Nicholas J. Laping

Development of a small molecule serum and glucocorticoid-regulated kinase 1 antagonist and its evaluation as a prostate cancer therapeutic

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference-mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer.

Gender Differences in Methamphetamine-Induced mRNA Associated with Neurodegeneration in the Mouse Nigrostriatal Dopaminergic System

In this report female and male CD-1 mice were treated with a neurotoxic regimen of methamphetamine (MA) to compare gender differences in striatal dopamine depletion and concordant changes in mRNA markers of the transforming growth factor-β injury response associated with neurodegeneration. Striatal dopamine concentrations of MA-treated female mice were less depleted and significantly greater than that of identically treated males. Associated with this gender difference in striatal dopamine depletion were significantly decreased mRNA levels of plasminogen activator inhibitor-1 and a trend for increased (p = 0.06) mRNA levels of glial fibrillary acidic protein within females. No statistically significant differences between MA-treated female and male mice were obtained in mRNA levels for transforming growth factor-β, transforming growth factor-β type 2 receptor, activin-like kinase-5 or fibronectin. These data demonstrate the presence of changes in two specific molecular markers of the transforming growth factor-β injury response which are in accordance with gender differences in MA-induced striatal dopamine depletion. The results suggest that the neuroprotective advantage displayed by females may in part be related to reductions in the transforming growth factor-β injury response as indicated by decreased mRNA plasminogen activator inhibitor-1 and an increased response of reactive astrocytes which promote neuronal survival as indicated by augmented glial fibrillary acidic protein mRNA levels.

Renoprotective effects of carvedilol in hypertensive‐stroke prone rats may involve inhibition of TGFβ expression

The effect of carvedilol on renal function, structure and expression of TGFβ and the matrix proteins fibronectin, collagen I and collagen III, was evaluated in spontaneously hypertensive stroke‐prone (SHR‐SP) rats fed a high fat, high salt diet. Carvedilol treatment for 11 to 18 weeks did not alter systolic blood pressure in SHR‐SP rats, however, it resulted in a significant reduction in heart rate. Carvedilol treatment reduced renal fibrosis and total, active and chronic renal damage to levels approaching those of WKY rats on a normal diet. Urinary protein excretion was higher in SHR‐SP rats (51±10 mg day−1) than WKY rats (18±2 mg day−1) and this was further increased when SHR‐SP rats were fed a high fat, high salt diet (251±120 mg day−1). Treatment with carvedilol resulted in significantly lower urinary protein excretion (37±15 mg day−1). The expression of TGFβ mRNA was significantly higher in SHR‐SP rats compared to WKY rats and a further increase was observed when rats were fed a high fat, high salt diet. Renal TGFβ expression was significantly reduced by treatment with carvedilol. The expression of fibronectin and collagen I and collagen III mRNA showed a pattern similar to that observed with TGFβ mRNA expression. Collagen I mRNA expression followed a pattern similar to renal fibrosis. These data indicate that carvedilol can provide significant renal protection in the absence of any antihypertensive activity and that the mechanisms involved in this action may include reduced expression of profibrotic factors such as TGFβ.

ALK5 inhibition in renal disease.

Recent advances have identified novel small molecule inhibitors of the transforming growth factor-beta (TGF-beta) type I receptor kinase as a potential therapy in organ remodeling diseases, such as chronic renal disease. Because TGF-beta is central to the progression of fibrosis, selective inhibition of this signaling pathway could provide a novel treatment in many fibrotic diseases. The rationale for inhibition of TGF-beta signaling in renal disease includes prevention of fibrosis, tubular dedifferentiation and vascular effects.

Inhibition of ALK5 Signaling Induces Physeal Dysplasia in Rats

TGF-|β|, and its type 1 (ALK5) receptor, are critical to the pathogenesis of fibrosis. In toxicologic studies of 4 or more days in 10-week-old Sprague–Dawley rats, using an ALK5 inhibitor (GW788388), expansion of hypertrophic and proliferation zones of femoral physes were noted. Subphyseal hyperostosis, chondrocyte hypertrophy/hyperplasia, and increased matrix were present. Physeal zones were laser microdissected from ALK5 inhibitor-treated and control rats sacrificed after 3 days of treatment. Transcripts for TGF-|β|1, TGF-|β|2, ALK5, IHH, VEGF, BMP-7, IGF-1, bFGF, and PTHrP were amplified by real-time PCR. IGF and IHH increased in all physis zones with treatment, but were most prominent in prehypertrophic zones. TGF-|β|2, bFGF and BMP7 expression increased in proliferative, pre- and hypertrophic zones. PTHrP expression was elevated in proliferative zones but decreased in hypertrophic zones. VEGF expression was increased after treatment in pre- and hypertrophic zones. ALK5 expression was elevated in prehypertrophic zones. Zymography demonstrated gelatinolytic activity was reduced after treatment. Apoptotic markers (TUNEL and caspase-3) were decreased in hypertrophic zones. Proliferation assessed by Topoisomerase II and Ki67 was increased in multiple zones. Movat stains demonstrated that proteoglycan deposition was altered. Physeal changes occurred at doses well above those resulting in fibrosis. Interactions of factors is important in producing the physeal dysplasia phenotype.

