Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity.

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable.

Synthesis, biological evaluation, and stability studies of raloxifene mono- and bis-sulfamates as dual-targeting agents.

All three possible sulfamate derivatives of the selective estrogen receptor modulator Raloxifene (bis-sulfamate 7 and two mono-sulfamates 8-9) were synthesized and evaluated as inhibitors of the clinical drug target steroid sulfatase (STS), both in cell-free and in cell-based assays, and also as estrogen receptor (ER) modulators. Bis-sulfamate 7 was the most potent STS inhibitor with an IC50 of 12.2 nM in a whole JEG3 cell-based assay, with the two mono-sulfamates significantly weaker. The estrogen receptor-modulating activities of 7-9 showed generally lower affinities compared to Raloxifene HCl, diethylstilbestrol and other known ligands, with mono-sulfamate 8 being the best ligand (Ki of 1.5 nM) for ERα binding, although 7 had a Ki of 13 nM and both showed desirable antagonist activity. The antiproliferative activities of the sulfamate derivatives against the T-47D breast cancer cell line showed 7 as most potent (GI50 = 7.12 µM), comparable to that of Raloxifene. Compound 7 also showed good antiproliferative potency in the NCI-60 cell line panel with a GI50 of 1.34 µM against MDA-MB-231 breast cancer cells. Stability testing of 7-9 showed that bis-sulfamate 7 hydrolyzed by desulfamoylation at a surprisingly rapid rate, initially leading selectively to 8 and finally to Raloxifene 3 without formation of 9. The mechanisms of these hydrolysis reactions could be extensively rationalized. Conversion of Raloxifene (3) into its bis-sulfamate (7) thus produced a promising drug lead with nanomolar dual activity as an STS inhibitor and ERα antagonist, as a potential candidate for treatment of estrogen-dependent breast cancer.

Depleting inositol pyrophosphate 5-InsP7 protected the heart against ischemia-reperfusion injury by elevating plasma adiponectin.

AIMS: Adiponectin is an adipocyte-derived circulating protein that exerts cardiovascular and metabolic protection. Due to the futile degradation of endogenous adiponectin and the challenges of exogenous administration, regulatory mechanisms of adiponectin biosynthesis are of significant pharmacological interest. METHODS AND RESULTS: Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) generated by inositol hexakisphosphate kinase 1 (IP6K1) governed circulating adiponectin levels via thiol-mediated protein quality control in the secretory pathway. IP6K1 bound to adiponectin and DsbA-L and generated 5-InsP7 to stabilize adiponectin/ERp44 and DsbA-L/Ero1-Lα interactions, driving adiponectin intracellular degradation. Depleting 5-InsP7 by either IP6K1 deletion or pharmacological inhibition blocked intracellular adiponectin degradation. Whole-body and adipocyte-specific deletion of IP6K1 boosted plasma adiponectin levels, especially its high molecular weight forms, and activated AMPK-mediated protection against myocardial ischemia-reperfusion injury. Pharmacological inhibition of 5-InsP7 biosynthesis in WT but not adiponectin knockout mice attenuated myocardial ischemia-reperfusion injury. CONCLUSIONS: Our findings revealed that 5-InsP7 is a physiological regulator of adiponectin biosynthesis that is amenable to pharmacological intervention for cardioprotection.

Crystal Structure and Enzymology of Solanum tuberosum Inositol Tris/Tetrakisphosphate Kinase 1 (StITPK1).

Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates.

Promising drug candidates for the treatment of polycystic ovary syndrome (PCOS) as alternatives to the classical medication metformin.

Polycystic ovary syndrome (PCOS) is a prevalent hormonal disorder that affects women of reproductive age. It is characterized by abnormal production of androgens, typically present in small quantities in females. This study aimed to investigate the therapeutic potential of Irosustat (STX64), STX140, and compound 1G as new drug candidates for the treatment of letrozole-induced PCOS in female Wistar rats. 36 rats were divided into six groups of equal size. PCOS was induced in all groups, except the normal control group, by administering letrozole orally (1 mg/kg/day for 35 days). The onset of abnormal estrous cycle was confirmed by examining daily vaginal smears under a microscope. Subsequently, each rat group was assigned to a different treatment regimen, including one control group, one letrozole group, one metformin group (500 mg/kg/day) as a reference drug, and the other groups received a different drug candidate orally for 30 days. After treatment, blood collection was performed for biochemical measurements and determination of oxidative stress markers. The rats were dissected to separate ovaries and uterus for morphological, histological, and western blotting studies. Treatment with the drug candidates improved the ovaries and uterus weight measurements compared to the untreated PCOS group. The three tested drug candidates demonstrated promising improvements in lipid profile, blood glucose level, testosterone, progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol levels. In addition, western blotting confirmed their promising effects on Akt, mTOR, and AMPK-α pathways. This study led to the discovery of three promising drug candidates for the management of PCOS as alternatives to metformin.

