Molecular architecture of zinc chelating small molecules that inhibit spliceosome assembly at an early stage

Abstract

The removal of intervening sequences (introns) from a primary RNA transcript is catalyzed by the spliceosome, a large ribonucleoprotein complex. At the start of each splicing cycle, the spliceosome assembles anew in a sequentially ordered manner on the pre-mRNA intron to be removed. We describe here the identification of a series of naphthalen-2-yl hydroxamate compounds that inhibit pre-mRNA splicing in vitro with mid-to high-micromolar values of IC 50 . These hydroxamates stall spliceosome assembly at the A complex stage. A structure-activity analysis of lead compounds revealed three pharmacophores that are essential for splicing inhibition. Specifically, a hydroxamate as a zinc-binding group and a 6-methoxynaphthalene cap group are both critical, and a linker chain comprising eight to nine methylene groups is also important, for the specific binding to the docking site of a target protein molecule and precise positioning of the zinc binding group. As we found no correlation between the inhibition patterns of known histone deacetylases on the one hand and pre-mRNA splicing on the other, we conclude that these compounds may function through the inhibition of the activities of other, at present, unknown spliceosomeassociated zinc metalloprotein(s).

Transcriptome profile analysis reflects rat liver and kidney damage following chronic ultra-low dose Roundup exposure

“…Indeed, glyphosate can interact at the substrate binding site and potentially inhibit mitochondrial succinate dehydrogenase [ 27 ]. Furthermore, as small molecule chelators of zinc can perturb spliceosome assembly and activity of chromatin modifying enzymes [ 28 ], glyphosate may also have exerted direct effects on spliceosome function due to its metal chelating properties (Patent No: US 3160632; [ 29 ]).…”

Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation

“…These include, among others: (i) Spliceostatin A, Pladionelide B, Sudemycin, GEX1A and related compounds that target the U2-associated SF3b protein complex ( Kaida et al, 2007 ; Kotake et al, 2007 ; Fan et al, 2011 ; Hasegawa et al, 2011 ) and stall splicing during the A complex stage ( Roybal and Jurica, 2010 ; Corrionero et al, 2011 ; Folco et al, 2011 ), (ii) isoginkgetin ( O'Brien et al, 2008 ) and (iii) 1,4 napthoquinones and 1,4-heterocyclic quinones, which block specifically the second catalytic step of splicing ( Berg et al, 2012 ). Several other splicing inhibitors have been reported ( Patil et al, 2012 ; Samatov et al, 2012 ; Effenberger et al, 2013 ; Pawellek et al, 2014 ; Effenberger et al, 2015 ), including inhibitors of protein acetylation/deacetylation ( Kuhn et al, 2009 ) and of DNA topoisomerase I ( Tazi et al, 2005 ). Finally, compounds that inhibit the kinase activity of members of the Clk/Sty, SRPK, or DYRK protein families, which phosphorylate serine-arginine-rich SR splicing factors and other spliceosomal proteins, were also identified and shown to modulate alternative splicing patterns in vivo (reviewed by Ohe and Hagiwara, 2015 ).…”