Abstract
Racemic mixtures and enantiomerically pure d-isomers of both myo-inositol 1,3,6-trisphosphorothioate [Ins(1,3,6)PS3] and myo-inositol 1,4,6-trisphosphorothioate [Ins(1,4,6)PS3], prepared by total synthesis, were examined in Ca2+ flux and binding assays. Both d-Ins(1,3,6)PS3 andd-Ins(1,4,6)PS3 were shown to be low intrinsic activity partial agonists at the platelet myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor, releasing less than 20% of the Ins(1,4,5)P3-sensitive Ca2+ store. d-Ins(1,4,6)PS3displaced specifically bound [3H]Ins(1,4,5)P3from rat cerebellar membranes, although displacement was some 34-fold weaker than by d-Ins(1,4,5)P3.d-Ins(1,4,6)PS3 displaced [3H]Ins(1,4,5)P3 from cerebellar membranes with roughly twice the affinity ofdl-Ins(1,4,6)PS3 (IC50 value = 1.4 ± 0.35 μM compared with 2.15 ± 0.13 μM), whereasd-Ins(1,3,6)PS3 displaced [3H]Ins(1,4,5)P3 with roughly twice the affinity of dl-Ins(1,3,6)PS3 (IC50value = 17.5 ± 5.8 μM compared with 34 ± 10 μM), confirming that the activity of both these phosphorothioates resides in their d-enantiomers. Increasing concentrations of either d-Ins(1,3,6)PS3 ord-Ins(1,4,6)PS3 were able to partially antagonize Ca2+ release induced by submaximal concentrations of Ins(1,4,5)P3, an inhibition that could be overcome by increasing the concentration of Ins(1,4,5)P3, suggesting competition for binding at the Ins(1,4,5)P3-R. The only low-efficacy partial agonists at the Ins(1,4,5)P3-R discovered to date have been phosphorothioates; the novel d-Ins(1,3,6)PS3and d-Ins(1,4,6)PS3 can now be added to this small group of analogs. However,d-Ins(1,4,6)PS3 has a relatively high affinity for the Ins(1,4,5)P3-R but maintains the lowest efficacy of all the partial agonists thus far identified. As such, it may be a useful tool for pharmacological intervention in the polyphosphoinositide pathway and an important lead compound for the development of further Ins(1,4,5)P3-R antagonists.
An elevated level of cytosolic Ca2+ is known to be a principle mediator of activation-response coupling in numerous cell types in response to a wide range of extracellular stimuli. In non–voltage-excitable cells, Ca2+ is elevated via two pathways: mobilization from the intracellular stores and influx across the plasma membrane (Berridge, 1993; Putney and Bird, 1993;Clapham, 1995). Agonist-receptor coupling activates the hydrolysis of phosphatidylinositol 4,5-bisphosphate, producing the signal molecule inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which, via ligation of specific receptors on Ins(1,4,5)P3-sensitive intracellular Ca2+ stores, induces Ca2+mobilization into the cytoplasm, (for review, see Patel et al., 1999). Three Ins(1,4,5)P3-receptor [Ins(1,4,5)P3-R] subtypes, together with splice variants of each of these, have now been identified and the genes cloned (Furuichi et al., 1989; Sudhof et al., 1991; Blondel et al., 1993). The Ins(1,4,5)P3-R is now known to exist as a heterotetrameric complex that forms the Ins(1,4,5)P3-gated Ca2+channel (Joseph et al., 1995; Monkawa et al., 1995; Wojcikiewicz and He, 1995). Expression of the Ins(1,4,5)P3-R was found to enhance both Ins(1,4,5)P3 binding and Ca2+-releasing activities in transfected cell lines, indicating expression of protein with both binding sites for Ins(1,4,5)P3 and Ca2+channel activity (Miyawaki et al., 1990).
To investigate the relative importance of Ins(1,4,5)P3-induced Ca2+release in mediating the physiological processes within cells, a specific, high-affinity Ins(1,4,5)P3-R antagonist is required. In the rational design of an Ins(1,4,5)P3-R antagonist or a low intrinsic activity partial agonist, extensive knowledge of the structure-activity relationships of Ins(1,4,5)P3 is required (for review, see Wilcox et al., 1998). At present, structure-activity studies using analogs of Ins(1,4,5)P3 have not identified any distinct structural motifs of Ins(1,4,5)P3 that are responsible solely for either its receptor binding capability or its Ca2+-releasing activity (Potter and Lampe, 1995), although the pivotal role of the vicinal 4,5-trisphosphate system augmented by other auxiliary motifs has long been recognized.
