Data-driven approach for the delineation of the irritative zone in epilepsy in MEG.

The reliable identification of the irritative zone (IZ) is a prerequisite for the correct clinical evaluation of medically refractory patients affected by epilepsy. Given the complexity of MEG data, visual analysis of epileptiform neurophysiological activity is highly time consuming and might leave clinically relevant information undetected. We recorded and analyzed the interictal activity from seven patients affected by epilepsy (Vectorview Neuromag), who successfully underwent epilepsy surgery (Engel > = II). We visually marked and localized characteristic epileptiform activity (VIS). We implemented a two-stage pipeline for the detection of interictal spikes and the delineation of the IZ. First, we detected candidate events from peaky ICA components, and then clustered events around spatio-temporal patterns identified by convolutional sparse coding. We used the average of clustered events to create IZ maps computed at the amplitude peak (PEAK), and at the 50% of the peak ascending slope (SLOPE). We validated our approach by computing the distance of the estimated IZ (VIS, SLOPE and PEAK) from the border of the surgically resected area (RA). We identified 25 spatiotemporal patterns mimicking the underlying interictal activity (3.6 clusters/patient). Each cluster was populated on average by 22.1 [15.0-31.0] spikes. The predicted IZ maps had an average distance from the resection margin of 8.4 ± 9.3 mm for visual analysis, 12.0 ± 16.5 mm for SLOPE and 22.7 ±. 16.4 mm for PEAK. The consideration of the source spread at the ascending slope provided an IZ closer to RA and resembled the analysis of an expert observer. We validated here the performance of a data-driven approach for the automated detection of interictal spikes and delineation of the IZ. This computational framework provides the basis for reproducible and bias-free analysis of MEG recordings in epilepsy.

Synthesis of the new nucleoside 5'-alpha-iminophosphates using Staudinger reaction.

Efficient protocols were developed for the synthesis of a new compounds - nucleoside 5'-α-iminophosphates using the Staudinger reaction. These substances are alpha-phosphate mimetic nucleotide in which an oxygen atom is replaced by a corresponding imino (=N-R)-group. Various 5'-iminomonophosphates of nucleosides were obtained. A chemical method for the synthesis of triphosphate derivatives based on the iminomonophosphates has been designed. Thymidine 5'-(1,3-dimethylimidazolidin-2-ylidene)-triphosphate (ppp(DMI)T) was synthesized, its hydrolytic stability and substrate properties in relation to some DNA polymerases was firstly studied. It was shown that ppp(DMI)T can serve as substrate for enzyme catalyzed template-independent DNA synthesis by human terminal deoxynucleotidyltransferase TdT.

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes.

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5' to an AP-site; can recognize and process some damaged nucleosides; and possess 3'-phosphodiesterase, 3'-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions' concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3'-5' exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.

Corrosion Behavior of Candidate Functional Materials for Molten Salts Reactors in LiF-NaF-KF Containing Actinide Fluoride Imitators.

Molten fluorides of alkali metals are considered a technological medium for molten salt reactors (MSRs). However, these media are known to be extremely corrosive. The successful implementation of high-temperature technological devices using molten alkali metal fluorides requires the selection of such structural materials that have high corrosion resistance in melts with compositional characteristic of MSRs. In this research, the corrosion behavior of 12Cr18Ni10Ti steel, the alloy Ni60Cr20Mo15, and the alloy Monel 404 (Ni50Cu50) was investigated in the LiF-NaF-KF eutectic melt, containing additions of CeF3 and NdF3 from 0 to 5 wt.% as imitator fluorides of actinides in an inert argon atmosphere at 550 °C for 100 h. Gravimetry, energy-dispersive X-ray (EDX) microanalysis of surfaces and cross-section of samples, and ICP-MS were used to establish the corrosion behavior of the investigated alloys. Corrosion resistance of the studied materials was found to decrease in a row from Monel 404 > Hastelloy C2000 > 12Cr18Ni10Ti. The addition of cerium fluoride into the melt resulted in the additional etching of the alloy surface. The addition of neodymium fluoride resulted in the formation of the point/inter-crystalline corrosion damages in the sample bulk. The samples of steel 12Cr18Ni10Ti were subjected to local cracking corrosion. The austenitic nickel-based alloys suffered specific local corrosion with formation of subsurface voids. Excellent corrosion resistance of the Monel alloy under the test conditions was found.

