Allosteric Site on SHIP2 Identified Through Fluorescent Ligand Screening and Crystallography: A Potential New Target for Intervention.

Src homology 2 domain-containing inositol phosphate phosphatase 2 (SHIP2) is one of the 10 human inositol phosphate 5-phosphatases. One of its physiological functions is dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, PtdIns(3,4,5)P3. It is therefore a therapeutic target for pathophysiologies dependent on PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Therapeutic interventions are limited by the dearth of crystallographic data describing ligand/inhibitor binding. An active site-directed fluorescent probe facilitated screening of compound libraries for SHIP2 ligands. With two additional orthogonal assays, several ligands including galloflavin were identified as low micromolar Ki inhibitors. One ligand, an oxo-linked ethylene-bridged dimer of benzene 1,2,4-trisphosphate, was shown to be an uncompetitive inhibitor that binds to a regulatory site on the catalytic domain. We posit that binding of ligands to this site restrains L4 loop motions that are key to interdomain communications that accompany high catalytic activity with phosphoinositide substrate. This site may, therefore, be a future druggable target for medicinal chemistry.

Inositol Adenophostin: Convergent Synthesis of a Potent Agonist of d-myo-Inositol 1,4,5-Trisphosphate Receptors.

d-myo-Inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ channels activated by the intracellular messenger inositol 1,4,5-trisphosphate (IP3, 1). The glyconucleotide adenophostin A (AdA, 2) is a potent agonist of IP3Rs. A recent synthesis of d-chiro-inositol adenophostin (InsAdA, 5) employed suitably protected chiral building blocks and replaced the d-glucose core by d-chiro-inositol. An alternative approach to fully chiral material is now reported using intrinsic sugar chirality to avoid early isomer resolution, involving the coupling of a protected and activated racemic myo-inositol derivative to a d-ribose derivative. Diastereoisomer separation was achieved after trans-isopropylidene group removal and the absolute ribose-inositol conjugate stereochemistry assigned with reference to the earlier synthesis. Optimization of stannylene-mediated regiospecific benzylation was explored using the model 1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol and conditions successfully transferred to one conjugate diastereoisomer with 3:1 selectivity. However, only roughly 1:1 regiospecificity was achieved on the required diastereoisomer. The conjugate regioisomers of benzyl derivatives 39 and 40 were successfully separated and 39 was transformed subsequently to InsAdA after amination, pan-phosphorylation, and deprotection. InsAdA from this synthetic route bound with greater affinity than AdA to IP3R1 and was more potent in releasing Ca2+ from intracellular stores through IP3Rs. It is the most potent full agonist of IP3R1 known and .equipotent with material from the fully chiral synthetic route.

Regioisomeric Family of Novel Fluorescent Substrates for SHIP2.

SHIP2 (SH2-domain containing inositol 5-phosphatase type 2) is a canonical 5-phosphatase, which, through its catalytic action on PtdInsP3, regulates the PI3K/Akt pathway and metabolic action of insulin. It is a drug target, but there is limited evidence of inhibition of SHIP2 by small molecules in the literature. With the goal to investigate inhibition, we report a homologous family of synthetic, chromophoric benzene phosphate substrates of SHIP2 that display the headgroup regiochemical hallmarks of the physiological inositide substrates that have proved difficult to crystallize with 5-phosphatases. Using time-dependent density functional theory (TD-DFT), we explore the intrinsic fluorescence of these novel substrates and show how fluorescence can be used to assay enzyme activity. The TD-DFT approach promises to inform rational design of enhanced active site probes for the broadest family of inositide-binding/metabolizing proteins, while maintaining the regiochemical properties of bona fide inositide substrates.

d-chiro-Inositol Ribophostin: A Highly Potent Agonist of d-myo-Inositol 1,4,5-Trisphosphate Receptors: Synthesis and Biological Activities.

