Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor ␤ (TGF-␤) is a member of a large family of pleiotropic cytokines that are involved in many biological processes, including growth control, differentiation, migration, cell survival, adhesion, and specification of developmental fate, in both normal and diseased states. TGF-␤ superfamily members signal through a receptor complex comprising a type II and type I receptor, both serine/threonine kinases. Here, we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-␤ type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7, which are very highly related to ALK5 in their kinase domains. It has no effect on the other, more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this, we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-␤ signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542, we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK, JNK, or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum.

Transforming growth factor ␤1 (TGF-␤1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-␤1 acts through the TGF-␤ type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-␤ type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC 50 of 94 nM. It inhibited TGF-␤1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC 50 values of 6 and 3 M, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 M to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-␤1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-␤1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-␤1-induced collagen I␣1 (col I␣1). These data indicate that some matrix markers that are stimulated by TGF-␤1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col I␣1).

The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-, which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2ϩ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC 50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4␣-phorbol 12,13-didecanoate (4␣-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4 ϩ/ϩ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4 Ϫ/Ϫ bladders. TRPV4 activation with GSK1016790A contracted TRPV4 ϩ/ϩ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4 Ϫ/Ϫ bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4␣-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4 ϩ/ϩ and TRPV4 Ϫ/Ϫ mice revealed an enhanced bladder capacity in the TRPV4 Ϫ/Ϫ mice. Infusion of GSK1016790A into the bladders of TRPV4 ϩ/ϩ mice induced bladder overactivity with no effect in TRPV4 Ϫ/Ϫ mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.Transient receptor potential (TRP) vanilloid 4 (TRPV4), a member of the TRP superfamily of cation channels, has been implicated in a number of physiological processes, including osmoregulation (Liedtke and Friedman, 2003;Mizuno et al., 2003), hearing (Tabuchi et al., 2005), thermal and mechaniThis work was supported by GlaxoSmithKline Pharmaceuticals. Article, publication date, and citation information can be found at

Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay.

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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 androgenregulated 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. [Cancer Res 2008;68(18):7475-83]

Clinically, there is a great need for small molecule inhibitors that could control pathogenic effects of transforming growth factor (TGF-beta) and/or modulate effects of TGF-beta in normal responses. Inhibition of TGF-beta signaling would be predicted to enhance re-epithelialization of cutaneous wounds and reduce scarring fibrosis. Selective small molecule inhibitors of the TGF-beta signaling pathway developed for therapeutics will also be powerful tools in experimentally dissecting this complex pathway, especially its cross-talk with other signaling pathways. In this study, we characterized 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124), a member of a new class of small molecule inhibitors related to imidazole inhibitors of p38, which inhibit the TGF-beta type I receptor serine/threonine kinase known as activin receptor-like kinase (ALK) 5. We demonstrate that this compound selectively and concentration-dependently inhibits ALK4-, ALK5-, and ALK 7-dependent activation of downstream cytoplasmic signal transducers, Smad2 and Smad3, and of TGF-beta-induced mitogen-activated protein kinase pathway components but does not alter ALK1, ALK2, ALK3 or ALK6-induced Smad signaling. SB-505124 also blocks more complex endpoints of TGF-beta action, as evidenced by its ability to abrogate cell death caused by TGF-beta1 treatment. SB-505124 is three to five times more potent than a related ALK5 inhibitor described previously, SB-431542.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.