New Insights into Oxidative and Reductive Stress Responses and Their Relation to the Anticancer Activity of Selenium-Containing Compounds as Hydrogen Selenide Donors.

Redox balance is important for the homeostasis of normal cells, but also for the proliferation, progression, and survival of cancer cells. Both oxidative and reductive stress can be harmful to cells. In contrast to oxidative stress, reductive stress and the therapeutic opportunities underlying the mechanisms of reductive stress in cancer, as well as how cancer cells respond to reductive stress, have received little attention and are not as well characterized. Therefore, there is recent interest in understanding how selective induction of reductive stress may influence therapeutic treatment and disease progression in cancer. There is also the question of how cancer cells respond to reductive stress. Selenium compounds have been shown to have chemotherapeutic effects against cancer, and their anticancer mechanism is thought to be related to the formation of their metabolites, including hydrogen selenide (H2Se), which is a highly reactive and reducing molecule. Here, we highlight recent reports on the molecular mechanism of how cells recognize and respond to oxidative and reductive stress (1) and the mechanisms through which different types of selenium compounds can generate H2Se (2) and thus selectively affect reductive stress under controlled conditions, which may be important for their anticancer effects.

Escherichia coli tRNA 2-Selenouridine Synthase (SelU): Elucidation of Substrate Specificity to Understand the Role of S-Geranyl-tRNA in the Conversion of 2-Thio- into 2-Selenouridines in Bacterial tRNA.

The bacterial enzyme tRNA 2-selenouridine synthase (SelU) is responsible for the conversion of 5-substituted 2-thiouridine (R5S2U), present in the anticodon of some bacterial tRNAs, into 5-substituted 2-selenouridine (R5Se2U). We have already demonstrated using synthetic RNAs that transformation S2U→Se2U is a two-step process, in which the S2U-RNA is geranylated and the resulting geS2U-RNA is selenated. Currently, the question is how SelU recognizes its substrates and what the cellular pathway of R5S2U→R5Se2U conversion is in natural tRNA. In the study presented here, we characterized the SelU substrate requirements, identified SelU-associated tRNAs and their specific modifications in the wobble position. Finally, we explained the sequence of steps in the selenation of tRNA. The S2U position within the RNA chain, the flanking sequence of the modification, and the length of the RNA substrate, all have a key influence on the recognition by SelU. MST data on the affinity of SelU to individual RNAs confirmed the presumed process. SelU binds the R5S2U-tRNA and then catalyzes its geranylation to the R5geS2U-tRNA, which remains bound to the enzyme and is selenated in the next step of the transformation. Finally, the R5Se2U-tRNA leaves the enzyme and participates in the translation process. The enzyme does not directly catalyze the R5S2U-tRNA selenation and the R5geS2U-tRNA is the intermediate product in the linear sequence of reactions.

Escherichia coli tRNA 2-selenouridine synthase SelU selects its prenyl substrate to accomplish its enzymatic function.

Bacterial tRNA 2-selenouridine synthase (SelU) in vitro converts S2U-RNA to its selenium analog (Se2U-RNA) in a two-step process: (i) geranylation of S2U-RNA (with geranyl pyrophosphate, gePP), and (ii) selenation of the resulting geS2U-RNA (with the selenophosphate anion, SePO33-). Using an S2U-containing anticodon stem-loop fragment derived from tRNALys (S2U-RNA) and recombinant SelU with an MBP tag, we found that only geranyl (C10) pyrophosphate is the substrate for this enzyme, while other pyrophosphates such as isopentenyl (C5), dimethylallyl (C5), farnesyl (C15) and geranylgeranyl (C20) are not. Interestingly, methyl (C1)- and C5-, C10-, and C15-prenyl-containing S2U-RNAs (which were chemically obtained) underwent the selenation reaction promoted by SelU, although the Se2U-RNA product was obtained in decreasing yields in the following order: geranyl ≥ farnesyl > dimethylallyl ≫ methyl. Microscale thermophoresis showed an affinity between gePP and SelU in the micromolar range, while the other pyrophosphates tested, such as isopentenyl, dimethylallyl, farnesyl and geranylgeranyl, either did not bind to the protein or their binding affinity was above 1 mM. These results agree well with the in silico analysis, with gePP being the best binding substrate (the lowest relative free energy of binding (ΔG) and a small solvent-accessible surface area (SASA)). These results suggest that SelU has high substrate specificity for the prenylation reaction (only gePP is accepted), whereas there is little discrimination for the selenation reaction. We therefore suggest that only gePP and the geranylated tRNA serve as substrates for the conversion of 2-thio-tRNAs to 2-seleno-tRNAs, as it is found in the bacterial system.

