A. S. Prakasha Gowda

An unprecedented nucleic acid capture mechanism for excision of DNA damage

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson–Crick base pairs without duplex intercalation.

Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway

A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma UACC903. Analogs 6, 11 and 13 displayed good in vitro anticancer activity on the HT29 human colon cancer cell line in the 1 μM range. Analogs 5, 9 and 12, containing a selenocyanate group in the alkyl chain were the most promising compounds on the breast cancer MCF-7 cell line. Biological assays relating to apoptosis were performed to understand the mechanism of action of these analogs. Compounds 5 and 6 were found to inhibit tubulin polymerization to the same extent as the anticancer drug vinblastine sulfate, but compounds 11 and 13 inhibited significantly better than vinblastine. Further western blot analysis suggested that compound 6 at 2 μM reduced both levels and phosphorylation state of Akt. Compounds 11 and 13 at 1 μM caused reduced Akt protein levels and strongly suppressed the phosphorylation of Akt. Therefore, 11 and 13 were demonstrated as efficient dual inhibitors of both tubulin polymerization and the Akt pathway and good candidates for further study. More importantly, the strategy of microtubule and Akt dual inhibitors might be a promising direction for developing novel drugs for cancer.

Structural Basis for the Adherence of Plasmodium falciparum-infected Erythrocytes to Chondroitin 4-Sulfate and Design of Novel Photoactivable Reagents for the Identification of Parasite Adhesive Proteins

A dodecasaccharide motif of the low-sulfated chondroitin 4-sulfate (C4S) mediate the binding of Plasmodium falciparum-infected red blood cells (IRBCs) in human placenta. Here we studied the detailed C4S structural requirements by assessing the ability of chemically modified C4S to inhibit IRBC binding to the placental chondroitin sulfate proteoglycan. Replacement of the N-acetyl groups with bulky N-acyl or N-benzoyl substituents had no effect on the inhibitory activity of C4S, whereas reduction of the carboxyl groups abrogated the activity. Dermatan sulfates showed ∼50% inhibitory activity when compared with C4Ss with similar sulfate contents. These data demonstrate that the C4S carboxyl groups and their equatorial orientation but not the N-acetyl groups are critical for IRBC binding. Conjugation of bulky substituents to the reducing end N-acetylgalactosamine residues of C4S dodecasaccharide had no effect on its inhibitory activity. Based on these results, we prepared photoaffinity reagents for the identification of the parasite proteins involved in C4S binding. Cross-linking of the IRBCs with a radioiodinated photoactivable C4S dodecasaccharide labeled a ∼22-kDa novel parasite protein, suggesting strongly for the first time that a low molecular weight IRBC surface protein rather than a 200–400-kDa PfEMP1 is involved in C4S binding. Conjugation of biotin to the C4S dodecasaccharide photoaffinity probe afforded a strategy for the isolation of the labeled protein by avidin affinity precipitation, facilitating efforts to identify the C4S-adherent IRBC protein(s). Our results also have broader implications for designing oligosaccharide-based photoaffinity probes for the identification of proteins involved in glycosaminoglycan-dependent attachment of microbes to hosts.

In vitro growth inhibition of human cancer cells by novel honokiol analogs

Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluated possible mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory activity in A549 human lung cancer cells and significant increase of cell population in the G0-G1 phase. Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both CDK1 and cyclin B1 protien levels in A549 cells.

Development of novel naphthalimide derivatives and their evaluation as potential melanoma therapeutics

Synthesis and anti-melanoma activity of various naphthalimide analogs, rationally modified by introducing isothiocyanate (ITC) and thiourea (TU) functionalities, found in well-known anti-cancer agents, is described. The structure-activity relationship comparison of the novel agents in inhibiting cancer cell growth was evaluated in various melanoma cell lines. Both ITC and TU analogs effectively inhibited cell viability and induced apoptosis in various human melanoma cells. Nitro substitution and increase in alkyl chain length, in general, enhanced the apoptotic activity of ITC derivatives. All the new compounds were well tolerated when injected intraperitoneal (i.p.) in mice at effective doses at which both the ITC and TU derivatives inhibited melanoma tumor growth in mice following i.p. xenograft. The nitro substituted naphthalimide-ITC derivative 3d was found to be the most effective in inducing apoptosis, and in inhibiting melanoma cell and tumor growth.

Formation of Purine–Purine Mispairs by Sulfolobus solfataricus DNA Polymerase IV

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.

