G. Michael Blackburn

The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design.

The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.

Nucleic acids in chemistry and biology

LIST OF CONTRIBUTORS NOMENCLATURE 1: INTRODUCTION AND OVERVIEW 1.1: The Biological Importance of DNA 1.2: The Origins of Nucleic Acids Research 1.3: Early Structural Studies on Nucleic Acids 1.4: The Discovery of the Structure of DNA 1.5: The Advent of Molecular Biology 1.6: The Partnership of Chemistry and Biology 1.7: Frontiers in Nucleic Acids Research 2: DNA AND RNA STRUCTURE 2.1: Structures of Components 2.2: Standard DNA Structures 2.3: Real DNA Structures 2.4: Structures of RNA Species 2.5: Dynamics of Nucleic Acid Structures 2.6: Higher-order DNA Structures 3: NUCLEOSIDES AND NUCLEOTIDES 3.1: Chemical Synthesis of Nucleosides 3.2: Chemistry of Esters and Anhydrides of Phosphorus Oxyacids 3.3: Nucleoside Esters of Polyphosphates 3.4: Biosynthesis of Nucleotides 3.5: Catabolism of Nucleotides 3.6: Polymerisation of Nucleotides 3.7: Therapeutic Applications of Nucleoside Analogues 4: SYNTHESIS OF OLIGONUCLEOTIDES 4.1: Synthesis of Oligodeoxyribonucleotides 4.2: Synthesis of Oligoribonucleotides 4.3: Enzymatic Synthesis of Oligonucleotides 4.4: Synthesis of Modified Oligonucleotides 5: NUCLEIC ACIDS IN BIOTECHNOLOGY 5.1: DNA Sequence Determination 5.2: Gene Cloning 5.3: Enzymes Useful in Gene Manipulation 5.4: Gene Synthesis 5.5: The Detection of Nucleic Acids Sequences by Hybridization 5.6: Gene Mutagenesis 5.7: Oligonucleotides as Reagents and Therapeutics 5.8: Footprinting 6: GENES AND GENOMES 6.1: Gene Structure 6.2: Gene Families 6.3: Intergenic DNA 6.4: Chromosomes 6.5: DNA Sequence and Bioinformatics 6.6: Copying DNA 6.7: DNA Mutation and Genome Repair 6.8: DNA Recombination 7: RNA STRUCTURE AND FUNCTION 7.1: Overview of RNA Structural Motifs 7.2: RNA Processing and Modification 7.3: RNAs in the Protein Factory: Translation 7.4: RNAs Involved in Export and Transport 7.5: RNAs and Epigenetic Phenomena 7.6: RNA Structure and Function in Viral Systems 8: COVALENT INTERACTIONS OF NUCLEIC ACIDS WITH SMALL MOLECULES AND THEIR REPAIR 8.1: Hydrolysis of Nucleosides, Nucleotides, and Nucleic Acids 8.2: Reduction of Nucleosides 8.3: Oxidation of Nucleosides, Nucleotides, and Nucleic Acids 8.4: Reactions with Nucleophiles 8.5: Reactions with Electrophiles 8.6: Reactions with Metabolically Activated Carcinogens 8.7: Reactions with Anti-cancer Drugs 8.8: Photochemical Modification of Nucleic Acids 8.9: Effects of Ionising Radiation on Nucleic Acids 8.10: Biological Consequences of DNA Alkylation 8.11: DNA Repair 9: REVERSIBLE SMALL MOLECULE-NUCLEIC ACID INTERACTIONS 9.1: Introduction 9.2: Binding Modes and Sites of Interaction 9.3: Counter-ion Condensation and Polyelectrolyte Theory 9.4: Non-specific Outside-edge Interactions 9.5: Hydration Effects and Water-DNA Interactions 9.6: DNA Intercalation 9.7: Interactions in the Minor Groove 9.8: Intercalation versus Minor Groove Binding 9.9: Co-operativity in Ligand-DNA Interactions 9.10: Small Molecule Interactions with Higher-order DNA 10: PROTEIN-NUCLEIC ACID INTERACTIONS 10.1: Structural Features of DNA Important in Protein Recognition 10.2: The Physical Chemistry of Protein-Nucleic Acid Interactions 10.3: Representative DNA Recognition Motifs 10.4: Kinetic and Thermodynamic Aspects of Protein-Nucleic Acid Interactions 10.5: The Specificity of DNA Enzymes 10.6: DNA Packaging 10.7: Polymerases 10.8: Machines that Manipulate Duplex DNA 10.9: RNA-Protein Interactions and RNA-mediated Assemblies 11: PHYSICAL AND STRUCTURAL TECHNIQUES APPLIED TO NUCLEIC ACIDS 11.1: Spectroscopic Techniques 11.2: Nuclear Magnetic Resonance 11.3: X-ray Crystallography 11.4: Hydrodynamic and Separation Methods 11.5: Microscopy 11.6: Mass Spectrometry 11.7: Molecular Modelling and Dynamics

Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(β,γ-dichloromethylene) triphosphate, by mammalian cells in vitro

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage‐like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5′‐(β,γ‐dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome‐encapsulated clodronate to the same ATP analog. Liposome‐encapsulated adenosine 5′‐(β,γ‐dichloromethylene) triphosphate was more potent than liposome‐encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome‐encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.

Crystal Structure and Functional Analysis of the Histone Methyltransferase Set7/9

Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.

Why did Nature select phosphate for its dominant roles in biology

Evolution has placed phosphate mono- and diesters at the heart of biology. The enormous diversity of their roles has called for the evolution of enzyme catalysts for phosphoryl transfer that are among the most proficient known. A combination of high-resolution X-ray structure analysis and 19F NMR definition of metal fluoride complexes of such enzymes, that are mimics of the transition state for the reactions catalysed, has delivered atomic detail of the nature of such catalysis for a range of phosphoryl transfer processes. The catalytic simplicity thus revealed largely explains the paradox of the contrast between the extreme stability of structural phosphate esters and the lability of phosphates in regulation and signalling processes. A brief survey of the properties of oxyacids and their esters for other candidate elements for these vital roles fails to identify a suitable alternative to phosphorus, thereby underpinning Todd’s Hypothesis “Where there’s life there’s phosphorus” as a statement of truly universal validity.

Isopolar vs Isosteric Phosphonate Analogues of Nucleotides

Abstract A range of β,γ-bridged phosphonate analogues of ATP and of β,β-bridged analogues of Ap4A has been synthesised. Some of their metal binding characteristics and inhibition of enzymatic phosphoryl transfer processes can be described in terms of the relative importance of steric and electronic features of the nucleotide analogues.

Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>1010 M−1) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.

Synthesis of α- and γ-fluoroalkylphosphonates

α-Fluorobenzylphosphonate esters are conveniently made by treating α-hydroxybenzylphosphonate esters with diethylaminosulphur trifluoride. The reaction is not subject to steric impedance and is extended to the α-fluorination of an α-hydroxybenzylphosphine oxide. For α-hydroxyallyl and α-hydroxycinnamylphosphonates, the replacement of the hydroxy group by fluorine proceeds via an SN2′ rearrangement to give the γ-fluoroalk-1-enylphosphonates exclusively. Dehydration rather than substitution occurs in the case of alcohols of secondary alkylphosphonates.

Transition State Analogue Structures of Human Phosphoglycerate Kinase Establish the Importance of Charge Balance in Catalysis.

Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF(3)(-)) and tetrafluoroaluminate (AlF(4)(-)) complexes of PGK have been determined using X-ray crystallography and (19)F-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the alpha-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF(3)(0)) complex. The AlF(3)(0) moiety retains the octahedral geometry observed within AlF(4)(-) TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF(3)(0) complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF(3)(-).

Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by phosphoranes.

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of β-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO 3 - , but is MgF 3 - (trifluoromagnesate). Although MgF 3 - complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using F 19 NMR. Measurements on three β - PGM - MgF 3 - -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F ⋯ H - N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the β - PGM - MgF 3 - - G 6 P complex. The measurements provide independent validation of the structural and isoelectronic MgF 3 - model of near-transition state conformations.