The first inositol phosphate demonstrated to be a partial agonist at the Ins(1,4,5)P3-R was the naturally occurring higher polyphosphate myo-inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4] in SH-SY5Y cells (Gawler et al., 1991), although this compound was a full agonist in rabbit platelets (Murphy et al., 1996).l-chiro-Inositol 2,3,5-trisphosphorothioate [l-chr-Ins(2,3,5)PS3] and d-6-deoxy-myo-inositol 1,4,5-trisphosphorothioate [6-deoxy-Ins(1,4,5)PS3] (Fig.1a) were found to be low-efficacy partial agonists at the Ins(1,4,5)P3-R (Safrany et al., 1993; Liu et al., 1994).l-chr-Ins(2,3,5)PS3and 6-deoxy-Ins(1,4,5)PS3 are the C-3- and C-6-modified analogs of Ins(1,4,5)P3,respectively, in addition to carrying phosphorothioate groups rather than phosphates at the 1-, 4- and 5-positions. A high intrinsic activity partial agonist scyllo-inositol 1,2,4,5-tetrakisphosphorothioate also combined phosphorothioate substitutions at the key vicinal 4,5-bisphosphate motif with further structural modifications (Wilcox et al., 1994). Replacement of all the phosphate groups on Ins(1,4,5)P3 with phosphorothioate groups, with no other perturbation of the molecular structure of Ins(1,4,5)P3, however, had no effect on efficacy and only a small decrease in affinity at the Ins(1,4,5)P3-R in numerous cell types (for review, see Potter and Nahorski, 1992). Most recently,d-3-fluoro-myo-inositol 1-phosphate-4,5-bisphosphorothioate [3F-Ins(1)P-(4,5)PS2] (Fig. 1a) was found to be only 10-fold less potent than Ins(1,4,5)P3 at displacing [3H]Ins(1,4,5)P3 from its receptor on pig cerebellum and to mobilize up to 60% of total Ca2+in permeabilized SH-SY5Y cells (Wilcox et al., 1997). Therefore, all of the partial agonists thus far described at the Ins(1,4,5)P3-R are Ins(1,4,5)P3 analogs and, with the exception ofscyllo-inositol 1,2,4,5-tetrakisphosphorothioate and Ins(1,3,4,6)P4, have combined modifications at C-3 or C-6 with phosphorothioate substitutions.
The Ca2+-mobilizing activity of Ins(1,3,4,6)P4 was rationalized by envisaging two alternative receptor-binding orientations in which the 1,6-vicinal bisphosphate of Ins(1,3,4,6)P4 mimics the normal 4,5-bisphosphate in the Ins(1,4,5)P3 binding orientation (although this did not explain its partial agonist properties). This model predicted that two Ins(1,4,5)P3 regioisomers (i.e.,d-myo-inositol 1,4,6-trisphosphate [d-Ins(1,4,6)P3] andd-myo-inositol 1,3,6-trisphosphate [d-Ins(1,3,6)P3{l-Ins(1,3,4)P3 1}]) should be able to mobilize Ca2+ and indeed this was confirmed (Murphy et al., 1996). Both of these active enantiomers possess one of the features found in the majority of partial agonists: a modification at either the C-3 or C-6 groups. The other characteristic feature found in common in the partial agonists is the replacement of the vicinal 4,5-bisphosphate group with phosphorothioate groups. To determine whether adoption of these minimal criteria, found in common with other partial agonists, was adequate in the rational design of a partial agonist, we replaced the phosphate groups of both Ins(1,3,6)P3 and Ins(1,4,6)P3 with phosphorothioates in the synthesis of Ins(1,3,6)PS3 and Ins(1,4,6)PS3. Preliminary data suggested that the racemic mixtures of the phosphorothioates Ins(1,4,6)PS3 and Ins(1,3,6)PS3 (Fig. 1a) were partial agonists at the Ins(1,4,5)P3-R in permeabilized rabbit platelets (Al-Hafidh et al., 1994; Mills et al., 1995). Using the same rationalization for the Ca2+-mobilizing activity of the partial agonist Ins(1,3,4,6)P3, we predicted that the two chiral phosphorothioate analogs,d-Ins(1,3,6)PS3 andd-Ins(1,4,6)PS3, were responsible for the observed partial agonist properties of their racemic mixtures. In this study, we demonstrate clearly that both of these phosphorothioate analogs are low-intrinsic-activity partial agonists at the Ins(1,4,5)P3-R and that one of them [d-Ins(1,4,6)PS3] possesses particularly promising potency coupled with very low intrinsic activity.
