Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs)

Brooke, Edward W.; Davies, Stephen G.; Mulvaney, Andrew W.; Okada, Minoru; Pompeo, Frédérique; Sim, Edith; Vickers, Richard; Westwood, Isaac M.

doi:10.1016/s0960-894x(03)00484-0

Cited by 65 publications

(30 citation statements)

References 19 publications

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“…This residue both stabilizes the catalytically important Asp 122 by forming a hydrogen bond and forms part of the loop containing Phe 125 , implicated in many substrate/inhibitor studies of NAT enzymes [5,6,29,48]. However, a consensus sequence for eukaryotic enzymes only extends further into the C-terminal domain and may reflect a greater similarity of function in this subgroup of NAT enzymes.…”

Section: Figure 5 Fully Conserved Residues Among Nat Sequencesmentioning

confidence: 99%

Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

Sandy

Mushtaq

Holton

et al. 2005

Biochemical Journal

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The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-HisAsp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly 126 , implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 Å (where 1 Å = 0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.

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Section: Figure 5 Fully Conserved Residues Among Nat Sequencesmentioning

confidence: 99%

Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

Sandy

Mushtaq

Holton

et al. 2005

Biochemical Journal

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“…We next examined the use of 1,2,3,4-benzothiatriazine-1,1(2H)-dioxides as a triazo substrate for an annulation reaction because of the medicinal importance of the resulting 1,2-benzothiazine-1,1(2H)-dioxide derivatives. [6] Herein, we report the enantioselective synthesis of substituted 3,4-dihydro-1,2-benzothiazine-1,1(2H)-dioxides by the nickel-catalyzed denitrogenative annulation of 1,2,3,4-benzothiatriazine-1,1(2H)-dioxides with allenes.…”

mentioning

confidence: 99%

Nickel‐Catalyzed Regio‐ and Enantioselective Annulation Reactions of 1,2,3,4‐Benzothiatriazine‐1,1(2H)‐dioxides with Allenes

et al. 2010

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Extrusion of N2: 1,2,3,4‐Benzothiatriazine‐1,1(2H)‐dioxides reacted with allenes in the presence of a nickel(0)/(R)‐quinap complex to produce a variety of substituted 3,4‐dihydro‐1,2‐benzothiazine‐1,1(2H)‐dioxides in a regio‐ and enantioselective fashion. An intermediate nickelacycle was generated through denitrogenative activation of the triazo moiety which allowed the intermolecular incorporation of an allene group. quinap=1‐(2‐diphenylphosphino‐1‐naphthyl)isoquinoline.

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“…4). A similar pattern of interaction between a series of substrates (4-alkoxyanilines) and NAT from M. smegmatis has also been observed (Brooke et al, 2003b). These NAT substrates/ligands were shown to have anti-tubercular activity although by an unexplained mechanism (Nodzu et al, 1954).…”

Section: Discussionmentioning

confidence: 57%

“…The sultam inhibitors, which were identified in the previous one-enzyme screen (Brooke et al, 2003b), did not appear as hits in this more discriminating five-enzyme screen. In order to confirm the inhibitory activity, the hits were re-screened using more stringent criteria against the same panel of five NAT enzymes.…”

Section: Screen Designmentioning

confidence: 75%

“…NAT isoenzymes have been characterized from a range of organisms and crystal structures have been obtained for the enzyme from Salmonella typhimurium (Sinclair et al, 2000), Pseudomonas aeruginosa (Westwood et al, 2005), the mycobacterial species Mycobacterium smegmatis (Sandy et al, 2002) and more recently from Mycobacterium marinum (Fullam et al, 2007;PDB code 2vfb), which is 74% identical to the enzyme from M. tuberculosis (Fullam et al, 2009). A preliminary manual screen of a limited range of compounds using the NAT enzyme from M. smegmatis had already proved successful in identifying NAT inhibitors (Brooke et al, 2003b). We report here that specific NAT inhibitors mimic the effects of deleting the nat gene in M. bovis BCG and that these compounds are also effective in inhibiting the growth of M. tuberculosis.…”

Section: Introductionmentioning

confidence: 99%

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Identification of arylamine N-acetyltransferase inhibitors as an approach towards novel anti-tuberculars

et al. 2010

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New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules-triazoles-in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.

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Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs)

Cited by 65 publications

References 19 publications

Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

Nickel‐Catalyzed Regio‐ and Enantioselective Annulation Reactions of 1,2,3,4‐Benzothiatriazine‐1,1(2H)‐dioxides with Allenes

Identification of arylamine N-acetyltransferase inhibitors as an approach towards novel anti-tuberculars

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