Modulation of bladder function by prostaglandin EP3 receptors in the central nervous system

Prostaglandin EP3 receptors in the central nervous system (CNS) may exert an excitatory effect on urinary bladder function via modulation of bladder afferent pathways. We have studied this action, using two EP3 antagonists, (2E)-3-{1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-yl}-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG041) and (2E)-N-{[5-bromo-2-(methyloxy)phenyl] sulfonyl}-3-[2-(2-naphthalenylmethyl)phenyl]-2-propenamide (CM9). DG041 and CM9 were proven to be selective EP3 antagonists with radioligand binding and functional fluorescent imaging plate reader (FLIPR) assays. Their effects on volume-induced rhythmic bladder contraction and the visceromotor reflex (VMR) response to urinary bladder distension (UBD) were evaluated in female rats after intrathecal or intracerebroventricular administration. Both DG041 and CM9 showed a high affinity for EP3 receptors at subnanomolar concentrations without significant selectivity for any splice variants. At the human EP3C receptor, both inhibited calcium influx produced by the nonselective agonist PGE2. After intrathecal or intracerebroventricular administration both CM9 and DG041 dose-dependently reduced the frequency, but not the amplitude, of the bladder rhythmic contraction. With intrathecal administration DG041 and CM9 produced a long-lasting and robust inhibition on the VMR response to UBD, whereas with intracerebroventricular injection both compounds elicited only a transient reduction of the VMR response to bladder distension. These data support the concept that EP3 receptors are involved in bladder micturition at supraspinal and spinal centers and in bladder nociception at the spinal cord. A centrally acting EP3 receptor antagonist may be useful in the control of detrusor overactivity and/or pain associated with bladder disorders.

An excitatory role for peripheral EP3 receptors in bladder afferent function

The excitatory roles of EP3 receptors at the peripheral afferent nerve innervating the rat urinary bladder have been evaluated by using the selective EP3 antagonist (2E)-3-[1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-yl]-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG-041). The bladder rhythmic contraction model and a bladder pain model measuring the visceromotor reflex (VMR) to urinary bladder distension (UBD) have been used to evaluate DG-041 in female rats. In addition, male rats [spontaneously hypertensive rat (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD)] were anesthetized with pentobarbital sodium, and primary afferent fibers in the L6 dorsal root were isolated for recording the inhibitory response to UBD following intravenous injection of DG-041. Intravenous injection of DG-041 (10 mg/kg), a peripherally restricted EP3 receptor antagonist, significantly reduced the frequency of bladder rhythmic contraction and inhibited the VMR response to bladder distension. The magnitude of reduction of the VMR response was not different in the different strains of rats (SD, SHR, and WKY). Furthermore, quantitative characterization of the mechanosensitive properties of bladder afferent nerves in SHR, WKY, and SD rats did not show the SHR to be supersensitive to bladder distension. DG-041 selectively attenuated responses of mechanosensitive afferent nerves to UBD, with strong suppression on the slow-conducting, high-threshold afferent fibers, with equivalent activity in the three strains. We conclude that sensitization of afferent nerve activity was not one of the mechanisms of bladder hypersensitivity in SHR. EP3 receptors are involved in the regulation of bladder micturition and bladder nociception at the peripheral level.

Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.

Hepatocyte growth factor : A regulator of extracellular matrix genes in mouse mesangial cells

The potential role of hepatocyte growth factor (HGF) in regulating extracellular matrix in mouse mesangial cells (MMC) was evaluated. Functional HGF receptors were deed in MMC by HGF-induced extracellular acidification, a response that was inhibited by the HGF inhibitor HGF/NK2, a splice variant expressing the N-terminal domain through the second kringle domain HGF also increased fibronectin and collagen alpha1 (IV) mRNA levels in these cells; the increases were associated with a concentration-dependent increase in transcriptional activity of the collagen alpha1 (IV) gene. HGF also stimulated fibronectin and collagen alpha1 (IV) mRNA levels in primary rabbit proximal tubule epithelial cells To evaluate the potential consequences of chronic elevation of HGF on renal fuction, HGF was administered continuously for 18 days to normal and diabetic C57BLKS/J lepr(db) mice. In the diabetic mice, HGF reduced creatinine clearance and increased microalbuminuria, indicating that chronic exposure to HGF impairs renal function. Thus, chronically elevated HGF may contribute to the progression of chronic renal disease in diabetes by decreasing the glomerular filtration rate and possibly promoting the accumulation of extracellular matrix.

Age-related expression of renal thrombospondin 1 mRNA in F344 rats: resemblance to diabetes-induced expression in obese Zucker rats.

Age-related increases occurred in renal thrombospondin 1 (TSP1) mRNA in F344 rats, resembling diabetes-induced TSP1 mRNA in the obese Zucker rat. TSP1 mRNA was 3.5-fold higher in 24-month-old than in 3-month-old F344 rats. TSP1 mRNA increased similarly in 5-month-old obese Zucker rats as compard with lean littermates and correlated positively with the extent of proteinuria (r = 0.71). In situ hybridization identified elevated TSP1 mRNA levels in epithelial cells of distended tubules as well as in interstitium near dilated tubules of both 24-month-old F344 rats and 5-month-old obese Zucker rats. Furthermore, thrombin increased TSP1 mRNA in mesangial and epithelial cells in culture, indicating that thrombin may contribute to elevated TSP1 expression in renal disease. Thrombin increased TSP1 mRNA within 30 min after treatment which required de novo synthesis of protein. The thrombin receptor tethered ligand peptide, SFLLRN, increased TSP1 mRNA, indicating that the thrombin-induced increase in TSP1 mRNA was due to direct thrombin receptor (PAR1) stimulation. These results show that increased TSP1 mRNA levels are a component of interstitial fibrosis seen in aged and diabetic kidneys and suggest that similar pathological changes occur in kidneys of aging and diabetic rats.