PDF file: EURASNET Annual Report 2008

variation and suitability for use within the context of the automated cell screening platform available at the University ofDundee Drug Hit Discovery Facility (http://www.drugdiscovery.dundee.ac.uk/shdf/overview/). Future work will include theuse of one or more of these cell lines for a new screen for splicing inhibitors.251 Evaluate the mechanism of splicing inhibition by small molecule effectors identified in screening initiative.This milestone is on target and has been partially achieved to date with other work still ongoing and also dependent uponoutput from activity 249 above. Work from the Lührmann group has identified the mechanism of action of a small moleculesplicing inhibitors (“Stalling of spliceosome assembly at distinct stages by small-molecule inhibitors of protein acetylationand deacetylation.” Kuhn et. al., RNA 2009 (epub Nov 2008) 15, 153-175). The Lührmann group have shown that severalsmall-molecule inhibitors of protein acetylation/deacetylation block the splicing cycle. Specifically, they found that threesmall-molecule inhibitors of histone acetyltransferases (HATs), as well as three small-molecule inhibitors of histonedeacetylases (HDACs), block pre-mRNA splicing in vitro. By purifying and characterizing the stalled spliceosomes, theyshowed that the splicing cycle is blocked at distinct stages by different inhibitors: two inhibitors allow only the formation ofA-like spliceosomes (as determined by the size of the stalled complexes and their snRNA composition), while the othercompounds inhibit activation for catalysis after incorporation of all U snRNPs into the spliceosome. Mass-spectrometricanalysis of affinity-purified stalled spliceosomes indicated that the intermediates differ in protein composition both fromeach other and from previously characterized native A and B splicing complexes. This suggests that the stalled complexesrepresent hitherto unobserved intermediates of spliceosome assembly.The Lamond group have identified two classes of small molecules that inhibit splicing of mRNA precursors in vitro andexperiments to analyse their mechanism of action will be undertaken in the next phase of the projectProblems and explanations for delays, postponements etc.249 Screen at least 10,000 compounds in vitro for inhibitory effects on splicing.This milestone has been the most problematic part of the project where delays in implementing the fully automated screenusing a highly selected collection of “drug-like” small molecules in a compound library available at the University ofDundee have been experienced. The major reason for the delay is that a large scale screen requires an automated assaysystem and cannot be commenced until a robust assay system has been fully validated. The main problem has beenexperienced in adapting the read-out from the assay to make it suitable for use with the plate reader technology available tous in the Hit Discovery Facility. Because of the constraints imposed by the use of the robotic platform, particularly withrespect to control of temperature, a larger degree of assay redesign was needed than originally anticipated. Another majorconstraint in this regard is the need to keep the unit cost down per assay to make the overall cost involved in screening alarge compound library economically feasible within the budget. This project thus involves a high overhead cost and thelimitations in the funding currently available for the project has had a detrimental effect on the rate of progress possible.74