Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae)

Plants in the genus Tripterygium, such as Tripterygium wilfordii Hook.f., have a long history of use in traditional Chinese medicine. In recent years there has been considerable interest in the use of Tripterygium extracts and of the main bioactive constituent, the diterpene triepoxide triptolide (1), to treat a variety of autoimmune and inflammation-related conditions. The main mode of action of the Tripterygium extracts and triptolide (1) is the inhibition of expression of proinflammatory genes such as those for interleukin-2 (IL-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2) and interferon-gamma (IFN-gamma). The efficacy and safety of certain types of Tripterygium extracts were confirmed in human clinical trials in the US and abroad. Over 300 compounds have been identified in the genus Tripterygium, and many of these have been evaluated for biological activity. The overall activity of the extract is based on the interaction between its components. Therefore, the safety and efficacy of the extract cannot be fully mimicked by any individual constituent. This review discusses the biochemical composition and biological and pharmacological activities of Tripterygium extracts, and their main bioactive components.     

Synthesis and use of cell-permeant cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca(2+). cADPR has a net negative charge at physiological pH making it not passively membrane permeant thereby requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been overcome by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl (AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca(2+). Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to cADPR-AM, whereas the response still occurred in the absence of extracellular Ca(2+). Combined, these data demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions.     

Calcium signaling by cyclic ADP-ribose and NAADP

Ca²⁺ mobilization as a signaling mechanism has been placed on center stage with the discovery of the first Ca²⁺ messenger, inositol trisphosphate (IP₃). This article focuses on two new Ca²⁺ release activators, which mobilize internal Ca²⁺ stores via mechanisms totally independent of IP₃. They are cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP), metabolites derived respectively from NAD and NADP. Major advances in the past decade in the understanding of these two novel signaling mechanisms are chronologically summarized.     

Vasodilation by the calcium-mobilizing messenger cyclic ADP-ribose

In artery smooth muscle, adenylyl cyclase-coupled receptors such as beta-adrenoceptors evoke Ca(2+) signals, which open Ca(2+)-activated potassium (BK(Ca)) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca(2+) signal is evoked via ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to the plasma membrane. We show here that cyclic adenosine diphosphate-ribose (cADPR), by activating RyRs, mediates, in part, hyperpolarization and vasodilation by beta-adrenoceptors. Thus, intracellular dialysis of cADPR increased the cytoplasmic Ca(2+) concentration proximal to the plasma membrane in isolated arterial smooth muscle cells and induced a concomitant membrane hyperpolarization. Smooth muscle hyperpolarization mediated by cADPR, by beta-adrenoceptors, and by cAMP, respectively, was abolished by chelating intracellular Ca(2+) and by blocking RyRs, cADPR, and BK(Ca) channels with ryanodine, 8-amino-cADPR, and iberiotoxin, respectively. The cAMP-dependent protein kinase A antagonist N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP, respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a downstream element in this signaling cascade. Importantly, antagonists of cADPR and BK(Ca) channels, respectively, inhibited beta-adrenoreceptor-induced artery dilation. We conclude, therefore, that relaxation of arterial smooth muscle by adenylyl cyclase-coupled receptors results, in part, from a cAMP-dependent and protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca(2+) release via RyRs, which leads to activation of BK(Ca) channels and membrane hyperpolarization.     

Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.     

Relating protein pharmacology by ligand chemistry

The identification of protein function based on biological information is an area of intense research. Here we consider a complementary technique that quantitatively groups and relates proteins based on the chemical similarity of their ligands. We began with 65,000 ligands annotated into sets for hundreds of drug targets. The similarity score between each set was calculated using ligand topology. A statistical model was developed to rank the significance of the resulting similarity scores, which are expressed as a minimum spanning tree to map the sets together. Although these maps are connected solely by chemical similarity, biologically sensible clusters nevertheless emerged. Links among unexpected targets also emerged, among them that methadone, emetine and loperamide (Imodium) may antagonize muscarinic M3, alpha2 adrenergic and neurokinin NK2 receptors, respectively. These predictions were subsequently confirmed experimentally. Relating receptors by ligand chemistry organizes biology to reveal unexpected relationships that may be assayed using the ligands themselves.     

NMR studies and semi-empirical energy calculations for cyclic ADP-ribose

A possible pH-dependent conformational switch was investigated for cyclic ADP-ribose. NMR signals for the exchangeable protons were observed in H2O at low temperature, but there was no direct evidence for the protonation of N-3 at neutral pH that has previously been postulated. MNDO calculations indicated that pH dependent 31P chemical shift changes are attributable to protonation of the phosphate adjacent to the N-1 of adenine, and not due to trans-annular hydrogen bonding with a protonated N-3.     

