Sudemycins, novel small molecule analogues of FR901464, induce alternative gene splicing

RNA splicing in mammalian cells is an exceedingly complex process that requires over 150 proteins, a panel of small nuclear ribonucleoprotein particles, and a series of small nuclear RNAs (snRNA). These molecules act in concert to achieve high fidelity editing of pre-mRNAs to yield bona fide mRNAs that are then subject to translation. Hence, the specificity and reproducibility of the events involved in the generation of these mature RNAs is likely to be highly regulated. Furthermore, due to the critical nature of these processes and their necessity to achieve accurate protein production, potentially the spliceosome represents a valid target for cytotoxic molecules. To this end, several natural products (pladienolide B and {"type":"entrez-nucleotide","attrs":{"text":"FR901464","term_id":"525229801"}}FR901464 (1, 2); Figure 1) have been identified that interact with, and disrupt the function of, the spliceosome. These compounds are both potent cytotoxins with IC50 values in the low nM range and the latter arrests cells in the G1 and G2/M phases of the cell cycle. The specific biological target for these drugs has been identified as the SF3b subunit of the spliceosome, a complex of at least six proteins and small nuclear RNAs (snRNA; 1, 2). It is thought that the entire SF3 subunit (including SF3a and SF3b) prevents inappropriate nucleophilic attack by other components of the spliceosome, prior to the initial transesterification reaction that must occur to achieve RNA splicing (3). Figure 1 Structures of pladienolide B, E7107, {"type":"entrez-nucleotide","attrs":{"text":"FR901464","term_id":"525229801"}}FR901464, Spliceostatin A and the Sudemycins. The lower structure indicates a summary of the design of concise analogs of {"type":"entrez-nucleotide","attrs":{"text":"FR901464","term_id":"525229801"}} ... Both pladienolide B and {"type":"entrez-nucleotide","attrs":{"text":"FR901464","term_id":"525229801"}}FR901464 are chemically complex and contain 9 and 10 stereocenters, respectively, such that they cannot be readily synthesized (the total synthesis of the latter molecule has recently been reported and requires greater than 40 independent reaction steps (4)). In addition, these molecules have limited chemical stability and are readily degraded in biological fluids. While a derivative E7107 (Figure 1) has been used in preclinical studies (2) , it is unclear whether the problems and liabilities associated with these natural products are predisposed for facile and effective use as therapeutic agents (5, 6). Therefore, we have developed a series of analogues (Sudemycins; Figure 1 (7, 8)), based upon the consensus pharmacophore obtained from pladienolide B and {"type":"entrez-nucleotide","attrs":{"text":"FR901464","term_id":"525229801"}}FR901464. The Sudemycins are structurally much less complex (demonstrating 6 stereocenters fewer than the natural products), show much better chemical stability (9), are not degraded in human plasma, exhibit IC50 values of ~80-500nM in a panel of human tumor cell lines, and have demonstrated activity in human lymphoma xenografts models (7, 8). We have also demonstrated that Sudemycin derivatives show the same effects as have been reported for the natural product spliceosome modulators including; inhibition of spicing in an in vitro cell-free splicing assay, inhibition of splicing in a cell-based dual reporter assay, cell cycle arrest, and alteration of the cellular localization of SF3b splicing factors (7, 8). Furthermore, a considerable amount of SAR analyses have been performed to identify key functionalities and chemotypes within the scaffold that are required for biological activity. Based upon these results, 3 analogues have been further developed namely Sudemycin C1, E and F, respectively (Figure 1). While Sudemycins and the associated natural products demonstrate significant potency toward tumor cells, in general, these molecules are much less toxic to normal human cells (1, 2, 7, 8). The mechanism for this selective toxicity is not understood since it would be anticipated that inhibition of the spliceosome would likely be a lethal event. We have therefore sought to evaluate the role of the Sudemycins in the alteration of RNA splicing in tumor cells, since this phenomenon has been found to be present in numerous tumor lines (10). Our results indicate that these compounds are potent modulators of alternative splicing using both endogenous genes and in mini gene constructs in tumor and normal cell lines. In addition, these effects can be demonstrated both in vitro and in vivo. We conclude that modulation of mRNA splicing is a key event in the toxicity of the Sudemycins.