Experimental Procedures
Materials
Chemically synthesized Ins(1,4,5)P3 was purchased from the Rhode Island Chemical Group (Kingston, RI). [3H]Ins(1,4,5)P3 (20–60 Ci/mmol, 10 μCi/ml) and45Ca2+ (5–50 mCi/mg Ca2+, 2 mCi/ml) were purchased from Amersham International (Buckinghamshire, UK). FP100 filters were purchased from Whatman (Clifton, NJ). Saponin A, oligomycin, leupeptin, pepstatin, and ATP were obtained from Sigma (St. Louis, MO), ionomycin was purchased from Calbiochem (San Diego, CA).dl-Ins(1,3,6)PS3 anddl-Ins(1,4,6)PS3 were synthesized as described by Mills et al. (1995).d-Ins(1,3,6)PS3 was synthesized from 1d-2,4,5-tri-O-benzyl-myo-inositol (Riley et al., 1994) andd-Ins(1,4,6)PS3 was synthesized from 1d-2,3,5-tri-O-benzylmyo-inositol (Mills and Potter, 1996) using methods similar to those described for the racemic mixture (Mills et al., 1995). All synthetic compounds were homogenous by 1H and31P NMR spectroscopy and mass spectroscopy after purification by ion-exchange chromatography. The compounds were quantified by total phosphate assay and then used as their triethylammonium salts.
Methods
Preparation of Platelets.
Washed rabbit platelets were prepared as described previously (Murphy et al., 1991). The resulting platelet pellet from this preparation was resuspended in HEPES-buffered Tyrode's solution (10 mM HEPES, 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM Na2HPO4, 5.5 mM glucose and 0.25% BSA, pH 7.4) before performing the following procedures.
45Ca2+ Release from Intracellular Stores.
Platelets were washed in high-K+ buffer A [120 mM KCl, 2 mM KH2PO4, 5 mM (CH2COONa)2.6H2O, 6 mM MgCl2, 20 mM HEPES, in MilliQ water; 5 mM ATP was added, pH adjusted to 6.9 and free Ca2+ concentration adjusted below 150 nM] and then suspended to 3 × 109/ml. The platelets were then permeabilized with 40 μg/ml saponin A, which was removed by further washing in buffer A. The intracellular Ca2+ stores were loaded with 45Ca2+(2 μCi/ml) for 1 h in the presence of 10 μg/ml oligomycin. Total release of 45Ca2+ from the stores was determined by a 3-min incubation with 75 μM ionomycin. Release of45Ca2+ from the intracellular stores at 4°C was determined 3 min after the addition of the inositol phosphate by separation of free and retained 45Ca2+ by filtration of cells using Whatman FP100 filters.45Ca2+ release was determined by liquid-scintillation counting (Murphy and Westwick, 1994).
Displacement of [3H]Ins(1,4,5)P3Binding to Specific Ins(1,4,5)P3 Receptors on Rat Cerebellar Membranes.
The preparation of rat cerebellar membranes and displacement of [3H]Ins(1,4,5)P3 bound to the Ins(1,4,5)P3 receptors on the membranes was performed as described previously (Challiss et al., 1991). Briefly, cerebella were removed from 6 rats (200–250 g) and homogenized (2 × 10 s, 4°C) in buffer C (20 mM Tris · HCl, 20 mM NaCl, 100 mM KCl, 1 mM EDTA, 1 mg/ml BSA, pH 7.7) containing the protease inhibitors 10 μM leupeptin and 10 μM pepstatin. After centrifugation (50,000g, 13 min, 4°C), the pellet was resuspended in buffer C, homogenized as above, and the protein content adjusted to 5 mg/ml. The cerebellar membranes were either used immediately or frozen (−80°C) until use. The binding assay mixture in a total volume of 250 μl contained 1 nM [3H]Ins(1,4,5)P3, and synthetic ligand diluted in buffer C at appropriate concentrations. Binding was initiated by the addition of 250 μg of the cerebellar membrane preparation. The assay tubes were incubated (4°C) for 10 min before termination of the reaction by centrifugation (10,000g, 4 min, 4°C). Nonspecific binding of [3H]Ins(1,4,5)P3 was assessed as the counts remaining upon inclusion of 10 μM cold Ins(1,4,5)P3 in the assay mixture. After centrifugation, the supernatant was carefully removed, the pellet resuspended, and radioactivity bound to the cerebellar membrane was determined by liquid scintillation counting.
