Signalling Properties of Inositol Polyphosphates

Several studies have identified specific signalling functions for inositol polyphosphates (IPs) in different cell types and have led to the accumulation of new information regarding their cellular roles as well as new insights into their cellular production. These studies have revealed that interaction of IPs with several proteins is critical for stabilization of protein complexes and for modulation of enzymatic activity. This has not only revealed their importance in regulation of several cellular processes but it has also highlighted the possibility of new pharmacological interventions in multiple diseases, including cancer. In this review, we describe some of the intracellular roles of IPs and we discuss the pharmacological opportunities that modulation of IPs levels can provide.


Ins(1,4,5)P 3 , a Bona Fide Second Messenger
Ins(1,4,5)P 3 was the first IP to be identified as a "second messenger" more than 35 years ago [12,13] and its identification as a key regulator of Ca 2+ release from intracellular stores sparked much interest on the role of inositol-derived molecules in signal transduction. Specifically, Ins(1,4,5)P 3 derives from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), which is catalyzed by members of the family of phospholipases C (PLC). Upon cellular stimulation and PLCs activation, the accumulated Ins(1,4,5)P 3 can bind to Ins(1,4,5)P 3 receptors which are expressed in most animal cells and are Ca 2+ channels located on intracellular organelles such as the endoplasmic reticulum (ER) and Golgi apparatus. Such binding mediates Ca 2+ release from intracellular stores, resulting in Ca 2+ accumulation in the cytosol and in organelles such as mitochondria and lysosomes. A schematic representation of the Ins(1,4,5)P 3 -mediated Ca 2+ release following activation of G protein-coupled receptors and of some members of the PLC family is depicted in Figure 2. The resulting decrease in ER luminal Ca 2+ concentration also triggers stromal interaction molecule 1, which then amasses at ER-plasma membrane (PM) junctions and opens Orai1, a hexameric Ca 2+ channel in the PM [14,15], causing store-operated Ca 2+ entry. Through these Ins(1,4,5)P 3 -mediated mechanisms, many extracellular stimuli induce redistribution of Ca 2+ from the ER to the cytosol or other organelles as well as extracellular Ca 2+ entry [16]. Ca 2+ regulates a multitude of intracellular processes including transcription, apoptosis, motility, excitability and exocytosis, mainly through modulation of protein conformations and charge [17]. Together with its direct role in signalling, Ins(1,4,5)P 3 serves as a precursor for the synthesis of several highly phosphorylated IPs, including Ins(1,3,4,5)P 4 , InsP 5 and InsP 6 . Importantly, a recent study has identified additional mechanisms that can lead to synthesis of higher phosphorylated IPs, independently from the Ins(1,4,5)P 3 pool derived from the lipid PtdIns(4,5)P 2 . Such an alternative route involves phosphorylation of inositol monophosphates (such as the previously mentioned Ins3P and Ins1P) by the kinase inositol tetrakisphosphate 1-kinase 1 and it depends on the metabolic status of the cells [9]. Whether the higher phosphorylated IPs can all be strictly considered "second messengers" has somehow been a matter of debate [9]. Nonetheless, their importance in regulation of a multitude of cellular processes is unquestionable.
as the previously mentioned Ins3P and Ins1P) by the kinase inositol tetrakisphosphate 1-kinase 1 and it depends on the metabolic status of the cells [9]. Whether the higher phosphorylated IPs can all be strictly considered "second messengers" has somehow been a matter of debate [9]. Nonetheless, their importance in regulation of a multitude of cellular processes is unquestionable.

Intracellular Processes Regulated by IPs Binding to Proteins
A key feature of IPs is their ability to bind to proteins and either contribute to maintain their structural fold or promote/stabilize their assembly into complexes or contribute to their activation, in the case of enzymes. Such binding properties are responsible for most of the intracellular roles that have been attributed to IPs. Indeed, IPs have been found to be involved in regulation of a vast array of very different intracellular processes. Just to mention an example of the diversity of intracellular roles that IPs can have, InsP6 has been implicated, amongst many other processes that we will mention later, in: (i) regulation of the activity of the RNA editing enzyme adenosine deaminase that act on RNA (ADAR)2 as well as adenosine deaminases that act on transfer RNA (ADAT)1 [18]; (ii) activation of Bruton's tyrosine kinase, a Tec-family tyrosine kinase that is essential for B-cell function [19]; (iii) regulation of casein kinase 2 [20], a ubiquitous protein kinase that can phosphorylate over 300 proteins involved in cell growth, development and several other cellular functions; (iv) allosteric regulation of Yersinia outer-protein J effectors, important to control their acetyltransferase activity [21]; (v) ubiquitylation, through regulation of the activity state of cullin-RING E3 ubiquitin ligases [22]; (vi) crotonylation, as indicated by data demonstrating that addition of InsP6 to the complex histone deacetylase (HDAC)1/CoREST/Lysine specific demethylase 1 increased its initial rate of decrotonylation by 1.8-fold [23].
In fact, IPs have been detected in several X-ray crystal structures and this suggests their involvement in many cellular processes [24]. As additional evidence of these interactions accumulates, the variety of cellular processes that relies on the correct balance between different IPs species is becoming more evident. Few examples of some of the first cellular functions that were attributed to IPs together with some of the most recently identified will be mentioned here. Attention

