Antitumoral Activity of Leptocarpha rivularis Flower Extracts against Gastric Cancer Cells.

Leptocarpha rivularis is a native South American plant used ancestrally by Mapuche people to treat gastrointestinal ailments. L. rivularis flower extracts are rich in molecules with therapeutic potential, including the sesquiterpene lactone leptocarpin, which displays cytotoxic effects against various cancer types in vitro. However, the combination of active molecules in these extracts could offer a hitherto unexplored potential for targeting cancer. In this study, we investigated the effect of L. rivularis flower extracts on the proliferation, survival, and spread parameters of gastric cancer cells in vitro. Gastric cancer (AGS and MKN-45) and normal immortalized (GES-1) cell lines were treated with different concentrations of L. rivularis flower extracts (DCM, Hex, EtOAc, and EtOH) and we determined the changes in proliferation (MTS assay, cell cycle analysis), cell viability/cytotoxicity (trypan blue exclusion assay, DEVDase activity, mitochondrial membrane potential MMP, and clonogenic ability), senescence (ß-galactosidase activity) and spread potential (invasion and migration assays using the Boyden chamber approach) in all these cells. The results showed that the DCM, EtOAc, and Hex extracts display a selective antitumoral effect in gastric cancer cells by affecting all the cancer parameters tested. These findings reveal an attractive antitumoral potential of L. rivularis flower extracts by targeting several acquired capabilities of cancer cells.

Photodynamic treatment with cationic Ir(III) complexes induces a synergistic antimicrobial effect with imipenem over carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Bacteria prevalent in the hospital environment have developed multi-drug resistance (MDR), such as the carbapenemase-producing Klebsiella pneumoniae (KPC+). Photodynamic therapy (PDT), which uses light-activated photosensitizer compounds (PSs), has emerged as an alternative to antibiotics. Cationic-PSs have a better bactericidal effect by interacting more closely with the bacterial envelope. METHODS: Two PSs based on cationic Ir (III) compounds (PSIR-1 and PSIR-2) were studied in photodynamic therapy against KPC+ and KPC- bacteria, and their PDT activities were compared with a cationic Ru(II) control compound (PS -Ru). RESULTS: Similar to the behavior of PS-Ru control, the cytotoxicity of PSIR-1 and 2, showing a bacterial inhibition growth of more than 3log10 (>99.9 % inactivation), at light fluency of 17 µW/cm2. The minimal dose to accomplish the inhibition in 3log10 was determined for PSIR-1 and PSIR-2 at 4 and 2 µg/mL, respectively and the lethality was 30 min of light exposure for both compounds. Notably, the PSIR-1 and 2 compounds showed a synergistic effect with imipenem by significantly increasing (up to 6 log10) the photodynamic bactericidal effect for KPC+ strains. This synergy is specific for PSIR-1 and 2 compounds, since it was not observed with the PS-Ru control. On normal gastric cells GES-1, both PSIR-1 and 2 showed significant cytotoxicity; however, the highest cytotoxicity was found in gastric tumor cells (AGS). CONCLUSION: The compounds PSIR-1 and 2 are bactericidal photosensitizers and represent a promising alternative for complementing the treatment of infections by MDR bacteria since they should not be toxic in the dark.

Minimal transition state charge stabilization of the oxyanion during peptide bond formation by the ribosome.

Peptide bond formation during ribosomal protein synthesis involves an aminolysis reaction between the aminoacyl α-amino group and the carbonyl ester of the growing peptide via a transition state with a developing negative charge, the oxyanion. Structural and molecular dynamic studies have suggested that the ribosome may stabilize the oxyanion in the transition state of peptide bond formation via a highly ordered water molecule. To biochemically investigate this mechanistic hypothesis, we estimated the energetic contribution to catalytic charge stabilization of the oxyanion using a series of transition state mimics that contain different charge distributions and hydrogen bond potential on the functional group mimicking the oxyanion. Inhibitors containing an oxyanion mimic that carried a neutral charge and a mimic that preserved the negative charge but could not form hydrogen bonds had less than a 3-fold effect on inhibitor binding affinity. These observations argue that the ribosome provides minimal transition state charge stabilization to the oxyanion during peptide bond formation via the water molecule. This is in contrast to the substantial level of oxyanion stabilization provided by serine proteases. This suggests that the oxyanion may be neutralized via a proton shuttle, resulting in an uncharged transition state.

Protection-free one-pot synthesis of 2'-deoxynucleoside 5'-triphosphates and DNA polymerization.

By differentiating the functional groups on nucleosides, we have designed and developed a one-pot synthesis of deoxyribonucleoside 5'-triphosphates without any protection on the nucleosides. A facile synthesis is achieved by generating an in situ phosphitylating reagent that reacts selectively with the 5'-hydroxyl groups of the unprotected nucleosides. The synthesized triphosphates are of high quality and can be effectively incorporated into DNAs by DNA polymerase. This novel approach is straightforward and cost-effective for triphosphate synthesis.

Transition state chirality and role of the vicinal hydroxyl in the ribosomal peptidyl transferase reaction.