Results
Ca2+ Release from Permeabilized Platelets.
Rabbit platelets permeabilized with saponin and in the presence of oligomycin displayed ATP-dependent45Ca2+ uptake into their nonmitochondrial stores. Uptake reached a steady state by 45 min and was monitored throughout the time course of the experiment and found to remain essentially unchanged. The ionomycin releasable component of accumulated45Ca2+ was found to be >92%; again, this was not found to change significantly throughout the time course of any of the45Ca2+ release experiments undertaken.
Treatment of permeabilized platelets withd-Ins(1,4,5)P3 (0.01–30 μM) for 3 min (4°C) caused a dose-dependent release of45Ca2+ from preloaded intracellular stores (Fig. 2).dl-Ins(1,3,6)PS3 (1–3000 μM) alone caused a dose-dependent release of45Ca2+ from the stores of permeabilized platelets. Maximal release, however, was only around 20% of the Ins(1,4,5)P3-sensitive Ca2+ pool, even at concentrations above 1 mM (some of which may have been caused by nonspecific release), demonstrating a very low efficacy fordl-Ins(1,3,6)PS3 at the Ins(1,4,5)P3-R of rabbit platelets (Fig. 2a). Treatment of permeabilized platelets with 1 μM Ins(1,4,5)P3, together with increasing concentrations of dl-Ins(1,3,6)PS3, caused an inhibition of Ins(1,4,5)P3-induced Ca2+-release (Fig. 2a). Ca2+ release induced by Ins(1,4,5)P3 was reduced as the concentration ofdl-Ins(1,3,6)PS3 increased, until release approached a level near the intrinsic efficacy ofdl-Ins(1,3,6)PS3 itself. The concentration of dl-Ins(1,3,6)PS3required to inhibit release of Ca2+ induced with 1 μM Ins(1,4,5)P3 by 50% (IC50) was >100 μM. However, increasing the concentration of dl-Ins(1,3,6)PS3 to more than 100 μM caused no further inhibition of Ca2+-release, suggesting that maximal inhibition of Ca2+-release induced with 1 μM Ins(1,4,5)P3 is reached by 100 μM (Fig. 2a).
dl-Ins(1,4,6)PS3 (0.03–300 μM) was also found to have a very low efficacy at the Ins(1,4,5)P3-R, releasing only ∼15% of the Ins(1,4,5)P3-sensitive Ca2+store at the highest concentration used (300 μM) (Fig. 2b). However, Ca2+ release induced by 1 μM Ins(1,4,5)P3 could be inhibited with increasing concentrations of dl-Ins(1,4,6)PS3(Fig. 2b). As described previously fordl-Ins(1,3,6)PS3, Ca2+ release induced by Ins(1,4,5)P3 was further inhibited as the concentration of dl-Ins(1,4,6)PS3increased, until release declined to a level near the intrinsic efficacy of dl-Ins(1,4,6)PS3 itself. The concentration of dl-Ins(1,4,6)PS3required to inhibit release of Ca2+ induced with 1 μM Ins(1,4,5)P3 by 50% (IC50) was found to be 56 ± 3.6 μM. Maximal inhibition of Ca2+-release induced by 1 μM Ins(1,4,5)P3 seems to have been achieved with 100 μM dl-Ins(1,4,6)PS3 as an increased Ca2+ release was observed with 300 μM (Fig. 2b).
Displacement of Specific [3H]Ins(1,4,5)P3Binding to Rat Cerebellar Membranes.
[3H]Ins(1,4,5)P3 was readily displaced from specific binding sites on rat cerebellar membranes by cold d-Ins(1,4,5)P3 with an IC50 of 0.043 ± 0.01 μM (Fig.3). The ability of dl- andd-Ins(1,3,6)PS3 and ofdl- and d-Ins(1,4,6)PS3to displace [3H]Ins(1,4,5)P3 from rat cerebellar membranes was also examined (Fig. 3).d-Ins(1,3,6)PS3 displaced specifically bound [3H]Ins(1,4,5)P3 from rat cerebellar membranes, although displacement byd-Ins(1,3,6)PS3 was 500 fold weaker than by d-Ins(1,4,5)P3 (Fig. 3a). However, comparing the IC50 value ford-Ins(1,3,6)PS3 (17.5 ± 5.8 μM) with that of the racemic mixturedl-Ins(1,3,6)PS3 (34 ± 10 μM), demonstrated thatd-Ins(1,3,6)PS3 was able to displace [3H]Ins(1,4,5)P3 with roughly twice the affinity of the racemic mixture, confirming that activity resides in the d-enantiomer (Fig. 3a).d-Ins(1,4,6)PS3 displaced specifically bound [3H]Ins(1,4,5)P3 from rat cerebellar membranes, although displacement byd-Ins(1,4,6)PS3 was some 34-fold weaker than by d-Ins(1,4,5)P3 (Fig.3a). d-Ins(1,4,6)PS3 was able to displace [3H]Ins(1,4,5)P3from cerebellar membranes with roughly twice the affinity of its racemic mixture (IC50 value of 1.4 ± 0.35 μM compared with an IC50 value of 2.15 ± 0.13 μM for the racemic mixture), indicating that the activity resides in the d-enantiomer (Fig. 3b).
