Bartholomew J. Votta

Inhibition of p38 MAP kinase as a therapeutic strategy

Since the discovery of p38 MAP kinase in 1994, our understanding of its biology has progressed dramatically. The key advances include (1) identification of p38 MAP kinase homologs and protein kinases that act upstream and downstream from p38 MAP kinase, (2) identification of interesting and potentially important substrates, (3) elucidation of the role of p38 MAP kinase in cellular processes and (4) the establishment of the mechanism by which the pyridinylimidazole p38 MAP kinase inhibitors inhibit enzyme activity. It is now known that there are four members of the p38 MAP kinase family. They differ in their tissue distribution, regulation of kinase activation and subsequent phosphorylation of downstream substrates. They also differ in terms of their sensitivities toward the p38 MAP kinase inhibitors. The best-studied isoform is p38 alpha, whose activation has been observed in many hematopoietic and non-hematopoietic cell types upon treatment with appropriate stimuli. The pyridinylimidazole compounds, exemplified by SB 203580, were originally prepared as inflammatory cytokine synthesis inhibitors that subsequently were found to be selective inhibitors of p38 MAP kinase. SB 203580 inhibits the catalytic activity of p38 MAP kinase by competitive binding in the ATP pocket. X-ray crystallographic studies of the target enzyme complexed with inhibitor reinforce the observations made from site-directed mutagenesis studies, thereby providing a molecular basis for understanding the kinase selectivity of these inhibitors. The p38 MAP kinase inhibitors are efficacious in several disease models, including inflammation, arthritis and other joint diseases, septic shock, and myocardial injury. In all cases, p38 activation in key cell types correlated with disease initiation and progression. Treatment with p38 MAP kinase inhibitors attenuated both p38 activation and disease severity. Structurally diverse p38 MAP kinase inhibitors have been tested extensively in preclinical studies.

N-((1S)-1-{[4-((2S)-2-{[(2,4-Dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a Novel and Potent Transient Receptor Potential Vanilloid 4 Channel Agonist Induces Urinary Bladder Contraction and Hyperactivity: Part I

Abstract The transient receptor potential vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. Here we describe a small molecule TRPV4 channel activator, GSK1016790A, which we have utilized as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2+ influx in mouse and human TRPV4 expressing HEK cells (EC 50 values of 18 and 2.1 nM, respectively), and evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast the TRPV4 activator 4α-phorbol 12,13-didecanoate (4α−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 +/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4 -/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4The 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-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 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 4α-phorbol 12,13-didecanoate (4α-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+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4−/− bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4−/− bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4α-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4−/− mice revealed an enhanced bladder capacity in the TRPV4−/− mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4−/− 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.

GSK1016790A, a Novel and Potent TRPV4 Channel Agonist Induces Urinary Bladder Contraction and Hyperactivity: Part I

Abstract The transient receptor potential vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. Here we describe a small molecule TRPV4 channel activator, GSK1016790A, which we have utilized as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2+ influx in mouse and human TRPV4 expressing HEK cells (EC 50 values of 18 and 2.1 nM, respectively), and evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast the TRPV4 activator 4α-phorbol 12,13-didecanoate (4α−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 +/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4 -/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4The 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-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 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 4α-phorbol 12,13-didecanoate (4α-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+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4−/− bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4−/− bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4α-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4−/− mice revealed an enhanced bladder capacity in the TRPV4−/− mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4−/− 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.

Systemic Activation of the Transient Receptor Potential Vanilloid Subtype 4 Channel Causes Endothelial Failure and Circulatory Collapse: Part 2

The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4−/− null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-l-arginine methyl ester; l-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (l-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type‐selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast‐mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast‐mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration‐dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast‐mediated assay in vitro. Cbz‐Leu‐Leu‐Leu‐H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)‐stimulated resorption in the FRLB assay with an IC‐50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC‐50 of 100 nM. In the adjuvant‐arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz‐Leu‐Leu‐Leu‐H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz‐Leu‐Leu‐Leu‐H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast‐mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Catalytic in vivo protein knockdown by small-molecule PROTACs

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the targets ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

Identification and Cloning of a Connective Tissue Growth Factor-like cDNA from Human Osteoblasts Encoding a Novel Regulator of Osteoblast Functions

We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity (∼60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA (∼1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a ∼26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.