The ribosomal peptidyl transferase is a biologically essential catalyst responsible for protein synthesis. The reaction is expected to proceed through a transition state approaching tetrahedral geometry with a specific chirality. To establish that stereospecificity, we synthesized two diastereomers of a transition state inhibitor with mimics for each of the four ligands around the reactive chiral center. Preferential binding of the inhibitor that mimics a transition state with S chirality establishes the spatial position of the nascent peptide and the oxyanion and places the amine near the critical A76 2'-OH group on the P-site tRNA. Another inhibitor series with 2'-NH 2 and 2'-SH substitutions at the critical 2'-OH group was used to test the neutrality of the 2'-OH group as predicted if the hydroxyl functions as a proton shuttle in the transition state. The lack of significant pH-dependent binding by these inhibitors argues that the 2'-OH group remains neutral in the transition state. Both of these observations are consistent with a proton shuttle mechanism for the peptidyl transferase reaction.

Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA.

The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.

Selenium derivatization of nucleic acids for crystallography.

The high-resolution structure of the DNA (5'-GTGTACA-C-3') with the selenium derivatization at the 2'-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 A resolution) with the 2'-Se modification in the minor groove is isomorphorous to the native structure (2.0 A). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 A resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization.

Efficient substrate cleavage catalyzed by hammerhead ribozymes derivatized with selenium for X-ray crystallography.

Because oxygen and selenium are in the same group (Family VI) in the periodic table, the site-specific mutagenesis at the atomic level by replacing RNA oxygen with selenium can provide insights on the structure and function of catalytic RNAs. We report here the first Se-derivatized ribozymes transcribed with all nucleoside 5'-(alpha-P-seleno)triphosphates (NTPalphaSe, including A, C, G, and U). We found that T7 RNA polymerase recognizes NTPalphaSe Sp diastereomers as well as the natural NTPs, whereas NTPalphaSe Rp diastereomers are neither substrates nor inhibitors. We also demonstrated the catalytic activity of these Se-derivatized hammerhead ribozymes by cleaving the RNA substrate, and we found that these phosphoroselenoate ribozymes can be as active as the native one. These hammerhead ribozymes site-specifically mutagenized by selenium reveal the close relationship between the catalytic activities and the replaced oxygen atoms, which provides insight on the participation of oxygen in catalysis or intramolecular interaction. This demonstrates a convenient strategy for the mechanistic study of functional RNAs. In addition, the active ribozymes site-specifically derivatized by selenium will allow for convenient MAD phasing in X-ray crystal structure studies.

Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion.

We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2'-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2'-methylseleno cytidine phosphoramidite and improved the accessibility of the 2'-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2'-selenium derivatization had no effect on the stability of the duplexes with the 3'-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3'-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2'-Se-C and 2'-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2'-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.

Synthesis of selenium-derivatized cytidine and oligonucleotides for X-ray crystallography using MAD.

Synthesis of the novel 2'-Se-cytidine phosphoramidite was achieved via transformation of the uridine analogue to the cytidine derivative in high yield. This 2'-Se-cytidine phosphoramidite was used to synthesize selenium-derivatized DNA and RNA oligonucleotides for X-ray crystallography using MAD. The nucleotide coupling yield using this novel phosphoramidite was over 99% when 5-benzylmercaptotetrazole (5-BMT) was used as the coupling reagent.

Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5'-(alpha-P-seleno)triphosphate and DNA polymerase for X-ray crystallography via MAD.

We report here the first study of enzymatic synthesis of two phosphoroselenoate (PSe) DNAs using the two alpha-Se-TTP diastereomers (Sp and Rp) and DNA polymerase. The experimental results indicate that Klenow equally recognizes the two individual diastereomers at the same level as natural TTP. The incorporations of the PSe groups at the expected sites have been confirmed by the digestion resistance to exonuclease III, and the different patterns of the digestion resistance of DNA I and II indicate the configurational differences of the PSe centers (Sp or Rp). Unlike chemical synthesis, which is limited to short DNAs and where the separation of the PSe DNA diastereomers is necessary, this enzymatic method can be used to prepare longer DNAs without diastereomer separation. This quantitative enzymatic approach is particular valuable for the synthesis of longer DNAs with multiple PSe groups in large scale for their X-ray crystal structure determination by the MAD phasing technique.

Covalent incorporation of selenium into oligonucleotides for X-ray crystal structure determination via MAD: proof of principle. Multiwavelength anomalous dispersion.

Selenium was incorporated into an oligodeoxynucleotide in the form of 2'-methylseleno-uridine (U(Se)). The X-ray crystal structure of the duplex left open bracket d(GCGTA)U(Se)d(ACGC) right open bracket (2) was determined by the multiwavelength anomalous dispersion (MAD) technique and refined to a resolution of 1.3 A, demonstrating that selenium can selectively substitute oxygen in DNA and that the resulting compounds are chemically stable. Since derivatization at the 2'-alpha-position with selenium does not affect the preference of the sugar for the C3'-endo conformation, this strategy is suitable for incorporating selenium into RNA. The availability of selenium-containing nucleic acids for crystallographic phasing offers an attractive alternative to the commonly used halogenated pyrimidines.

Internal derivatization of oligonucleotides with selenium for X-ray crystallography using MAD.

We have developed a route for the synthesis of 2'-selenium uridine analogues and oligonucleotides containing selenium labels, and have demonstrated for the first time a new strategy to covalently derivatize nucleotides with selenium for phase and structure determination in X-ray crystallography.