Therefore, the ability ofd-Ins(1,3,6)PS3 andd-Ins(1,4,6)PS3 to displace [3H]Ins(1,4,5)P3 from specific binding sites on rat cerebellar membranes contrasted with their relative inability to release45Ca2+ from the intracellular stores of permeabilized platelets.
Effect of Trisphosphorothioates on Ins(1,4,5)P3-Induced Ca2+ Release.
From the binding studies, it seems thatd-Ins(1,3,6)PS3 is the active component of the racemic mixturedl-Ins(1,3,6)PS3, whereasd-Ins(1,4,6)PS3 is the active component of the racemic mixture ofdl-Ins(1,4,6)PS3. As for the racemic mixtures, both d-Ins(1,3,6)PS3 andd-Ins(1,4,6)PS3 were also found to have a very low efficacy at the Ins(1,4,5)P3–receptor of platelets, releasing only a small percentage (<15%) of the Ins(1,4,5)P3-sensitive Ca2+store even at 300 μM (Fig. 4a). It is possible that the release of Ca2+ by high concentrations of bothd-Ins(1,3,6)PS3 andd-Ins(1,4,6)PS3 may be nonspecific.
Increasing concentrations ofd-Ins(1,3,6)PS3 (30, 100, and 300 μM) were able to partially antagonize Ca2+release induced by submaximal concentrations of Ins(1,4,5)P3 (0.1–3 μM); however, by increasing the concentration of Ins(1,4,5)P3 (10 and 30 μM), this inhibition was no longer observed, suggesting competition for binding at the Ins(1,4,5)P3-R (Fig. 4b). The IC50 value for the inhibition of Ca2+ elevation induced by 1 μM Ins(1,4,5)P3 was >100 μM ford-Ins(1,3,6)PS3. Competitive partial antagonism of Ins(1,4,5)P3-induced Ca2+ release was also observed withd-Ins(1,4,6)PS3 (10, 30, and 100 μM) (Fig. 4c). The IC50 value ofd-Ins(1,4,6)PS3 for Ca2+ release induced by 1 μM Ins(1,4,5)P3 was 27 ± 8.1 μM, approximately half that required of the racemic mixture (56 ± 3.6 μM).
Discussion
Structure-activity studies performed to date using Ins(1,4,5)P3analogs have concluded that the vicinal 4,5-bisphosphate configuration plays the key role in receptor recognition and mediation of Ca2+ release from intracellular stores (for reviews, see Potter and Nahorski, 1992; Potter and Lampe, 1995; Wilcox et al., 1998). Other structural requirements for Ca2+ release include an additional phosphate group at the 1-position (but it can be tolerated at the 2-position), which increases affinity at the Ins(1,4,5)P3-R (Potter and Lampe, 1995). The importance of the hydroxyl groups of Ins(1,4,5)P3 is well characterized, with modification of the three hydroxyl groups at either the 2-, 3- or 6- position of Ins(1,4,5)P3 varying the impact on Ca2+ release and the binding of Ins(1,4,5)P3 to its receptor (for review, seePotter and Lampe, 1995).
