The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction. Importantly, compounds of this family specifically inhibit exonic splicing enhancer (ESE)-dependent splicing, because they interact directly and selectively with members of the serine-arginine-rich protein family. Treatment of cells expressing reporter constructs with ESE sequences demonstrated that selected indole derivatives mediate inhibition of ESE usage in vivo and prevent early splicing events required for HIV replication. This discovery opens the exciting possibility of a causal pharmacological treatment of aberrant splicing in human genetic disorders and development of new antiviral therapeutic approaches.splicing correction ͉ exonic splicing enhancer ͉ small chemicals ͉ pathologic splicing R emoval of introns from newly transcribed RNA polymerase II precursors (pre-mRNA) during splicing not only is an essential step for the expression of most genes in higher eukaryotic cells but also constitutes an important mechanism for generation of protein diversity and regulation of gene expression (1, 2). It is estimated that Ͼ70% of human genes are subjected to alternative splicing, and it is not surprising that many point mutations causing human diseases are associated with aberrant splicing (3, 4).Current models of constitutive and a fortiori alternative splicing suggest that splice site recognition is strongly modulated by the interaction of specific exonic and intronic pre-mRNA sequences with at least two classes of nonspliceosomal nuclear RNA-binding proteins: serine-arginine-rich (SR) proteins (5-7) and heterogeneous nuclear ribonucleoproteins (8-10). These proteins interact with spliceosomal components (5-7) and either activate or prevent the use of degenerate splice sites in their vicinity. Thus, binding of SR proteins to exonic splicing enhancers (ESE) through their RNA-recognition motif (RRM) promotes exon definition by recruiting constitutive factors via protein-protein interactions mediated by their arginine-serine-rich (RS) domain and prevents the action of nearby splicing silencers (4, 6, 11).Mutations causing human diseases may affect splice sites as well as regulatory sequences leading to the production of defective proteins (4, 11). Thus, targeting either the mutated sequences or the factors that bind them may prove to be a valuable strategy to correct aberrant splicing. Recently, antisense strategies targeting ESEdependent mechanisms have been used to induce skipping of exons containing nonsense mutations or, conversely, to restore exon inclusion by synthetic exon-specific effectors (bifunctional antisense peptide molecules or tailed antisense oligonucleotides) or spliceosome-mediat...

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