A Fluorescent Probe Identifies Active Site Ligands of Inositol Pentakisphosphate 2-Kinase

Inositol pentakisphosphate 2-kinase catalyzes the phosphorylation of the axial 2-OH of myo-inositol 1,3,4,5,6-pentakisphosphate for de novo synthesis of myo-inositol hexakisphosphate. Disruption of inositol pentakisphosphate 2-kinase profoundly influences cellular processes; from nuclear mRNA export and phosphate homeostasis in yeast and plants, to establishment of left-right asymmetry in zebra fish. We elaborate an active site fluorescent probe that allows high throughput screening of Arabidopsis inositol pentakisphosphate 2-kinase. We show that the probe has a binding constant comparable to the Km values of inositol phosphate substrates of this enzyme, and can be used to prospect for novel substrates and inhibitors of inositol phosphate kinases. We identify several micromolar Ki inhibitors and validate this approach by solving the crystal structure of protein in complex with purpurogallin. We additionally solve structures of protein in complexes with epimeric higher inositol phosphates. This probe may find utility in characterization of a wide family of inositol phosphate kinases

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

17β-Hydroxysteroid dehydrogenase type 3 (17β-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 17β-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 17β-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 17β-HSD3 and as inhibitors of prostate cancer cell growth

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

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 synthesise 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.Fondation Philippe Wiener-Maurice Anspach Royal Society Fellowship Queens’ College, Cambridge Belgian FRS-FNR

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.</p