Analogues of the Ca2+-releasing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [1, Ins(1,4,5)P3] are important synthetic targets. Replacement of the α-glucopyranosyl motif in the natural product mimic adenophostin 2 by d-chiro-inositol in d-chiro-inositol adenophostin 4 increased the potency. Similar modification of the non-nucleotide Ins(1,4,5)P3 mimic ribophostin 6 may increase the activity. d-chiro-Inositol ribophostin 10 was synthesized by coupling as building blocks suitably protected ribose 12 with l-(+)-3-O-trifluoromethylsulfonyl-6-O-p-methoxybenzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 11. Separable diastereoisomeric 3-O-camphanate esters of (±)-6-O-p-methoxy-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol allowed the preparation of 11. Selective trans-isopropylidene deprotection in coupled 13, then monobenzylation gave separable regioisomers 15 and 16. p-Methoxybenzyl group deprotection of 16, phosphitylation/oxidation, then deprotection afforded 10, which was a full agonist in Ca2+-release assays; its potency and binding affinity for Ins(1,4,5)P3R were similar to those of adenophostin. Both 4 and 10 elicited a store-operated Ca2+ current ICRAC in patch-clamped cells, unlike Ins(1,4,5)P3 consistent with resistance to metabolism. d-chiro-Inositol ribophostin is the most potent small-molecule Ins(1,4,5)P3 receptor agonist without a nucleobase yet synthesized.

A synthetic cyclitol-nucleoside conjugate polyphosphate is a highly potent second messenger mimic.

Reactions that form sec-sec ethers are well known, but few lead to compounds with dense functionality around the O-linkage. Replacement of the α-glucopyranosyl unit of adenophostin A, a potent d-myo-inositol 1,4,5-trisphosphate (IP3R) agonist, with a d-chiro-inositol surrogate acting substantially as a pseudosugar, leads to "d-chiro-inositol adenophostin". At its core, this cyclitol-nucleoside trisphosphate comprises a nucleoside sugar linked via an axial d-chiro-inositol 1-hydroxyl-adenosine 3'-ribose ether linkage. A divergent synthesis of d-chiro-inositol adenophostin has been achieved. Key features of the synthetic strategy to produce a triol for phosphorylation include a new selective mono-tosylation of racemic 1,2:4,5-di-O-isopropylidene-myo-inositol using tosyl imidazole; subsequent conversion of the product into separable camphanate ester derivatives, one leading to a chiral myo-inositol triflate used as a synthetic building block and the other to l-5-O-methyl-myo-inositol [l-(+)-bornesitol] to assign the absolute configuration; the nucleophilic coupling of an alkoxide of a ribose pent-4-ene orthoester unit with a structurally rigid chiral myo-inositol triflate derivative, representing the first sec-sec ether formation between a cyclitol and ribose. Reaction of the coupled product with a silylated nucleobase completes the assembly of the core structure. Further protecting group manipulation, mixed O- and N-phosphorylation, and subsequent removal of all protecting groups in a single step achieves the final product, avoiding a separate N6 protection/deprotection strategy. d-chiro-Inositol adenophostin evoked Ca2+ release through IP3Rs at lower concentrations than adenophostin A, hitherto the most potent known agonist of IP3Rs.

Real-time monitoring through the use of technology to enhance performances throughout HIV cascades.

PURPOSE OF REVIEW: Controlling the HIV epidemic requires strong linkages across a 'cascade' of prevention, testing, and treatment services. Information and communications technology (ICT) offers the potential to monitor and improve the performance of this HIV cascade in real time. We assessed recent (<18 months) peer-reviewed publications regarding uses of ICT to improve performance through expanded and targeted reach, improved clinical service delivery, and reduced loss to follow-up. RECENT FINDINGS: Research on ICT has tended to focus on a specific 'silo' of the HIV cascade rather than on tracking individuals or program performance across the cascade. Numerous innovations have been described, including use of social media to expand reach and improve programmatic targeting; technology in healthcare settings to strengthen coordination, guide clinical decision-making and improve clinical interactions; and telephone-based follow-up to improve treatment retention and adherence. With exceptions, publications have tended to be descriptive rather than evaluative, and the evidence-base for the effectiveness of ICT-driven interventions remains mixed. SUMMARY: There is widespread recognition of the potential for ICT to improve HIV cascade performance, but with significant challenges. Successful implementation of real-time cascade monitoring will depend upon stakeholder engagement, compatibility with existing workflows, appropriate resource allocation, and managing expectations.

Electronic health, telemedicine, and new paradigms for training and care.