Fluorination Influences the Bioisostery of Myo-Inositol Pyrophosphate Analogs.

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.

2-Methoxyestradiol-3,17-O,O-bis-sulfamate (STX140) Inhibits Proliferation and Invasion via Senescence Pathway Induction in Human BRAFi-Resistant Melanoma Cells.

The endogenous estradiol derivative 2-Methoxyestradiol (2-ME) has shown good and wide anticancer activity but suffers from poor oral bioavailability and extensive metabolic conjugation. However, its sulfamoylated derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (STX140), has superior potential as a therapeutic agent, acts by disrupting microtubule polymerization, leading to cell cycle arrest and apoptosis in cancer cells and possesses much better pharmaceutical properties. This study investigated the antiproliferative and anti-invasive activities of STX140 in both SKMEL-28 naïve melanoma (SKMEL28-P) cells and resistant melanoma cells (SKMEL-28R). STX140 inhibited cell proliferation in the nanomolar range while having a less pronounced effect on human melanocytes. Additionally, STX140 induced cell cycle arrest in the G2/M phase and sub-G1, reduced migration, and clonogenic potential in monolayer models, and inhibited invasion in a 3D human skin model with melanoma cells. Furthermore, STX140 induced senescence features in melanoma and activated the senescence machinery by upregulating the expression of senescence genes and proteins related to senescence signaling. These findings suggest that STX140 may hold potential as a therapeutic agent for melanoma treatment.

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

2-Methoxyestradiol-3,17-O,O-bis-sulfamate inhibits store-operated Ca2+ entry in T lymphocytes and prevents experimental autoimmune encephalomyelitis.

Ca2+ signaling is one of the essential signaling systems for T lymphocyte activation, the latter being an essential step in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Store-operated Ca2+ entry (SOCE) ensures long lasting Ca2+ signaling and is of utmost importance for major downstream T lymphocyte activation steps, e.g. nuclear localization of the transcription factor 'nuclear factor of activated T cells' (NFAT). 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol (E2), blocks nuclear translocation of NFAT. The likely underlying mechanism is inhibition of SOCE, as shown for its synthetic sulfamate ester analogue 2-ethyl-3-sulfamoyloxy-17ß-cyanomethylestra-1,3,5(10)-triene (STX564). Here, we demonstrate that another synthetic bis-sulfamoylated 2ME2 derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE, STX140), an orally bioavailable, multi-targeting anticancer agent and potent steroid sulfatase (STS) inhibitor, antagonized SOCE in T lymphocytes. Downstream events, e.g. secretion of the pro-inflammatory cytokines interferon-γ and interleukin-17, were decreased by STX140 in in vitro experiments. Remarkably, STX140 dosed in vivo completely blocked the clinical disease in both active and transfer experimental autoimmune encephalomyelitis (EAE) in Lewis rats, a T cell-mediated animal model for MS, at a dose of 10 mg/kg/day i.p., whereas neither 2ME2 nor Irosustat, a pure STS inhibitor, showed any effect. The STS inhibitory activity of STX140 is therefore not responsible for its activity in this model. Taken together, inhibition of SOCE by STX140 resulting in full antagonism of clinical symptoms in EAE in the Lewis rat, paired with the known excellent bioavailability and pharmaceutical profile of this drug, open potentially new therapeutic avenues for the treatment of MS.

Expedient synthesis and luminescence sensing of the inositol pyrophosphate cellular messenger 5-PP-InsP5.

Inositol pyrophosphates are important biomolecules associated with apoptosis, cell growth and kinase regulation, yet their exact biological roles are still emerging and probes do not exist for their selective detection. We report the first molecular probe for the selective and sensitive detection of the most abundant cellular inositol pyrophosphate 5-PP-InsP5, as well as an efficient new synthesis. The probe is based on a macrocyclic Eu(iii) complex bearing two quinoline arms providing a free coordination site at the Eu(iii) metal centre. Bidentate binding of the pyrophosphate group of 5-PP-InsP5 to the Eu(iii) ion is proposed, supported by DFT calculations, giving rise to a selective enhancement in Eu(iii) emission intensity and lifetime. We demonstrate the use of time-resolved luminescence as a bioassay tool for monitoring enzymatic processes in which 5-PP-InsP5 is consumed. Our probe offers a potential screening methodology to identify drug-like compounds that modulate the activity of enzymes of inositol pyrophosphate metabolism.

Itraconazole inhibits endothelial cell migration by disrupting inositol pyrophosphate-dependent focal adhesion dynamics and cytoskeletal remodeling.

The antifungal drug itraconazole has been repurposed to anti-angiogenic agent, but the mechanisms of action have been elusive. Here we report that itraconazole disrupts focal adhesion dynamics and cytoskeletal remodeling, which requires 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP7, which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP7, thus augments 5-InsP7-mediated inhibition of Arp2/3 complex and reduces 5-InsP7-mediated FAK dimerization. Itraconazole-treated cells display reduced focal adhesion dynamics and actin cytoskeleton remodeling. Accordingly, itraconazole severely disrupts cell motility, an essential component of angiogenesis. These results demonstrate critical roles of IP6K1-generated 5-InsP7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility.

Novel Anti-Acanthamoebic Activities of Irosustat and STX140 and Their Nanoformulations.

Pathogenic Acanthamoeba produce keratitis and fatal granulomatous amoebic encephalitis. Treatment remains problematic and often ineffective, suggesting the need for the discovery of novel compounds. For the first time, here we evaluated the effects of the anticancer drugs Irosustat and STX140 alone, as well as their nanoformulations, against A. castellanii via amoebicidal, excystment, cytopathogenicity, and cytotoxicity assays. Nanoformulations of the compounds were successfully synthesized with high encapsulation efficiency of 94% and 82% for Irosustat and STX140, respectively. Nanoparticles formed were spherical in shape and had a unimodal narrow particle size distribution, mean of 145 and 244 nm with a polydispersity index of 0.3, and surface charge of -14 and -15 mV, respectively. Irosustat and STX140 exhibited a biphasic release profile with almost 100% drug released after 48 h. Notably, Irosustat significantly inhibited A. castellanii viability and amoebae-mediated cytopathogenicity and inhibited the phenotypic transformation of amoebae cysts into the trophozoite form, however their nanoformulations depicted limited effects against amoebae but exhibited minimal cytotoxicity when tested against human cells using lactate dehydrogenase release assays. Accordingly, both compounds have potential for further studies, with the hope of discovering novel anti-Acanthamoeba compounds, and potentially developing targeted therapy against infections of the central nervous system.

Diversification in the inositol tris/tetrakisphosphate kinase (ITPK) family: crystal structure and enzymology of the outlier AtITPK4.

Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Šresolution that, along with a description of the enantiospecificity of the enzyme, affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a KM for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologues in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.

2-Difluoromethoxy-Substituted Estratriene Sulfamates: Synthesis, Antiproliferative SAR, Antitubulin Activity, and Steroid Sulfatase Inhibition.

2-Difluoromethoxyestratriene derivatives were designed to improve potency and in vivo stability of the drug candidate 2-methoxyestradiol (2ME2). Compound evaluation in vitro against the proliferation of MCF-7 and MDA MB-231 breast cancer cells, as inhibitors of tubulin polymerisation and also steroid sulfatase (STS) both in cell lysates and in whole cells, showed promising activities. In antiproliferative assays 2-difluoromethoxyestradiol was less potent than 2ME2, but its sulfamates were often more potent than their corresponding non-fluorinated analogues. The fluorinated bis-sulfamate is a promising antiproliferative agent in MCF-7 cells (GI50 0.28 µM) vs the known 2-methoxyestradiol-3,17-O,O-bissulfamate (STX140, GI50 0.52 µM), confirming the utility of our approach. Compounds were also evaluated in the NCI 60-cell line panel and the fluorinated bis-sulfamate derivative displayed very good overall activities with a sub-micromolar average GI50 . It was a very potent STS inhibitor in whole JEG-3 cells (IC50 3.7 nM) similar to STX140 (4.2 nM) and additionally interferes with tubulin assembly in vitro and colchicine binding to tubulin. An X-ray study of 2-difluoromethoxy-3-benzyloxyestra-1,3,5(10)-trien-17-one examined conformational aspects of the fluorinated substituent. The known related derivative 2-difluoromethyl-3-sulfamoyloxyestrone was evaluated for STS inhibition in whole JEG-3 cells and showed an excellent IC50 of 55 pM.

Cell Fate following Irradiation of MDA-MB-231 and MCF-7 Breast Cancer Cells Pre-Exposed to the Tetrahydroisoquinoline Sulfamate Microtubule Disruptor STX3451.

A tetrahydroisoquinoline (THIQ) core is able to mimic the A and B rings of 2-methoxyestradiol (2ME2), an endogenous estrogen metabolite that demonstrates promising anticancer properties primarily by disrupting microtubule dynamic instability parameters, but has very poor pharmaceutical properties that can be improved by sulfamoylation. The non-steroidal THIQ-based microtubule disruptor 2-(3-bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline (STX3451), with enhanced pharmacokinetic and pharmacodynamic profiles, was explored for the first time in radiation biology. We investigated whether 24 h pre-treatment with STX3451 could pre-sensitize MCF-7 and MDA-MB-231 breast cancer cells to radiation. This regimen showed a clear increase in cytotoxicity compared to the individual modalities, results that were contiguous in spectrophotometric analysis, flow cytometric quantification of apoptosis induction, clonogenic studies and microscopy techniques. Drug pre-treatment increased radiation-induced DNA damage, with statistically more double-strand (ds) DNA breaks demonstrated. The latter could be due to the induction of a radiation-sensitive metaphase block or the increased levels of reactive oxygen species, both evident after compound exposure. STX3451 pre-exposure may also delay DNA repair mechanisms, as the DNA damage response element ataxia telangiectasia mutated (ATM) was depressed. These in vitro findings may translate into in vivo models, with the ultimate aim of reducing both radiation and drug doses for maximal clinical effect with minimal adverse effects.

A structural exposé of noncanonical molecular reactivity within the protein tyrosine phosphatase WPD loop.

Structural snapshots of protein/ligand complexes are a prerequisite for gaining atomic level insight into enzymatic reaction mechanisms. An important group of enzymes has been deprived of this analytical privilege: members of the protein tyrosine phosphatase (PTP) superfamily with catalytic WPD-loops lacking the indispensable general-acid/base within a tryptophan-proline-aspartate/glutamate context. Here, we provide the ligand/enzyme crystal complexes for one such PTP outlier: Arabidopsis thaliana Plant and Fungi Atypical Dual Specificity Phosphatase 1 (AtPFA-DSP1), herein unveiled as a regioselective and efficient phosphatase towards inositol pyrophosphate (PP-InsP) signaling molecules. Although the WPD loop is missing its canonical tripeptide motif, this structural element contributes to catalysis by assisting PP-InsP delivery into the catalytic pocket, for a choreographed exchange with phosphate reaction product. Subsequently, an intramolecular proton donation by PP-InsP substrate is posited to substitute functionally for the absent aspartate/glutamate general-acid. Overall, we expand mechanistic insight into adaptability of the conserved PTP structural elements.

Synthesis and In Vitro Antimicrobial SAR of Benzyl and Phenyl Guanidine and Aminoguanidine Hydrazone Derivatives.

A series of benzyl, phenyl guanidine, and aminoguandine hydrazone derivatives was designed and in vitro antibacterial activities against two different bacterial strains (Staphylococcus aureus and Escherichia coli) were determined. Several compounds showed potent inhibitory activity against the bacterial strains evaluated, with minimal inhibitory concentration (MIC) values in the low µg/mL range. Of all guanidine derivatives, 3-[2-chloro-3-(trifluoromethyl)]-benzyloxy derivative 9m showed the best potency with MICs of 0.5 µg/mL (S. aureus) and 1 µg/mL (E. coli), respectively. Several aminoguanidine hydrazone derivatives also showed good overall activity. Compounds 10a, 10j, and 10r-s displayed MICs of 4 µg/mL against both S. aureus and E. coli. In the aminoguanidine hydrazone series, 3-(4-trifluoromethyl)-benzyloxy derivative 10d showed the best potency against S. aureus (MIC 1 µg/mL) but was far less active against E. coli (MIC 16 µg/mL). Compound 9m and the para-substituted derivative 9v also showed promising results against two strains of methicillin-resistant Staphylococcus aureus (MRSA). These results provide new and potent structural leads for further antibiotic optimisation strategies.

Small Molecule CD38 Inhibitors: Synthesis of 8-Amino-N1-inosine 5'-monophosphate, Analogues and Early Structure-Activity Relationship.

Although a monoclonal antibody targeting the multifunctional ectoenzyme CD38 is an FDA-approved drug, few small molecule inhibitors exist for this enzyme that catalyzes inter alia the formation and metabolism of the N1-ribosylated, Ca2+-mobilizing, second messenger cyclic adenosine 5'-diphosphoribose (cADPR). N1-Inosine 5'-monophosphate (N1-IMP) is a fragment directly related to cADPR. 8-Substituted-N1-IMP derivatives, prepared by degradation of cyclic parent compounds, inhibit CD38-mediated cADPR hydrolysis more efficiently than related cyclic analogues, making them attractive for inhibitor development. We report a total synthesis of the N1-IMP scaffold from adenine and a small initial compound series that facilitated early delineation of structure-activity parameters, with analogues evaluated for inhibition of CD38-mediated hydrolysis of cADPR. The 5'-phosphate group proved essential for useful activity, but substitution of this group by a sulfonamide bioisostere was not fruitful. 8-NH2-N1-IMP is the most potent inhibitor (IC50 = 7.6 µM) and importantly HPLC studies showed this ligand to be cleaved at high CD38 concentrations, confirming its access to the CD38 catalytic machinery and demonstrating the potential of our fragment approach.

Substituted Aryl Benzylamines as Potent and Selective Inhibitors of 17ß-Hydroxysteroid Dehydrogenase Type 3.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is expressed at high levels in testes and seminal vesicles; it is also present in prostate tissue and involved in gonadal and non-gonadal testosterone biosynthesis. The enzyme is membrane-bound, and a crystal structure is not yet available. Selective aryl benzylamine-based inhibitors were designed and synthesised as potential agents for prostate cancer therapeutics through structure-based design, using a previously built homology model with docking studies. Potent, selective, low nanomolar IC50 17ß-HSD3 inhibitors were discovered using N-(2-([2-(4-chlorophenoxy)phenylamino]methyl)phenyl)acetamide (1). The most potent compounds have IC50 values of approximately 75 nM. Compound 29, N-[2-(1-Acetylpiperidin-4-ylamino)benzyl]-N-[2-(4-chlorophenoxy)phenyl]acetamide, has an IC50 of 76 nM, while compound 30, N-(2-(1-[2-(4-chlorophenoxy)-phenylamino]ethyl)phenyl)acetamide, has an IC50 of 74 nM. Racemic C-allyl derivative 26 (IC50 of 520 nM) was easily formed from 1 in good yield and, to determine binding directionality, its enantiomers were separated by chiral chromatography. Absolute configuration was determined using single crystal X-ray crystallography. Only the S-(+)-enantiomer (32) was active with an IC50 of 370 nM. Binding directionality was predictable through our in silico docking studies, giving confidence to our model. Importantly, all novel inhibitors are selective over the type 2 isozyme of 17ß-HSD2 and show <20% inhibition when tested at 10 µM. Lead compounds from this series are worthy of further optimisation and development as inhibitors of testosterone production by 17ß-HSD3 and as inhibitors of prostate cancer cell growth.

Quantal Ca2+ release mediated by very few IP3 receptors that rapidly inactivate allows graded responses to IP3.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that link extracellular stimuli to Ca2+ signals. Ca2+ release from intracellular stores is "quantal": low IP3 concentrations rapidly release a fraction of the stores. Ca2+ release then slows or terminates without compromising responses to further IP3 additions. The mechanisms are unresolved. Here, we synthesize a high-affinity partial agonist of IP3Rs and use it to demonstrate that quantal responses do not require heterogenous Ca2+ stores. IP3Rs respond incrementally to IP3 and close after the initial response to low IP3 concentrations. Comparing functional responses with IP3 binding shows that only a tiny fraction of a cell's IP3Rs mediate incremental Ca2+ release; inactivation does not therefore affect most IP3Rs. We conclude, and test by simulations, that Ca2+ signals evoked by IP3 pulses arise from rapid activation and then inactivation of very few IP3Rs. This allows IP3Rs to behave as increment detectors mediating graded Ca2+ release.