Intracellular Processes Regulated by IPs Binding to Proteins
A key feature of IPs is their ability to bind to proteins and either contribute to maintain their structural fold or promote/stabilize their assembly into complexes or contribute to their activation, in the case of enzymes. Such binding properties are responsible for most of the intracellular roles that have been attributed to IPs. Indeed, IPs have been found to be involved in regulation of a vast array of very different intracellular processes. Just to mention an example of the diversity of intracellular roles that IPs can have, InsP 6 has been implicated, amongst many other processes that we will mention later, in: (i) regulation of the activity of the RNA editing enzyme adenosine deaminase that act on RNA (ADAR)2 as well as adenosine deaminases that act on transfer RNA (ADAT)1 [18]; (ii) activation of Bruton's tyrosine kinase, a Tec-family tyrosine kinase that is essential for B-cell function [19]; (iii) regulation of casein kinase 2 [20], a ubiquitous protein kinase that can phosphorylate over 300 proteins involved in cell growth, development and several other cellular functions; (iv) allosteric regulation of Yersinia outer-protein J effectors, important to control their acetyltransferase activity [21]; (v) ubiquitylation, through regulation of the activity state of cullin-RING E3 ubiquitin ligases [22]; (vi) crotonylation, as indicated by data demonstrating that addition of InsP 6 to the complex histone deacetylase (HDAC)1/CoREST/Lysine specific demethylase 1 increased its initial rate of decrotonylation by 1.8-fold [23].
In fact, IPs have been detected in several X-ray crystal structures and this suggests their involvement in many cellular processes [24]. As additional evidence of these interactions accumulates, the variety of cellular processes that relies on the correct balance between different IPs species is becoming more evident. Few examples of some of the first cellular functions that were attributed to IPs together with some of the most recently identified will be mentioned here. Attention is focused on processes that can provide examples of the rationale behind the design of potential therapeutic strategies based on exploitation of the role of IPs.

Endo and Exocytosis
The identification of proteins able to bind to InsP 6 , such as clathrin assembly proteins (AP)-2 [25] and AP-3 [26] and arrestin [27] suggested very early a role for this IP in endocytosis and exocytosis. Indeed, InsP 6 was proposed to be involved in the regulation of both processes in pancreatic β cells, where it appeared to be the most abundant IP [28]. Specifically, InsP 6 was shown to regulate insulin exocytosis by enhancing the activity of the voltage-dependent Ca 2+ channels through deactivation of Serine/Threonine protein phosphatases and activation of Serine/Threonine kinases [28,29]. In addition, InsP 6 was also reported to be involved in the regulation of calcineurin-dependent endocytosis in β cells [30]. Over the years, roles for different IPs, in particular PP-IPs, in the complex process of insulin secretion have been described more precisely [31,32]. A wider role for InsP 6 in regulation of endocytosis was further indicated by data supporting its involvement in regulation of this process in synaptic vesicle. This was suggested by the identification of its role in phosphorylation of a protein kinase responsible for regulation of the synaptic vesicle-associated protein pacsin/syndapin I and the demonstration that such InsP 6 -regulated phosphorylation increased the interaction between dynamin and pacsin/syndapin I [33]. Importantly, a specific role for InsP 6 (and isomers of the PP-IP InsP 7 ) was reported in this study, with no phosphorylation detected using lower IPs, in particular InsP 3 or InsP 4 [33].

Nuclear Functions
Ins(1,4,5,6)P 4 has emerged as a key regulator of some members of the family of class I HDACs, enzymes that regulate lysine acetylation in histone tails and are involved in epigenetic regulation of genes [34]. The role of Ins(1,4,5,6)P 4 was first suggested by its presence in the crystal structure of HDAC3 and its co-repressor silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), specifically in a binding pocket at the interface between the enzyme and the co-repressor [35]. Later studies corroborated that the inositol phosphate-binding pocket was present in other class I HDAC co-repressor complexes, and, indeed, it was reported that Ins(1,4,5,6)P 4 enhances the deacetylase activity of both the HDAC3:SMRT and HDAC1:SMRT complexes [36]. The stereochemical requirement for binding and activation by IPs has also been described [37]. Furthermore, mutations that abolish InsP 4 binding decrease the activity of HDAC1/2 in vivo [38]. These data indicate a role for Ins(1,4,5,6)P 4 in histone acetylation and chromatin condensation and contribute to the growing interest around the role of IPs and PIs in epigenetic regulation.
A role for InsP 6 in the nucleus has been suggested by many lines of evidence, including the identification of a well-ordered InsP 6 molecule in the crystal structure of sister chromatid cohesion protein Pds5 homolog B (Pds5B), a subunit of the cohesin complex, which controls transcription, chromosome segregation and DNA repair [39]. Additional studies have indicated that InsP 6 has a role in DNA repair, in particular non-homologous end-joining mediated by DNA-dependent protein kinase [40] and nuclear mRNA export [41], acting together with nucleoporin Gle1 [42,43]. A role for InsP 5 and InsP 6 in chromatin remodelling was also reported [44,45].

Platelet Aggregation
A recent study has reported the increase of InsP 6 in platelets upon stimulation with thrombin, collagen I and ADP [46]. Importantly, this study has revealed a role for InsP 6 in regulation of platelet aggregate size, in a mechanism involving its interaction with fibrinogen. Although previous evidence identified both InsP 5 and InsP 6 as binding partners of fibrinogen [47], InsP 5 did not have a role in regulation of aggregation, which appeared specific for InsP 6 [46]. The authors suggested a role for InsP 6 in supporting and stabilizing the crosslinking between fibrinogen and platelets, identifying this IP as a novel potential player in regulation of platelet functions [46].

Reactive Oxygen Species (ROS) Formation and Drug Sensitivity
A very recent study reported that InsP 4 selectively binds to the enzyme NADPH oxidase 4 (NOX4) which is important for ROS generation [48]. Such a direct interaction competes with NADPH binding to NOX4 and inhibits the enzyme, resulting in reduced ROS production. Importantly, the authors showed that downregulation of ITPKB, sensitized cisplatin-resistant cancer cells to cisplatin treatment both in vitro and in vivo. This was likely due to the reduced levels of InsP 4 , which would result in increased ROS production and therefore increased cisplatin-induced ROS production [48]. Consistent with this, the authors reported that expression of ITPKB and cisplatin resistance positively correlated in 22 human cancer cell lines and 13 patient-derived xenograft tumours of head and neck squamous cell carcinoma (HNSCC), lung cancer and ovarian cancer. In addition, it was shown that expression of the enzyme was higher in primary tumours from HNSCC patients who had recurrent disease compared with tumours from responsive patients [48]. The observation that chemical inhibition of ITPKB also sensitized cells to cisplatin treatment both in vitro and in vivo confirms how manipulation of IPs levels can have an important therapeutic value [49].

Viral Replication
Several lines of evidence indicate that InsP 6 plays a crucial role during HIV-1 infection, being involved in some of the several capsid transformations that occur during viral replication. Specifically, it has been reported that HIV-1 recruits InsP 6 into virions using two Lysine rings in its immature hexamers [50] and that InsP 6 in turn promotes the assembly and maturation of the mature capsid lattice [51][52][53]. Furthermore, binding of InsP 6 increases HIV-1 capsid stability from minutes to hours, enabling freshly synthesized DNA to gather inside the capsid during reverse transcription [54]. These data indicate that InsP 6 is important in HIV-1 assembly and during viral entry by stabilizing the capsid while moving towards the nucleus, making it a critical cofactor for HIV replication (Figure 3).