Pre-Steady-State Kinetics of the SARS-CoV-2 Main Protease as a Powerful Tool for Antiviral Drug Discovery.

The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, Mpro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific Mpro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of Mpro-targeting molecules is urgently needed. Here, we propose a pre-steady-state kinetic analysis of the interaction of Mpro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type Mpro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of Mpro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre-steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A Mpro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A Mpro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 Mpro.

Common Kinetic Mechanism of Abasic Site Recognition by Structurally Different Apurinic/Apyrimidinic Endonucleases.

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site-containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes' intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.

Lesion Recognition and Cleavage of Damage-Containing Quadruplexes and Bulged Structures by DNA Glycosylases.

Human telomeres as well as more than 40% of human genes near the promoter regions have been found to contain the sequence that may form a G-quadruplex structure. Other non-canonical DNA structures comprising bulges, hairpins, or bubbles may have a functionally important role during transcription, replication, or recombination. The guanine-rich regions of DNA are hotspots of oxidation that forms 7,8-dihydro-8-oxoguanine, thymine glycol, and abasic sites: the lesions that are handled by the base excision repair pathway. Nonetheless, the features of DNA repair processes in non-canonical DNA structures are still poorly understood. Therefore, in this work, a comparative analysis of the efficiency of the removal of a damaged nucleotide from various G-quadruplexes and bulged structures was performed using endonuclease VIII-like 1 (NEIL1), human 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III (NTH1), and prokaryotic formamidopyrimidine-DNA glycosylase (Fpg), and endonuclease VIII (Nei). All the tested enzymes were able to cleave damage-containing bulged DNA structures, indicating their important role in the repair process when single-stranded DNA and intermediate non-B-form structures such as bubbles and bulges are formed. Nevertheless, our results suggest that the ability to cleave damaged quadruplexes is an intrinsic feature of members of the H2tH structural family, suggesting that these enzymes can participate in the modulation of processes controlled by the formation of quadruplex structures in genomic DNA.

Activity of Human Apurinic/Apyrimidinic Endonuclease APE1 Toward Damaged DNA and Native RNA With Non-canonical Structures.

The primary role of apurinic/apyrimidinic (AP) endonuclease APE1 in human cells is the cleavage of the sugar phosphate backbone 5' to an AP site in DNA to produce a single-strand break with a 5'-deoxyribose phosphate and 3'-hydroxyl end groups. APE1 can also recognize and incise some damaged or modified nucleotides and possesses some minor activities: 3'-5' exonuclease, 3'-phosphodiesterase, 3'-phosphatase, and RNase H. A molecular explanation for the discrimination of structurally different substrates by the single active site of the enzyme remains elusive. Here, we report a mechanism of target nucleotide recognition by APE1 as revealed by the results of an analysis of the APE1 process involving damaged DNA and native RNA substrates with non-canonical structures. The mechanism responsible for substrate specificity proved to be directly related to the ability of a target nucleotide to get into the active site of APE1 in response to an enzyme-induced DNA distortion.

The Role of Active-Site Plasticity in Damaged-Nucleotide Recognition by Human Apurinic/Apyrimidinic Endonuclease APE1.

Human apurinic/apyrimidinic (AP) endonuclease APE1 hydrolyzes phosphodiester bonds on the 5' side of an AP-site, and some damaged nucleotides such as 1,N6-ethenoadenosine (εA), α-adenosine (αA), and 5,6-dihydrouridine (DHU). To investigate the mechanism behind the broad substrate specificity of APE1, we analyzed pre-steady-state kinetics of conformational changes in DNA and the enzyme during DNA binding and damage recognition. Molecular dynamics simulations of APE1 complexes with one of damaged DNA duplexes containing εA, αA, DHU, or an F-site (a stable analog of an AP-site) revealed the involvement of residues Asn229, Thr233, and Glu236 in the mechanism of DNA lesion recognition. The results suggested that processing of an AP-site proceeds faster in comparison with nucleotide incision repair substrates because eversion of a small abasic site and its insertion into the active site do not include any unfavorable interactions, whereas the insertion of any target nucleotide containing a damaged base into the APE1 active site is sterically hindered. Destabilization of the α-helix containing Thr233 and Glu236 via a loss of the interaction between these residues increased the plasticity of the damaged-nucleotide binding pocket and the ability to accommodate structurally different damaged nucleotides. Nonetheless, the optimal location of εA or αA in the binding pocket does not correspond to the optimal conformation of catalytic amino acid residues, thereby significantly decreasing the cleavage efficacy for these substrates.

