Interlocking activities of DNA polymerase ß in the base excision repair pathway.

SignificanceBase excision repair (BER) is one of the major DNA repair pathways used to fix a myriad of cellular DNA lesions. The enzymes involved in BER, including DNA polymerase ß (Polß), have been identified and characterized, but how they act together to efficiently perform BER has not been fully understood. Through gel electrophoresis, mass spectrometry, and kinetic analysis, we discovered that the two enzymatic activities of Polß can be interlocked, rather than functioning independently from each other, when processing DNA intermediates formed in BER. The finding prompted us to hypothesize a modified BER pathway. Through conventional and time-resolved X-ray crystallography, we solved 11 high-resolution crystal structures of cross-linked Polß complexes and proposed a detailed chemical mechanism for Polß's 5'-deoxyribose-5-phosphate lyase activity.

Elongation Factor P Modulates the Incorporation of Structurally Diverse Noncanonical Amino Acids into Escherichia coli Dihydrofolate Reductase.

The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide/protein structure and mechanism. In addition to numerous nonproteinogenic α-l-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. This finding prompted us to investigate the properties of this protein factor with a broad variety of structurally diverse amino acid analogues using an optimized suppressor tRNAPro that we designed. While these analogues can generally be incorporated into proteins only in systems containing modified ribosomes specifically selected for their incorporation, we found that EF-P could significantly enhance their incorporation into model protein dihydrofolate reductase using wild-type ribosomes. Plausibly, the increased yields observed in the presence of structurally diverse amino acid analogues may result from the formation of a stabilized ribosomal complex in the presence of EF-P that provides more favorable conditions for peptide bond formation. This finding should enable the facile incorporation of a much broader structural variety of amino acid analogues into proteins and peptides using native ribosomes.

Facilitated synthesis of proteins containing modified dipeptides.

The elaboration of peptides and proteins containing non-proteinogenic amino acids has been realized using several complementary strategies, including chemical synthesis, ribosome- or non-ribosome-mediated elaboration, intein-mediated polypeptide rearrangements, or some combination of these strategies. All of these have strengths and limitations, and significant efforts have been focused on minimizing the effects of limitations, to improve the overall utility of individual strategies. Our laboratory has studied ribosomally mediated peptide and protein synthesis involving a wide variety of non-proteinogenic amino acids, and in recent years we have described a novel strategy for the selection of modified bacterial ribosomes. These modified ribosomes have enabled the incorporation into peptides and proteins of numerous modified amino acids not accessible using wild-type ribosomes. This has included d-amino acids, ß-amino acids, dipeptides and dipeptidomimetic species, as well as phosphorylated amino acids. Presently, we have considered novel strategies for incorporating non-proteinogenic amino acids in improved yields. This has included the incorporation of non-proteinogenic amino acids into contiguous positions, a transformation known to be challenging. We demonstrate the preparation of this type of protein modification by utilizing a suppressor tRNACUA activated with a dipeptide consisting of two identical non-proteinogenic amino acids, in the presence of modified ribosomes selected to recognize such dipeptides. Also, we demonstrate that the use of bis-aminoacylated suppressor tRNAs, shown previously to increase protein yields significantly in vitro, can be extended to the use of non-proteinogenic amino acids.

Alteration of Transcriptional Regulator Rob In Vivo: Enhancement of Promoter DNA Binding and Antibiotic Resistance in the Presence of Nucleobase Amino Acids.