Intracellular HINT1-Assisted Hydrolysis of Nucleoside 5'-O-Selenophosphate Leads to the Release of Hydrogen Selenide That Exhibits Toxic Effects in Human Cervical Cancer Cells.

In this study, we present a new selenium derivative, 2'-deoxyguanosine-5'-O-selenophosphate (dGMPSe), synthesized by the oxathiaphospholane method and adapted here for the synthesis of nucleoside selenophosphates. Using biochemical assays (HPLC- and fluorescence-based), we investigated the enzymatic activity of HINT1 towards dGMPSe in comparison with the corresponding thiophosphate nucleoside, i.e., dGMPS. Both substrates showed similar kcat and a small difference in Km, and during the reactions the release of reducing agents such as H2Se and H2S were expected and detected. MTT viability assay and microscopic analysis showed that dGMPSe was toxic to HeLa cancer cells, and this cytotoxicity was due to the release of H2Se. The release of H2Se or H2S in the living cells after administration of dGMPSe and/or dGMPS, both without carrier and by electroporation, was observed using a fluorescence assay, as previously for NMPS. In conclusion, our comparative experiments with dGMPSe and dGMPS indicate that the HINT1 enzyme is capable of converting (d)NMPSe to (d)NMP and H2Se, both in vitro and intracellularly. Since the anticancer activity of various selenium compounds depends on the formation of hydrogen selenide, the actual inducer of cell death, we propose that selenium-containing nucleotides represent another option as novel compounds with anticancer therapeutic potential.

Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful in Regenerative Medicine.

The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO- as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1-33 and 2.1-2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1ß1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.

Biochemical, crystallographic and biophysical characterization of histidine triad nucleotide-binding protein 2 with different ligands including a non-hydrolyzable analog of Ap4A.

BACKGROUND: Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional structure of the full-length protein have not been identified yet. METHODS: An HPLC assay was used to test the hydrolytic activity of HINT2 against various adenosine, guanosine, and 2'-deoxyguanosine derivatives containing phosphate bonds of different types and different leaving groups. Data on binding affinity were obtained by microscale thermophoresis (MST). Crystal structures of HINT2, in its apo form and with a dGMP ligand, were resolved to atomic resolution. RESULTS: HINT2 substrate specificity was similar to that of HINT1, but with the major exception of remarkable discrimination against substrates lacking the 2'-hydroxyl group. The biochemical results were consistent with binding affinity measurements. They showed a similar binding strength of AMP and GMP to HINT2, and much weaker binding of dGMP, in contrast to HINT1. A non-hydrolyzable analog of Ap4A (JB419) interacted with both proteins with similar Kd and Ap4A is the signaling molecule that can interact with hHINT1 and regulate the activity of some transcription factors. CONCLUSIONS: Several forms of homo- and heterodimers of different lengths of N-terminally truncated polypeptides resulting from degradation of the full-length protein were described. Ser144 in HINT2 appeared to be functionally equivalent to Ser107 in HINT1 by supporting the protonation of the leaving group in the hydrolytic mechanism of HINT2. SIGNIFICANCE: Our results should be considered in future studies on the natural function of HINT2 and its role in nucleotide prodrug processing.

P-stereocontrolled synthesis of oligo(nucleoside N3'→O5' phosphoramidothioate)s - opportunities and limitations.

