Human histamine N-methyltransferase pharmacogenetics: common genetic polymorphisms that alter activity.

Histamine N-methyltransferase (HNMT) catalyzes a major pathway in histamine metabolism. Levels of HNMT activity in humans are regulated by inheritance. We set out to study the molecular basis for this genetic regulation. Northern blot analysis showed that HNMT is highly expressed in the kidney, so we determined levels of enzyme activity and thermal stability in 127 human renal biopsy samples. DNA was isolated from 12 kidney samples with widely different HNMT phenotypes, and exons of the HNMT gene were amplified with the polymerase chain reaction. In these 12 samples, we observed a C314T transition that resulted in a Thr105Ile change in encoded amino acid, as well as an A939G transition within the 3'-untranslated region. All remaining renal biopsy samples then were genotyped for these two variant sequences. Frequencies of the alleles encoding Thr105 and Ile105 in the 114 samples studied were 0.90 and 0.10, respectively, whereas frequencies for the nucleotide A939 and G alleles were 0.79 and 0.21, respectively. Kidney samples with the allele encoding Ile105 had significantly lower levels of HNMT activity and thermal stability than did those with the allele that encoded Thr105. These observations were confirmed by transient expression in COS-1 cells of constructs that contained all four alleles for these two polymorphisms. COS-1 cells transfected with the Ile105 allele had significantly lower HNMT activity and immunoreactive HNMT protein than did those transfected with the Thr105 allele. These observations will make it possible to test the hypothesis that genetic polymorphisms for HNMT may play a role in the pathophysiology of human disease.

Arylacetamide deacetylase activity towards monoacetyldapsone. Species comparison, factors that influence activity, and comparison with 2-acetylaminofluorene and p-nitrophenyl acetate hydrolysis.

The deacetylation of monoacetyldapsone (MADDS) was examined in liver microsomes and cytosol from male Sprague-Dawley rats, Golden Syrian hamsters, and Swiss Albino mice. All three rodent species demonstrated greater MADDS deacetylation activity in liver microsomes than in liver cytosol. Further investigations were conducted in hamsters. The velocity of MADDS deacetylation in major organs in the hamster was greatest in the intestine, followed by the liver and kidney. The effect of pretreatment with common inducers on liver microsomal deacetylation activity was also examined in the hamster. Phenobarbital, 100 mg/kg/day x 3 days, did not alter activity, while dexamethasone at the same dose reduced 2-acetylaminofluorene (2-AFF), MADDS, and p-nitrophenyl acetate (NPA) hydrolysis by at least 50%. Due to a previous report that KI activated the deacetylation of an arylacetamide in vitro (Khanna et al., J Pharmacol Exp Ther 262: 1225-1231, 1992), the effects of the halides KF, KCl, KBr and KI on MADDS hydrolysis in vitro were tested. Of the halides studied, only KF altered MADDS hydrolysis, resulting in an almost complete inhibition of deacetylase activity at 50 mM (with the initial concentration of MADDS at 0.6 mM) with an IC50 = 0.16 mM. Cornish-Bowden and Dixon plots indicated that the inhibition exerted by KF was non-competitive. The rank order of inhibitor potencies was constructed using phenylmethylsulfonyl fluoride (PMSF), bis(p-nitrophenyl)phosphate (BNPP), physostigmine, and KF with 2-AFF, MADDS, and NPA as substrates. Different rank order potencies were obtained for each of the substrates tested. The substrates 2-AFF, MADDS, and NPA did not act as competitive inhibitors on the hydrolysis rates of each other. Liver microsomal arylacetamide deacetylase activity was greater in male hamsters than in females with either MADDS or 2-AAF as substrates; however, hydrolysis of NPA was similar in both male and female hamsters. These data support the hypothesis that the enzyme which catalyzes the hydrolysis of MADDS differs from that catalyzing either 2-AAF or NPA hydrolysis.

Synthesis and evaluation of unsymmetrically substituted polyamine analogues as modulators of human spermidine/spermine-N1-acetyltransferase (SSAT) and as potential antitumor agents.

Spermidine/spermine-N1-acetyltransferase (SSAT), the rate-limiting step in polyamine catabolism, is critical for the interconversion and modulation of cellular polyamines. Inhibitor-initiated induction of this enzyme also appears to correlate with the sensitivity of tumor cells to a class of novel polyamine analogues, the bis(ethyl)polyamines. Thus, terminally alkylated polyamines which modulate the cellular level of SSAT could be of great value for understanding the role of this enzyme both in analogue-mediated cytotoxicity and in overall cellular polyamine metabolism. Such analogues could also become important therapeutic agents by disrupting cellular polyamine metabolism. The structure-activity relationships defining the interaction of polyamine analogues with SSAT have not been fully elucidated, and, in particular, unsymmetrically alkylated polyamines have not been synthesized and evaluated as modulators of SSAT. To this end, we now report the synthesis and preliminary biological evaluation of N1-ethyl-N11-propargyl-4,8-diazaundecane and N1-ethyl-N11-((cyclopropyl)methyl)-4,8-diazaundecane via a synthetic pathway which represents an efficient route to a variety of unsymmetrically substituted polyamine analogues. The title compounds act as effective inhibitors of isolated human SSAT and produce a differential superinduction of SSAT in situ which appears to be associated with a cell specific cytotoxic response in two human lung cancer cell lines. In so doing, these analogues exhibit promising antitumor activity against cultured human lung cancer cells.