The identification of proteins that bind selectively to nucleic acid sequences is an ongoing challenge. We previously synthesized nucleobase amino acids designed to replace proteinogenic amino acids; these were incorporated into proteins to bind specific nucleic acids predictably. An early example involved selective cell free binding of the hnRNP LL RRM1 domain to its i-motif DNA target via Watson-Crick-like H-bonding interactions. In this study, we employ the X-ray crystal structure of transcriptional regulator Rob bound to its micF promoter, which occurred without DNA distortion. Rob proteins modified in vivo with nucleobase amino acids at position 40 exhibited altered DNA promoter binding, as predicted on the basis of their Watson-Crick-like H-bonding interactions with promoter DNA A-box residue Gua-6. Rob protein expression ultimately controls phenotypic changes, including resistance to antibiotics. Although Rob proteins with nucleobase amino acids were expressed in Escherichia coli at levels estimated to be only a fraction of that of the wild-type Rob protein, those modified proteins that bound to the micF promoter more avidly than the wild type in vitro also produced greater resistance to macrolide antibiotics roxithromycin and clarithromycin in vivo, as well as the ß-lactam antibiotic ampicillin. Also demonstrated is the statistical significance of altered DNA binding and antibiotic resistance for key Rob analogues. These preliminary findings suggest the ultimate utility of nucleobase amino acids in altering and controlling preferred nucleic acid target sequences by proteins, for probing molecular interactions critical to protein function, and for enhancing phenotypic changes in vivo by regulatory protein analogues.

Effects of Nucleobase Amino Acids on the Binding of Rob to Its Promoter DNA: Differential Alteration of DNA Affinity and Phenotype.

Nucleic acid binding proteins have been studied extensively, but the nature of the interactions that control their affinity, selectivity, and DNA and RNA functions is still not well understood. To understand the nature and functional consequences of such interactions, we introduced nucleobase amino acids at specific positions of the transcriptional regulator Rob protein in vivo and succeeded in demonstrating that an alteration of the protein-DNA affinity can affect specific phenotypes associated with Rob protein-DNA interactions. Previously, we inserted different nucleobase amino acids in lieu of Arg40; this residue is known (via X-ray crystallography) to interact with the micF DNA promoter A-box residue Gua6. The interactions predominantly involved Watson-Crick-like H bonding. The present study focused primarily on the micF DNA promoter B-box; the crystallographically determined interaction involves H bonding between the agmatine moiety of Arg90 within an HTH motif of Rob and a phosphate oxygen anion to the 5'-side of Thy14. We had two main goals, the first of which was to demonstrate enhanced Rob-binding to the micF promoter DNA and the functional consequences resulting from the interaction of micF DNA with Rob analogues containing Arg90 nucleobase mimics. The second was to explore the possible functional consequences of enhancing the protein-DNA affinity with nucleobase replacements, which mechanistically mediate interactions differently than those reported to be operative for specific protein-DNA interactions. Nucleobase replacement at position 90 with Arg isosteres enhanced the Rob protein-micF DNA affinity in parallel with increasing antibiotic and Hg2+ resistance, while aromatic amino acid replacements increased the affinity but not the antibiotic or Hg2+ resistance. The demonstration of an increased affinity through strong base stacking interactions was notable.

Protein synthesis with conformationally constrained cyclic dipeptides.

We have synthesized several conformationally constrained dipeptide analogues as possible substrates for incorporation into proteins. These have included three cyclic dipeptides formed from Boc derivatives of 2,4-diaminobutyric acid, ornithine and lysine, having 5-, 6-, and 7-membered lactam rings, respectively. These dipeptides were used to activate a suppressor tRNA transcript, the latter of which had been prepared by in vitro transcription. Using modified E. coli ribosomes described previously, these activated suppressor tRNAs enabled the incorporation of the three cyclic dipeptides into dihydrofolate reductase (DHFR) at positions 18 and 49. The suppression yields increased with increasing lactam ring size and were found to proceed in suppression yields ranging from 3.4 to 8.9% at two different protein sites for the 5-, 6- and 7-membered lactam dipeptides. The greater facility of incorporation of the 7-membered lactam prompted us to prepare two 7-membered cyclic acylhydrazides (4 and 5) by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)-mediated cyclization of amino acids having selectively protected hydrazine functional groups in their side chains. In common with the lactam dipeptides, acylhydrazide dipeptides 4 and 5 could be used to activate the same suppressor tRNA transcript and to incorporate the cyclic dipeptides into DHFR. They were incorporated into the same two DHFR sites in suppression yields ranging from 8.3 to 11.2%.