Human Tyrosyl-DNA Phosphodiesterase 1 Possesses Transphosphooligonucleotidation Activity With Primary Alcohols.

Human tyrosyl-DNA phosphodiesterase 1 (TDP1) belongs to the phospholipase D superfamily, whose members contain paired catalytic histidine and lysine residues within two conserved motifs and hydrolyze phosphodiester bonds. TDP1 is a DNA repair enzyme that processes 3' DNA end blocking lesions and a wide range of synthetic DNA adducts as a substrate. TDP1 hydrolyzes DNA-adducts via two coordinated SN2 nucleophilic attacks mediated by the action of two histidine residues and leads to the formation of the covalent intermediate. Hydrolysis of this intermediate is proposed to be carried out by a water molecule that is activated by the His493 residue acting as a general base. It was known that phospholipase D enzymes are able to catalyze not only hydrolysis but also a transphosphatidylation reaction in the presence of primary alcohols in which they transfer the substrate to the alcohol instead of water. Here, we first demonstrated that TDP1 is able to undergo a "transphosphooligonucleotidation" reaction, transferring the substrate residue to the alcohol, thus inducing the formation of covalent DNA adducts with different primary alcohol residues. Such adducts can be accumulated in the conditions of high concentration of alcohol. We demonstrated that glycerol residue was efficiently cleaved from the 3'-end by TDP1 but not by its mutant form associated with the disease spinocerebellar ataxia with axonal neuropathy. Therefore, the second reaction step can be carried out not only by a water molecule but also by the other small nucleophilic molecules, e.g., glycerol and ethanol. Thus, in some cases, TDP1 can be regarded not only as a repair enzyme but also as a source of DNA damage especially in the case of mutation. Such damages can make a negative contribution to the stability of cell vitality.

Roles of Active-Site Amino Acid Residues in Specific Recognition of DNA Lesions by Human 8-Oxoguanine-DNA Glycosylase (OGG1).

Human 8-oxoguanine-DNA glycosylase (hOGG1) possesses very high specificity for 8-oxoguanine (oxoG), even though this damaged base differs from normal guanine by only two atoms. Our aim was to determine the roles of certain catalytically important amino acid residues in the hOGG1 enzymatic pathway and describe their involvement in the mechanism of DNA lesion recognition. Molecular dynamic simulation and pre-steady-state fluorescence kinetics were performed to analyze the conformational behavior of wild-type hOGG1 and mutants G42S, D268A, and K249Q, as well as damaged and undamaged DNA. A loss of electrostatic interactions in the K249Q mutant leads to the disruption of specific contacts in the active site of the enzyme and the loss of catalytic activity. The absence of residue Asp-268 abrogates the ability of the enzyme to fully flip out the oxoG base from the double helix, thereby disrupting proper positioning of the damaged base in the active site. Furthermore, substitution of Gly-42 with Ser, which forms a damage-specific H-bond with the N7 atom of the oxoG base, creates a stable H-bond between N7 of undamaged G and Oγ of Ser-42. Nevertheless, positioning of the undamaged base in the active site is unsuitable for catalytic hydrolysis of the N-glycosidic bond.

Substrate specificity of human apurinic/apyrimidinic endonuclease APE1 in the nucleotide incision repair pathway.

Human apurinic/apyrimidinic (AP) endonuclease APE1 catalyses the hydrolysis of phosphodiester bonds on the 5' side of an AP-site (in the base excision repair pathway) and of some damaged nucleotides (in the nucleotide incision repair pathway). The range of substrate specificity includes structurally unrelated damaged nucleotides. Here, to examine the mechanism of broad substrate specificity of APE1, we performed pulsed electron-electron double resonance (PELDOR) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer (FRET) detection of DNA conformational changes during DNA binding and lesion recognition. Equilibrium PELDOR and kinetic FRET data revealed that DNA binding by APE1 leads to noticeable damage-dependent bending of a DNA duplex. Molecular dynamics simulations showed that the damaged nucleotide is everted from the DNA helix and placed into the enzyme's binding pocket, which is formed by Asn-174, Asn-212, Asn-229, Ala-230, Phe-266 and Trp-280. Nevertheless, no damage-specific contacts were detected between these amino acid residues in the active site of the enzyme and model damaged substrates containing 1,N6-ethenoadenosine, α-adenosine, 5,6-dihydrouridine or F-site. These data suggest that the substrate specificity of APE1 is controlled by the ability of a damaged nucleotide to flip out from the DNA duplex in response to an enzyme-induced DNA distortion.

