Functional consequence of SNPs on the Tuberculosis drug metabolising enzyme, human arylamine N-acetyltransferase 1

The human arylamine N-acetyltransferase 1 (NAT1) plays a vital role in determining the duration of action and pharmacokinetics of amine-containing drugs such as para-aminosalicylic acid used in clinical therapy, as well as influencing the balance between detoxification and metabolic activation of these drugs. Single nucleotide polymorphisms (SNPs) in this enzyme are continuously being detected and show inter-ethnic and inter-individual variation. Administrating tuberculosis (TB) treatment in the absence of genotypic information for drug metabolizing enzymes can limit the successful eradication of the disease from a patient. Recent studies have shown that loss of H-bonds affects protein function.

Results: In this study, the eects of 11 novel non-synonymous SNPs (nsSNPs) on the structure and function of NAT1 was tested computationally using SIFT and POLYPHEN-2 algorithms and structural analyses methods including loss of hydrogen-bonding, stability calculation, solvent accessibility and sequence conservation. Four out of 11 nsSNPs (Q210P, D229H, V231G and V235A) were predicted to aect protein function using both algorithms. Two of these four SNPs showed a loss of 2-4 hydrogen bonds and in most cases a destabilized protein structure. Another two SNPs (F202V, N245I) were predicted to aect protein function using both algorithms but without any loss of hydrogen-bonds. Three additional nsSNPs (T240S, S259R, T193S) were predicted to be benign with either a loss of three hydrogen bonds or no loss of hydrogen-bonds. The remaining two nsSNPs (E264K and R242M) showed conflicting results between SIFT and POLYPHEN-2 and both cases showed stable Gibbs free energy. No correlation could be identified between the predicted functional eects from SIFT and POLYPHEN-2, and the stability calculations and the hydrogen-bonding analyses. However, the structural effects of modifying an amino acid together with the conficting results from both algorithms warrant experimental testing to resolve the consequences of these 11 novel nsSNPs on NAT1.

Conclusion: The nsSNPs that aect protein function and/or have a destabilized structure provides a prioritized list of SNPs that will be tested in the laboratory by creating a SNP construct that will be cloned into an expression vector. These ndings will inform a strategy of incorporating genotypic data (i.e, functional SNP alleles) with phenotypic information (slow or fast acetylators) to better prescribe effective tuberculosis treatment.

Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolising enzymes (XMEs) that infuence the biological activity and toxicity of certain compounds by catalysing the acetyl-CoA dependent N-acetylation (usually deactivation) of primary aromatic amines and hydrazines, as well as O-acetylation (usually activation) of their N-hydroxylated metabolites. Generally, N-acetylation leads to stable products while O-acetylation can lead to nucleophilic nitrineum ions capable of binding DNA bases or proteins. In humans, there are two functional enzymes; N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2), each exhibit single nucleotide polymorphisms (SNPs) and show inter-individual and inter-ethnic differences. Polymorphisms in these enzymes result in rapid, intermediate or normal and slow acetylator phenotypes. The proportions of rapid and slow acetylators vary remarkably in dierent ethnic and/or geographic-origin populations. For example, the percentage of slow acetylators among Canadian Eskimos is 5%; whereas it rises to over 80% among Egyptians and 90% among Moroccans [1]. Most populations in Europe and North America have 40 to 70% slow acetylators; Asian populations, only 10 to 20% slow acetylators [1]. Because NATs catalyse the N-acetylation and O-acetylation of aromatic and heterocyclic amine carcinogens [2{5] , genetic polymorphisms in NAT1 and/or NAT2 modify risk associated with carcinogen exposures [6].