An assay for DNA polymerase ß lyase inhibitors that engage the catalytic nucleophile for binding.

DNA polymerase ß (Pol ß) repairs cellular DNA damage. When such damage is inflicted upon the DNA in tumor cells treated with DNA targeted antitumor agents, Pol ß thus diminishes their efficacy. Accordingly, this enzyme has long been a target for antitumor therapy. Although numerous inhibitors of the lyase activity of the enzyme have been reported, none has yet proven adequate for development as a therapeutic agent. In the present study, we developed a new strategy to identify lyase inhibitors that critically engage the lyase active site primary nucleophile Lys72 as part of the binding interface. This involves a parallel evaluation of the effect of the inhibitors on the wild-type DNA polymerase ß (Pol ß) and Pol ß modified with a lysine analogue at position 72. A model panel of five structurally diverse lyase inhibitors identified in our previous studies (only one of which has been published) with unknown modes of binding were used for testing, and one compound, cis-9,10-epoxyoctadecanoic acid, was found to have the desired characteristics. This finding was further corroborated by in silico docking, demonstrating that the predominant mode of binding of the inhibitor involves an important electrostatic interaction between the oxygen atom of the epoxy group and Nε of the main catalytic nucleophile, Lys72. The strategy, which is designed to identify compounds that engage certain structural elements of the target enzyme, could find broader application for identification of ligands with predetermined sites of binding.

Expanding the Scope of Protein Synthesis Using Modified Ribosomes.

The ribosome produces all of the proteins and many of the peptides present in cells. As a macromolecular complex composed of both RNAs and proteins, it employs a constituent RNA to catalyze the formation of peptide bonds rapidly and with high fidelity. Thus, the ribosome can be argued to represent the key link between the RNA World, in which RNAs were the primary catalysts, and present biological systems in which protein catalysts predominate. In spite of the well-known phylogenetic conservation of rRNAs through evolutionary history, rRNAs can be altered readily when placed under suitable pressure, e.g. in the presence of antibiotics which bind to functionally critical regions of rRNAs. While the structures of rRNAs have been altered intentionally for decades to enable the study of their role(s) in the mechanism of peptide bond formation, it is remarkable that the purposeful alteration of rRNA structure to enable the elaboration of proteins and peptides containing noncanonical amino acids has occurred only recently. In this Perspective, we summarize the history of rRNA modifications, and demonstrate how the intentional modification of 23S rRNA in regions critical for peptide bond formation now enables the direct ribosomal incorporation of d-amino acids, ß-amino acids, dipeptides and dipeptidomimetic analogues of the normal proteinogenic l-α-amino acids. While proteins containing metabolically important functional groups such as carbohydrates and phosphate groups are normally elaborated by the post-translational modification of nascent polypeptides, the use of modified ribosomes to produce such polymers directly is also discussed. Finally, we describe the elaboration of such modified proteins both in vitro and in bacterial cells, and suggest how such novel biomaterials may be exploited in future studies.

In Cellulo Synthesis of Proteins Containing a Fluorescent Oxazole Amino Acid.