3'-N-(2-Thio-1,3,2-oxathiaphospholane) derivatives of 5'-O-DMT-3'-amino-2',3'-dideoxy-ribonucleosides (NOTP-N), that bear a 4,4-unsubstituted, 4,4-dimethyl, or 4,4-pentamethylene substituted oxathiaphospholane ring, were synthesized. Within these three series, NOTP-N differed by canonical nucleobases (i.e., AdeBz, CytBz, GuaiBu, or Thy). The monomers were chromatographically separated into P-diastereomers, which were further used to prepare NNPSN' dinucleotides (3), as well as short P-stereodefined oligo(deoxyribonucleoside N3'→O5' phosphoramidothioate)s (NPS-) and chimeric NPS/PO- and NPS/PS-oligomers. The condensation reaction for NOTP-N monomers was found to be 5-6 times slower than the analogous OTP derivatives. When the 5'-end nucleoside of a growing oligomer adopts a C3'-endo conformation, a conformational 'clash' with the incoming NOTP-N monomer takes place, which is a main factor decreasing the repetitive yield of chain elongation. Although both isomers of NNPSN' were digested by the HINT1 phosphoramidase enzyme, the isomers hydrolyzed at a faster rate were tentatively assigned the R P absolute configuration. This assignment is supported by X-ray analysis of the protected dinucleotide DMTdGiBu NPSMeTOAc, which is P-stereoequivalent to the hydrolyzed faster P-diastereomer of dGNPST.

The role of the Hint1 protein in the metabolism of phosphorothioate oligonucleotides drugs and prodrugs, and the release of H2S under cellular conditions.

Phosphorothioate oligonucleotides (PS-oligos) containing sulfur atom attached in a nonbridging position to the phosphorus atom at one or more internucleotide bond(s) are often used in medicinal applications. Their hydrolysis in cellular media proceeds mainly from the 3'-end, resulting in the appearance of nucleoside 5'-O-phosphorothioates ((d)NMPS), whose further metabolism is poorly understood. We hypothesize that the enzyme responsible for (d)NMPS catabolism could be Hint1, an enzyme that belongs to the histidine triad (HIT) superfamily and is present in all organisms. We previously found that (d)NMPS were desulfurated in vitro to yield (d)NMP and H2S in a Hint1-assisted reaction. Here, we demonstrate that AMPS/GMPS/dGMPS introduced into HeLa/A549 cells are intracellularly converted into AMP/GMP/dGMP and H2S. The level of the released H2S was relative to the concentration of the compounds used and the reaction time. Using RNAi technology, we have shown decreased levels of AMPS/GMPS desulfuration in HeLa/A549 cells with reduced Hint1 levels. Finally, after transfection of a short Rp-d(APSAPSA) oligomer into HeLa cells, the release of H2S was observed. These results suggest that the metabolic pathway of PS-oligos includes hydrolysis into (d)NMPS (by cellular nucleases) followed by Hint1-promoted conversion of the resulting (d)NMPS into (d)NMP accompanied by H2S elimination. Our observations may be also important for possible medicinal applications of (d)NMPS because H2S is a gasotransmitter involved in many physiological and pathological processes.

Diaryl ethers with carboxymethoxyphenacyl motif as potent HIV-1 reverse transcriptase inhibitors with improved solubility.

In search of new non-nucleoside reverse transcriptase inhibitors (NNRTIs) with improved solubility, two series of novel diaryl ethers with phenacyl moiety were designed and evaluated for their HIV-1 reverse transcriptase inhibition potentials. All compounds exhibited good to excellent results with IC50 at low micromolar to submicromolar concentrations. Two most active compounds (7e and 7 g) exhibit inhibitory potency comparable or even better than that of nevirapine and rilpivirine. Furthermore, SupT1 and CD4+ cell infectivity assays for the most promising (7e) have confirmed its strong antiviral potential while docking studies indicate a novel binding interactions responsible for high activity.

New interactions between tumor suppressor Fhit protein and a nonhydrolyzable analog of its AP4 A substrate.

