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The first small-molecule inhibitors of members of the ribonuclease E family.

Kime L, Vincent HA, Gendoo DM, Jourdan SS, Fishwick CW, Callaghan AJ, McDowall KJ - Sci Rep (2015)

Bottom Line: Homologues of RNase E can be found in many bacterial families including important pathogens, but no homologues have been identified in humans or animals.RNase E represents a potential target for the development of new antibiotics to combat the growing number of bacteria that are resistant to antibiotics in use currently.Potent small molecule inhibitors that bind the active site of essential enzymes are proving to be a source of potential drug leads and tools to dissect function through chemical genetics.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

ABSTRACT
The Escherichia coli endoribonuclease RNase E is central to the processing and degradation of all types of RNA and as such is a pleotropic regulator of gene expression. It is essential for growth and was one of the first examples of an endonuclease that can recognise the 5'-monophosphorylated ends of RNA thereby increasing the efficiency of many cleavages. Homologues of RNase E can be found in many bacterial families including important pathogens, but no homologues have been identified in humans or animals. RNase E represents a potential target for the development of new antibiotics to combat the growing number of bacteria that are resistant to antibiotics in use currently. Potent small molecule inhibitors that bind the active site of essential enzymes are proving to be a source of potential drug leads and tools to dissect function through chemical genetics. Here we report the use of virtual high-throughput screening to obtain small molecules predicted to bind at sites in the N-terminal catalytic half of RNase E. We show that these compounds are able to bind with specificity and inhibit catalysis of Escherichia coli and Mycobacterium tuberculosis RNase E and also inhibit the activity of RNase G, a paralogue of RNase E.

No MeSH data available.


Related in: MedlinePlus

Structure of the RNase E catalytic domain and compound docking.(a) A top elevation of a principal dimer of the N-terminal catalytic domain of E. coli RNase E with bound RNA (green). The dimer is shown as a surface representation with the two protomers superimposed as a cartoon diagram. Red, blue, gold and grey colouring identifies the DNase I, S1, 5′ sensor and RNase H domains, respectively. The zinc and magnesium ions are shown as grey and magenta spheres, respectively. (b) The catalytic site. The DNase I side of each of the two channels presents a magnesium ion that is co-ordinated by the carboxylates of aspartic acid residues 303 and 346. The base of the nucleotide at the +2 position relative to the site of RNA cleavage is partitioned into a recess on the surface of the S1 domain. The nucleotide base is held by hydrophobic interactions with a phenylalanine at position 67 and the aliphatic portion of a lysine at position 112. The exocyclic oxygen of the base of the nucleotide immediately 5′ forms a hydrogen bond with a lysine at position 106, also in the S1 domain. (c) The pocket for 5′-monophosphorylated ends contacts both the monophosphate group and the base of the terminal nucleotide. The monophosphate group is hydrogen bonded by the side-chain and peptide amide of a threonine at position 170 and the guanidino group of an arginine at 169: the latter interaction is supported by a hydrogen bond to the peptide backbone of a glycine at position 124. The aromatic ring of the base of the terminal nucleotide is contacted via hydrophobic interaction with the side chain of a valine at 128. (d) The site of catalysis, with predicted docking of compound M5. (e) The 5′-monophosphate binding pocket, with predicted docking of compound P11. The binding of compounds M5 and P11 sterically hinder binding of the RNA molecule.
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f1: Structure of the RNase E catalytic domain and compound docking.(a) A top elevation of a principal dimer of the N-terminal catalytic domain of E. coli RNase E with bound RNA (green). The dimer is shown as a surface representation with the two protomers superimposed as a cartoon diagram. Red, blue, gold and grey colouring identifies the DNase I, S1, 5′ sensor and RNase H domains, respectively. The zinc and magnesium ions are shown as grey and magenta spheres, respectively. (b) The catalytic site. The DNase I side of each of the two channels presents a magnesium ion that is co-ordinated by the carboxylates of aspartic acid residues 303 and 346. The base of the nucleotide at the +2 position relative to the site of RNA cleavage is partitioned into a recess on the surface of the S1 domain. The nucleotide base is held by hydrophobic interactions with a phenylalanine at position 67 and the aliphatic portion of a lysine at position 112. The exocyclic oxygen of the base of the nucleotide immediately 5′ forms a hydrogen bond with a lysine at position 106, also in the S1 domain. (c) The pocket for 5′-monophosphorylated ends contacts both the monophosphate group and the base of the terminal nucleotide. The monophosphate group is hydrogen bonded by the side-chain and peptide amide of a threonine at position 170 and the guanidino group of an arginine at 169: the latter interaction is supported by a hydrogen bond to the peptide backbone of a glycine at position 124. The aromatic ring of the base of the terminal nucleotide is contacted via hydrophobic interaction with the side chain of a valine at 128. (d) The site of catalysis, with predicted docking of compound M5. (e) The 5′-monophosphate binding pocket, with predicted docking of compound P11. The binding of compounds M5 and P11 sterically hinder binding of the RNA molecule.

Mentions: The N-terminal catalytic half (NTH) of RNase E is a tetramer, which is best described as a dimer of dimers1415. Each dimer contributes a pair of equivalent antiparallel channels that include the sites of catalysis. Each channel binds a single-stranded RNA segment and has an adjacent pocket for binding 5′ termini that are both monophosphorylated and unpaired (Fig. 1a–c)16. Such termini are found on the downstream products of cleavage and may provide a means of prolonging contact such that additional cleavages can occur17. Each single-stranded RNA-binding channel16 is formed on one side by a domain that closely resembles the RNA-binding domain of S118 and on the other side by one that resembles the catalytic domain of DNase I19. The latter reveals an unexpected link in the evolution of RNA and DNA nucleases. Aspartic acid residues at 303 and 346 in the DNase I domain may act as general bases to activate the hydroxyl group of a water molecule coordinated to the magnesium to attack the scissile phosphate16. The scissile phosphodiester bond in the RNA is the point at which cleavage occurs (the bases 3′ to the site of RNA cleavage are defined as +1, +2..., and those 5′ are −1, −2...). The contacts between RNase E and bound RNA are described in the legend to Fig. 1a–c. Recent evidence suggests that simultaneous recognition of two or more unpaired regions may provide a sufficiently stable interaction for efficient cleavage by RNase E to occur62021. However, it is also clear that the efficient cleavage of some substrates requires them to be 5′ monophosphorylated172223. A simple explanation for these observations is that because of structural constraints not all RNAs present a combination of unpaired regions that can cooperate in binding RNase E, but for some of these RNAs a 5′-monophosphorylated end provides an additional foothold for RNase E that allows binding and cleavage of a co-accessible unpaired region621.


The first small-molecule inhibitors of members of the ribonuclease E family.

Kime L, Vincent HA, Gendoo DM, Jourdan SS, Fishwick CW, Callaghan AJ, McDowall KJ - Sci Rep (2015)

Structure of the RNase E catalytic domain and compound docking.(a) A top elevation of a principal dimer of the N-terminal catalytic domain of E. coli RNase E with bound RNA (green). The dimer is shown as a surface representation with the two protomers superimposed as a cartoon diagram. Red, blue, gold and grey colouring identifies the DNase I, S1, 5′ sensor and RNase H domains, respectively. The zinc and magnesium ions are shown as grey and magenta spheres, respectively. (b) The catalytic site. The DNase I side of each of the two channels presents a magnesium ion that is co-ordinated by the carboxylates of aspartic acid residues 303 and 346. The base of the nucleotide at the +2 position relative to the site of RNA cleavage is partitioned into a recess on the surface of the S1 domain. The nucleotide base is held by hydrophobic interactions with a phenylalanine at position 67 and the aliphatic portion of a lysine at position 112. The exocyclic oxygen of the base of the nucleotide immediately 5′ forms a hydrogen bond with a lysine at position 106, also in the S1 domain. (c) The pocket for 5′-monophosphorylated ends contacts both the monophosphate group and the base of the terminal nucleotide. The monophosphate group is hydrogen bonded by the side-chain and peptide amide of a threonine at position 170 and the guanidino group of an arginine at 169: the latter interaction is supported by a hydrogen bond to the peptide backbone of a glycine at position 124. The aromatic ring of the base of the terminal nucleotide is contacted via hydrophobic interaction with the side chain of a valine at 128. (d) The site of catalysis, with predicted docking of compound M5. (e) The 5′-monophosphate binding pocket, with predicted docking of compound P11. The binding of compounds M5 and P11 sterically hinder binding of the RNA molecule.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4306137&req=5

