The Natural Product N-Palmitoyl-l-leucine Selectively Inhibits Late Assembly of Human Spliceosomes.

The spliceosome is a dynamic complex of five structural RNAs and dozens of proteins, which assemble together to remove introns from nascent eukaryotic gene transcripts in a process called splicing. Small molecules that target different components of the spliceosome represent valuable research tools to investigate this complicated macromolecular machine. However, the current collection of spliceosome inhibitors is very limited. To expand the toolkit we used a high-throughput in vitro splicing assay to screen a collection of pre-fractions of natural compounds derived from marine bacteria for splicing inhibition. Further fractionation of initial hits generated individual peaks of splicing inhibitors that interfere with different stages of spliceosome assembly. With additional characterization of individual peaks, we identified N-palmitoyl-l-leucine as a new splicing inhibitor that blocks a late stage of spliceosome assembly. Structure-activity relationship analysis of the compound revealed that length of carbon chain is important for activity in splicing, as well as for effects on the cytological profile of cells in culture. Together these results demonstrate that our combination of in vitro splicing analysis with complex natural product libraries is a powerful strategy for identifying new small molecule tools with which to probe different aspects of spliceosome assembly and function.

Abyssomicin 2 reactivates latent HIV-1 by a PKC- and HDAC-independent mechanism.

Screening of a marine natural products library afforded three new analogues of the tetronic acid containing polyketide abyssomicin family and identified abyssomicin 2 as a selective reactivator of latent HIV virus. Examination of the mode of action of this new latent HIV reactivating agent demonstrated that it functions via a distinct mechanism compared to that of existing reactivating agents and is effective at reactivating latent virus in a subset of primary patient cell lines.

Insights into the capillary electrophoresis separation of heparin disaccharides from nuclear magnetic resonance, pKa, and electrophoretic mobility measurements.

Determination of the pK(a) values of heparin disaccharide functional groups can provide insights into the nature of glycosaminoglycan (GAG)-protein interactions and prove useful for optimization of the charged-based separations typically used in GAG analysis. In order to gain a better understanding into the capillary electrophoresis (CE) separation process, the pK(a) values of the carboxylate and primary amine moieties of 11 heparin disaccharide standards were determined through (1)H NMR detected pH titrations. These pK(a) values were used to calculate the effective net charge of each disaccharide and compared to the electrophoretic mobilities measured by CE. Although a different migration order had been reported by other researchers, our results indicate a strong positive correlation between the two measurements, consistent with the migration order observed in our CE separations. The effect of mutarotation was also examined by (1)H NMR. Mutarotation equilibrium constants favored the alpha anomer over the beta conformation. pK(a) values determined for both anomers of the four disaccharide standards containing a GlcN primary amine indicated that the beta anomer of the GlcN residue was more acidic. Partial separation of these anomers was achieved in CE separations using either formic acid or phosphate buffer.