SAR optimization studies on modified salicylamides as a potential treatment for acute myeloid leukemia through inhibition of the CREB pathway.

Disruption of cyclic adenosine monophosphate response element binding protein (CREB) provides a potential new strategy to address acute leukemia, a disease associated with poor prognosis, and for which conventional treatment options often carry a significant risk of morbidity and mortality. We describe the structure-activity relationships (SAR) for a series of XX-650-23 derived from naphthol AS-E phosphate that disrupts binding and activation of CREB by the CREB-binding protein (CBP). Through the development of this series, we identified several salicylamides that are potent inhibitors of acute leukemia cell viability through inhibition of CREB-CBP interaction. Among them, a biphenyl salicylamide, compound 71, was identified as a potent inhibitor of CREB-CBP interaction with improved physicochemical properties relative to previously described derivatives of naphthol AS-E phosphate.

Niclosamide suppresses acute myeloid leukemia cell proliferation through inhibition of CREB-dependent signaling pathways.

CREB (cAMP Response Element Binding protein) is a transcription factor that is overexpressed in primary acute myeloid leukemia (AML) cells and associated with a decreased event-free survival and increased risk of relapse. We recently reported a small molecule inhibitor of CREB, XX-650-23, which inhibits CREB activity in AML cells. Structure-activity relationship analysis for chemical compounds with structures similar to XX-650-23 led to the identification of the anthelminthic drug niclosamide as a potent anti-leukemic agent that suppresses cell viability of AML cell lines and primary AML cells without a significant decrease in colony forming activity of normal bone marrow cells. Niclosamide significantly inhibited CREB function and CREB-mediated gene expression in cells, leading to apoptosis and G1/S cell cycle arrest with reduced phosphorylated CREB levels. CREB knockdown protected cells from niclosamide treatment-mediated cytotoxic effects. Furthermore, treatment with a combination of niclosamide and CREB inhibitor XX-650-23 showed an additive anti-proliferative effect, consistent with the hypothesis that niclosamide and XX-650-23 regulate the same targets or pathways to inhibit proliferation and survival of AML cells. Niclosamide significantly inhibited the progression of disease in AML patient-derived xenograft (PDX) mice, and prolonged survival of PDX mice. Niclosamide also showed synergistic effects with chemotherapy drugs to inhibit AML cell proliferation. While chemotherapy antagonized the cytotoxic potential of niclosamide, pretreatment with niclosamide sensitized cells to chemotherapeutic drugs, cytarabine, daunorubicin, and vincristine. Therefore, our results demonstrate niclosamide as a potential drug to treat AML by inducing apoptosis and cell cycle arrest through inhibition of CREB-dependent pathways in AML cells.

Inositol Pyrophosphate Specificity of the SPX-Dependent Polyphosphate Polymerase VTC.

The free energy of nucleotide hydrolysis depends on phosphate concentration. Cells regulate cytosolic phosphate levels by orchestrating phosphate acquisition and storage through inositol pyrophosphates (PP-InsP) and SPX domains. Here, we report the synthesis of the novel 5-PPP-InsP5 containing a triphosphate subunit. Using this and a series of synthetic PP-InsP, we examined the ligand specificity of the SPX domain in the PP-InsP-controlled yeast polyphosphate polymerase VTC. SPX decodes the relative positioning of the phosphoric anhydrides, their structure (diphosphate vs triphosphate), and the presence of other phosphates on the inositol ring. Despite the higher potency of 1,5-(PP)2-InsP4, 5-PP-InsP5 is the primary activator of VTC in cells, indicating that its higher concentration compensates for its lower potency. 1,5-(PP)2-InsP4 levels rise and could become relevant under stress conditions. Thus, SPX domains may integrate PP-InsP dependent signaling to adapt cytosolic phosphate concentrations to different metabolic situations.

Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S](2+) cluster which inhibits inositol pyrophosphate 1-phosphatase activity.

Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron-sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1(371-920)), were each heterologously expressed in Escherichia coli. The phosphatase activity is exquisitely specific: it hydrolyzes the 1-diphosphate from just two members of the inositol pyrophosphate (PP-InsP) signaling family, namely, 1-InsP7 and 1,5-InsP8. We demonstrate that Asp1 does not hydrolyze either InsP6, 2-InsP7, 3-InsP7, 4-InsP7, 5-InsP7, 6-InsP7, or 3,5-InsP8. We also recorded 1-phosphatase activity in a human homologue of Asp1, hPPIP5K1, which was heterologously expressed in Drosophila S3 cells with a biotinylated N-terminal tag, and then isolated from cell lysates with avidin beads. Purified, recombinant Asp1(371-920) contained iron and acid-labile sulfide, but the stoichiometry (0.8 atoms of each per protein molecule) indicates incomplete iron-sulfur cluster assembly. We reconstituted the Fe-S cluster in vitro under anaerobic conditions, which increased the stoichiometry to approximately 2 atoms of iron and acid-labile sulfide per Asp1 molecule. The presence of a [2Fe-2S](2+) cluster in Asp1(371-920) was demonstrated by UV-visible absorption, resonance Raman spectroscopy, and electron paramagnetic resonance spectroscopy. We determined that this [2Fe-2S](2+) cluster is unlikely to participate in redox chemistry, since it rapidly degraded upon reduction by dithionite. Biochemical and mutagenic studies demonstrated that the [2Fe-2S](2+) cluster substantially inhibits the phosphatase activity of Asp1, thereby increasing its net kinase activity.

VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis.

Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.

Desymmetrization of myo-inositol derivatives by lanthanide catalyzed phosphitylation with C2-symmetric phosphites.

Desymmetrization by phosphorylation represents a promising method with potential impact in many different areas of research. C2-Symmetric phosphoramidites have been used to desymmetrize myo-inositol derivatives by functionalization at different positions. With this method, 1:1 mixtures of diastereomers are obtained that can be separated subsequently. In this work, activation of a C2-symmetric phosphoramidite is achieved by addition of pentafluorophenol (PFP) and leads to a reactive PFP phosphite, which can then be coupled to protected myo-inositol derivatives with reactive OH groups at the 1, 3, 4 and 6 positions. This strategy enhances the diastereoselectivity of the coupling reaction with a preference towards phosphitylation at position 6 (up to 3:1) or position 3 (up to 2:1). The concept of attenuative activation of phosphoramidites via in situ generated pentafluorophenol phosphite triesters is thus proven in these studies. It is further shown that Lewis-Acid catalysis enhances the rate of phosphite triester coupling without affecting the diastereoselectivity. This novel strategy improves access to different phosphorylated myo-inositol derivatives and will thus enable further studies into the function of these important intracellular second messengers.

Synthesis of densely phosphorylated bis-1,5-diphospho-myo-inositol tetrakisphosphate and its enantiomer by bidirectional P-anhydride formation.

The ubiquitous mammalian signaling molecule bis-diphosphoinositol tetrakisphosphate (1,5-(PP)2 -myo-InsP4 , or InsP8 ) displays the most congested three-dimensional array of phosphate groups found in nature. The high charge density, the accumulation of unstable P-anhydrides and P-esters, the lack of UV absorbance, and low levels of optical rotation constitute severe obstacles to its synthesis, characterization, and purification. Herein, we describe the first procedure for the synthesis of enantiopure 1,5-(PP)2 -myo-InsP4 and 3,5-(PP)2 -myo-InsP4 utilizing a C2 -symmetric P-amidite for desymmetrization and concomitant phosphitylation followed by a one-pot bidirectional P-anhydride-forming reaction that combines sixteen chemical transformations with high efficiency. The configuration of these materials is unambiguously shown by subsequent X-ray analyses of both enantiomers after being individually soaked into crystals of the kinase domain of human diphosphoinositol pentakisphosphate kinase 2.

Elucidating diphosphoinositol polyphosphate function with nonhydrolyzable analogues.

The diphosphoinositol polyphosphates (PP-IPs) represent a novel class of high-energy phosphate-containing messengers which control a wide variety of cellular processes. It is thought that PP-IPs exert their pleiotropic effects as allosteric regulators and through pyrophosphorylation of protein substrates. However, most details of PP-IP signaling have remained elusive because of a paucity of suitable tools. We describe the synthesis of PP-IP bisphosphonate analogues (PCP-IPs), which are resistant to chemical and biochemical degradation. While the two regioisomers 1PCP-IP5 and 5PCP-IP5 inhibited Akt phosphorylation with similar potencies, 1PCP-IP5 was much more effective at inhibiting its cognate phosphatase hDIPP1. Furthermore, the PCP analogues inhibit protein pyrophosphorylation because of their inability to transfer the ß-phosphoryl group, and thus enable the distinction between PP-IP signaling mechanisms. As such, the PCP analogues will find widespread applications for the structural and biochemical characterization of PP-IP signaling properties.

Development of endocannabinoid-based chemical probes for the study of cannabinoid receptors.

We report the synthesis of new chemical probes (1a,b, 2a-c, 3a-c) based on the structure of the main endocannabinoids for their use in biological systems directly or via click chemistry. As proof of concept, 2-arachidonyl glyceryl ether based biotinylated 3b enables direct visualization of CB(1) receptor in cells. These results represent the starting point for the development of advanced small molecule chemical probes able to generate valuable information about the cannabinoid receptors.