Genetic code expansion has enabled many noncanonical amino acids to be incorporated into proteins in vitro and in cellulo. These have largely involved α-l-amino acids, reflecting the substrate specificity of natural aminoacyl-tRNA synthetases and ribosomes. Recently, modified E. coli ribosomes, selected using a dipeptidylpuromycin analogue, were employed to incorporate dipeptides and dipeptidomimetics. Presently, we report the in cellulo incorporation of a strongly fluorescent oxazole amino acid (lacking an asymmetric center or α-amino group) by using modified ribosomes and pyrrolysyl-tRNA synthetase (PylRS). Initially, a plasmid encoding the RRM1 domain of putative transcription factor hnRNP LL was cotransformed with plasmid pTECH-Pyl-OP in E. coli cells, having modified ribosomes able to incorporate dipeptides. Cell incubation in a medium containing oxazole 2 resulted in the elaboration of RRM1 containing the oxazole. Green fluorescent protein, previously expressed in vitro with several different oxazole amino acids at position 66, was also expressed in cellulo containing oxazole 2; the incorporation was verified by mass spectrometry. Finally, oxazole 2 was incorporated into position 13 of MreB, a bacterial homologue of eukaryotic cytoskeletal protein actin F. Modified MreB expressed in vitro and in cellulo comigrated with wild type. E. coli cells expressing the modified MreB were strongly fluorescent and retained the E. coli cell rod-like phenotype. For each protein studied, the incorporation of oxazole 2 strongly increased oxazole fluorescence, suggesting its potential utility as a protein tag. These findings also suggest the feasibility of dramatically increasing the repertoire of amino acids that can be genetically encoded for protein incorporation in cellulo.

Study of the Lyase Activity of Human DNA Polymerase ß Using Analogues of the Intermediate Schiff Base Complex.

DNA polymerase ß (Pol ß) participates in mammalian base excision repair. The enzyme has a two-domain architecture, reflecting its dual functionality. The polymerase activity, which replaces damaged nucleosides removed during an initial excision process, is within the C-terminal 31 kDa domain, while the N-terminal 8 kDa domain participates in a lyase function, working to remove a 5'-deoxyribose phosphate (5'-dRP) moiety from the damaged DNA substrate. The currently accepted mechanism of the lyase reaction involves a transient covalent enzyme-DNA intermediate in the form of a Schiff base connecting Lys72 of the enzyme with the 5'-dRP moiety. The Schiff base intermediate is resolved via a ß-elimination reaction, initiated by abstraction of a C2'-H atom from the 5'-dRP moiety. Presently, we describe the preparation of three Pol ß enzymes modified at position 72 with aminooxy or hydrazinyl analogues of lysine. These enzymes form transient covalent bonds with the 5'-dRP moiety of the damaged DNA, in the form of an oxime or hydrazone, respectively. Both types of enzyme DNA intermediates are ultimately resolved by the lyase activities of each of the modified enzymes. Unsurprisingly, the formation and resolution of these E-S complexes proceed with diminished kinetics, and with an altered pH profile. The performed experiments provide additional support for Schiff base formation as an obligatory intermediate on the pathway to DNA repair, as well as for the proposed participation of Lys72 in effecting opening of the 5'-dRP ring via protonation of the ring oxygen atom, and for complex resolution via a ß-elimination reaction.

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich's ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.

Oligomeric amyloid ß preferentially targets neuronal and not glial mitochondrial-encoded mRNAs.

INTRODUCTION: Our laboratories have demonstrated that accumulation of oligomeric amyloid ß (OAß) in neurons is an essential step leading to OAß-mediated mitochondrial dysfunction. METHODS: Alzheimer's disease (AD) and matching control hippocampal neurons, astrocytes, and microglia were isolated by laser-captured microdissection from the same subjects, followed by whole-transcriptome sequencing. Complementary in vitro work was performed in OAß-treated differentiated SH-SY5Y, followed by the use of a novel CoQ10 analogue for protection. This compound is believed to be effective both in suppressing reactive oxygen species and also functioning in mitochondrial electron transport. RESULTS: We report decreases in the same mitochondrial-encoded mRNAs in Alzheimer's disease laser-captured CA1 neurons and in OAß-treated SH-SY5Y cells, but not in laser-captured microglia and astrocytes. Pretreatment with a novel CoQ10 analogue, protects neuronal mitochondria from OAß-induced mitochondrial changes. DISCUSSION: Similarity of expression changes in neurons from Alzheimer's disease brain and neuronal cells treated with OAß, and the effect of a CoQ10 analogue on the latter, suggests a pretreatment option to prevent OAß toxicity, long before the damage is apparent.

Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase ß with Unnatural Lysine Analogues.

DNA polymerase ß (Pol ß) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5'-deoxyribose phosphate (dRP) lyase and "gap-filling" DNA polymerase activities. The lyase reaction is believed to occur via a ß-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the ε-amino group of Lys72. To probe the steric constraints on the formation and subsequent resolution of the putative Schiff base intermediate within the lyase catalytic pocket, Lys72 was replaced with each of several nonproteinogenic lysine analogues. The modified Pol ß enzymes were produced by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNACUA transcript, suppression of the UAG codon in the transcribed mRNA led to elaboration of full length Pol ß having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of N-methyllysine (4) affected mainly the stability of the Schiff base intermediate and resulted in relatively moderate inhibition of lyase activity and BER. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine (6) at position 72, apparently shifted the amino group to a position less favorable for Schiff base formation. Interestingly, this effect was attenuated when the side chain was elongated by replacing one side-chain methylene group with a bridging S atom (thialysine, 2). In comparison, replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an ε-amino group (homoarginine, 5) or a sterically constrained secondary amine (piperidinylalanine, 3) led to almost complete suppression of dRP excision activity and the ability of Pol ß to support BER. These results help to define the tolerance of Pol ß to subtle local structural and functional alterations.

Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids.

Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

Incorporation of Phosphorylated Tyrosine into Proteins: In Vitro Translation and Study of Phosphorylated IκB-α and Its Interaction with NF-κB.

Phosphorylated proteins play important roles in the regulation of many different cell networks. However, unlike the preparation of proteins containing unmodified proteinogenic amino acids, which can be altered readily by site-directed mutagenesis and expressed in vitro and in vivo, the preparation of proteins phosphorylated at predetermined sites cannot be done easily and in acceptable yields. To enable the synthesis of phosphorylated proteins for in vitro studies, we have explored the use of phosphorylated amino acids in which the phosphate moiety bears a chemical protecting group, thus eliminating the negative charges that have been shown to have a negative effect on protein translation. Bis-o-nitrobenzyl protection of tyrosine phosphate enabled its incorporation into DHFR and IκB-α using wild-type ribosomes, and the elaborated proteins could subsequently be deprotected by photolysis. Also investigated in parallel was the re-engineering of the 23S rRNA of Escherichia coli, guided by the use of a phosphorylated puromycin, to identify modified ribosomes capable of incorporating unprotected phosphotyrosine into proteins from a phosphotyrosyl-tRNACUA by UAG codon suppression during in vitro translation. Selection of a library of modified ribosomal clones with phosphorylated puromycin identified six modified ribosome variants having mutations in nucleotides 2600-2605 of 23S rRNA; these had enhanced sensitivity to the phosphorylated puromycin. The six clones demonstrated some sequence homology in the region 2600-2605 and incorporated unprotected phosphotyrosine into IκB-α using a modified gene having a TAG codon in the position corresponding to amino acid 42 of the protein. The purified phosphorylated protein bound to a phosphotyrosine specific antibody and permitted NF-κB binding to a DNA duplex sequence corresponding to its binding site in the IL-2 gene promoter. Unexpectedly, phosphorylated IκB-α also mediated the exchange of exogenous DNA into an NF-κB-cellular DNA complex isolated from the nucleus of activated Jurkat cells.

Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.

Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.

Lipophilic methylene violet analogues as modulators of mitochondrial function and dysfunction.

In an effort to identify methylene blue analogues having improved antioxidant activity, a series of new methylene violet analogues have been designed and synthesized. The analogues were prepared following a synthetic route that is more efficient than the previously reported methods, both in terms of yield and purity of the final products. The route involves the Smiles rearrangement as one of the crucial steps. Smiles rearrangement of suitably substituted diphenyl sulfide intermediates afforded the corresponding phenothiazine analogues in high yields, which were subsequently converted to the final products. The methylene violet analogues were evaluated for their ability to preserve mitochondrial function in Friedreich's ataxia (FRDA) lymphocytes. The analogues were shown to be efficient ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I. The analogues also preserved mitochondrial membrane potential and augmented ATP production. The analogues were found to be better antioxidants than the parent compounds methylene blue and methylene violet.