PURPOSE OF REVIEW: HIV prevention and care is changing rapidly; guideline revisions and programmatic scale-up require innovative approaches to in-service training and care extension to improve provider practice and care access. We assessed recent (≤12 months) peer-reviewed publications on electronic health (eHealth), telemedicine, and other innovative provider-targeted interventions for HIV-related care. RECENT FINDINGS: Key developments included systems merging electronic medical records (EMR) with provider clinical decision aids to prompt action, demonstration eHealth, and telemedicine projects, reviews or descriptions of technology to improve connectivity in lower resource settings, and a few trials on provider-centered interventions. Most publications were program reports and few data were available regarding efficacy of eHealth interventions for providers on patient HIV-related outcomes, notably identification and management of antiretroviral treatment failure in Kenya. Better evidence is needed for strategies to train providers and care extenders with the goal to improve impact of HIV prevention and care interventions. SUMMARY: Rapid technology introduction and expansion may change the paradigm for improving provider knowledge and practice. Although new, the developments are promising for HIV provider-targeted eHealth and innovations for traditional training. More rigorous testing with randomized trials is needed to demonstrate impact on services for people living with HIV.

Crystal Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family.

The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3',4,4',5,5'-hexakisphosphate [BiPh(3,3',4,4',5,5')P6, IC50 5.5 µM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3',4,4',5,5')P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 µM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3',4,4',5,5')P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B-BiPh(3,3',4,4',5,5')P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed "moving metal" mechanism.

The "Other" Inositols and Their Phosphates: Synthesis, Biology, and Medicine (with Recent Advances in myo-Inositol Chemistry).

Cell signaling via inositol phosphates, in particular via the second messenger myo-inositol 1,4,5-trisphosphate, and phosphoinositides comprises a huge field of biology. Of the nine 1,2,3,4,5,6-cyclohexanehexol isomers, myo-inositol is pre-eminent, with "other" inositols (cis-, epi-, allo-, muco-, neo-, L-chiro-, D-chiro-, and scyllo-) and derivatives rarer or thought not to exist in nature. However, neo- and d-chiro-inositol hexakisphosphates were recently revealed in both terrestrial and aquatic ecosystems, thus highlighting the paucity of knowledge of the origins and potential biological functions of such stereoisomers, a prevalent group of environmental organic phosphates, and their parent inositols. Some "other" inositols are medically relevant, for example, scyllo-inositol (neurodegenerative diseases) and d-chiro-inositol (diabetes). It is timely to consider exploration of the roles and applications of the "other" isomers and their derivatives, likely by exploiting techniques now well developed for the myo series.

Designer small molecules to target calcium signalling.

Synthetic compounds open up new avenues to interrogate and manipulate intracellular Ca2+ signalling pathways. They may ultimately lead to drug-like analogues to intervene in disease. Recent advances in chemical biology tools available to probe Ca2+ signalling are described, with a particular focus on those synthetic analogues from our group that have enhanced biological understanding or represent a step towards more drug-like molecules. Adenophostin (AdA) is the most potent known agonist at the inositol 1,4,5-trisphosphate receptor (IP3R) and synthetic analogues provide a binding model for receptor activation and channel opening. 2-O-Modified inositol 1,4,5-trisphosphate (IP3) derivatives that are partial agonists at the IP3R reveal key conformational changes of the receptor upon ligand binding. Biphenyl polyphosphates illustrate that simple non-inositol surrogates can be engineered to give prototype IP3R agonists or antagonists and act as templates for protein co-crystallization. Cyclic adenosine 5'-diphosphoribose (cADPR) can be selectively modified using total synthesis, generating chemically and biologically stable tools to investigate Ca2+ release via the ryanodine receptor (RyR) and to interfere with cADPR synthesis and degradation. The first neutral analogues with a synthetic pyrophosphate bioisostere surprisingly retain the ability to release Ca2+, suggesting a new route to membrane-permeant tools. Adenosine 5'-diphosphoribose (ADPR) activates the Ca2+-, Na+- and K+-permeable transient receptor potential melastatin 2 (TRPM2) cation channel. Synthetic ADPR analogues provide the first structure-activity relationship (SAR) for this emerging messenger and the first functional antagonists. An analogue based on the nicotinic acid motif of nicotinic acid adenine dinucleotide phosphate (NAADP) antagonizes NAADP-mediated Ca2+ release in vitro and is effective in vivo against induced heart arrhythmia and autoimmune disease, illustrating the therapeutic potential of targeted small molecules.

A synthetic polyphosphoinositide headgroup surrogate in complex with SHIP2 provides a rationale for drug discovery.

