Bernard Spiess

Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug–RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (Kd ∼ 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (Kd ∼ 1.6 µM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop–loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

Ionization state of myo-inositol 1,4,5-trisphosphate: correlation with binding properties

The comparison between the ionization state and the binding properties on brain membrane receptors of the synthetized myo-inositol 1,4,5-trisphosphate lead to the conclusion that the biological active species may be either the monoprotonated or the fully deprotonated trisphosphate.

scyllo-Inositol Pentakisphosphate as an Analogue of myo-Inositol 1,3,4,5,6-Pentakisphosphate: Chemical Synthesis, Physicochemistry and Biological Applications

myo‐Inositol 1,3,4,5,6‐pentakisphosphate (Ins(1,3,4,5,6)P5), an inositol polyphosphate of emerging significance in cellular signalling, and its C‐2 epimer scyllo‐inositol pentakisphosphate (scyllo‐InsP5) were synthesised from the same myo‐inositol‐based precursor. Potentiometric and NMR titrations show that both pentakisphosphates undergo a conformational ring‐flip at higher pH, beginning at pH 8 for scyllo‐InsP5 and pH 9 for Ins(1,3,4,5,6)P5. Over the physiological pH range, however, the conformation of the inositol rings and the microprotonation patterns of the phosphate groups in Ins(1,3,4,5,6)P5 and scyllo‐InsP5 are similar. Thus, scyllo‐InsP5 should be a useful tool for identifying biologically relevant actions of Ins(1,3,4,5,6)P5, mediated by specific binding sites, and distinguishing them from nonspecific electrostatic effects. We also demonstrate that, although scyllo‐InsP5 and Ins(1,3,4,5,6)P5 are both hydrolysed by multiple inositol polyphosphate phosphatase (MINPP), scyllo‐InsP5 is not dephosphorylated by PTEN or phosphorylated by Ins(1,3,4,5,6)P5 2‐kinases. This finding both reinforces the value of scyllo‐InsP5 as a biological control and shows that the axial 2‐OH group of Ins(1,3,4,5,6)P5 plays a part in substrate recognition by PTEN and the Ins(1,3,4,5,6)P5 2‐kinases.

Complexation studies on inositol-phosphates. V. Cu2+, Zn2+, Fe2+ and Fe3+ complexes of some myo-inositol triphosphates

Potentiometry and 31P NMR spectroscopy have been used to study the protonation and complexation properties of some myo-inositol triphosphates (Ins(1,2,6)P3, Ins(1,3,5)P3, and Ins(2,4,6)P3) with Cu2+, Zn2+, Fe2+, and Fe3+. The study was performed for all the ligands at 25°C in a 0.1 M tetrabutylammonium bromide solution (medium 1) and in addition, for Ins(1,2,6)P3, at 37°C in a 0.2 M KCl medium (medium 2). The position of the phosphate groups around the inositol ring largely influences the basicity of the ligand. Log β011 has the highest value for Ins(1,2,6)P3 which displays three vicinal phosphates. When no interfering cations such as K+ are present, M2HL, M2L, MHL, ML, and MOHL complexes were found for most of the systems. Although the stability of the complexes follows the Irving-Williams rule, i.e., Cu(II) > Zn(II) > Fe(II), no real selectivity of complexation is observed. A 31P NMR titration was performed for the Zn2+-H+-Ins(1,2,6)P3 system. The resulting titration curves, compared with those obtained in the absence of metal showed two opposite effects: i) a downfield shift due to the competition of the metal with the proton, ii) an upfield shift corresponding to the binding of Zn2+. From these curves it can, in addition, be concluded that the cation in the mononuclear species is mainly coordinated to P1 and P2 whereas the second cation of the homodinuclear complex is stabilized by P6.

Total syntheses of chiral sn-myo-inositol-1,4,5-trisphosphate1 and its enantiomer

Abstract sn-myo-Inositol-1,4,5-trisphophate (Ins(1,4,5)P3) and its enantiomers are prepared by synthesis of suitably protected myo-inositols, separation of enantiomers via the formation of D-mannose diastereomeric derivatives and selective phosphorylations.

Complexation studies on inositol-phosphates. II. Alkali-metal complexes of D-myo-inositol 1,2,6 trisphosphate

The complexation properties of the D-myo-inositol 1,2,6 trisphosphate (Ins(1,2,6)P3) towards Li+, Na+, K+, Rb+, and Cs+ cations were studied at 25 degrees C in a 0.1 M tetra-n-butylammonium bromide medium. For all cations, mononuclear and protonated species were found. For smaller cations (Li+, Na+, and K+) a dinuclear complex was also put into evidence. The main characteristic of the complexes is its high stability; and of the ligand, its nonselectivity. The Ins(1,2,6)P3-K system was ascertained using Sammartanos method which additionally enabled the influence of various K+ concentrations on the protonations constants to be considered.

Complexation studies on inositol-phosphates, VI. Al3+ complexes of DL-myo-inositol 1,4,5-triphosphate and D-myo-inositol 1,2,6-triphosphate

Abstract Aluminum complexes of D- myo -inositol 1,2,6-triphosphate (Ins(1,2,6)P 3 ) and DL- myo -inositol 1,4,5-triphosphate (Ins(1,4,5)P 3 ) have been studied by potentiometry at 25°C in a 0.1 M tetrabutylammonium bromide medium. Both ligands form AlHL, AlL, and AlOHL species whereas Ins(1,2,6)P 3 forms, in addition, an Al 2 L complex and Ins(1,4,5)P 3 an Al(OH) 2 L species. In an attempt to assess the biological significance of the Al 3+ binding to Ins(1,4,5)P 3 , the results were compared to the Al 3+ -ATP complexes that have been found in similar medium conditions. Taking into account the relative stability of the complexes of both systems, it appears likely that the intracellular second messenger system involving Ins(1,4,5)P 3 may be disturbed by the presence of the aluminum cation.

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 31P 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 °C, and 1H 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.

COMPLEXATION OF SPERMINE AND SPERMIDINE BY MYO-INOSITOL 1,4,5-TRIS(PHOSPHATE) AND RELATED COMPOUNDS : BIOLOGICAL SIGNIFICANCE

D myo-inositol 1,4,5-tris(phosphate) (Ins(1,4,5)P3) displays a multicoordination site arrangement that allows strong interactions with polycationic species such as the naturally occurring polyamines spermine and spermidine. In the present work, the complexation of these polyamines by Ins(1,4,5)P3 and related compounds was quantitatively investigated. The study was performed in a 0.1 M tetramethylammonium p-toluenesulfonate (Me4NOTs) solution at 25 degrees C. For purpose of comparison, the complexation of the polyamine-ATP systems were also considered in the same experimental conditions. 31P-NMR experiments showed for Ins(1,4,5)P3 and its analogues, the formation of complexes of a 1:1 stoichiometry. As expected, the most stable complexes are formed between the most charged partners. In addition, the basicity of the phosphate groups seems to govern the stability of the complexes. If both ATP and Ins(1,4,5)P3 are present at the same concentration, the latter interacts preferably with the polyamines. Ins(1,4,5)P3-spermine complex formation provides a possible simple explanation for the inhibition by spermine of Ins(1,4,5)P3-induced Ca2+ release. Spermine will undoubtedly compete with metallic cations such as Ca2+ in the intracellular medium and consequently, may play a regulatory role in the signal transduction mediated by Ins(1,4,5)P3.

Complexation studies on inositol-phosphates: IV. Ca(II) complexes of myo-inositol 1,4,5-trisphosphate

The stability constants of the complexes formed between Ca2+ and the myo-inositol 1,4,5-triphosphate (Ins(1,4,5)P3) were determined by potentiometric titration in two different media and temperature conditions (medium 1: I = 0.1 M But4NBr, 25 degrees C; medium 2: I = 0.2 M KCl, 37 degrees C). Mainly because of the presence of potassium the results obtained in these media show large differences in both the nature and the stability of the complexes. In medium 1, MH2L and M2L species are formed along with the ML and MHL species which also exist in medium 2. In addition, the stability of the latter species decreases by more than one log unit in going from medium 1 to medium 2. In an attempt to assess the biological significance of the metal binding to Ins(1,4,5)P3, the results were compared to the Ca2+-ATP complexes that form in the same media conditions. Taking into account the relative stability of the complexes of both systems, it is likely that the action or metabolism of Ins(1,4,5)P3 may be influenced by coordination of either alkali or alkali-earth cations.