Chondroprotective role of the osmotically sensitive ion channel transient receptor potential vanilloid 4: Age- and sex-dependent progression of osteoarthritis in Trpv4-deficient mice

OBJECTIVE Mechanical loading significantly influences the physiology and pathology of articular cartilage, although the mechanisms of mechanical signal transduction are not fully understood. Transient receptor potential vanilloid 4 (TRPV4) is a Ca(++)-permeable ion channel that is highly expressed by articular chondrocytes and can be gated by osmotic and mechanical stimuli. The goal of this study was to determine the role of Trpv4 in the structure of the mouse knee joint and to determine whether Trpv4(-/-) mice exhibit altered Ca(++) signaling in response to osmotic challenge. METHODS Knee joints of Trpv4(-/-) mice were examined histologically and by microfocal computed tomography for osteoarthritic changes and bone structure at ages 4, 6, 9, and 12 months. Fluorescence imaging was used to quantify chondrocytic Ca(++) signaling within intact femoral cartilage in response to osmotic stimuli. RESULTS Deletion of Trpv4 resulted in severe osteoarthritic changes, including cartilage fibrillation, eburnation, and loss of proteoglycans, that were dependent on age and male sex. Subchondral bone volume and calcified meniscal volume were greatly increased, again in male mice. Chondrocytes from Trpv4(+/+) mice demonstrated significant Ca(++) responses to hypo-osmotic stress but not to hyperosmotic stress. The response to hypo-osmotic stress or to the TRPV4 agonist 4α-phorbol 12,13-didecanoate was eliminated in Trpv4(-/-) mice. CONCLUSION Deletion of Trpv4 leads to a lack of osmotically induced Ca(++) signaling in articular chondrocytes, accompanied by progressive, sex-dependent increases in bone density and osteoarthritic joint degeneration. These findings suggest a critical role for TRPV4-mediated Ca(++) signaling in the maintenance of joint health and normal skeletal structure.

IL‐1‐ and TNF‐induced bone resorption is mediated by p38 mitogen activated protein kinase

We have previously shown that p38 mitogen‐activated protein kinase (MAPK) inhibitors, which block the production and action of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin‐1 (IL‐1), are effective in models of bone and cartilage degradation. To further investigate the role of p38 MAPK, we have studied its activation in osteoblasts and chondrocytes, following treatment with a panel of proinflammatory and osteotropic agents. In osteoblasts, significant activation of p38 MAPK was observed following treatment with IL‐1 and TNF, but not parathyroid hormone, transforming growth factor‐β (TGF‐β), 1,25(OH)2D3, insulin‐like growth factor‐1 (IGF‐1), or IGF‐II. Similar results were obtained using primary bovine chondrocytes and an SV40‐immortalized human chondrocyte cell line, T/C28A4. SB 203580, a selective inhibitor of p38 MAPK, inhibited IL‐1 and TNF‐induced p38 MAPK activity and IL‐6 production (IC50s 0.3–0.5 μM) in osteoblasts and chondrocytes. In addition, IL‐1 and TNF also activated p38 MAPK in fetal rat long bones and p38 MAPK inhibitors inhibited IL‐1‐ and TNF‐stimulated bone resorption in vitro in a dose‐dependent manner (IC50s 0.3–1 μM). These data support the contention that p38 MAPK plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. Furthermore, the strong correlation between inhibition of kinase activity and IL‐1 and TNF‐stimulated biological responses indicates that selective inhibition of the p38 MAPK pathway may have therapeutic utility in joint diseases such as rheumatoid arthritis (RA).

CKβ‐8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues

We have previously demonstrated that a tartrate‐resistant acid phosphatase (TRAP)‐positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKβ‐8, RANTES, and MIP‐1α elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP‐1β, MCP‐1, MCP‐2, MCP‐3, MCP‐4, HCC‐1, eotaxin‐2, PARC, SLC, ELC) and 3 CXC chemokines (IL‐8, GROα, SDF‐1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG‐63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross‐desensitization studies demonstrate that RANTES and MIP‐1α can partially inhibit the chemotactic response elicited by CKβ‐8. CKβ‐8, the most potent of the active CC chemokines (ECmax 0.1–0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKβ‐8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKβ‐8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKβ‐8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption. J. Cell. Physiol. 183:196–207, 2000.