f1: Structure of the RNase E catalytic domain and compound docking.(a) A top elevation of a principal dimer of the N-terminal catalytic domain of E. coli RNase E with bound RNA (green). The dimer is shown as a surface representation with the two protomers superimposed as a cartoon diagram. Red, blue, gold and grey colouring identifies the DNase I, S1, 5′ sensor and RNase H domains, respectively. The zinc and magnesium ions are shown as grey and magenta spheres, respectively. (b) The catalytic site. The DNase I side of each of the two channels presents a magnesium ion that is co-ordinated by the carboxylates of aspartic acid residues 303 and 346. The base of the nucleotide at the +2 position relative to the site of RNA cleavage is partitioned into a recess on the surface of the S1 domain. The nucleotide base is held by hydrophobic interactions with a phenylalanine at position 67 and the aliphatic portion of a lysine at position 112. The exocyclic oxygen of the base of the nucleotide immediately 5′ forms a hydrogen bond with a lysine at position 106, also in the S1 domain. (c) The pocket for 5′-monophosphorylated ends contacts both the monophosphate group and the base of the terminal nucleotide. The monophosphate group is hydrogen bonded by the side-chain and peptide amide of a threonine at position 170 and the guanidino group of an arginine at 169: the latter interaction is supported by a hydrogen bond to the peptide backbone of a glycine at position 124. The aromatic ring of the base of the terminal nucleotide is contacted via hydrophobic interaction with the side chain of a valine at 128. (d) The site of catalysis, with predicted docking of compound M5. (e) The 5′-monophosphate binding pocket, with predicted docking of compound P11. The binding of compounds M5 and P11 sterically hinder binding of the RNA molecule.
Mentions: The N-terminal catalytic half (NTH) of RNase E is a tetramer, which is best described as a dimer of dimers1415. Each dimer contributes a pair of equivalent antiparallel channels that include the sites of catalysis. Each channel binds a single-stranded RNA segment and has an adjacent pocket for binding 5′ termini that are both monophosphorylated and unpaired (Fig. 1a–c)16. Such termini are found on the downstream products of cleavage and may provide a means of prolonging contact such that additional cleavages can occur17. Each single-stranded RNA-binding channel16 is formed on one side by a domain that closely resembles the RNA-binding domain of S118 and on the other side by one that resembles the catalytic domain of DNase I19. The latter reveals an unexpected link in the evolution of RNA and DNA nucleases. Aspartic acid residues at 303 and 346 in the DNase I domain may act as general bases to activate the hydroxyl group of a water molecule coordinated to the magnesium to attack the scissile phosphate16. The scissile phosphodiester bond in the RNA is the point at which cleavage occurs (the bases 3′ to the site of RNA cleavage are defined as +1, +2..., and those 5′ are −1, −2...). The contacts between RNase E and bound RNA are described in the legend to Fig. 1a–c. Recent evidence suggests that simultaneous recognition of two or more unpaired regions may provide a sufficiently stable interaction for efficient cleavage by RNase E to occur62021. However, it is also clear that the efficient cleavage of some substrates requires them to be 5′ monophosphorylated172223. A simple explanation for these observations is that because of structural constraints not all RNAs present a combination of unpaired regions that can cooperate in binding RNase E, but for some of these RNAs a 5′-monophosphorylated end provides an additional foothold for RNase E that allows binding and cleavage of a co-accessible unpaired region621.

Bottom Line: Homologues of RNase E can be found in many bacterial families including important pathogens, but no homologues have been identified in humans or animals.RNase E represents a potential target for the development of new antibiotics to combat the growing number of bacteria that are resistant to antibiotics in use currently.Potent small molecule inhibitors that bind the active site of essential enzymes are proving to be a source of potential drug leads and tools to dissect function through chemical genetics.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

ABSTRACT
The Escherichia coli endoribonuclease RNase E is central to the processing and degradation of all types of RNA and as such is a pleotropic regulator of gene expression. It is essential for growth and was one of the first examples of an endonuclease that can recognise the 5'-monophosphorylated ends of RNA thereby increasing the efficiency of many cleavages. Homologues of RNase E can be found in many bacterial families including important pathogens, but no homologues have been identified in humans or animals. RNase E represents a potential target for the development of new antibiotics to combat the growing number of bacteria that are resistant to antibiotics in use currently. Potent small molecule inhibitors that bind the active site of essential enzymes are proving to be a source of potential drug leads and tools to dissect function through chemical genetics. Here we report the use of virtual high-throughput screening to obtain small molecules predicted to bind at sites in the N-terminal catalytic half of RNase E. We show that these compounds are able to bind with specificity and inhibit catalysis of Escherichia coli and Mycobacterium tuberculosis RNase E and also inhibit the activity of RNase G, a paralogue of RNase E.

No MeSH data available.


Related in: MedlinePlus