Fragile histidine triad protein (Fhit) is a protein which primarily hydrolyses dinucleoside polyphosphates. To investigate possible interactions between the protein and a substrate, we used a nonhydrolyzable phosphorothioate analog of Ap4 A, containing 5-bromo-2'-deoxyuridine instead of one adenosine residue. Photocrosslinking, followed by LC-MS experiments, determined a complex in which the probe was covalently linked to the NDSIYEELQK peptide (residues 110-119). The peptide was located within the 'disordered' region, which is invisible in the known crystal structures of Fhit. This invisible and flexible part seems to play a role in the stabilization of the Fhit-substrate complex, which may be important for its tumor suppressor activity.

Phosphorothioate analogs of P1,P3-di(nucleosid-5'-yl) triphosphates: Synthesis, assignment of the absolute configuration at P-atoms and P-stereodependent recognition by Fhit hydrolase.

Di(nucleosid-5'-yl) polyphosphates (NPnN) are involved in various biological processes, and constitute signaling molecules in the intermolecular purinergic systems. They exert tumor suppression function and are substrates for specific hydrolases (e.g., HIT proteins). Their structural analogs may serve as molecular probes and potential therapeutic agents. Three P1,P3-bis-thio-analogs of symmetrical di(nucleosid-5'-yl) triphosphates (NP3N) bearing adenosine, guanosine or ribavirin residues (6, 7 and 8, respectively), were obtained by direct condensation of corresponding base-protected nucleoside-5'-O-(2-thio-1,3,2-oxathiaphospholane) with anhydrous phosphoric acid in the presence of DBU. Deprotected products 6 and 8 were separated into individual P-diastereoisomers, whereas 7 was partially separated to yield diastereomerically enriched fractions. The absolute configuration at P-stereogenic centers in the separated diastereoisomers was assigned by RP-HPLC analysis of the products of enzymatic digestion with snake venom phosphodiesterase. The Fhit-assisted hydrolysis rates for 6 and 7 are by 2-3 orders of magnitude lower than that for the reference AP3A, and depend on the configuration of the stereogenic phosphorus atoms, while 8 occurred to be resistant to this cleavage.

Crystallographic studies of the complex of human HINT1 protein with a non-hydrolyzable analog of Ap4A.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine triad proteins superfamily. HINT1 plays an important role in various biological processes, and it has been found in many species. Here, we report the first structure (at a 2.34Å resolution) of a complex of human HINT1 with a non-hydrolyzable analog of an Ap4A dinucleotide, containing bis-phosphorothioated glycerol mimicking a polyphosphate chain, obtained from a primitive monoclinic space group P21 crystal. In addition, the apo form of hHINT1 at the space group P21 refined to 1.92Å is reported for comparative studies.

Searching for novel scaffold of triazole non-nucleoside inhibitors of HIV-1 reverse transcriptase.

Azoles are a promising class of the new generation of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). From thousands of reported compounds, many possess the same basic structure of an aryl substituted azole ring linked by a thioglycolamide chain with another aromatic ring. In order to find novel extensions for this basic scaffold, we explored the 5-position substitution pattern of triazole NNRTIs using molecular docking followed by the synthesis of selected compounds. We found that heterocyclic substituents in the 5-position of the triazole ring are detrimental to the inhibitory activity of compounds with four-membered thioglycolamide linker and this substitution seems to be viable only for compounds with shorter two-membered linker. Promising compound, N-(4-carboxy-2-chlorophenyl)-2-((4-benzyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl)acetamide, with potent inhibitory activity and acceptable aqueous solubility has been identified in this study that could serve as lead scaffold for the development of novel water-soluble salts of triazole NNRTIs.

LNA units present in the (2'-OMe)-RNA strand stabilize parallel duplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA and parallel triplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription.

Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA→DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases.

Interactions of cellular histidine triad nucleotide binding protein 1 with nucleosides 5'-O-monophosphorothioate and their derivatives - Implication for desulfuration process in the cell.

