2,5-Diketopiperazines: A New Class of Poly(ADP-ribose)polymerase Inhibitors.

We show for the first time that natural 2,5-diketopiperazines (cyclic dipeptides) can suppress the activity of the important anticancer target poly(ADP-ribose)polymerase (PARP). Cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) can interact with the key residues of the PARP-1 active site, as demonstrated using docking and molecular dynamics simulations. One of the amide groups of cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) forms hydrogen bonds with the Gly863 residue, while the second amide group can form a hydrogen bond with the catalytic residue Glu988, and the side chain can make a hydrophobic contact with Ala898. Newly identified diketopiperazine inhibitors are promising basic structures for the design of more effective inhibitors of PARP family enzymes. The piperazine core with two chiral centers provides many opportunities for structural optimization.

A rapid fluorescent method for the real-time measurement of poly(ADP-ribose) polymerase 1 activity.

Poly(ADP-ribose) polymerase 1 (PARP1) is a key enzyme that regulates important cellular processes, including DNA repair. PARP1 binds to a DNA damage site and synthesizes poly(ADP-ribose) chains (PARs), which serve as a signal of DNA damage for other DNA repair enzymes. PARP1 is a recognized target for the development of anti-cancer drugs. In this work, a method is developed that makes it possible to investigate the complex formation of PARP1 with DNA as well as its dissociation by detecting the fluorescence anisotropy of this complex during the poly(ADP-ribose) synthesis. The method allows investigation of the inhibition of PARP1 activity in the presence of its inhibitors. In this work, we demonstrated that PARP1 is activated by DNA duplexes containing a damage and a fluorophore at the 3'-end of one of the DNA duplex chains. The effects of the clinical inhibitor olaparib on the activity of PARP1 was studied. It was shown that olaparib has no influence on the binding of PARP1 to the model DNA structures used, but it significantly inhibits the poly(ADP-ribosyl)ation of PARP1. The proposed convenient method for the real-time determination of the PARP1 activity can be used to screen PARP1 inhibitors with the calculation of quantitative inhibition parameters.

[Improved procedure of the search for poly(ADP-ribose) polymerase-1 potential inhibitors with use of molecular docking approach].

A search for poly(ADP-ribose) polymerase-1 inhibitors by virtual screening of a chemical compound database and a subsequent experimental verification of their activities have been done. It was shown that the most efficient method to predict inhibitory properties implies a combinatorial approach joining molecular docking capabilities with structural filtration. Among more than 300000 database chemicals 9 PARP1 inhibitors were revealed; the most active ones, namely: STK031481, STK056130, and STK265022,--displayed biological effect at a micro-molar concentration (IC50 = 2.0 microM, 1.0 microM and 2.6 microM, respectively).

Interaction of poly(ADP-ribose) polymerase 1 with apurinic/apyrimidinic sites within clustered DNA damage.

To study the interaction of poly(ADP-ribose) polymerase 1 (PARP1) with apurinic/apyrimidinic sites (AP sites) within clustered damages, DNA duplexes were created that contained an AP site in one strand and one of its analogs situated opposite the AP site in the complementary strand. Residues of 3-hydroxy-2-hydroxymethyltetrahydrofuran (THF), diethylene glycol (DEG), and decane-1,10-diol (DD) were used. It is shown for the first time that apurinic/apyrimidinic endonuclease 1 (APE1) cleaves the DNA strands at the positions of DEG and DD residues, and this suggests these groups as AP site analogs. Insertion of DEG and DD residues opposite an AP site decreased the rate of AP site hydrolysis by APE1 similarly to the effect of the THF residue, which is a well-known analog of the AP site, and this allowed us to use such AP DNAs to imitate DNA with particular types of clustered damages. PARP1, isolated and in cell extracts, efficiently interacted with AP DNA with analogs of AP sites producing a Schiff base. PARP1 competes with APE1 upon interaction with AP DNAs, decreasing the level of its cross-linking with AP DNA, and inhibits hydrolysis of AP sites within AP DNAs containing DEG and THF residues. Using glutaraldehyde as a linking agent, APE1 is shown to considerably decrease the amount of AP DNA-bound PARP1 dimer, which is the catalytically active form of this enzyme. Autopoly(ADP-ribosyl)ation of PARP1 decreased its inhibitory effect. The possible involvement of PARP1 and its automodification in the regulation of AP site processing within particular clustered damages is discussed.

Apurinic/apyrimidinic (AP) site recognition by the 5'-dRP/AP lyase in poly(ADP-ribose) polymerase-1 (PARP-1).

The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP-1 was only weakly activated to conduct poly(ADP-ribose) synthesis upon binding to AP site-containing DNA, but was strongly activated for poly(ADP-ribose) synthesis upon strand incision by AP endonuclease 1 (APE1). By virtue of its binding to AP sites, PARP-1 could be poised for its role in base excision repair, pending DNA strand incision by APE1 or the 5'-dRP/AP lyase activity in PARP-1.

Influence of poly(ADP-ribose) polymerase-1 and its apoptotic 24-kD fragment on repair of DNA duplexes in bovine testis nuclear extract.

Effects of exogenous proteins poly(ADP-ribose) polymerase-1 (PARP1) and its 24-kD proteolytic fragment (p24) on the repair of DNA duplexes containing a one nucleotide gap with furan phosphate or phosphate group at the 5'-end of the downstream primer were studied in bovine testis nuclear extract. These damaged DNAs are repaired by the long-patch or short-patch subpathways of base excision repair (BER), respectively. Exogenous PARP1 and p24 decreased the efficiency of gap filling DNA synthesis for both duplexes, but did not influence the ligation stage in the repair of DNA duplex by the short-patch subpathway. Under the same conditions, these proteins inhibited strand-displacement DNA synthesis and decreased the efficiency of the flap endonuclease 1 (FEN1)-catalyzed endonuclease reaction in the nuclear extract, blocking repair of DNA duplex by the long-patch subpathway. Addition of exogenous PARP1 and p24 also reduced the efficiency of UV light crosslinking of extract BER proteins to the photoreactive BER intermediates carrying a nick. Thus, PARP1 and p24 interact with DNA intermediates of BER and compete with nuclear extract proteins for binding to DNA. The interaction of PARP1 and p24 with DNA intermediates of the long-patch subpathway of BER resulted in inhibition of subsequent stages of the repair mediated by this mechanism. However, on recovery of the intact structure of DNA duplex by the short-patch subpathway, PARP1 and p24 suppressed the repair of the one nucleotide gap less efficiently and failed to influence the final stage of the repair, ligation.

3'-5' exonuclease activity of human apurinic/apyrimidinic endonuclease 1 towards DNAs containing dNMP and their modified analogs at the 3 end of single strand DNA break.

Human DNA apurinic/apyrimidinic (AP-) endonuclease 1 (APE1) is involved in the base excision repair (BER) pathway. The enzyme hydrolyzes DNA from the 5 side of the AP site. In addition to endonuclease activity, APE1 also possesses other slight activities including 3 -5 exonuclease activity. The latter is preferentially exhibited towards mispaired (non-canonical) nucleotides, this being the reason why APE1 is considered as a proofreading enzyme correcting the misincorporations introduced by DNA polymerase beta. We have studied 3 -5 exonuclease activity of APE1 towards dCMP and dTMP residues and modified dCMP analogs with photoreactive groups at the 3 end of the nicked DNA. Photoreactive dNMP residues were incorporated at the 3 end of the lesion using DNA polymerase beta and photoreactive dNTPs. The dependence of exonuclease activity on the "canonicity" of the base pair formed by dNMP flanking the nick at the 3 end, on the nature of the group flanking the nick at the 5 end, and on the reaction conditions has been determined. Optimal reaction conditions for the 3 -5 exonuclease hydrolysis reaction catalyzed by APE1 in vitro have been established, and conditions when photoreactive residues are not removed by APE1 have been chosen. These reaction conditions are suitable for using photoreactive nicked DNAs bearing 3 -photoreactive dNMP residues for photoaffinity labeling of proteins in cellular/nuclear extracts and model APE1-containing systems. We recommend using FAPdCTP for photoaffinity modification in APE1-containing systems because the FAPdCMP residue is less prone to exonuclease degradation, in contrast to FABOdCTP, which is not recommended.

[Poly(ADP-ribose) polymerase-1 as a regulator of protein-nucleic acid interactions in the processes responding to genotoxic action]. / Poli(ADP-ribozo)polimeraza-1-reguliator belkovo-nukleinovykh vzaimodeistvii v protsessakh voznikaiushchikh pri genotoksicheskom vozdeistvii.

Poly(ADP-ribose) polymerase-1 (PARP-1), nuclear protein of higher eukaryotes, specifically detects strand breaks in DNA. When bound to DNA strand breaks, PARP-1 is activated and catalyzes synthesis of poly(ADP-ribose) covalently attached to the row of nuclear proteins, with the main acceptor being PARP-1 itself. This protein participates in a majority of DNA dependent processes: repair, recombination; replication: cell death: apoptosis and necrosis. Poly(ADP-ribosyl)ation of proteins is considered as mechanism, which signals about DNA damage and modulate protein functioning in response to genotoxic impact. The main emphasis is made on the roles of PARP-1 and poly(ADP-ribosyl)ation in base excision repair (BER), the process, which provides repair of DNA breaks. The main proposed functions of PARP-1 in this process are: factor initiating assemblage of protein complex of BER; temporary protection of DNA ends; modulation of chromatin structure via poly(ADP-ribosyl)ation of histones; signaling function in detection of the levels of DNA damage in cell.

Poly(ADP-ribose) polymerase-1 inhibits strand-displacement synthesis of DNA catalyzed by DNA polymerase beta.

Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.

[Experience in using acupuncture reflexotherapy combined with weight-reducing diet therapy in hypertension]. / Opyt ispol'zovaniia pri gipertonicheskoi bolezni iglorefleksoterapii v sochetanii s razgruzochno-dieticheskoi terapiei.

AIM: To study the effectiveness of acupuncture in combination with unload diet against hypertension. MATERIALS AND METHODS: 137 patients with mild hypertension have undergone unload diet therapy or therapeutic fasting in combination with acupuncture. Acupuncture therapy was carried out according to the physiological model. The unload diet therapy was performed according to the method of Iu. S. Nikolaev and consisted of voluntary fasting. RESULTS: The treatment resulted in a decrease of cholesterol levels and blood pressure, positive trend in ECG. For 3 years 20 patients with mild hypertension had normal blood pressure. They kept on diet, had short-term courses of fasting, exercised in free-choice regimen. CONCLUSION: Acupuncture in combination with diet therapy are recommended for treatment of blood hypertension. They can be used alone or in combination with drug therapy.

A peculiarity of the reaction of tRNA aminoacylation catalyzed by phenylalanyl-tRNA synthetase from the extreme thermophile Thermus thermophilus.

It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low: the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNAPhe but also Escherichia coli tRNAPhe and E. coli tRNAPhe transcript, indicating that the presence of modified nucleotides is not necessary for tRNAPhe overcharging. Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases. Aminoacylated tRNAPhe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed.