Active metabolites early screening

In an effort to uncover anticancer active metabolites in Brazilian plants, in early 1993 we initiated active collaboration with several Brazilian natural products chemists. Out of the large number of extracts brought to our laboratory by Dr. Vanderlan da S. Bolzani and screened in our mechanism-based yeast bioassay, extracts derived from two leguminous plants were selected for further studies. Based on preliminary investigation the bioactive methanolic chloroform extract of the leaves of Cassia leptophylla which was shown to contain bioactive alkaloidal constituents was further processed for alkaloids. The alkaloid fraction thus obtained was fractionated by column chromatography on alumina, followed by Silica gel preparative TLC and reversed-phase TLC to afford seven piperidine alkaloids [55] (-)-spectaline (68), (-)-spectalinine (69), canavaline (70), leptophyllin A (71), 3-acetylleptophyllin A (72), iso-... 

Potential Effects on Transcription or Translation To complement direct polymerase intaaction assays, the potential effects of nonnatural monomeric metaboUles on transcription and translation processes should also be considered. Cell-free recombinant transcriplion/translation kits are commercially available (bacterial or mammalian origin) to enable the characterization of individual monomeric metabolites. In principle, any bad actor chemically modified nucleotide could be identified via alterations to overall RNA yield/integrity. Again, these assays could be conducted in competition-type formats (modified NTP spiked into endogenous NTP pool) mimicking relative exposure ratios in vivo. For translation, synthetic RNA templates could be synthesized that contain the chemical modification in question to see whether the presence of that modification in an RNA template alters overall protein yield. As an early hazard ID-type screen, any nucleotide modifications that behave as chain terminators or have some other inhibitory activity should be readily identified. 

A number of inborn errors of metabolism are due to an enzyme deficiency which leads to the accumulation of the precursor of that particular pathway. This type of clinical expression was among the first to be described, since simple clinical screening tests often uncovered the increased excretion in the urine, or accumulation in the blood, of early metabolites of the pathway which had been blocked. An important example of such a defect is homocystinuria, where both methionine and homocystine accumulate as the result of the deficiency of activity of cystathionine synthase (Mudd and Levy, 1978). A variant of the foregoing occurs when both the precursor of the main pathway and metab-... 

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