BACKGROUND: One of the activities of histidine triad nucleotide-binding protein 1 (Hint1) under in vitro conditions is the conversion of nucleoside 5'-O-phosphorothioate (NMPS) to its 5'-O-phosphate (NMP), which is accompanied by the release of hydrogen sulfide. METHODS: Non-hydrolyzable derivatives of AMPS and dCMPS, each containing the residue able to form a covalent bond in nucleic acid-protein complexes via photocrosslinking (at 308nm), were applied at the complexing experiments with recombinant and cellular Hint1. The cellular lysates prepared after RNAi-mediated knockdown of Hint1 were incubated with AMPS and the level of desulfuration was measured. RESULTS: Recombinant Hint1 and Hint1 present in the cellular lysate of A549 cells, formed complexes with the used substrate analogs. Computer modeling experiments, in which the ligand was docked at the binding pocket, confirmed that direct interactions between Hint1 and the screened analogs are possible. Using RNAi technology, we demonstrated lowered levels of AMPS substrate desulfuration in reactions that employed the cell lysates with a reduced Hint1 level. CONCLUSIONS: The enzymatic conversion of AMPS to AMP occurred with the participation of cellular Hint1, the protein, which is present in all organisms. GENERAL SIGNIFICANCE: The intracellular Hint1 could be responsible for the in vivo desulfuration of nucleosides-5'-monophosphorothioate, thus it can contribute to the phosphorothioate oligonucleotides metabolism. H2S released during this process may participate in several physiological processes, thus NMPSs can be precursors/donors of H2S in vivo and can be used to study the effects of this gas in biological systems. Moreover, the controlled delivery of (d)NMPSs into cells may be of medicinal utility.

N-Acyl-phosphoramidates as potential novel form of gemcitabine prodrugs.

Gemcitabine (dFdC) is a cytidine analog remarkably active against a wide range of solid tumors. Inside a cell, gemcitabine is phosphorylated by deoxycytidine kinase to yield gemcitabine monophosphate, further converted to gemcitabine di- and triphosphate. The most frequent form of acquired resistance to gemcitabine in vitro is the deoxycytidine kinase deficiency. Thus, proper prodrugs carrying the 5'-pdFdC moiety may help to overcome this problem. A series of new derivatives of gemcitabine possessing N-acyl(thio)phosphoramidate moieties were prepared and their cytotoxic properties were determined. N-Acyl-phosphoramidate derivatives of gemcitabine have similar cytotoxicity as gemcitabine itself, and have been found accessible to the cellular enzymes. The nicotinic carboxamide derivative of gemcitabine 5'-O-phosphorothioate occurred to be the best inhibitor of bacterial DNA polymerase I and human DNA polymerase α.

A new crystal form of human histidine triad nucleotide-binding protein 1 (hHINT1) in complex with adenosine 5'-monophosphate at 1.38†Šresolution.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch of the histidine triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the structure of the human HINT1-adenosine 5'-monophosphate (AMP) complex at 1.38 Šresolution obtained from a new monoclinic crystal form is reported. The final structure has R(cryst) = 0.1207 (R(free) = 0.1615) and the model exhibits good stereochemical quality. Detailed analysis of the high-resolution data allowed the details of the protein structure to be updated in comparison to the previously published data.

[Histidine triad protein superfamily--biological function and enzymatic activity]. / Rodzina bialek triady histydynowej (HIT)--aktywnosc enzymatyczna a funkcja biologiczna.

The HIT superfamily consists of proteins that share the histidine triad motif, His-X-His-X-His-X-X (where X is a hydrophobic amino acid), which constitutes enzymatic catalytic center. These enzymes act as nucleotidylyl hydrolase or transferase, and the mutation of the second histidine in the triad abolishes their activity. HIT proteins were found ubiquitous in all organisms and they were classified into 5 branches, which are represented by human proteins: HINT1, FHIT, Aprataxin, GALT and DCPS. Because HINT1 orthologs, which belong to the evolutionally oldest family branch, were found from prokaryotes to eukaryotes, it is clear that HIT motif was conserved during the evolution what means that the enzymatic activity is necessary for functions of these proteins. However, in few cases, e.g. HINT1 and FHIT, the connection between the biological function and the enzymatic activity is still obscure. In this review, the relations between biology and activity for 7 HIT proteins, which were found in human, are highlighted.

Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis.

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 µM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.