Preliminary evaluation of the cytotoxicity of a series of tris-2-aminoethylamine (Tren) based hexadentate heterocyclic donor agents

Tachpyridine is a cytotoxic metal chelator with potential anti-tumor activity. The synthesis and evaluation of a set of derivatives of the related hexadentate heterocyclic donor agents tris-2-aminoethylamine (tren) and tris[N-(2-pyridylmethylene)-2-aminoethyl]amine (trenpyr) was performed to compare their cytotoxic activity to tachpyridine in HeLa tumor cells. Methyl groups were added to the pyridyl ring of trenpyr, and the effects of alkyl group substitution on cell survival were assessed. Profound cytotoxicity was observed and IC50 data were obtained in ascending order from those compounds substituted with a methyl group at the 3-, 4-, or 5-position and lastly by the 6-methyl derivative. These results suggest that analogous derivatives with substitution at the 3-position of the pyridyl ring deserve further exploration.     

Oxathiolene oxide synthesis via chelation-controlled addition of organometallic reagents to alkynols followed by addition of sulfur electrophiles and evaluation of oxathiolene oxides as anticarcinogenic enzyme inducers

A number of alkynols have been prepared by Sonogashira coupling of propargyl alcohol to aromatic halides. Chelation-controlled addition of organometallic nucleophiles to these alkynols was then effected followed by the addition of the sulfur electrophiles, sulfur dioxide or thionyl chloride. This methodology was used to prepare a number of oxathiolene oxides, which have been screened as NQO1 (quinone oxidoreductase) inducers.     

A selective role for phosphatidylinositol 3,4,5-trisphosphate in the Gi-dependent activation of platelet Rap1B

The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G(i)-dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fcgamma receptor IIA (FcgammaRIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G(i)-dependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of FcgammaRIIA, by stimulation of the P2Y(12) receptor for ADP, or by epinephrine. However, in platelets from PI3Kgamma-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3Kgamma did not prevent the ability of epinephrine to potentiate platelet aggregation through a G(i)-dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), but not PtdIns(3,4)P(2), although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P(3) was able to relieve the inhibitory effect of apyrase on FcgammaRIIA-mediated platelet aggregation. We conclude that stimulation of a G(i)-dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P(3), but not PtdIns(3,4)P(2), and that this process may contribute to potentiation of platelet aggregation.     

Poly(ADP-ribose) degradation by post-nuclear extracts from human cells

The nuclear metabolism of poly(ADP-ribose) is mainly regulated by poly(ADP-ribose) polymerase-1 (PARP-1) and by poly(ADP-ribose) glycohydrolase (PARG). A PARP-like enzyme, V-PARP, and a PARG isoform are present in the extra-nuclear compartment of mammalian cells, even if poly(ADP-ribose) has never been detected therein. In this work, we demonstrate the ability of post-nuclear extracts from HeLa and HL60 cells to degrade synthetic 32P-polymers of ADP-ribose to ADP-ribose and, further, to AMP. This reaction implies the combined action of PARG and of an ADP-ribose-degrading activity, possibly corresponding to a phosphodiesterase and/or to an ADP-ribose pyrophosphatase. The inhibition of PARG or ADP-ribose-degrading enzymes allowed the demonstration that in vitro synthesized 32P-poly(ADP-ribose) is first digested to ADP-ribose monomers by a typical PARG reaction, and that ADP-ribose is further rapidly converted into AMP by an Mg(2+)-dependent activity. Collectively, our results demonstrate the ability of the human cell post-nuclear fraction to convert synthetic poly(ADP-ribose) into utilizable AMP units by the concerted action of PARG and ADP-ribose-degrading activities.     

Active dendrites,potassium channels and synaptic plasticity

The dendrites of CA1 pyramidal neurons in the hippocampus express numerous types of voltage-gated ion channel, but the distributions or densities of many of these channels are very non-uniform. Sodium channels in the dendrites are responsible for action potential (AP) propagation from the axon into the dendrites (back-propagation); calcium channels are responsible for local changes in dendritic calcium concentrations following back-propagating APs and synaptic potentials; and potassium channels help regulate overall dendritic excitability. Several lines of evidence are presented here to suggest that back-propagating APs, when coincident with excitatory synaptic input, can lead to the induction of either long-term depression (LTD) or long-term potentiation (LTP). The induction of LTD or LTP is correlated with the magnitude of the rise in intracellular calcium. When brief bursts of synaptic potentials are paired with postsynaptic APs in a theta-burst pairing paradigm, the induction of LTP is dependent on the invasion of the AP into the dendritic tree. The amplitude of the AP in the dendrites is dependent, in part, on the activity of a transient, A-type potassium channel that is expressed at high density in the dendrites and correlates with the induction of the LTP. Furthermore, during the expression phase of the LTP, there are local changes in dendritic excitability that may result from modulation of the functioning of this transient potassium channel. The results support the view that the active properties of dendrites play important roles in synaptic integration and synaptic plasticity of these neurons.     

Hydroxynaphthaldehyde phosphate derivatives as potent covalent Schiff base inhibitors of fructose-1,6-bisphosphate aldolase

Interactions of phosphate derivatives of 2,6-dihydroxynaphthalene (NA-P(2)) and 1,6-dihydroxy-2-naphthaldehyde (HNA-P, phosphate at position 6) with fructose-1,6-bisphosphate aldolase from rabbit muscle were analyzed by enzyme kinetics, difference spectroscopy, site-directed mutagenesis, mass spectrometry, and molecular dynamics. Enzyme activity was competitively inhibited by NA-P(2), whereas HNA-P exhibited slow-binding inhibition with an overall inhibition constant of approximately 24 nM. HNA-P inactivation was very slowly reversed with t(1/2) approximately 10 days. Mass spectrometry and spectrophotometric absorption indicated that HNA-P inactivation occurs by Schiff base formation. Rates of enzyme inactivation and Schiff base formation by HNA-P were identical and corresponded to approximately 4 HNA-P molecules bound par aldolase tetramer at maximal inhibition. Site-directed mutagenesis of conserved active site lysine residues 107, 146, and 229 and Asp-33 indicated that Schiff base formation by HNA-P involved Lys-107 and was promoted by Lys-146. Titration of Lys-107 by pyridoxal 5-phosphate yielded a microscopic pK(a) approximately 8 for Lys-107, corroborating a role as nucleophile at pH 7.6. Site-directed mutagenesis of Ser-271, an active site residue that binds the C(1)-phosphate of dihydroxyacetone phosphate, diminished HNA-P binding and enabled modeling of HNA-P in the active site. Molecular dynamics showed persistent HNA-P phosphate interactions with the C(1)-phosphate binding site in the noncovalent adduct. The naphthaldehyde hydroxyl, ortho to the HNA-P aldehyde, was essential for promoting carbinolamine precursor formation by intramolecular catalysis. The simulations indicate a slow rate of enzyme inactivation due to competitive inhibition by the phenate form of HNA-P, infrequent nucleophilic attack in the phenol form, and significant conformational barrier to bond formation as well as electrostatic destabilization of protonated ketimine intermediates. Solvent accessibility by Lys-107 Nz was reduced in the covalent Schiff base complex, and in those instances where water molecules interacted with Lys-107 in the simulations, Schiff base hydrolysis was not mechanistically favorable. The findings at the molecular level corroborate the observed mechanism of slow-binding tight inhibition by HNA-P of muscle aldolase and should serve as a blueprint for future aldolase inhibitor design.     

Thermodynamic studies of the mechanism of metal binding to the Escherichia coli zinc transporter YiiP

Sequence homology of the Escherichia coli YiiP places it within the family of cation diffusion facilitators, a family of membrane transporters that play a central role in regulating cellular zinc homeostasis. Here we describe the first thermodynamic and mechanistic studies of metal binding to a cation diffusion facilitator. Isothermal titration calorimetric analyses of the purified YiiP and binding competitions among Zn(2+), Cd(2+), and Hg(2+) revealed a mutually competitive binding site common to three metal ions and a set of noncompetitive binding sites, including one Cd(2+) site, one Hg(2+) site, and at least one Zn(2+) site, to which the binding of Zn(2+) exhibited partial inhibitions of both Cd(2+) and Hg(2+) bindings. Lowering the pH from 7.0 to 5.5 inhibited binding of Zn(2+) and Cd(2+) to the common site. Further, the enthalpy change of the Cd(2+) binding to the common site was found to be related linearly to the ionization enthalpy of the pH buffer with a slope corresponding to the release of 1.23 H(+) for each Cd(2+) binding. These H(+) effects are consistent with a coupled deprotonation process upon binding of Zn(2+) and Cd(2+). Modification of histidine residues by diethyl pyrocarbonate specifically inhibited Zn(2+) binding to the common binding site, indicating that the mechanism of binding-deprotonation coupling involves a histidine residue(s).     

N,N-dialkyl-4-[(8-azabicyclo[3.2.1]-oct-3-ylidene)phenylmethyl]benzamides, potent, selective delta opioid agonists

A series of N,N-dialkyl-4-(9-aryltropanylidenemethyl)benzamides was prepared. The lead compounds, 15a and 15c, exhibited extremely high affinity for the delta opioid receptor with excellent selectivity versus the micro opioid receptor. They were full agonists at the delta opioid receptor, as assessed by stimulation of GTPgammaS binding, and displayed antinociceptive activity.