At present very few partial agonists at the Ins(1,4,5)P3-R have been reported; these include Ins(1,3,4,6)P4 (Gawler et al., 1991),l-chr-Ins(2,3,5)PS3, and 6-deoxy-Ins1,4,5)PS3 (Safrany et al., 1993),scyllo-inositol 1,2,4,5-tetrakisphosphorothioate (Wilcox et al., 1994), and 3F-Ins(1)P-(4,5)PS2 (Wilcox et al., 1997). By using rapid kinetic measurements of45Ca2+ mobilization, Ins(2,4,5)P3 has also been demonstrated to be a partial agonist at hepatic Ins(1,4,5)P3-Rs (Marchant et al., 1997). Because of the quantal mechanism of Ca2+ release, whereby even partial agonists may completely empty the Ins(1,4,5)P3-sensitive Ca2+ stores, albeit at slower rates than Ins(1,4,5)P3, it is possible that the partial agonist properties of other inositol phosphates may be distinguished under high temporal resolution (Menza and Michelangeli, 1998).d-3-Amino-3-deoxy-Ins(1,4,5)P3has also been described as a partial agonist in SH-SY5Y neuroblastoma cells, but increasing pH from 6.8 to 7.2 negates the partial agonist properties (Kozikowski et al., 1994).
In a previous study (Murphy et al., 1996), we rationalized how Ins(1,3,4,6)P4 elicits Ca2+release by envisaging two alternative receptor binding orientations, where the 1,6-vicinal bisphosphate is presumed to mimic the normal 4,5-bisphosphate of Ins(1,4,5)P3. As either the 4-phosphate or the 3-phosphate of Ins(1,3,4,6)P4could mimic the 1-phosphate of Ins(1,4,5)P3, it is likely that Ins(1,3,4,6)P4 evokes Ca2+ release by a similar binding mechanism to Ins(1,4,5)P3. We went on to show that two related trisphosphates [d-Ins(1,4,6)P3 andd-Ins(1,3,6)P3] were also able to displace [3H]Ins(1,4,5)P3from the Ins(1,4,5)P3-R and to possess Ca2+ mobilization ability, whereas their enantiomers were inactive (Murphy et al., 1996). Noting that Ins(1,4,6)P3 and Ins(1,3,6)P3 possessed one of the features common to the known partial agonist, namely modification at some of the positions corresponding to C-2, C-3, or C-6 of Ins(1,4,5)P3, we went on to replace their phosphate groups with phosphorothioates, giving Ins(1,4,6)PS3 and Ins(1,3,6)PS3. From the preceding structure-activity arguments, we predicted thatd-Ins(1,4,6)PS3 andd-Ins(1,3,6)PS3 would show partial agonist properties, whereas their enantiomers would be inactive.
Both the racemic trisphosphorothioatesdl-Ins(1,3,6)PS3 anddl-Ins(1,4,6)PS3 were found to have very low efficacy at the Ins(1,4,5)P3-R of rabbit platelets. Taken in isolation, this result does not show that either of these compounds is a partial agonist; an extremely-low-potency full agonist could give similar results. However, when platelets were treated with 1 μM Ins(1,4,5)P3, together with increasing concentrations of eitherdl-Ins(1,3,6)PS3 ordl-Ins(1,4,6)PS3, a definite inhibition of Ins(1,4,5)P3-stimulated Ca2+ release was observed, demonstrating that both dl-Ins(1,3,6)PS3 anddl-Ins(1,4,6)PS3 were acting as true partial agonists.
It was assumed that the partial agonist activity of racemicdl-Ins(1,4,6)PS3 anddl-Ins(1,3,6)PS3 resided in thed-enantiomers. These isomers have in common the possession of a “pseudo” vicinal d-4,5-bisphosphate motif of the same absolute stereochemistry as that found in Ins(1,4,5)P3. On the other hand, neither thel-enantiomer of Ins(1,4,6)PS3 nor thel-enantiomer of Ins(1,3,6)PS3possesses this motif; rather, they are similar tol-Ins(1,4,5)P3. To examine this theory, the ability of bothd-Ins(1,4,6)PS3 andd-Ins(1,3,6)PS3 to displace [3H]Ins(1,4,5)P3 from its binding site on rat cerebellar membranes was compared with their respective racemic mixtures. Both the d-isomer of Ins(1,4,6)PS3 and the d-isomer of Ins(1,3,6)PS3 were found to have roughly twice the affinity for the Ins(1,4,5)P3-R of their racemic mixtures. This confirms that the activity of the racemic mixtures resides with the enantiomers possessing a vicinal bisphosphate of the correct absolute stereochemistry.
Compared with d-Ins(1,3,6)PS3,d-Ins(1,4,6)PS3 showed a higher affinity for the Ins(1,4,5)P3-R in binding studies. In d-Ins(1,4,6)PS3, the orientation of the 5-OH [which mimics the 6-OH of Ins(1,4,5)P3] is equatorial [as ind-Ins(1,4,5)P3], whereas the OH-group corresponding to the 3-OH is axial rather than equatorial (Fig. 1a). The 2-OH [which mimics the 6-OH of Ins(1,4,5)P3] is reoriented to axial ind-Ins(1,3,6)PS3 and is therefore different from that ind-Ins(1,4,5)P3, whereas the OH group corresponding to the 3-OH of Ins(1,4,5)P3 remains equatorial. From structure-activity studies, the 3-OH group of Ins(1,4,5)P3 seems to have only a minor role in receptor recognition (Hirata et al., 1989; Seewald et al., 1990); thus, reorientation of the OH-group on the “pseudo” 3-position (actually the 2-position) of the inositol ring [as in Ins(1,4,6)PS3] might not be expected to have a significant effect on Ins(1,4,5)P3 binding. However, modification at the 6-OH group [as in Ins(1,3,6)PS3] would be expected to reduce binding and activity (Polokoff et al., 1988; Safrany et al., 1991). The finding that d-Ins(1,4,6)PS3 is more potent than d-Ins(1,3,6)PS3 at displacing [3H]Ins(1,4,5)P3 conforms with these structural requirements and confirms the conclusion that the 6-OH group of Ins(1,4,5)P3 is more important for binding than the 3-OH group (Hirata et al., 1993; Murphy et al., 1996).
Increasing concentrations of eitherd-Ins(1,3,6)PS3 ord-Ins(1,4,6)PS3 were able to partially antagonize Ca2+ release induced by submaximal concentrations of Ins(1,4,5)P3. However, by increasing the concentration of Ins(1,4,5)P3, this inhibition was no longer observed, suggesting competition for binding at the Ins(1,4,5)P3-R (Fig. 4b). The IC50 value for the inhibition of Ca2+ elevation induced by 1 μM Ins(1,4,5)P3 was >100 μM for both racemic andd-Ins(1,3,6)PS3 and was 27 ± 8.1 μM for d-Ins(1,4,6)PS3, approximately half that of the racemic mixture.
The only two low-intrinsic-activity partial agonists described previously arel-chr-Ins(2,3,5)PS3 and 6-deoxy-Ins(1,4,5)PS3, which were found to release 34 and 42% of Ca2+ respectively in SH-SY5Y cells (Safrany et al., 1993). It is interesting to note that the only structural difference betweend-Ins(1,4,6)PS3 andl-chr-Ins(2,3,5)PS3is that the hydroxyl group that mimics the 2-OH of Ins(1,4,5)P3, is reoriented from axial to equatorial in Ins(1,4,6)PS3 relative tol-chr-Ins(2,3,5)PS3(see Fig. 1b). In structure-activity studies, the 2-OH group has been shown to have the least importance in receptor recognition (Hirata et al., 1989; Wilcox et al., 1994), yet this reorientation seems to contribute both to lower efficacy ofd-Ins(1,4,6)PS3 and an increase in its affinity for the receptor [l-chr-Ins(2,3,5)PS3was found to have some 100-fold lower affinity for the Ins(1,4,5)P3-R in bovine adrenal cortical membranes (Safrany et al., 1993), whereas the affinity of Ins(1,4,6)PS3 was only 34-fold lower than Ins(1,4,5)P3 in rat cerebellar membranes]. There are two differences between 6-deoxy-Ins(1,4,5)PS3and d-Ins(1,3,6)PS3: first, the 6-hydroxyl group is deleted in 6-deoxy-Ins(1,4,5)PS3, whereas the “pseudo” 6-OH is axial in Ins(1,3,6)PS3; second, the 2-OH group is axial in 6-deoxy-Ins(1,4,5)PS3 [as in Ins(1,4,5)P3], whereas the “pseudo” 2-OH is equatorial in Ins(1,3,6)PS3. These differences cause a 2-fold increase in the affinity for the receptor and reduce the efficacy from 42% to less than 20% (Safrany et al., 1993).