Adp-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. a primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction is unique to pulmonary arteries and serves to match lung perfusion to ventilation. However, in disease states this process can promote hypoxic pulmonary hypertension. Hypoxic pulmonary vasoconstriction is associated with increased NADH levels in pulmonary artery smooth muscle and with intracellular Ca(2+) release from ryanodine-sensitive stores. Because cyclic ADP-ribose (cADPR) regulates ryanodine receptors and is synthesized from beta-NAD(+), we investigated the regulation by beta-NADH of cADPR synthesis and metabolism and the role of cADPR in hypoxic pulmonary vasoconstriction. Significantly higher rates of cADPR synthesis occurred in smooth muscle homogenates of pulmonary arteries, compared with homogenates of systemic arteries. When the beta-NAD(+):beta-NADH ratio was reduced, the net amount of cADPR accumulated increased. This was due, at least in part, to the inhibition of cADPR hydrolase by beta-NADH. Furthermore, hypoxia induced a 10-fold increase in cADPR levels in pulmonary artery smooth muscle, and a membrane-permeant cADPR antagonist, 8-bromo-cADPR, abolished hypoxic pulmonary vasoconstriction in pulmonary artery rings. We propose that the cellular redox state may be coupled via an increase in beta-NADH levels to enhanced cADPR synthesis, activation of ryanodine receptors, and sarcoplasmic reticulum Ca(2+) release. This redox-sensing pathway may offer new therapeutic targets for hypoxic pulmonary hypertension.     

Ca2+ release induced by cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is the most potent Ca²⁺-mobilizing agent known. It has been found in many different cell types, where it is synthesized from its precursor NAD⁺ by ADP-ribosyl cyclases. cADPR binds to Ca²⁺ channels in the endoplasmic reticulum membrane to activate a Ca²⁺-release mechanism. This release is itself potentiated by elevated cytoplasmic Ca²⁺ concentrations. Thus, cADPR may function as an endogenous regulator of Ca²⁺-induced Ca²⁺ release, and there is excitement that it may also function as a Ca²⁺-mobilizing second messenger.     

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn-dependent ADP-ribose/CDP-alcohol pyrophosphatase

Cyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N¹-glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N¹-(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.     

Abscisic acid signaling through cyclic ADP-ribose in hydroid regeneration

Cyclic ADP-ribose (cADPR) is an intracellular calcium (Ca(2+)(i)) mobilizer involved in fundamental cell functions from protists to higher plants and mammals. Biochemical similarities between the drought-signaling cascade in plants and the temperature-sensing pathway in marine sponges suggest an ancient evolutionary origin of a signaling cascade involving the phytohormone abscisic acid (ABA), cADPR, and Ca(2+)(i). In Eudendrium racemosum (Hydrozoa, Cnidaria), exogenously added ABA stimulated ADP-ribosyl cyclase activity via a protein kinase A (PKA)-mediated phosphorylation and increased regeneration in the dark to levels observed under light conditions. Light stimulated endogenous ABA synthesis, which was conversely inhibited by the inhibitor of plant ABA synthesis Fluridone. The signal cascade of light-induced regeneration uncovered in E. racemosum: light --> increasing ABA --> PKA --> cyclase activation --> increasing [cADPR](i) --> increasing [Ca(2+)](i) --> regeneration is the first report of a complete signaling pathway in Eumetazoa involving a phytohormone.     

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.     

A novel fluorescent cell membrane-permeable caged cyclic ADP-ribose analogue

Cyclic adenosine diphosphate ribose is an endogenous Ca(2+) mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca(2+) increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca(2+) release from endoplasmic reticulum, with concomitant Ca(2+) influx in Jurkat cells. Ca(2+) release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca(2+) influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca(2+) release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca(2+) release from endoplasmic reticulum via RyRs and triggers Ca(2+) influx via TRPM2.     

Determination of endogenous levels of cyclic ADP-ribose in rat tissues

Cyclic ADP-ribose (cADPR) is a potent mediator of calcium mobilization in sea urchin eggs. The cADPR synthesizing enzyme is present not only in the eggs but also in various mammalian tissue extracts. The purpose of this study was to ascertain whether cADPR is a naturally occurring nucleotide in mammalian tissues. Rat tissues were frozen and powdered in liquid N2, followed by extraction with perchloric acid at -10 degrees C. [32P]cADPR was prepared and used as a tracer. The acid extracts were chromatographed on a Mono-Q column and cADPR in the fractions were determined by its ability to release Ca2+ from egg homogenates. That the release was mediated by cADPR and not inositol trisphosphate (IP3) in the extracts was shown by the fact that the homogenates, subsequent to Ca2+ release induced by active fractions, were desensitized to authentic cADPR but not to IP3. Furthermore, the Ca2+ release activity was shown to co-elute with [32P]cADPR. The endogenous level of cADPR determined in rat liver is 3.37 +/- 0.64 pmol/mg, in heart is 1.04 +/- 0.08 pmol/mg and in brain is 2.75 +/- 0.35 pmol/mg. These results indicate cADPR is a naturally occurring nucleotide and suggest that it may be a general second messenger for mobilizing intracellular Ca2+.     

Sympathetic potentiation of cyclic ADP-ribose formation in rat cardiac myocytes

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.     

