kohkura@cis.ac.jp

N-acetyl transferase 2 polymorphism and drug metabolism

Kazuto OHKURA*1, Katsumi FUKINO1, Yasuo SHINOHARA2 and Hitoshi HORI3

1Faculty of Pharmacy, Chiba Institute of Science, 3 Shiomi-cho, Choshi, Chiba, 288-0025; 2Institute for Genome Research, The University of Tokushima, 3-8 Kuramoto-cho, Tokushima 770-8503; 3Department of Biological Science and Technology, Faculty of Engineering, The

University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan

1. Introduction In the NAT2 gene, 36 haplotypes have been identified so far. NAT2s are classified into three groups, which are named rapid (RA), intermediate (IA), and slow acetylators (SA). Phenotypes of NAT2s depend on the number of active alleles (wild NAT2*4); RA, IA, and SA type has two, one, and no active alleles, respectively. In SA type patients, isoniazid (INH) metabolism are slow and serum INH level become high by a standard amount administration of INH. In contrast, RA type patients might be administered an increased dosage of INH to achieve sufficient therapeutic serum level. Polymorphisms of NAT2 alleles had were reported as follows: I114 to S (NAT2*5), R197 to Q (NAT2*6), G286 to E (NAT2*7). RA type have NAT2*4/*4 alleles. IA type subjects have any of the following alleles; NAT2*4/*5, *4/*6, or *4/*7. SA type did not have NAT2*4 allele, and it is constructed with NAT2*5, *6 or *7 allele. The main metabolic pathway of INH is the metabolism of INH to acetyl isoniazid (AcINH) by NAT2 (Fig. 1). AcINH produced in this pathway is then hydrolyzed by amidase to acetyl hydradine (AcHz). On the other hand, there is also a pathway in which INH is hydrolyzed by amidase into hydrazine (Hz). Hz is one of the hepatotoxic factors. We analyzed the correlation between genotype-involved molecular features of NAT2 and serum INH, Hz, AcINH, AcHz level, and discussed the effect of site mutation in NAT2 molecule. 2. Method Molecular model of these NAT2*5, *6, and *7 were constructed based on the X-ray data of human wild NAT2*4 (2PFR). Electrostatic potential fields of NAT2s were calculated, and the +1.0 kT/e (gray) and -1.0 kT/e (dark gray) contour was displayed. The z-matrix data for single nucleotide polymorphism (SNP) involved regions of NAT2*4, *5, *6, *7 (Region 1: L110 ~ N118, Region 2: T193 ~ S201, and Region 3: K282 ~ I290) were extracted from each NAT2, and molecular orbital analysis was performed. Solvation free energies (dGW) were determined from MO parameters. Nonbinding energies between ligands (acetyl CoA (2PFR), isoniazid (1W6F)) and NAT2s were determined. Population of NAT2 gene was analyzed for 129 tuberculosis (TB) patients. After INH administration, the serum concentration of INH and INH metabolites (Hz, AcINH, AcHz) in TB patients were determined (1).

Figure 1. Metabolic pathways of isoniazid in human. Table 1. Genotype distribution of NAT2 in tuberculosis patients.

KP-25

3. Results and Discussion Genotype distribution of NAT2s. NAT2 gene distribution of Japanese tuberculosis patients were summarized (Table 1) (1). The percentages of RA, IA, and SA were 46.5, 44.2, and 9.3%. In each acetylator type, the serum level of INH, Hz, AcINH, and AcHz were summarized as Table 2. In SA type patients, Hz accumulation was observed, and 35.23+14.44 ng/mL of Hz was detected. Table 2. Serum concentration of INH and metabolites.

Molecular features of NAT2s. Solvation free energy of three mutation sites of NAT2*4 were -1132.1 (site 1), -1900.5 (site 2), -1498.7 (site 3) kJ/mol (Table 3). In NAT2*5, *6, *7 mutation sites, their dGW values decreased to -1155.4, -2370.6, -1843.2 kJ/mol and the hydrophobicity of these sites significantly increased. The differences of dGWs between wild NAT2*4 and mutated NAT2*5, *6, *7 were 23.3, 470.1, 344.5 kJ/mol, respectively. The absolute differential dGW value between CH4 and C120H242 were 6.616 kJ/mol, thus the dGW difference between wild and mutant NAT2s were very large. Table 3. Solvation free energy (dGW) of NAT2 mutation sites.

The wild NAT2*4 was almost occupied by positive electrostatic potential field, and negative field was covered by positive field (Fig. 2). The CoA molecule located in the boundary of positive and negative electrostatic potential field. Ile144 and Arg197, which are mutation sites of NAT2*4 to *5 and *6, were located in the peripheral part of positive field. Gly286, the mutation site from NAT2*4 to *7, was located near CoA. NAT2*5, *6, and *7 molecule had same electrostatic potential field in each model. Nonbinding energy between wild NAT2*4 and CoA, which is the native cofactor for NAT2, was -41.7 kcal/mol (Table 4). The nonbinding energies between mutated NAT2*5, *6, *7 and CoA was the same as that of NAT2*4. The mutation of these sites did not affect the NAT2 reactivity to CoA molecule. Nonbinding energies between NAT2s and isoniazid were larger than those of CoA, and the order of energy was NAT2*4 (1.91 x 103) < *5, *6 (8.83 x 105) < *7 (1.34 x 106 kcal/mol). Thus the molecular polymorphism influenced to the reactivity between NAT2 and external ligand.

Figure 2. Electrostatic potential field of NAT2*4. Table 4. Nonbinding energies between NAT2 and ligands. References 1. Fukino K. et al. J Toxicol Sci. 2008, 33, 237-40.