Wilcox et al. (1997) investigated the three compoundsd-3-fluoro-3-deoxy-myo-inositol 1,5-bisphosphate-4-phosphorothioate,d-3-fluoro-3-deoxy-myo-inositol 1,4-bisphosphate-5-phosphorothioate, and 3F-Ins(1)P-(4,5)PS2 for partial agonist activity (Wilcox et al., 1997). Similarly tod-Ins(1,4,6)PS3, these compounds possessed a structural perturbation at the hydroxyl group that mimics the 3-OH of Ins(1,4,5)P3. This was achieved by the replacement of the native 3-OH with a fluorine group. Again, liked-Ins(1,4,6)PS3, these compounds had phosphorothioate substitutions, although only one, 3F-Ins(1)P-(4,5)PS2, had phosphorothioate substitutions at both members of the crucial vicinal 4,5-bisphosphate motif. Of these compounds, 3F-Ins(1)P-(4,5)PS2was the only one identified as a partial agonist, able to inhibit Ca2+ mobilization induced by submaximal concentrations of Ins(1,4,5)P3 (Wilcox et al., 1997). It was also demonstrated to be an antagonist of receptor-mediated Ca2+ signaling (Davis et al., 1998). Compared with 3F-Ins(1)P-(4,5)PS2,d-Ins(1,4,6)PS3 has a lower affinity for the Ins(1,4,5)P3-R [it is ∼34-fold weaker than Ins(1,4,5)P3 at displacing [3H]Ins(1,4,5)P3], whereas 3F-Ins(1)P-(4,5)PS2 was only 10-fold weaker (Wilcox et al., 1997). Of all the phosphorothioate-containing partial agonists, 3-F-Ins(1)P-(4,5)P2 is the only one that has not substituted the (“pseudo”)-1-phosphate with a phosphorothioate; this may account for its increased affinity above the other partial agonists described so far. However, compared withd-Ins(1,4,6)PS3, 3F-Ins(1)P-(4,5)PS2, has a relatively high efficacy, causing over 60% of Ca2+ to be released. Therefore, although 3F-Ins(1)P-(4,5)PS2has a high affinity, its potential as a partial antagonist and a lead compound is reduced by its higher efficacy. It is possible, therefore, that the lower affinity ofd-Ins(1,4,6)PS3 at the Ins(1,4,5)P3-R compared with 3F-Ins(1)P-(4,5)PS2 is because it has three phosphorothioates, one of which is at the 1-position. Thus a version ofd-Ins(1,4,6)PS3 with a vicinal bisphosphorothioate and a pseudo-1-phosphate [namelyd-Ins(4)P-(1,6)PS2] may combine the high affinity of 3F-Ins(1)P-(4,5)PS2 and the low efficacy of d-Ins(1,4,6)PS3.
The only low-efficacy partial agonists at the Ins(1,4,5)P3-R discovered to date have been phosphorothioates; d-Ins(1,3,6)PS3and d-Ins(1,4,6)PS3 now expand this small group of such analogs. However,d-Ins(1,4,6)PS3 in particular has a relatively high affinity for the Ins(1,4,5)P3receptor and yet maintains very low efficacy. Thusd-Ins(1,4,6)PS3 may be a useful tool for pharmacological intervention in the polyphosphoinositide pathway and an important lead compound for the development of Ins(1,4,5)P3 receptor antagonists. Indeed, it has recently been successfully employed via microinjection to inhibit Ins(1,4,5)P3-induced Ca2+mobilization in intact Jurkat T-lymphocytes (Guse et al., 1997, 1999).
Footnotes
- Received May 20, 1999.
- Accepted November 16, 1999.
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Send reprint requests to: Dr. C. T. Murphy, Department of Pharmacy & Pharmacology, University of Bath, Bath, BA2 7AY UK. E-mail: c.t.murphy{at}bath.ac.uk
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↵1 Alternative nomenclature ford-Ins(1,3,6)P3 isl-Ins(1,3,4)P3.
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This work was supported by the British Heart Foundation (JW, CTM) and The Wellcome Trust for Project (BVLP, JW) and Programme (BVLP: 045491) Grants.
Abbreviations
- Ins(1,4,5)P3
- myo-inositol 1,4,5-trisphosphate
- Ins(1,3,6)PS3
- myo-inositol 1,3,6-trisphosphorothioate
- Ins(1,4,6)PS3
- myo-inositol 1,4,6-trisphosphorothioate
- Ins(1,4,6)P3
- myo-inositol 1,4,6-trisphosphate
- Ins(1,3,6)P3
- myo-inositol 1,3,6-trisphosphate
- Ins(1,4,5)P3-R
- myo-Ins(1,4,5)P3-receptor
- Ins(1,3,4,6)P4
- myo-inositol 1,3,4,6-tetrakisphosphate
- l-chr-Ins(2,3,5)PS3
- l-chiro-inositol 2,3,5-trisphosphorothioate
- 3F-Ins(1)P-(4,5)PS2
- d-3-fluoro-3-deoxy-myo-inositol 1-phosphate-4,5-bisphosphorothioate
- The American Society for Pharmacology and Experimental Therapeutics