Overall, S-CR8 showed more effective cell cycle blockade compared with R-roscovitine (Fig.?4A). Open in a separate Rabbit Polyclonal to CDH11 window Figure?4. renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD. and models of PKD.27-30 Mechanistic studies demonstrated that roscovitine inhibited cystogenesis through cell cycle arrest, transcriptional regulation and inhibition of apoptosis. Importantly, roscovitine treatment suppressed cAMP and aquaporin 2 in the cystic kidneys, suggesting that CDK inhibition targets the most proximal step in cystogenesis.31 To further validate CDK inhibition as an approach to treat ADPKD, preclinical efficacy needs to be established in an orthologous model. The goals of this study were to confirm efficacy of R-roscovitine in an orthologous Gosogliptin mouse model of ADPKD with a conditionally inactivated gene (cKO)32 and to assess the efficacy of the second generation analog of roscovitine, S-CR8, a more potent and selective CDK inhibitor.33 We demonstrate effective inhibition of both renal and hepatic cystogenesis with R-roscovitine and S-CR8 compounds. Mode of action studies demonstrate that both compounds act through blockade of cell cycle and proliferation and attenuation of apoptosis. Results CDK inhibitor S-CR8 potently inhibits cystogenesis in vitro To improve drug-like properties of R-roscovitine (metabolic stability, potency and selectivity), extensive medicinal chemistry studies identified a new and improved analog S-CR8, shown in Figure?1A.33,34 We have used a standard assay of MDCK cystogenesis in vitro to assess potency of S-CR8 as described previously.29,35 R-roscovitine was tested in parallel for comparison. MDCK cysts were grown in 96-well plates containing collagen gel with FBS-containing media for 4 d. Increasing concentrations of compounds were added to cysts and incubated for additional 4 d. Percent of inhibition of cystogenesis by each compound was measured by standard Alamar Blue assay (Fig.?1B) and confirmed by visual observation of cultured cysts under light microscope (not shown). The assay showed that both R-roscovitine and S-CR8 compounds reduce cyst formation in vitro in a dose-dependent manner with an IC50 of 16 M and 0.2 M, respectively. These data indicate that S-CR8 is approximately 80-fold more potent than R-roscovitine in cellular assay. This observation is in agreement with previously published data suggesting greater anti-tumor potency for S-CR8 compared with R-roscovitine in multiple cell lines (100-fold on the average of more than 65 cell lines).33 Open in a separate window Figure?1. Comparative analysis of inhibitory activities of CDK inhibitors S-CR8 and R-roscovitine on cystogenesis in vitro. (A) Chemical structures of R-roscovitine and its derivative, S-CR8. (B) In vitro inhibition of cystic growth in MDCK 3D collagen-based assay. Values were measured in quadruplets in two independent experiments. R-roscovitine and S-CR8 effectively inhibit renal cystic disease progression in gene at day Gosogliptin 5 results in a rapid onset PKD that is gender-independent.32 Cysts in the liver are also observed in this model. Similar to other models with conditionally inactivated gene, the majority of cysts originate from distal nephron segments and collecting ducts.17 In the current study, cystogenesis was induced with tamoxifen at postnatal day 5. Animals received daily injections of either R-roscovitine (100 mg/kg IP, once a day) or vehicle control from day 7C33 (Fig.?2A). The R-roscovitine-treated group showed a significant inhibition of PKD, evident by a decrease in kidney to body weight ratio, cystic volume and blood urea nitrogen (BUN) (Fig.?2B and Table 1). Effective reduction of cystic tissue in a representative R-roscovitine treated kidney is illustrated in Figure?2C. Open in a separate window Figure?2. CDK inhibitors R-roscovitine and S-CR8 inhibit renal cystogenesis in deletion with tamoxifen and schedule of treatment with R-roscovitine and S-CR8. (B) Quantitative analysis of Gosogliptin effect of R-roscovitine and S-CR8 on cystogenesis in kidney measured as kidney/body weight (BW) ratio, cystic volume and blood urea nitrogen (BUN); * p 0.05 compared with vehicle control. Error bars indicate SEM; (C) Representative kidney sections (H&E staining) from treated mice and vehicle control suggest preservation of kidney parenchyma in animals treated with CDK inhibitors as compared with vehicle-treated group. Table?1. Anti-cystic effect of CDK inhibitors R-roscovitine and S-CR8 in cKO mice cKO mice develop liver cysts.
- Post author:groundwater2011
- Post published:February 2, 2023
- Post category:Glutamate (Metabotropic) Group I Receptors