ABSTRACT
Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising â¼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1's ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5'-TTTTT/AA-3'. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN's sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes 'compression' near the cleavage site. This provides multiple advantages for L1-EN's role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.
Subject(s)
DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA/chemistry , Deoxyribonuclease I/chemistry , Base Sequence , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , DNA/genetics , DNA/metabolism , DNA Cleavage , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Genome, Human , Genomic Instability , Humans , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , ThermodynamicsABSTRACT
A specific, sensitive, rapid and reproducible method for the determination of flomoxef in human plasma using high-performance liquid chromatography-tandem mass spectrometry was developed and validated. Flomoxef was detected using an electrospay ionization method operated in negative-ion mode. Chromatographic separation was performed in gradient elution mode on a Luna® C18(2) column (3 µM, 20 × 4.0 mm) at a flow rate of 1 mL/min and runtime 3.5 min. The mobile phase consisted of acetonitrile and water containing 0.1% formic acid as additive. Extraction of flomoxef from plasma and precipitation of plasma proteins was performed with acetonitrile with an absolute recovery of 86.4 ± 1.6%. The calibration curve was linear with a correlation coefficient of 0.999 over the concentration range 10-5000 ng/mL and the lower limit of quantification was 10 ng/mL. The intra- and inter-day precisions were <11.8%, while the accuracy ranged from 99.6 to 109.0%. A stability study of flomoxef revealed that it could be successfully analyzed at 4 ºÐ¡ over 24 h, but it was unstable in solutions at room temperature during short-term storage (4 h) and several freeze-thaw cycles.
Subject(s)
Cephalosporins/blood , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Cephalosporins/chemistry , Chromatography, Liquid , Drug Stability , Humans , Least-Squares Analysis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A general pharmachophore model for various types of Ser/Thr kinases was developed. Search for the molecules fitting to this pharmacophore among ASINEX proprietary library revealed a number of compounds, which were tested and appeared to possess some activity against several Ser/Thr kinases such as Aurora A, Aurora B and Haspin. The possibility of performing the fine-tuning of the general Ser/Thr pharmacophore to desired types of kinase to get active and selective inhibitors was exemplified by Aurora A kinase. As a result, several hits in 3-5 nm range of activity against Aurora A kinase with rather good selectivity and ADME properties were obtained.
Subject(s)
Aurora Kinase A , Models, Molecular , Protein Kinase Inhibitors/chemistry , Aurora Kinase A/antagonists & inhibitors , Aurora Kinase A/chemistry , HumansABSTRACT
Based on the data for compounds known from the literature to be active against various types of Ser/Thr kinases, a general pharmachophore model for these types of kinases was developed. The search for the molecules fitting to this pharmacophore among the ASINEX proprietary library revealed a number of compounds, which were tested and appeared to possess some activity against Ser/Thr kinases such as Aurora A, Aurora B and Haspin. Our work on the optimization of these molecules against Aurora A kinase allowed us to achieve several hits in a 3-5 nM range of activity with rather good selectivity and Absorption, Distribution, Metabolism, and Excretion (ADME) properties, and cytotoxicity against 16 cancer cell lines. Thus, we showed the possibility to fine-tune the general Ser/Thr pharmacophore to design active and selective compounds against desired types of kinases.