Synthesis of Trichodermin Derivatives and Their Antimicrobial and Cytotoxic Activities.

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9, 13, and 15, which presented average IC50 values of 2 µg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes).

Affinity labeling the ribosome with eukaryotic-specific antibiotics: (bromoacetyl)trichodermin.

Trichodermin, a eukaryotic-specific antibiotic, inhibits protein synthesis in Drosophila cells. We have synthesized a 14C-labeled bromoacetyl derivative of trichodermin that binds to Drosophila 80S ribosomes and once bound reacts covalently with ribosomal proteins. It does not react with rRNA. Three large-subunit proteins (L1, L3, and L24) and three small-subunit proteins (S3/S5, 2/3S, and S8) are labeled by [14C] (bromoacetyl)trichodermin. Reaction with each of these proteins can be competed by an excess of unmodified trichodermin, indicating that the labeling has occurred from the native binding site of the parent drug. One of the (bromoacetyl)trichodermin-labeled proteins (S8) is also labeled by photoactivated puromycin in the A site. A second protein (S3/S5) is found to be labeled by a P-site affinity reagent. The results suggest that the trichodermin binding site spans both the small and large subunits and portions of both the A and P sites. These data combined with previous studies on the A and P sites of Drosophila ribosomes have allowed us to construct a model of the protein locations in this important active site.

Competition between trichodermin and several other sesquiterpene antibiotics for binding to their receptor site(s) on eukaryotic ribosomes.

1. Of the five sesquiterpene antibiotics tested and found to inhibit protein synthesis in yeast spheroplasts, trichothecin, trichodermol or trichodermin stabilized polyribosomes whereas, in contrast, verrucarin A or T-2 toxin induced 'run off' of polyribosomes with a corresponding increase in 80S monoribosomes. The effect of fusarenon X on the system could not be determined as the drug failed to enter the cells. 2. [acetyl-14C]Trichodermin bound to yeast polyribosomes with a dissociation constant of 2.10 muM and to yeast 'run off' ribosomes with a dissociation constant of 0.72 muM. 3. Trichothecin, trichodermol, fusarenon X, T-2 toxin and verrucarin A competed with [acetyl-14C]trichodermin for binding to its receptor site on 'run off' ribosomes. The observed competition was quantitatively similar for all drugs tested. In contrast, the five drugs competed to different extents with trichodermin for binding to its receptor site on polyribosomes. Thus trichothecin competed with relative efficiency, whereas verrucarin A competed poorly, and the other drugs occupied intermediate positions between these two extremes. 4. Studies were also carried out with yeast 'run off' ribosomes prepared from both a wild-type strain and a strain resistant to trichodermin. Competition experiments between verrucarin A and [3H]anisomycin indicated that verrucarin A bound to 'run off' ribosomes from the mutant strain less efficiently than to those from the wild-type.