Robert W. Marquis

NOVEL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) GAMMA AND PPARDELTA LIGANDS PRODUCE DISTINCT BIOLOGICAL EFFECTS

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARα, PPARδ, and PPARγ. PPARγ has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARγ and PPARδ that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARγ and PPARδ directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARγ agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabeticdb/db mice all PPARγ agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selectivein vivo activation of PPARδ did not significantly affect these parameters. In vivo PPARα activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARγ and PPARδ; 2) ligand-dependent activation of PPARδ involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARγ activation (but not PPARδ or PPARα activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARγ agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARα activation is sufficient to affect triglyceride metabolism, PPARδ activation does not appear to modulate glucose or triglyceride levels.

Activation of PPARδ alters lipid metabolism in db/db mice

Peroxisome proliferator‐activated receptors (PPARs) are nuclear receptors, which heterodimerize with the retinoid X receptor and bind to peroxisome proliferator response elements in the promoters of regulated genes. Despite the wealth of information available on the function of PPARα and PPARγ, relatively little is known about the most widely expressed PPAR subtype, PPARδ. Here we show that treatment of insulin resistant db/db mice with the PPARδ agonist L‐165 041, at doses that had no effect on either glucose or triglycerides, raised total plasma cholesterol concentrations. The increased cholesterol was primarily associated with high density lipoprotein (HDL) particles, as shown by fast protein liquid chromatography analysis. These data were corroborated by the chemical analysis of the lipoproteins isolated by ultracentrifugation, demonstrating that treatment with L‐165 041 produced an increase in circulating HDL without major changes in very low or low density lipoproteins. White adipose tissue lipoprotein lipase activity was reduced following treatment with the PPARδ ligand, but was increased by a PPARγ agonist. These data suggest both that PPARδ is involved in the regulation of cholesterol metabolism in db/db mice and that PPARδ ligands could potentially have therapeutic value.

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.

Potent and Selective Inhibition of Human Cathepsin K Leads to Inhibition of Bone Resorption In Vivo in a Nonhuman Primate

Cathepsin K is a cysteine protease that plays an essential role in osteoclast‐mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB‐357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB‐357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin‐releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB‐357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N‐terminal telopeptides (NTx) and C‐terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.

Inhibitors of cysteine protease

The roles of cysteine proteases (CP) as protein degrading and protein processing enzymes both in physiological and pathological processes of mammals are well known. Furthermore, the key roles of CP;s in the life cycles of infectious agents like protozoa and viruses turn them into new important targets for anti-infective drugs. Thus, the effective inhibition of pathologically relevant cysteine proteases has raised increasing interest in drug development. One strategy to create CP inhibitors is the use of electrophilic moieties, which covalently bind to the cysteine residue of the active site of the target protease. In a previous approach we have selected the aziridine-2,3-dicarboxylic acid as weak electrophilic inhibitor fragment. In order to achieve effective enzyme inhibition this electrophile was incorporated into peptidic or peptidomimetic sequences addressing the substrate binding sites of the protease. High selectivity could be obtained with compounds, which bind into both the primed and non-primed substrate binding pockets. In a second approach the alpha,beta-unsaturated ketone of the well-known diuretic drug ethacrynic acid was found to be another appropriate electrophilic moiety. Derivatives thereof turned out to be new non-peptidic CP inhibitors. Results of inhibition assays obtained with these two inhibitor series on various proteases of human, protozoan, and viral origin, theoretical studies to investigate binding modes and inhibition mechanisms, and structure-activity relationships are presented. Furthermore, the results of in vitro assays on respective pathogens as well as the results of first toxicity studies are summarized.

Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis.

Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.

Potent dipeptidylketone inhibitors of the cysteine protease cathepsin K

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor.

Potent and selective cathepsin L inhibitors do not inhibit human osteoclast resorption in vitro.

Cathepsins K and L are related cysteine proteases that have been proposed to play important roles in osteoclast-mediated bone resorption. To further examine the putative role of cathepsin L in bone resorption, we have evaluated selective and potent inhibitors of human cathepsin L and cathepsin K in an in vitro assay of human osteoclastic resorption and an in situ assay of osteoclast cathepsin activity. The potent selective cathepsin L inhibitors (K i = 0.0099, 0.034, and 0.27 nm) were inactive in both the in situcytochemical assay (IC50 > 1 μm) and the osteoclast-mediated bone resorption assay (IC50 > 300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC50 = 63 nm) and resorption (IC50 = 71 nm) assays. A recently reported dipeptide aldehyde with activity against cathepsins L (K i = 0.052 nm) and K (K i = 1.57 nm) was also active in both assays (IC50 = 110 and 115 nm, respectively) These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.