Phosphoinositides regulate many cellular processes, and cellular levels are controlled by kinases and phosphatases. SHIP2 (SH2 (Src homology 2)-domain-containing inositol-phosphatase-2) plays a critical role in phosphoinositide signaling, cleaving the 5-phosphate from phosphatidylinositol 3,4,5-trisphosphate. SHIP2 is thought to be involved in type-2 diabetes and obesity, conditions that could therefore be open to pharmacological modulation of the enzyme. However, rational design of SHIP2 inhibitors has been limited by the absence of a high-resolution structure. Here, we present a 2.1 Å resolution crystal structure of the phosphatase domain of SHIP2 bound to the synthetic ligand biphenyl 2,3',4,5',6-pentakisphosphate (BiPh(2,3',4,5',6)P(5)). BiPh(2,3',4,5',6)P(5) is not a SHIP2 substrate but inhibits Ins(1,3,4,5)P(4) hydrolysis with an IC(50) of 24.8 ± 3.0 µM, (K(m) for Ins(1,3,4,5)P(4) is 215 ± 28 µM). Molecular dynamics simulations suggest that when BiPh(2,3',4,5',6)P(5) binds to SHIP2, a flexible loop folds over and encloses the ligand. Compounds targeting such a closed conformation might therefore deliver SHIP2-specific drugs.

Fibrinogen - a possible extracellular target for inositol phosphates.

A potential extracellular target for inositol phosphates and analogues with anticancer properties is identified. Proteins from detergent-solubilised HeLa cell lysates bound to a novel affinity column of myo-inositol 1,3,4,5,6-pentakisphosphate (InsP5) coupled to Affigel-10. One high-affinity ligand was fibrinogen Bß. Inositol phosphates and analogues were able to elute purified fibrinogen from this matrix. InsP5 and the inositol phosphate mimic biphenyl 2,3',4,5',6-pentakisphosphate (BiPhP5) bind fibrinogen in vitro, and block the effects of fibrinogen in A549 cell-based assays of proliferation and migration. They are also able to prevent the fibrinogen-mediated activation of phosphatidylinositol 3-kinase. These effects of fibrinogen appear to be mediated through the intercellular adhesion molecule-1 (ICAM-1), as cells not expressing ICAM-1 fail to respond. In contrast, myo-inositol hexakisphosphate and the epimeric scyllo-inositol 1,2,3,4,5-pentakisphosphate were without effect. These findings are consistent with earlier reports that higher inositol phosphates have anticancer properties. This new mechanism of action and target for these extracellular inositol phosphates to have their effects allows a re-evaluation of earlier data.

Multivalent benzene polyphosphate derivatives are non-Ca2+-mobilizing Ins(1,4,5)P3 receptor antagonists.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P31] mobilizes intracellular Ca2+ through the Ins(1,4,5)P3 receptor [InsP3R]. Although some progress has been made in the design of synthetic InsP3R partial agonists and antagonists, there are still few examples of useful small molecule competitive antagonists. A "multivalent" approach is explored and new dimeric polyphosphorylated aromatic derivatives were designed, synthesized and biologically evaluated. The established weak InsP3R ligand benzene 1,2,4-trisphosphate [Bz(1,2,4)P32] is dimerized through its 5-position in two different ways, first directly as the biphenyl derivative biphenyl 2,2',4,4',5,5'-hexakisphosphate, [BiPh(2,2',4,4',5,5')P68] and with its regioisomeric biphenyl 3,3',4,4',5,5'-hexakisphosphate [BiPh(3,3',4,4',5,5')P611]. Secondly, a linker motif is introduced in a flexible ethylene-bridged dimer (9) with its corresponding 1,2-bisphosphate dimer (10), both loosely analogous to the very weak antagonist 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA 7). In permeabilized L15 fibroblasts overexpressing type 1 InsP3R, BiPh(2,2',4,4',5,5')P6 (8) inhibits Ins(1,4,5)P3-induced Ca2+ release in a apparently competitive fashion [IC50 187 nM] and the Bz(1,2,4)P3 dimer (9) is only slightly weaker [IC50 380 nM]. Compounds were also evaluated against type I Ins(1,4,5)P3 5-phosphatase. All compounds are resistant to dephosphorylation, with BiPh(2,2',4,4',5,5')P6 (8), being the most effective inhibitor of any biphenyl derivative synthesized to date [IC50 480 nM] and the Bz(1,2,4)P3 ethylene dimer (9) weaker [IC50 3.55 µM]. BiPh(3,3',4,4',5,5')P6 (11) also inhibits 5-phosphatase [IC50 730 nM] and exhibits unexpected Ca2+ releasing activity [EC50 800 nM]. Thus, relocation of only a single mirrored phenyl phosphate group in (11) from that of antagonist (8) does not markedly change enzyme inhibitory activity, but elicits a dramatic switch in Ca2+-releasing activity. Such new agents demonstrate the power of the multivalent approach and may be useful to investigate the chemical biology of signaling through InsP3R and as templates for further design.