Low Fidelity Bypass of O2-(3-Pyridyl)-4-oxobutylthymine, the Most Persistent Bulky Adduct Produced by the Tobacco Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone by Model DNA Polymerases

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most important human carcinogens. It is metabolized to produce a variety of methyl and 4-(3-pyridyl)-4-oxo-butyl (POB) DNA adducts. A potentially important POB adduct is O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT) because it is the most abundant POB adduct in NNK-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past both O(2)-POB-dT and O(2)-methylthymidine (O(2)-Me-dT) by two model polymerases, E. coli DNA polymerase I (Klenow fragment) with the proofreading exonuclease activity inactivated (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4). We found that the size of the alkyl chain only marginally affected the reactivity and that the specificity of adduct bypass was very low. The k(cat)/K(m) for the Kf catalyzed incorporation opposite and extension past the adducts was reduced ∼10(6)-fold when compared to undamaged DNA. Dpo4 catalyzed the incorporation opposite and extension past the adducts approximately 10(3)-fold more slowly than undamaged DNA. The dNTP specificity was less for Dpo4 than for Kf. In general, dA was the preferred base pair partner for O(2)-Me-dT and dT the preferred base pair partner for O(2)-POB-dT. With enzyme in excess over DNA, the time courses of the reactions showed a biphasic kinetics that indicates the formation inactive binary and ternary complexes.

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

Background: DNA polymerase ν is a low fidelity polymerase with an unknown function. Results: The kinetic mechanism of correct and incorrect base pair formation was determined. Conclusion: Phosphodiester bond formation is rapid for both correct and mispair formation. Significance: The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a Kd for DNA of 9.2 nm, with an off-rate constant of 0.013 s−1and an on-rate constant of 14 μm−1 s−1. dNTP binding is rapid with Kd values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a Kd value for PPi of 3.7 mm and a maximal rate constant of 11 s−1. Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s−1; rapid phosphodiester bond formation occurs with a Keq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s−1, for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.

Honokiol inhibits DNA polymerases β and λ and increases bleomycin sensitivity of human cancer cells.

A major concept to sensitize cancer cells to DNA damaging agents is by inhibiting proteins in the DNA repair pathways. X-family DNA polymerases play critical roles in both base excision repair (BER) and nonhomologous end joining (NHEJ). In this study, we examined the effectiveness of honokiol to inhibit human DNA polymerase β (pol β), which is involved in BER, and DNA polymerase λ (pol λ), which is involved in NHEJ. Kinetic analysis with purified polymerases showed that honokiol inhibited DNA polymerase activity. The inhibition mode for the polymerases was a mixed-function noncompetitive inhibition with respect to the substrate, dCTP. The X-family polymerases, pol β and pol λ, were slightly more sensitive to inhibition by honokiol based on the Ki value of 4.0 μM for pol β, and 8.3 μM for pol λ, while the Ki values for pol η and Kf were 20 and 26 μM, respectively. Next we extended our studies to determine the effect of honokiol on the cytotoxicity of bleomycin and temozolomide in human cancer cell lines A549, MCF7, PANC-1, UACC903, and normal blood lymphocytes (GM12878). Bleomycin causes both single strand DNA damage that is repaired by BER and double strand breaks that are repaired by NHEJ, while temozolomide causes methylation damage repaired by BER and O6-alkylguanine-DNA alkyltransferase. The greatest effects were found with the honokiol and bleomycin combination in MCF7, PANC-1, and UACC903 cells, in which the EC50 values were decreased 10-fold. The temozolomide and honokiol combination was less effective; the EC50 values decreased three-fold due to the combination. It is hypothesized that the greater effect of honokiol on bleomycin is due to inhibition of the repair of the single strand and double strand damage. The synergistic activity shown by the combination of bleomycin and honokiol suggests that they can be used as combination therapy for treatment of cancer, which will decrease the therapeutic dosage and side effects of bleomycin.

Design, Synthesis and Antibacterial Activity Studies of Model Peptides of Proline / Arginine-Rich Region in Bactenecin7

Bactenecin 7 (Bac7), a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa = hydrophobic residue). To investigate the structure and property of a Pro/Arg-rich region, e synthesized a series of peptides, Xaa-Pro-Arg-Pro (Xaa = Gly, Arg, Leu, Ile, and Phe) as models and characterized . The conformational preferences of these peptides in water and trifluoroethanol were examined by circular dichroism. The results suggest the presence of largely poly(Pro)-II helical conformation in aqueous and trifluoroethanol solutions. Their antibacterial activity against gram-negative bacteria such as Escherichia coli, Klebsiella Pneumoniae, Pseudomonas aeruginosa, and Escherichia coliHB101, and gram-positive bacteria such as Staphylococcus aureus were measured at various peptide concentrations. Two of our synthetic tetrapeptide fragments containing Gly and Arg were efficiently killed with gram-positive bacteria, Staphylococcus aureus, at the concentration level of 200 microg/mL.