Alpha-methylated polyamines as potential drugs and experimental tools in enzymology.

We describe synthesis of alpha-methylated analogues of the natural polyamines and their use as tools in unraveling polyamine functions. Experiments with alpha-methylated spermidine and spermine revealed that the polyamines are exchangeable in supporting cellular growth. Degradation of the analogues by polyamine oxidase disclosed hidden, aldehyde-guided stereospecificity of the enzyme.

Alpha-methyl polyamines: efficient synthesis and tolerance studies in vivo and in vitro. First evidence for dormant stereospecificity of polyamine oxidase.

Efficient syntheses of metabolically stable alpha-methylspermidine 1, alpha-methylspermine 2, and bis-alpha,alpha'-methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven spermidine/spermine N(1)-acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in vitro with the immortalized fibroblasts derived from these animals. Enantiomers of previously unknown 1-amino-8-acetamido-5-azanonane dihydrochloride 4 were synthesized starting from enantiomerically pure (R)- and (S)-alaninols. The studies with recombinant human polyamine oxidase (PAO) showed that PAO (usually splits achiral substrates) strongly favors the (R)-isomer of 4 that demonstrates for the first time that the enzyme has hidden potency for stereospecificity.

Genetic manipulation of polyamine catabolism in rodents.

Activation of polyamine catabolism through the overexpression of spermidine/spermine N1-acetyltransferase (SSAT) in transgenic rodents does not only lead to distorted tissue polyamine homeostasis, manifested as striking accumulation of putrescine, appearance N1-acetylspermidine and reduction of tissue spermidine and/or spermine pools, but likewise creates striking phenotypic changes. The latter include loss of hair, lipoatrophy and female infertility. Forced expression of SSAT modulates skin, prostate and intestinal carcinogenesis, induces acute pancreatitis and blocks early liver regeneration. Although many of these features are directly attributable to altered tissue polyamine pools, some of them are more likely related to the greatly accelerated flux of the polyamines caused by activated catabolism and compensatorily enhanced biosynthesis.

Predicting quantitative genetic interactions by means of sequential matrix approximation.

Despite the emerging experimental techniques for perturbing multiple genes and measuring their quantitative phenotypic effects, genetic interactions have remained extremely difficult to predict on a large scale. Using a recent high-resolution screen of genetic interactions in yeast as a case study, we investigated whether the extraction of pertinent information encoded in the quantitative phenotypic measurements could be improved by computational means. By taking advantage of the observation that most gene pairs in the genetic interaction screens have no significant interactions with each other, we developed a sequential approximation procedure which ranks the mutation pairs in order of evidence for a genetic interaction. The sequential approximations can efficiently remove background variation in the double-mutation screens and give increasingly accurate estimates of the single-mutant fitness measurements. Interestingly, these estimates not only provide predictions for genetic interactions which are consistent with those obtained using the measured fitness, but they can even significantly improve the accuracy with which one can distinguish functionally-related gene pairs from the non-interacting pairs. The computational approach, in general, enables an efficient exploration and classification of genetic interactions in other studies and systems as well.

Guide molecule-driven stereospecific degradation of alpha-methylpolyamines by polyamine oxidase.

FAD-dependent polyamine oxidase (PAO; EC 1.5.3.11) is one of the key enzymes in the catabolism of polyamines spermidine and spermine. The natural substrates for the enzyme are N1-acetylspermidine, N1-acetylspermine, and N1,N12-diacetylspermine. Here we report that PAO, which normally metabolizes achiral substrates, oxidized (R)-isomer of 1-amino-8-acetamido-5-azanonane and N1-acetylspermidine as efficiently while (S)-1-amino-8-acetamido-5-azanonane was a much less preferred substrate. It has been shown that in the presence of certain aldehydes, the substrate specificity of PAO and the kinetics of the reaction are changed to favor spermine and spermidine as substrates. Therefore, we examined the effect of several aldehydes on the ability of PAO to oxidize different enantiomers of alpha-methylated polyamines. PAO supplemented with benzaldehyde predominantly catalyzed the cleavage of (R)-isomer of alpha-methylspermidine, whereas in the presence of pyridoxal the (S)-alpha-methylspermidine was preferred. PAO displayed the same stereospecificity with both singly and doubly alpha-methylated spermine derivatives when supplemented with the same aldehydes. Structurally related ketones proved to be ineffective. This is the first time that the stereospecificity of FAD-dependent oxidase has been successfully regulated by changing the supplementary aldehyde. These findings might facilitate the chemical regulation of stereospecificity of the enzymes.

Animal disease models generated by genetic engineering of polyamine metabolism.

The polyamines putrescine, spermidine and spermine are natural components of all living cells. Although their exact cellular functions are still largely unknown, a constant supply of these compounds is required for mammalian cell proliferation to occur. Studies with animals displaying genetically altered polyamine metabolism have shown that polyamines are intimately involved in the development of diverse tumors, putrescine apparently has specific role in skin physiology and neuroprotection and the higher polyamines spermidine and spermine are required for the maintenance of pancreatic integrity and liver regeneration. In the absence of ongoing polyamine biosynthesis, murine embryogenesis does not proceed beyond the blastocyst stage. The last years have also witnessed the appearance of the first reports linking genetically altered polyamine metabolism to human diseases.

Metabolic stability of alpha-methylated polyamine derivatives and their use as substitutes for the natural polyamines.

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues.

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.