Kinetic Features of 3'-5' Exonuclease Activity of Human AP-Endonuclease APE1.

Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5' to the damage. In addition to the AP-endonuclease activity, APE1 possesses 3'-5' exonuclease activity, which presumably is responsible for cleaning up nonconventional 3' ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3'-end nucleotide removal in the 3'-5' exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5' dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3' end of the short oligonucleotide. The impact of the 3'-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3'-5' exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3' nucleotide removal by APE1 in the 3'-5' exonuclease process is the release of the detached nucleotide from the enzyme's active site.

Pre-steady-state kinetic analysis of damage recognition by human single-strand selective monofunctional uracil-DNA glycosylase SMUG1.

In all organisms, DNA glycosylases initiate base excision repair pathways resulting in removal of aberrant bases from DNA. Human SMUG1 belongs to the superfamily of uracil-DNA glycosylases catalyzing the hydrolysis of the N-glycosidic bond of uridine and uridine lesions bearing oxidized groups at C5: 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), and 5-hydroxyuridine (5hoU). An apurinic/apyrimidinic (AP) site formed as the product of an N-glycosylase reaction is tightly bound to hSMUG1, thus inhibiting the downstream action of AP-endonuclease APE1. The steady-state kinetic parameters (kcat and KM; obtained from the literature) correspond to the enzyme turnover process limited by the release of hSMUG1 from the complex with the AP-site. In the present study, our objective was to carry out a stopped-flow fluorescence analysis of the interaction of hSMUG1 with a DNA substrate containing a dU:dG base pair to follow the pre-steady-state kinetics of conformational changes in both molecules. A comparison of kinetic data obtained by means of Trp and 2-aminopurine fluorescence and Förster resonance energy transfer (FRET) detection allowed us to elucidate the stages of specific and nonspecific DNA binding, to propose the mechanism of damaged base recognition by hSMUG1, and to determine the true rate of the catalytic step. Our results shed light on the kinetic mechanism underlying the initiation of base excision repair by hSMUG1 using the "wedge" strategy for DNA lesion search.

Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site.

Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII.

Escherichia coli endonuclease VIII (Endo VIII) is a DNA glycosylase with substrate specificity for a wide range of oxidatively damaged pyrimidine bases. Endo VIII catalyzes hydrolysis of the N-glycosidic bond and ß, δ-elimination of 3'- and 5'-phosphate groups of an apurinic/apyrimidinic site. Single mutants of Endo VIII L70S, L70W, Y71W, F121W, F230W, and P253W were analyzed here with the aim to elucidate the kinetic mechanism of protein conformational adjustment during damaged-nucleotide recognition and catalytic-complex formation. F121W substitution leads to a slight reduction of DNA binding and catalytic activity. F230W substitution slows the rate of the δ-elimination reaction indicating that interaction of Phe230 with a 5'-phosphate group proceeds in the latest catalytic step. P253W Endo VIII has the same activity as the wild type (WT) enzyme. Y71W substitution slightly reduces the catalytic activity due to the effect on the later steps of catalytic-complex formation. Both L70S and L70W substitutions significantly decrease the catalytic activity, indicating that Leu70 plays an important role in the course of enzyme-DNA catalytic complex formation. Our data suggest that Leu70 forms contacts with DNA earlier than Tyr71 does. Therefore, most likely, Leu70 plays the role of a DNA lesion "sensor", which is used by Endo VIII for recognition of a DNA damage site.

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3'-end-blocking modifications, possesses DNA and RNA 3'-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3'-end of DNA have been used to prevent 3'-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3'-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3'-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3'-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.

Trimeprazine increases IRS2 in human islets and promotes pancreatic ß cell growth and function in mice.

The capacity of pancreatic ß cells to maintain glucose homeostasis during chronic physiologic and immunologic stress is important for cellular and metabolic homeostasis. Insulin receptor substrate 2 (IRS2) is a regulated adapter protein that links the insulin and IGF1 receptors to downstream signaling cascades. Since strategies to maintain or increase IRS2 expression can promote ß cell growth, function, and survival, we conducted a screen to find small molecules that can increase IRS2 mRNA in isolated human pancreatic islets. We identified 77 compounds, including 15 that contained a tricyclic core. To establish the efficacy of our approach, one of the tricyclic compounds, trimeprazine tartrate, was investigated in isolated human islets and in mouse models. Trimeprazine is a first-generation antihistamine that acts as a partial agonist against the histamine H1 receptor (H1R) and other GPCRs, some of which are expressed on human islets. Trimeprazine promoted CREB phosphorylation and increased the concentration of IRS2 in islets. IRS2 was required for trimeprazine to increase nuclear Pdx1, islet mass, ß cell replication and function, and glucose tolerance in mice. Moreover, trimeprazine synergized with anti-CD3 Abs to reduce the progression of diabetes in NOD mice. Finally, it increased the function of human islet transplants in streptozotocin-induced (STZ-induced) diabetic mice. Thus, trimeprazine, its analogs, or possibly other compounds that increase IRS2 in islets and ß cells without adverse systemic effects might provide mechanism-based strategies to prevent the progression of diabetes.

Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1.

Here, we used stopped-flow fluorescence techniques to conduct a comparative kinetic analysis of the conformational transitions in human apurinic/apyrimidinic endonuclease 1 (APE1) and in DNA containing an abasic site in the course of their interaction. Effects of monovalent (K(+)) and divalent (Mg(2+), Mn(2+), Ca(2+), Zn(2+), Cu(2+), and Ni(2+)) metal ions on DNA binding and catalytic stages were studied. It was shown that the first step of substrate binding (corresponding to formation of a primary enzyme-substrate complex) does not depend on the concentration (0.05-5.0 mM) or the nature of divalent metal ions. In contrast, the initial DNA binding efficiency significantly decreased at a high concentration (5-250 mM) of monovalent K(+) ions, indicating the involvement of electrostatic interactions in this stage. It was also shown that Cu(2+) ions abrogated the DNA binding ability of APE1, possibly, due to a strong interaction with DNA bases and the sugar-phosphate backbone. In the case of Ca(2+) ions, the catalytic activity of APE1 was lost completely with retention of binding potential. Thus, the enzymatic activity of APE1 is increased in the order Zn(2+) < Ni(2+) < Mn(2+) < Mg(2+). Circular dichroism spectra and calculation of the contact area between APE1 and DNA reveal that Mg(2+) ions stabilize the protein structure and the enzyme-substrate complex.

New oligonucleotide derivatives as unreactive substrate analogues and potential inhibitors of human apurinic/apyrimidinic endonuclease APE1.

Human apurinic/apyrimidinic endonuclease APE1 is one of the key enzymes of the base excision DNA repair system. The main biological function of APE1 is the hydrolysis of the phosphodiester bond on the 5'-side of an apurinic/apyrimidinic site (AP-site) to give the 5'-phosphate and 3'-hydroxyl group. It has long been known that AP-sites have mutagenic and cytotoxic effects and their accumulation in DNA is a potential hazard to the cell lifecycle. The structural and biochemical studies of APE1 are complicated by its high catalytic activity towards the AP-site and its cyclic or acyclic analogues. This work has focussed on the design, synthesis and analysis of oligonucleotide derivatives as potentially unreactive APE1 substrates. We have shown that the replacement of oxygen atoms in the phosphate group on the 5'-side from the AP-site analogue tetrahydrofuran (F) considerably decreases the rate of enzymatic hydrolysis of modified oligonucleotides. We have calculated that a N3'-P5' phosphoramidate linkage is hydrolysed about 30 times slower than the native phosphodiester bond while phosphorothioate or primary phosphoramidate linkages are cleaved more than three orders of magnitude slower. The value of IC50 of the oligonucleotide duplex containing a primary phosphoramidate linkage is 2.5 × 10(-7) M, which is in accordance with the APE1 association constant of DNA duplexes containing AP-sites. Thus, it is demonstrated that oligonucleotide duplexes with chemical modifications could be used as unreactive substrates and potential competitive inhibitors of APE1.