Reactive Oxygen Species (ROS) Formation and Drug Sensitivity
A very recent study reported that InsP4 selectively binds to the enzyme NADPH oxidase 4 (NOX4) which is important for ROS generation [48]. Such a direct interaction competes with NADPH binding to NOX4 and inhibits the enzyme, resulting in reduced ROS production. Importantly, the authors showed that downregulation of ITPKB, sensitized cisplatin-resistant cancer cells to cisplatin treatment both in vitro and in vivo. This was likely due to the reduced levels of InsP4, which would result in increased ROS production and therefore increased cisplatin-induced ROS production [48]. Consistent with this, the authors reported that expression of ITPKB and cisplatin resistance positively correlated in 22 human cancer cell lines and 13 patient-derived xenograft tumours of head and neck squamous cell carcinoma (HNSCC), lung cancer and ovarian cancer. In addition, it was shown that expression of the enzyme was higher in primary tumours from HNSCC patients who had recurrent disease compared with tumours from responsive patients [48]. The observation that chemical inhibition of ITPKB also sensitized cells to cisplatin treatment both in vitro and in vivo confirms how manipulation of IPs levels can have an important therapeutic value [49].

Viral Replication
Several lines of evidence indicate that InsP6 plays a crucial role during HIV-1 infection, being involved in some of the several capsid transformations that occur during viral replication. Specifically, it has been reported that HIV-1 recruits InsP6 into virions using two Lysine rings in its immature hexamers [50] and that InsP6 in turn promotes the assembly and maturation of the mature capsid lattice [51][52][53]. Furthermore, binding of InsP6 increases HIV-1 capsid stability from minutes to hours, enabling freshly synthesized DNA to gather inside the capsid during reverse transcription [54]. These data indicate that InsP6 is important in HIV-1 assembly and during viral entry by stabilizing the capsid while moving towards the nucleus, making it a critical cofactor for HIV replication (Figure 3). Indeed, cells lacking IPPK, the enzyme that catalyzes the addition of the final phosphate on the position 2 of InsP5, produced much fewer infectious HIV-1 particles [51]. Importantly, however, infectious particles were still being produced in these cells albeit at a much-reduced rate [51]. In line Indeed, cells lacking IPPK, the enzyme that catalyzes the addition of the final phosphate on the position 2 of InsP 5 , produced much fewer infectious HIV-1 particles [51]. Importantly, however, infectious particles were still being produced in these cells albeit at a much-reduced rate [51]. In line with this, a recent study revealed that, in the absence of InsP 6 , as achieved upon genetic knockout of IPMK and IPPK, HIV-1 packages InsP 5 and it can substitute InsP 5 for InsP 6 during viral production without affecting its infectivity [50]. Indeed, InsP 5 was found to be able to stimulate immature HIV-1 assembly in vitro, although less efficiently that InsP 6 [50,51,54]. A very recent study has demonstrated that the almost complete ablation of both InsP 6 and InsP 5 induced a 1000-fold reduction (i.e., an almost abrogation) in the production of HIV-1 infectious particle and virus release, establishing an absolute requirement for these IPs in HIV-1 viral production [55]. Furthermore, the authors demonstrated that ablation of InsP 6 and InsP 5 in viral target cells did not affect permissivity to HIV-1 infection [55]. Importantly, recent evidence suggests a conserved role for InsP 6 in lentiviral assembly, as indicated by its ability to stimulate the in vitro assembly of immature particles of many other retroviruses in the lentivirus genus [56]. On the other hand, ablation of IPPK only modestly reduced the production of infectious particles by other retroviruses, such as a gammaretrovirus, a betaretrovirus and two non-primate lentiviruses. This study in particular demonstrated that only the primate (macaque) lentivirus simian immunodeficiency virus displayed a similar dependence on InsP 6 /InsP 5 as HIV-1 [55].

Exogenous IPs
Until now, the therapeutic potential of IPs has been mainly tested through administration of exogenous IPs. In some cases, this strategy aims to inhibit PIs-dependent signaling pathways through competition between exogenous IPs and endogenous PIs towards same protein domains. The best example of this is provided by proteins containing pleckstrin homology (PH) domains, protein modules that can bind to IPs and PIs [57][58][59]. Twenty years ago, we hypothesized that water soluble IPs could be delivered intracellularly to compete with PIs for PH domain binding and therefore they could inhibit activation of proteins that rely on PIs/PH domain interaction [2]. Over the following years, we and others demonstrated that this strategy was correct and it held potential therapeutic value. Work in our lab was mostly focused on Akt PH domain, whose interaction with phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) is critical for its translocation to the plasma membrane and enzymatic activation. (Figure 4).
with this, a recent study revealed that, in the absence of InsP6, as achieved upon genetic knockout of IPMK and IPPK, HIV-1 packages InsP5 and it can substitute InsP5 for InsP6 during viral production without affecting its infectivity [50]. Indeed, InsP5 was found to be able to stimulate immature HIV-1 assembly in vitro, although less efficiently that InsP6 [50,51,54]. A very recent study has demonstrated that the almost complete ablation of both InsP6 and InsP5 induced a 1000-fold reduction (i.e., an almost abrogation) in the production of HIV-1 infectious particle and virus release, establishing an absolute requirement for these IPs in HIV-1 viral production [55]. Furthermore, the authors demonstrated that ablation of InsP6 and InsP5 in viral target cells did not affect permissivity to HIV-1 infection [55]. Importantly, recent evidence suggests a conserved role for InsP6 in lentiviral assembly, as indicated by its ability to stimulate the in vitro assembly of immature particles of many other retroviruses in the lentivirus genus [56]. On the other hand, ablation of IPPK only modestly reduced the production of infectious particles by other retroviruses, such as a gammaretrovirus, a betaretrovirus and two non-primate lentiviruses. This study in particular demonstrated that only the primate (macaque) lentivirus simian immunodeficiency virus displayed a similar dependence on InsP6/InsP5 as HIV-1 [55].

Exogenous IPs
Until now, the therapeutic potential of IPs has been mainly tested through administration of exogenous IPs. In some cases, this strategy aims to inhibit PIs-dependent signaling pathways through competition between exogenous IPs and endogenous PIs towards same protein domains. The best example of this is provided by proteins containing pleckstrin homology (PH) domains, protein modules that can bind to IPs and PIs [57][58][59]. Twenty years ago, we hypothesized that water soluble IPs could be delivered intracellularly to compete with PIs for PH domain binding and therefore they could inhibit activation of proteins that rely on PIs/PH domain interaction [2]. Over the following years, we and others demonstrated that this strategy was correct and it held potential therapeutic value. Work in our lab was mostly focused on Akt PH domain, whose interaction with phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is critical for its translocation to the plasma membrane and enzymatic activation. (Figure 4)  As it was known that InsP 5 and, to a slightly lesser extent, Ins(1,4,5,6)P 4 , were also able to bind Akt PH domain [57], we hypothesized that these IPs could compete with PtdIns(3,4,5)P 3 for Akt PH domain binding, resulting in inhibition of Akt translocation to the plasma membrane. A schematic representation of the mechanism of Akt activation upon growth factors-mediated activation of receptor Tyrosine kinases as well as the inhibitory role of exogenous IPs (in particular of InsP 5 ) are shown in Figure 4. Indeed, supporting our hypothesis of a potential competition between the two groups of compounds, we observed that plasma membrane translocation of a Green fluorescent protein-tagged Akt PH domain was inhibited by exogenous Ins(1,4,5,6)P 4 [60]. Later additional evidence of such an inhibition appeared, including data showing that a cell permeant Ins(1,3,4,5)P 4 was able to inhibit a receptor-dependent plasma membrane translocation of AKT PH domain in neutrophils [61]. More important, impairment of Akt plasma membrane translocation resulted in inhibition of its activation, as indicated by our subsequent studies showing that exogenously added InsP 5 inhibited phosphorylation of Akt in ovarian cancer line SKOV-3 cells [62], in PTEN -/embryonic stem cells [62], in human umbilical vein endothelial cells (HUVEC) activated with basic fibroblast growth factor (FGF-2) [63] and in dissected tumours from a xenograft model of SKOV-3 cells [63]. Notably, our studies confirmed that the IPs-mediated inhibition of Akt phosphorylation was able to affect Akt-dependent cellular processes. Specifically, the addition of exogenous Ins(1,4,5,6)P 4 and InsP 5 resulted in inhibition of proliferation/growth of breast cancer cell line MCF7, small cell lung cancer cell line H69 and SKOV-3 cells [60]. Furthermore, InsP 5 induced apoptosis in H69 cells and SKOV-3 cells [62] and inhibited FGF-2-induced cell survival, cell migration and capillary tube formation in HUVECs as well as inhibiting angiogenic response in vivo [63]. Additional evidence of the anti-angiogenic properties of InsP 5 has been provided by a recent study reporting that InsP 5 can also inhibit vascular endothelial growth factor production by fibroblasts and neural cells in a mechanism involving degradation of hypoxia inducible factor 1α [64]. The definite demonstration that such a strategy could represent an important potential therapeutic strategy came from the observation that InsP 5 reduced tumour growth in a xenograft model of SKOV-3 cells [63]. Interestingly, in this experimental setting, treatment with InsP 6 did not reduce tumour growth or Akt activation in dissected tumours [63].
The potential beneficial effects of exogenous IPs in cancer might go beyond their inhibition of PIs-mediated processes. For instance, InsP 6 has been reported to have anti-cancer activity in several in vitro and in vivo models [65], including prostate [66][67][68], breast [69,70], bladder [71] and colon [72,73] cancer. Some very preliminary clinical results are also appearing [65]. Whether InsP 6 exerts its anti-cancer activity by interfering with PIs-mediated activation of proteins is not established, as it has been reported that its inhibition of cancer cell proliferation, cancer cell survival and metastasis [64,74,75] is a result of inhibition of several pathways, including the phosphoinositide 3-kinase [PI3K)/Akt pathway [67,73,76]. The impact of InsP 6 can be very diverse: recently, for instance, its involvement in regulation of microRNA levels, in particular miR-155, in human colon cancer cells has also been reported [77], suggesting additional mechanisms by which this IP might exert its anti-cancer activity. Indeed, InsP 6 therapeutic potential has been mainly related to its anti-oxidant properties and its ability to oppose many carcinogens [65]. It must be noted that concentrations of exogenously added InsP 6 reported to have anti-cancer activity in vitro were usually in the millimolar range (1-5 mM), i.e., concentrations that can induce metals chelation and changes in the pH in cellular medium. To avoid this problem, a recent study has reported the synthesis of a prodrug of InsP 6 which retains the pro-apoptotic activity in vitro as well as anti-cancer activity in vivo in a mouse model of adult T-cell leukemia [78]. Whether the anti-cancer activity is due to InsP 6 itself or to its rapid conversion to other IPs, namely InsP 5 and InsP 4 , remains a matter of debate. Analysis of the IPs in extracts of intact HeLa cells incubated with 3 H-InsP 6 revealed that 3 H-InsP 3 , 3 H-InsP 4 and 3 H-InsP 5 accumulated inside the cells, confirming that internalized InsP 6 was dephosphorylated into lower forms [79]. Moreover, these authors reported that both Ins(1,4,5,6)P 4 and InsP 5 were more active than InsP 6 in inducing apoptosis [79]. Consistent with this, recent evidence indicated that InsP 6 derivatives, resulting from partial degradation of ingested InsP 6 by phytase into hydrolysates, also possess anti-cancer activity.
For instance, it has been reported that partially degraded InsP 6 inhibited proliferation of HCT116 colon carcinoma cells [80]. Similarly, a study demonstrated that InsP 6 hydrolysates inhibited proliferation of the colorectal cancer cell line SW620, with evidence suggesting that InsP 4 and InsP 5 were the likely primary constituents of the hydrolysates responsible for the anti-proliferative activity via Akt inhibition [72]. In contrast to InsP 6 , we found that exogenous InsP 5 not only was quickly and systematically internalized by cells but it was also converted slowly into different metabolites [63], supporting the conclusion that its anti-tumour effects were due to its activity and were not mediated by conversion to different phosphorylated forms.
Potential beneficial effects of exogenous IPs have also been explored in other diseases including diabetes [81], which, possibly, is not surprising considering that IPs and PP-IPs have been involved in insulin secretion, as previously mentioned.

Interfering with IPs/Proteins Binding
As we discussed above, many of the cellular roles of IPs depend on their ability to bind proteins and modulate their assembly into complexes or their activation. This raises the interesting possibility of inhibiting selective cellular processes by modulating intracellular levels of IPs through blockade of one of the enzymes responsible for their interchange or by developing compounds that might interfere with the selective IPs/proteins binding. For instance, small molecules might be designed to interfere with the recruitment of InsP 6 in the immature hexamers or possibly compete with InsP 6 and therefore they might block HIV viral particles production. Similar compounds might interfere with InsP 6 to fibrinogen and therefore possibly be beneficial to destabilize thrombi, as hypothesized recently [46].
However, even though this strategy is potentially interesting, possible limitations need to be taken into accountthe use of IPs as novel therapeutics should consider the variety of roles that they have in cell homeostasis, such as insulin signaling and nuclear processes [82], that could potentially generate off-target effects and hence side effects.

Conclusions
The family of IPs has been implicated in a multitude of intracellular functions and new evidence constantly appears identifying their contribution to additional processes. Such a variety and versatility of roles mean that several opportunities exist to exploit these molecules from a therapeutic point of view, either by delivering IPs and/or their analogues intracellularly or by manipulating the enzymes that control their interchanges or by interfering with their interaction with their effector proteins. In this respect, additional studies to shed light into the specific mechanisms of interaction between IPs and their effector proteins (such as X-ray crystal structure and modelling) might provide important information to design novel strategies to interfere with their cellular processes.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.