Influence of substituent heteroatoms on the cytoprotective properties of pyrimidinol antioxidants.

Recently, we described the optimization of novel pyrimidinol-based antioxidants as potential therapeutic molecules for targeting mitochondrial diseases. That study focused on improving the potency and metabolic stability of pyrimidinol antioxidants. This led us to consider the possibility of altering the positions of the exocyclic alkoxy and alkylamino substituents on the pyrimidinol scaffold. Twelve new analogues were prepared and their biological activities were investigated. The metabolic stability of the prepared regioisomers was also assessed in vitro using bovine liver microsomes. Unexpectedly, the 2-alkoxy-4-alkylamino substituted pyrimidinol antioxidants were found to have properties in protecting mitochondrial function superior to the isomeric 4-alkoxy-2-alkylamino substituted pyrimidinols evaluated in all earlier studies. This observation suggests a possible mode of action involving the intermediacy of an ortho-iminoquinone, a species not previously associated with mitochondrial respiratory chain function.

Inhibition of Human Cancer Cell Growth by Analogues of Antimycin A.

In a recent study, several new derivatives of antimycin A (AMA) were produced by means of a novel transacylation reaction, and these were shown to mediate selective toxicity toward cultured A549 human lung epithelial adenocarcinoma cells, as compared with WI-38 normal human lung fibroblasts. The purpose of our study was to investigate whether the analogues all expressed their cytotoxicity by the same mechanism. This was done by studying the effects of the compounds in several types of cell lines. In comparison with 2-O-methylantimycin, which acts at the locus of Bcl-2, none of the new derivatives exhibited a difference in cytotoxicity toward cells expressing different levels of Bcl-2. In cell lines that over- or underexpress estrogen or Her2 receptors, AMA analogue 2 exhibited Her2 receptor dependency at low concentration. Three compounds (1, 4, and 6) exhibited concentration-dependent increases in reactive oxygen species, with 6 being especially potent. Compounds 5 and 6 diminished mitochondrial membrane potential more potently than AMA, and 1 also displayed enhanced activity relative to 2-4. Interestingly, only 1 and AMA displayed strong inhibition of the respiratory chain, as measured by monitoring NADH (reduced nicotinamide adenine dinucleotide) oxidase. Because four of the analogues have positively charged substituents, two of these (4 and 6) were studied to see whether the observed effects were due to much higher level of accumulation within the mitochondria. Their presence in the mitochondria was not dramatically enhanced. Neither of the two presently characterized mechanisms of cell killing by AMA can fully account for the observed results.

Nuclear but not mitochondrial-encoded oxidative phosphorylation genes are altered in aging, mild cognitive impairment, and Alzheimer's disease.

INTRODUCTION: We have comprehensively described the expression profiles of mitochondrial DNA and nuclear DNA genes that encode subunits of the respiratory oxidative phosphorylation (OXPHOS) complexes (I-V) in the hippocampus from young controls, age matched, mild cognitively impaired (MCI), and Alzheimer's disease (AD) subjects. METHODS: Hippocampal tissues from 44 non-AD controls (NC), 10 amnestic MCI, and 18 AD cases were analyzed on Affymetrix Hg-U133 plus 2.0 arrays. RESULTS: The microarray data revealed significant down regulation in OXPHOS genes in AD, particularly those encoded in the nucleus. In contrast, there was up regulation of the same gene(s) in MCI subjects compared to AD and ND cases. No significant differences were observed in mtDNA genes identified in the array between AD, ND, and MCI subjects except one mt-ND6. DISCUSSION: Our findings suggest that restoration of the expression of nuclear-encoded OXPHOS genes in aging could be a viable strategy for blunting AD progression.