Clients of female sex workers as a bridging population in Vietnam.

Understanding bridging behaviors of clients of female sex workers (FSWs) is important for projecting and intervening in the spread of sexually transmitted infections in Vietnam. The goals of the study were to determine HIV/STI prevalence amongst different bridging groups, identify factors associated with being potential and active bridgers, and assess the association of drug use and unsafe sex with HIV and/or STI prevalence. In April, 2007, 292 clients were anonymously interviewed at sex venues in a two-stage time-location cluster sampling survey, followed by HIV, syphilis, and HSV-2 testing. Based on condom use with both high-risk (FSWs) and low-risk (wives/girlfriends) sexual partners, clients were classified as unlikely, potential, or active bridgers. The majority of clients were potential or active bridgers (55.8%) who had a significantly higher prevalence of herpes simplex type 2 (HSV-2) (21% and 33%, respectively) than unlikely bridgers (8.7%). HIV seropositivity was 4.4-fold (95% CI 1.1-17.1) higher among those who were HSV-2-positive. Clients of FSWs may be playing a major bridging role in transmitting HIV and sexually transmitted infections (STIs) in Vietnam. An observed synergistic interaction between drug use and condom slippage/breakage emphasizes the importance of proper condom use, particularly among drug users.

Benzene polyphosphates as tools for cell signalling: inhibition of inositol 1,4,5-trisphosphate 5-phosphatase and interaction with the PH domain of protein kinase Balpha.

Novel benzene polyphosphates were synthesised as inositol polyphosphate mimics and evaluated against type-I inositol 1,4,5-trisphosphate 5-phosphatase, which only binds soluble inositol polyphosphates, and against the PH domain of protein kinase Balpha (PKBalpha), which can bind both soluble inositol polyphosphates and inositol phospholipids. The most potent trisphosphate 5-phosphatase inhibitor is benzene 1,2,4-trisphosphate (2, IC(50) of 14 microM), a potential mimic of D-myo-inositol 1,4,5-trisphosphate, whereas the most potent tetrakisphosphate Ins(1,4,5)P(3) 5-phosphatase inhibitor is benzene 1,2,4,5-tetrakisphosphate, with an IC(50) of 4 microM. Biphenyl 2,3',4,5',6-pentakisphosphate (4) was the most potent inhibitor evaluated against type I Ins(1,4,5)P(3) 5-phosphatase (IC(50) of 1 microM). All new benzene polyphosphates are resistant to dephosphorylation by type I Ins(1,4,5)P(3) 5-phosphatase. Unexpectedly, all benzene polyphosphates studied bind to the PH domain of PKBalpha with apparent higher affinity than to type I Ins(1,4,5)P(3) 5-phosphatase. The most potent ligand for the PKBalpha PH domain, measured by inhibition of biotinylated diC(8)-PtdIns(3,4)P(2) binding, is biphenyl 2,3',4,5',6-pentakisphosphate (4, K(i)=27 nm). The approximately 80-fold enhancement of binding relative to parent benzene trisphosphate is explained by the involvement of a cation-pi interaction. These new molecular tools will be of potential use in structural and cell signalling studies.

Novel inositol phospholipid headgroup surrogate crystallized in the pleckstrin homology domain of protein kinase Balpha.

Protein kinase B (PKB/Akt) plays a key role in cell signaling. The PH domain of PKB binds phosphatidylinositol 3,4,5-trisphosphate translocating PKB to the plasma membrane for activation by 3-phosphoinositide-dependent protein kinase 1. The crystal structure of the headgroup inositol 1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P4-PKB complex facilitates in silico ligand design. The novel achiral analogue benzene 1,2,3,4-tetrakisphosphate (Bz(1,2,3,4)P4) possesses phosphate regiochemistry different from that of Ins(1,3,4,5)P4 and surprisingly binds with similar affinity as the natural headgroup. Bz(1,2,3,4)P4 co-crystallizes with the PKBalpha PH domain in a fashion also predictable in silico. The 2-phosphate of Bz(1,2,3,4)P4 does not interact with any residue, and the D5-phosphate of Ins(1,3,4,5)P4 is not mimicked by Bz(1,2,3,4)P4. Bz(1,2,3,4)P4 is an example of a simple inositol phosphate surrogate crystallized in a protein, and this approach could be applied to design modulators of inositol polyphosphate binding proteins.