Mythobotany,pharmacology, and chemistry of thujone-containing plants and derivatives

Thujone, C₁₀H₁₆O, is the primary constituent of essential oils derived from a variety of plants, including wormwood, Artemisia absinthium; mugwort, Artemisia vulgaris; sage, Salvia officinalis; clary, Salvia sclarea; tansy, Tanacetum vulgare; and yellow cedar or the tree of life, Thuja occidentalis. While oils derived from the individual species may vary in the modifying constituents which they contain, the pharmacological effects common to thujone–containing plants are generally considered to include the following: 1 ) anthelmintic, 2) psychedelic, 3) uterine, 4) antidote to opium and other central nervous system depressant poisons. The effects delineated by modern experimentation correlate remarkably well with those ascribed to the plants in mythology, folklore, and other pre–contemporary documents.     

Medicinal chemistry approaches to avoid aldehyde oxidase metabolism

Aldehyde oxidase (AO) is a molybdenum-containing enzyme distributed throughout the animal kingdom and capable of metabolising a wide range of aldehydes and N-heterocyclic compounds. Although metabolism by this enzyme in man is recognised to have significant clinical impact where human AO activity was not predicted by screening in preclinical species, there is very little reported literature offering real examples where drug discoverers have successfully designed away from AO oxidation. This article reports on some strategies adopted in the Pfizer TLR7 agonist programme to successfully switch off AO metabolism that was seen principally in the rat.     

Medicinal chemistry and chemical biology of new generation taxane antitumor agents

P-glycoprotein (P-GP)-based multidrug resistance (MDR) and undesirable side effects are significant drawbacks to the clinical use of paclitaxel and docetaxel. Extensive SAR studies of taxanes in these laboratories led to the discovery of new generation taxanes that are highly active against not only drug-sensitive but also drug-resistant human cancer cell lines as well as tumor xenografts in mice. One of these second generation taxanes, SB-T-110131 (IDN5109), exhibited excellent pharmacological profile in the preclinical studies and has been selected for clinical development (recoded as Bay 59-8862), which is currently in the phase II clinical trials. Bay 59-8862 is orally active with high bioavailability, showing excellent activity against a variety of drug-resistant tumors. "Advanced second generation taxanes" show essentially no difference in cytotoxicity against drug-resistant and drug-sensitive cell lines, virtually overcoming MDR. Photoaffinity labeling of P-GP using photoreactive radiolabeled paclitaxel analogs has disclosed the paclitaxel-binding domain of P-GP. Highly efficient taxane-based MDR reversal agents (TRAs) have also been developed, which can recover the cytotoxicity of paclitaxel to practically the original level against paclitaxel-resistant MDR expressing cancer cells. Highly promising results have emerged from the study of taxane-monoclonal antibody (MAb) immunoconjugates, which have been proved to specifically deliver extremely cytotoxic agents to tumor in an animal model.     

Mechanism-based inhibitors of CD38: a mammalian cyclic ADP-ribose synthetase

The soluble domain of human CD38 catalyzes the conversion of NAD(+) to cyclic ADP-ribose and to ADP-ribose via a common covalent intermediate [Sauve, A. A., Deng, H. T., Angelletti, R. H., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 7855-7859]. Here we establish that mechanism-based inhibitors can be produced by chemical stabilization of this intermediate. The compounds nicotinamide 2'-deoxyriboside (1), 5-methylnicotinamide 2'-deoxyriboside (2), and pyridyl 2'-deoxyriboside (3) were synthesized and evaluated as inhibitors for human CD38. The nicotinamide derivatives 1 and 2 were inhibitors of the enzyme as determined by competitive behavior in CD38-catalyzed conversion of nicotinamide guanine dinucleotide (NGD(+)) to cyclic GDP-ribose. The K(i) values for competitive inhibition were 1.2 and 4.0 microM for 1 and 2, respectively. Slow-onset characteristics of reaction progress curves indicated a second higher affinity state of these two inhibitors. Inhibitor off-rates were slow with rate constants k(off) of 1.5 x 10(-5) s(-1) for 1 and 2.5 x 10(-5) s(-1) for 2. Apparent dissociation constants K(i(total)) for 1 and 2 were calculated to be 4.5 and 12.5 nM, respectively. The similar values for k(off) are consistent with the hydrolysis of common enzymatic intermediates formed by the reaction of 1 and 2 with the enzyme. Both form covalently attached deoxyribose groups to the catalytic site nucleophile. Chemical evidence for this intermediate is the ability of nicotinamide to rescue enzyme activity after inactivation by either 1 or 2. A covalent intermediate is also indicated by the ability of CD38 to catalyze base exchange, as observed by conversion of 2 to 1 in the presence of nicotinamide. The deoxynucleosides 1 and 2 demonstrate that the chemical determinants for mechanism-based inhibition of CD38 can be satisfied by nucleosides that lack the 5'-phosphate, the adenylate group, and the 2'-hydroxyl moiety. In addition, these compounds reveal the mechanism of CD38 catalysis to proceed by the formation of a covalent intermediate during normal catalytic turnover with faster substrates. The covalent 2'-deoxynucleoside inactivators of CD38 are powerful inhibitors by acting as good substrates for formation of the covalent intermediate but are poor leaving groups from the intermediate complex because hydrolytic assistance of the 2'-hydroxyl group is lacking. The removal of the adenylate nucleophile required for the cyclization reaction provides slow hydrolysis as the only exit from the covalent complex.