Mikhail S. Shchepinov

Molecular interactions on microarrays

The structural features of nucleic acid probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications for the hybridization process. We discuss how arrays of oligonucleotides provide powerful tools to study the molecular basis of these interactions on a scale which is impossible using conventional analysis.

Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis

Significance Ferroptosis is a regulated form of cell death induced by loss of glutathione peroxidase 4 (GPX4) phospholipid peroxidase activity and lethal accumulation of reactive oxygen species. Small-molecule inhibitors of GPX4 induce ferroptosis; however, the interaction between these inhibitors and GPX4 has remained elusive, as has the identity of the reactive oxygen species that drive execution of ferroptosis. We identified here a ligand-binding site on GPX4 and determined the specific lipids oxidized during ferroptosis. We further identified two key drivers of lipid peroxidation during ferroptosis: lipoxygenases and phosphorylase kinase G2. These findings reveal a previously enigmatic mechanism of ferroptotic lipid peroxide generation and suggest new strategies for pharmacological control of ferroptosis and diseases associated with this mode of cell death. Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts.

Small Amounts of Isotope-reinforced Polyunsaturated Fatty Acids Suppress Lipid Autoxidation

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast.

Trityl Tags for Encoding in Combinatorial Synthesis

Abstract New tags and an encoding strategy for combinatorial synthesis are described. Combinatorial libraries of short oligonucleotides attached to TentaGel beads were synthesised by a split-and-mix strategy using 5′-DMTr or 5′-Fmoc-protected nucleoside phosphoramidites. Trityl moieties with different masses were used to tag the nature and position of monomer units (bases) coupled at each step in the synthesis. Beads with a specific oligonucleotide were selected by hybridisation from combinatorial libraries. Tags orthogonal to the added nucleotides were produced by coupling amines of different molecular masses to an activated carboxyl group(s) on the trityl moiety. The tags may be released from the support by an acidic treatment or laser irradiation and then analysed by (MA)LDI-TOF. These properties make trityl-based tags promising for encoding in strategies not involving strong acids, such as oligonucleotide and peptide synthesis and small molecule combinatorial libraries.

Unusual kinetic isotope effects of deuterium reinforced polyunsaturated fatty acids in tocopherol-mediated free radical chain oxidations.

Substitution of -CD2- at the reactive centers of linoleic and linolenic acids reduces the rate of abstraction of D by a tocopheryl radical by as much as 36-fold, compared to the abstraction of H from a corresponding -CH2- center. This H atom transfer reaction is the rate-determining step in the tocopherol-mediated peroxidation of lipids in human low-density lipoproteins, a process that has been linked to coronary artery disease. The unanticipated large kinetic isotope effects reported here for the tocopherol-mediated oxidation of linoleic and linolenic acids and esters suggests that tunneling makes this process favorable.

Isotope-reinforced polyunsaturated fatty acids protect mitochondria from oxidative stress.

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure-activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D1-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D2-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20-50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function.

DESIGN OF MULTIDYE SYSTEMS FOR FRET-BASED APPLICATIONS

A new solid phase approach, based on orthogonal protective group strategy utilizing Fmoc and DMTr groups, was used to assemble linear polymeric chains with pending groups at desired locations. A compound synthesized using four different fluorophores with consequently overlapping absorption and emission spectra (pyrene, perylene, fluorescein and TAMRA) was shown to fluoresce at 570 nm when excited at 330 nm, demonstrating sequential energy transfer across four chromophores.

Tritylisation of pyrene, perylene and coronene: a new family of switchable fluorescent labels

Abstract The synthesis of novel fluorescent labels based on pyrene, perylene and coronene is described. Due to the trityl-type structure, their fluorescence may be reversibly switched on and off by changing the pH. This property can be used to expand the palette of fluorophores available for multicolour DNA detection on DNA chips. Some FRET and surface chemistry applications are also demonstrated.

Deuterium Protection of Polyunsaturated Fatty Acids against Lipid Peroxidation: A Novel Approach to Mitigating Mitochondrial Neurological Diseases

Oxidative stress is associated with numerous neurological disorders. Mitochondrial malfunction contributes to generation of reactive oxygen species (ROS) in proximity to mitochondrial membranes rich in polyunsaturated fatty acids (PUFAs). ROS initiate PUFA autoxidation, a chain reaction that alters membrane fluidity and promotes toxic reactive carbonyl products (RCP) that damage proteins and DNA. Due to the stochastic nature of ROS generation, increased antioxidants cannot prevent such injury. However, deuterium-for-hydrogen substitution at bis-allylic sites reduces the rate-limiting step of autoxidation and results in inhibition of the subsequent PUFA oxidation chain reaction, decreasing toxic RCP levels. We propose a novel approach, using such isotopic reinforcement, to alleviate oxidative injury and have pre-clinical evidence demonstrating neuroprotection in an oxidative stress model of Parkinson’s disease. We hypothesize that site-specific PUFA deuteration will mitigate progression of degeneration in other disorders that manifest oxidative injury either as a primary or secondary insult.

Control of lysyl oxidase activity through site-specific deuteration of lysine.

Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as β-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d(2)-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V(max)/K(m) as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D(2)Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies.