Novel Small Molecules Targeting the Spliceosome for Cancer Therapy 1. Background Targeting the spliceosome to fight cancer: The spliceosome is fundamental for the generation of mature mRNA and the regulation of the gene expression in eukaryotic cells. Importantly, its biological mechanism (splicing) contributes to proteome complexity. 1 However, some human diseases, including cancer, are linked to splicing mis-regulation, particularly for alternatively spliced genes. A recent comparative study over 8,705 patients and 32 different types of cancer has revealed that there are up to 30% more mis-regulation of alternative splicing events in tumours than in normal tissues. 2 Thus, the splicing machinery represents a solid target for developing new effective anticancer therapies. Cancer and Spliceosome: Abnormal spliced mRNA isoforms have been found in different types of cancer and have been proven to play a crucial role in tumour progression and invasion, as well as in cell proliferation, apoptosis, angiogenesis, and metabolism.3 When the tumour is generated by these isoforms, the clinical outcome of traditional cancer therapies can be compromised, as demonstrated for some types of lung, ovarian and breast cancer, or melanoma, and several other cancer types.4-6 Interestingly, it has been shown that small molecules acting on cancer-specific splicing factors can help overcoming this kind of drug resistance.7 Also, preclinical studies have shown that such molecules preferentially target cancer cells rather than healthy cells.8 However, these molecules can still interfere with off-target proteins in healthy tissues, with the possibility of severe side effects.9 For these reasons, specific spliceosome modulators are urgently needed. State of the art of spliceosome inhibitors for cancer: small molecules spliceosome inhibitors can be classified into two main categories10: the first category is constituted by spliceosomal assembly inhibitors, which block the formation of the spliceosome active complex. These molecules bind specific splicing factors, overexpressed in tumoral cells, interrupting the spliceosomal assembly and so its activity. The second category of inhibitors target spliceosome-related enzymes (overexpressed in various human cancer cells) involved in the recognition between the pre-RNA and the spliceosome complex. These enzymes play an important role on exon definition via binding to enhance or silence sequences in the pre-RNA.11 Although the inhibitors presented in literature have shown antitumoral activity, with promising preliminary results both in vitro and in vivo, only a very few, at the best of our knowledge, have entered into clinical studies.3 It is important to note that these compounds operate on components of the spliceosome machinery and not on the active core of the spliceosome itself. Interestingly, the first small molecule acting on subunit-RNAs located in the proximity of the spliceosome catalytic core has been recently approved by the FDA to treat Spinal Muscular Atrophy (SMA).12 The discovery of such a drug through cell-based assays, represents a milestone on RNAdirected drug discovery. Small molecules modulating RNA therefore play a crucial role in modern drug discovery. Importantly, the complex atomic structure of the spliceosome’s catalytic site was enlightened only recently when high-resolution crystal structures of the major spliceosomal complex Ci 13 and the minor spliceosomal complex Bact 14 have been finally solved. These structural data, together with phylogenetic analysis, chemical mapping, enzymatic and biological assays, are the bases to crucially expand the chemical space of RNA-directed small molecules. For all these reasons, the development of novel, potent, selective, and safer anti-cancer drugs targeting the spliceosome’s active core is a therapeutic strategy with great potential.15 In this context, here we propose a structure-based drug design strategy to discover and optimize novel small molecules capable of modulating the spliceosome activity in tumoral cells and fight cancer.