Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca²⁺-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.     

Novel boronated chlorin e6-based photosensitizers: Synthesis,binding to albumin and antitumour efficacy

Chlorins, a class of plant porphyrins, are perspective as photosensitizing agents due to light absorption in the long wavelength spectral region and deeper photodamage of tissues. Aiming at optimization of antitumour properties of chlorins, we synthesized a series of boronated derivatives of chlorin e₆ and their complexes containing Zn(II), Pd(II) or Sn(IV). The compounds were synthesized by alkylation of amino or hydroxy derivatives of chlorin e₆ with 1-trifluoromethanesulfonylmethyl-o-carborane. Chlorin e₆ 13(1)-N-{2-[N-(o-carboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester (compound 5) formed complexes with serum albumin, a major porphyrin carrier. The binding constant of these complexes was ～4 times bigger than the respective value for the complexes of albumin with boron-free aminochlorin e₆. Compound 5 potently sensitized rat fibroblasts to illumination with monochromatic red light: >98% of cells were necrotic by 24 h post-illumination with 1 μM of 5. This compound demonstrated high efficacy in photodynamic therapy of rat M-1 sarcoma. After PDT with 25 mg/kg of 5 the residual tumours were significantly smaller than in animals subjected to PDT with equal concentration of boron-free aminochlorin e₆. No signs of general toxicity were detectable after PDT with 5. Thus, boronation can enhance the potency of chlorins in PDT, in particular, due to an increased binding to albumin. Our data expand the therapeutic applicability of boronated chlorins beyond boron neutron capture therapy; these agents emerge as dual efficacy photoradiosensitizers.     

The Effect of Gold Nanoparticle Surface Modification with Polyethylene Glycol on the Absorbed Dose Distribution upon Irradiation with 137Cs and 60Co Photons

Biophysics - Abstract—Modification of the surface of gold nanoparticles with polyethylene glycol (PEG) is widely used to investigate radiosensitization in vivo. This modification may lead to...     

Synthesis and cytotoxicity of oligomycin A derivatives modified in the side chain

A novel way of chemical modification of the macrolide antibiotic oligomycin A (1) at the side chain was developed. Mesylation of 1 with methane sulfonyl chloride in the presence of 4-dimethylaminopyridine produced 33-O-mesyl oligomycin in 56% yield. Reactions of this intermediate with sodium azide produced the key derivative 33-azido-33-deoxy-oligomycin A in 60% yield. 1,3-Dipolar cycloaddition reaction with propiolic acid, methyl ester of propiolic acid, and phenyl acetylene resulted in 33-deoxy-33-(1,2,3-triazol-1-yl)oligomycin A derivatives substituted at N4 of the triazole cycle. The mesylated oligomycin A and 33-deoxy-33-azidooligomycin A did not inhibit F₀F₁ ATFase ATPase; however, 33-azido-33-deoxy-oligomycin A and the derivatives containing 4-phenyltriazole, 4-methoxycarbonyl-triazole and 3-dimethylaminoethyl amide of carboxyltriazole substituents demonstrated a high cytotoxicity against K562 leukemia and HCT116 human colon carcinoma cell lines whereas non-malignant skin fibroblasts were less sensitive to these compounds. Novel series of oligomycin A derivatives allow for the search of intracellular molecules beyond F₀F₁ ATP synthase relevant to the cytotoxic properties of this perspective chemical class.     

Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione

Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethyl)amino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (K_ass∼10⁶ M⁻¹) for human telomeric antiparallel quadruplex d(TTAGGG)₄ and duplex d(TTAGGG)₄∶d(CCCTAA)₄. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na⁺ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K⁺ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.     

Naphthoindole-based analogues of tryptophan and tryptamine: synthesis and cytotoxic properties

The efficacy of anthracycline based anticancer drugs is limited by pleiotropic drug resistance of tumor cells. Aiming at the design of anthracyclinone congeners capable of circumventing drug resistance, we synthesized naphthoindole containing derivatives of tryptophan and tryptamine. In doing so we adapted the traditional, gramine based approach for tryptophan and tryptamine synthesis. The most potent new compound, 3-(2-aminoethyl)-4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione (16), was equally cytotoxic (IC(50) within low micromolar concentrations) for human K562 leukemia and HCT116 colon carcinoma cell lines and their isogenic sublines with genetically defined determinants of altered drug response, that is, the expression of the multidrug transporter P-glycoprotein and loss of pro-apoptotic p53. Each of these mechanisms conferred resistance to the reference drug adriamycin. In contrast, naphthotryptamine 16, although less potent than adriamycin, was equally toxic for wild type cell lines and drug resistant counterparts. Moreover, at 3-5 microM 16 inhibited topoisomerase I in vitro. Thus, our novel naphthoindole based derivative of tryptamine gained new activities important for anticancer therapy, namely, suppression of topoisomerase I and the ability to overcome resistance mediated by P-glycoprotein expression and p53 dysfunction.     

Novel boronated derivatives of 5,10,15,20-tetraphenylporphyrin: synthesis and toxicity for drug-resistant tumor cells

We have developed the synthesis of boronated porphyrins for potential application in cancer treatment, based on the functional derivatives of 5,10,15,20-tetraphenylporphyrin. Boronated amide derivatives starting from 5,10,15,20-tetra(p-aminophenyl)porphyrin and 9-o- and 9-m-carborane carboxylic acid chlorides were prepared. Also, the reaction of 2-formyl-5,10,15,20-tetraphenylporphyrin with closo-C-lithium-o- and m-carboranes, as well as with closo-C-lithium monocarbon carborane, yielded neutral and anionic boronated hydroxy derivatives of 5,10,15,20-tetraphenylporphyrin, respectively. Water-soluble forms of neutral compounds were prepared by deboronation of closo-polyhedra with Bu4NF into nido-7,8- and nido-7,9-dicarbaundecaborate anions. Monocarbon carborane conjugated with copper (II) complex of 5,10,15,20-tetraphenylporphyrin was active for a variety of tumor cell lines (IC50 approximately 5 microM after 48-72 h of exposure) but was inert for non-malignant fibroblasts at up to 100 microM. At low micromolar concentrations, this compound caused the death of cells that express P-glycoprotein and other mechanisms of resistance to conventional anticancer drugs.     

5,10,15,20-Tetra-(N-methyl-3-pyridyl)porphyrin destabilizes the antiparallel telomeric quadruplex d(TTAGGG)<Subscript>4</Subscript>

5,10,15,20-Tetra-(N-methyl-3-pyridyl)porphyrin (TMPyP3) is a DNA-binding derivative of porphyrins. A comparative study of the binding of this ligand to biologically significant DNA structures was performed. For this purpose, the interactions of TMPyP3 with the antiparallel telomeric G-quadruplex d(TTAGGG)₄, oligonucleotide dTTAGGGTTAGAG(TTAGGG)₂ (not forming a quadruplex structure), double-stranded d(AC)₈ · d(GT)₈, and single-stranded d(AC)₈ and d(GT)₈ DNA molecules have been studied. Analysis of absorption isotherms has demonstrated that the binding constants and the number of binding sites for the complexes TMPyP3: DNA increase in the following order: d(AC)₈ < d(GT)₈ < d(AC)₈ · d(GT)₈ = d(TTAGGG)₄ < dTTAGGGTTAGAG(TTAGGG)₂. It has been for the first time demonstrated that the constant for TMPyP3 binding to unfolded dTTAGGGTTAGAG(TTAGGG)₂ strand (1.3 × 10⁷ M⁻¹) is approximately threefold higher than for the G-quadruplex d(TTAGGG)₄ (4.7 × 10⁶ M⁻¹). Binding of two TMPyP3 molecules to d(TTAGGG)₄ decreases the thermostability of G-quadruplex (ΔT_m = −8°C). Circular dichroism spectra of the TMPyP3 complexes with d(TTAGGG)₄ suggest that the ligand partially unfolds the G-quadruplex structure. Structural destabilization of the telomeric G-quadruplex by TMPyP3 can explain the relatively low activity of this ligand as a telomerase inhibitor and a low cytotoxicity for cultured tumor cells.