Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy
<p>Reactivation of latent infection is the major cause of tuberculosis (TB). Cholesterol is a critical carbon source during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids.</p> <p>Arylamine...
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| Format: | Thesis |
| Language: | English |
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2013
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| _version_ | 1826316421010817024 |
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| author | Abuhammad, A |
| author2 | Sim, E |
| author_facet | Sim, E Abuhammad, A |
| author_sort | Abuhammad, A |
| collection | OXFORD |
| description | <p>Reactivation of latent infection is the major cause of tuberculosis (TB). Cholesterol is a critical carbon source during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids.</p> <p>Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the sterol-ring degradation and is essential for intracellular survival. NAT from M. tuberculosis (TBNAT) can utilise propionyl-CoA and therefore was proposed as a target for TB-drug development. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. NAT inhibitors, including the piperidinol class, were identified by high-throughput screening. The insolubility of recombinant TBNAT has been a major limitation in pursuing it as a drug target.</p> <p>Subcloning tbnat into a pVLT31 vector resulted in a yield of 6-16 mg/litre-bacterial-culture of pure-soluble recombinant TBNAT. The increased yield allowed for extensive screening for crystallisation conditions. However, since a structure was not obtained, the model NAT from M. marinum (MMNAT) was employed to further understand NAT as a target. Screening against a panel of Acyl-CoA cofactors showed that MMNAT can also utilise propionyl-CoA.</p> <p>The MMNAT structure in complex with the high affinity substrate hydralazine was determined (2.1 Å) and the architecture of the arylamine pocket was delineated. A novel mechanism for the acetylation reaction of hydralazine has emerged. It is proposed that the acetyl group is transferred from acetyl-CoA to the heterocyclic aromatic nitrogen of hydralazine, which explains the immediate cyclisation of the acetylated metabolite into an N-methyltriazolophthalazine.</p> <p>By employing mass spectroscopy, enzyme assays, computational docking and structural studies, a covalent mechanism of inhibition by the piperidinol class was established, and the inhibitor-binding pocket was identified. Inhibitors with new scaffolds were identified using the in silico 3D-shape screening and thermal shift assay.</p> |
| first_indexed | 2024-03-06T21:14:37Z |
| format | Thesis |
| id | oxford-uuid:3f571661-7b51-4fa8-bf5e-2adff9269c59 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:44:50Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:3f571661-7b51-4fa8-bf5e-2adff9269c592024-12-07T16:26:47ZArylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapyThesishttp://purl.org/coar/resource_type/c_db06uuid:3f571661-7b51-4fa8-bf5e-2adff9269c59PharmacologyMedical sciencesEnglishOxford University Research Archive - Valet2013Abuhammad, ASim, EGarman, E<p>Reactivation of latent infection is the major cause of tuberculosis (TB). Cholesterol is a critical carbon source during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids.</p> <p>Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the sterol-ring degradation and is essential for intracellular survival. NAT from M. tuberculosis (TBNAT) can utilise propionyl-CoA and therefore was proposed as a target for TB-drug development. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. NAT inhibitors, including the piperidinol class, were identified by high-throughput screening. The insolubility of recombinant TBNAT has been a major limitation in pursuing it as a drug target.</p> <p>Subcloning tbnat into a pVLT31 vector resulted in a yield of 6-16 mg/litre-bacterial-culture of pure-soluble recombinant TBNAT. The increased yield allowed for extensive screening for crystallisation conditions. However, since a structure was not obtained, the model NAT from M. marinum (MMNAT) was employed to further understand NAT as a target. Screening against a panel of Acyl-CoA cofactors showed that MMNAT can also utilise propionyl-CoA.</p> <p>The MMNAT structure in complex with the high affinity substrate hydralazine was determined (2.1 Å) and the architecture of the arylamine pocket was delineated. A novel mechanism for the acetylation reaction of hydralazine has emerged. It is proposed that the acetyl group is transferred from acetyl-CoA to the heterocyclic aromatic nitrogen of hydralazine, which explains the immediate cyclisation of the acetylated metabolite into an N-methyltriazolophthalazine.</p> <p>By employing mass spectroscopy, enzyme assays, computational docking and structural studies, a covalent mechanism of inhibition by the piperidinol class was established, and the inhibitor-binding pocket was identified. Inhibitors with new scaffolds were identified using the in silico 3D-shape screening and thermal shift assay.</p> |
| spellingShingle | Pharmacology Medical sciences Abuhammad, A Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy |
| title | Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy |
| title_full | Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy |
| title_fullStr | Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy |
| title_full_unstemmed | Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy |
| title_short | Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy |
| title_sort | arylamine n acetyltransferases from mycobacteria investigations of a potential yarget for anti tubercular therapy |
| topic | Pharmacology Medical sciences |
| work_keys_str_mv | AT abuhammada arylaminenacetyltransferasesfrommycobacteriainvestigationsofapotentialyargetforantituberculartherapy |