Article Snippet: Twenty-four hours after plating, cells were changed into either media supplemented with 5% FBS or 5% CSS was added to the cells, and were treated with either 50 μM, 100 μM or 200 μM riociguat (Cayman chemical, 9000554) or DMSO vehicle control (Sigma, D8418).

Techniques: Staining, Marker, Two Tailed Test, Irradiation, Activity Assay