Biphenyl 2,3',4,5',6-pentakisphosphate, a novel inositol polyphosphate surrogate, modulates Ca2+ responses in rat hepatocytes.

Benzene polyphosphates containing phosphate groups on one ring are Ins(1,4,5)P3 5-phosphatase inhibitors when evaluated against type-I Ins(1,4,5)P3 5-phosphatase. A novel biphenyl derivative, biphenyl 2,3',4,5',6-pentakisphosphate, with five phosphate groups on two rings was synthesized: It inhibited the activity of two inositol 5-phosphatases: type I and SHIP2 with Ins(1,3,4,5)P4 as substrate. The inhibition was competitive with respect to the substrate. IC50 value measured in rat hepatocytes, which contains the native Ins(1,4,5)P3 5-phosphatase, was in the micromolar range at 1.0 microM Ins(1,4,5)P3 as substrate. Biphenyl 2,3',4,5',6-pentakisphosphate did not affect the activity of Ins(1,4,5)P3 3-kinase A in the 5-100 microM range. Surprisingly, experimental evidence supports an effect of biphenyl 2,3',4,5',6-pentakisphosphate at the level of the Ins(1,4,5)P3 receptor. Finally, when injected into rat hepatocytes, the analog affected the frequency of Ca2+ oscillations in a positive or negative way depending on its concentration. At very high concentrations of the analog, Ca2+ oscillations were even suppressed. These data were interpreted as a dual effect of the biphenyl 2,3',4,5',6-pentakisphosphate on cytosolic [Ca2+] increases: an activation effect through an increase in Ins(1,4,5)P3 level via Ins(1,4,5)P3 5-phosphatase inhibition and an inhibitory effect, which was exerted directly on the Ins(1,4,5)P3 receptor. Thus, our data show for the first time that the frequency of Ca2+ oscillations in response to a Ca2+-mobilizing agonist can be controlled by inhibitors of type-I Ins(1,4,5)P3 5-phosphatase.

3-hydroxybenzene 1,2,4-trisphosphate, a novel second messenger mimic and unusual substrate for type-I myo-inositol 1,4,5-trisphosphate 5-phosphatase: Synthesis and physicochemistry.

3-Hydroxybenzene 1,2,4-trisphosphate 4 is a new myo-inositol 1,4,5-trisphosphate analogue based on the core structure of benzene 1,2,4-trisphosphate 2 with an additional hydroxyl group at position-3, and is the first noninositol based compound to be a substrate for inositol 1,4,5-trisphosphate 5-phosphatase. In physicochemical studies on 2, when three equivalents of protons were added, the (31)P NMR spectrum displayed monophasic behaviour in which phosphate-1 and phosphate-2 behaved independently in most of the studied pH range. For compound 4, phosphate-2 and phosphate-4 interacted with the 3-OH group, which does not titrate at physiological pH, displaying complex biphasic behaviour which demonstrated co-operativity between these groups. Phosphate-1 and phosphate-2 strongly interacted with each other and phosphate-4 experienced repulsion because of the interaction of the 3-OH group. Benzene 1,2,4-trisphosphate 2 is resistant to inositol 1,4,5-trisphosphate type I 5-phosphatase catalysed dephosphorylation. However, surprisingly, 3-hydroxybenzene 1,2,4-trisphosphate 4 was dephosphorylated by this 5-phosphatase to give the symmetrical 2,3-dihydroxybenzene 1,4-bisphosphate 16. The extra hydroxyl group is shown to form a hydrogen bond with the vicinal phosphate groups at -15 degrees C, and (1)H NMR titration of the ring and hydroxyl protons in 4 shows the OH proton to be strongly stabilized as soon as the phosphate groups are deprotonated. The effect of the phenolic 3-OH group in compound 4 confirms a critical role for the 6-OH group of the natural messenger in the dephosphorylation mechanism that persists even in radically modified analogues.

Interaction of the catalytic domain of inositol 1,4,5-trisphosphate 3-kinase A with inositol phosphate analogues.

The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3-kinase A in Escherichia coli as a His- and S-tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D-2-deoxy-Ins(1,4,5)P3 was devised. D-2-Deoxy-Ins(1,4,5)P3 and D-3-deoxy-Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D-2-deoxy-Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3-kinase. Therefore, the axial 2-hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3-hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3-hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding.