Structural evidence of the species dependent albumin binding of the modified cyclic phosphatidic acid with cytotoxic properties

Cyclic phosphatidic acids (cPAs) are naturally occurring, very active signalling molecules, which are involved in several pathological states, such as cancer, diabetes or obesity. As molecules of highly lipidic character found in the circulatory system, cPAs are bound and transported by the main extracellular lipid binding protein–serum albumin. Here, we present the detailed interactions between human serum albumin (HSA) and equine serum albumin (ESA) with a derivative of cPA, 1-O-myristoyl-sn-glycerol-2,3-cyclic phosphorodithioate (Myr-2S-cPA). Initial selection of the ligand used for the structural study was made by the analysis of the therapeutically promising properties of the sulfur containing analogues of cPA in respect to the unmodified lysophospholipids (LPLs). Substitution of one or two non-bridging oxygen atoms in the phosphate group with one or two sulfur atoms increases the cytotoxic effect of cPAs up to 60% on the human prostate cancer (PC) cells. Myr-2S-cPA reduces cancer cell viability in a dose-dependent manner, with IC(50) value of 29.0 μM after 24 h incubation, which is almost 30% lower than IC(50) of single substituted phosphorothioate cPA. Although, the structural homology between HSA and ESA is big, their crystal complexes with Myr-2S-cPA demonstrate significantly different mode of binding of this LPL analogue. HSA binds three molecules of Myr-2S-cPA, whereas ESA only one. Moreover, none of the identified Myr-2S-cPA binding sites overlap in both albumins

Backbone-modified oligonucleotides containing a butanediol-1,3 moiety as a 'vicarious segment' for the deoxyribosyl moiety--synthesis and enzyme studies.

Sequential single replacement of nucleosides within the decanucleotide d[GGGAATTCCC] (7) by means of a butanediol-1,3 residue allowed us to obtain a set of ten decanucleotides containing 'vicarious' (V) carbon-phosphate fragments. These analogues were further used as substrates for svPDE, nuclease PI and EcoR1 endonuclease. Interestingly, replacement of any of the nucleosides within the canonical sequence ...GAATTC... by the butanediol-1,3 'vicarious' segment discriminates such constructs 8c-h as substrates for Eco-R1 enzyme

Cyclic phosphatidic acid and its analogues can regulate ERK1/2 activity via LPA receptors

Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3 cyclic phosphate, cPA) and lysophosphatidic acid (LPA) are bioactive lipid mediators belonging to the small glycerophospholipids. Recent studies have shown that these compounds possess contrasting biological activities: LPA induces tumor cell proliferation and motility, while cPA suppresses metastasis and invasion of cancer cells. There are known at least eight LPA receptors involved in LPA signaling, however selective receptors of cPA are still unknown

Binding of phosphorothioate oligodeoxynucleotides to basic fibroblast growth factor, recombinant soluble CD4, laminin and fibronectin is P-chirality independent.

Antisense oligodeoxynucleotides can selectively inhibit the expression of individual genes and thus have potential applications in anticancer and antiviral therapy. A critical prerequisite to their use as therapeutic agents is the understanding of their non-specific interactions with biological structures, e.g. proteins. In this study we examined the interactions of P-chiral phosphorothioate oligodeoxynucleotides with several proteins. The Rp- and Sp- diastereomers, and racemic machine-made mixtures, or M-oligodeoxynucleotides were used independently as competitors of the binding of a probe, phosphodiester oligodeoxynucleotide bearing a 5' alkylating moiety, to rsCD4, bFGF and laminin. These oligodeoxynucleotides were also used as competitors of the binding of a non-alkylating probe M-phosphorothioate oligodeoxynucleotide, 5'-32P-SdT18 to fibronectin. The average values of and quantitative estimates for the IC50 of competition and the constant of competition (Kc) of Rp-, Sp- and M-stereoisomers of several homo- and heteropolymer oligodeoxynucleotides were determined and compared. Surprisingly, in the proteins we studied, the values of IC50 and Kc for the Rp-, Sp- and M-oligodeoxynucleotides were essentially identical. Thus, the ability of the phosphorothioate oligodeoxynucleotides we employed, to bind to the proteins studied in this work, is virtually independent of P-chirality. Our results also imply that the role of the purine and pyrimidine bases in oligodeoxynucleotide-protein interactions, as well as the nature of the contact points (sulfur versus oxygen) between the oligomer and the protein, may be relatively unimportant

Oligonucleotide N3'-->P5' phosphoramidates as antisense agents.

Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3'-oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents