tag:blogger.com,1999:blog-1136153439451224584.post1588651089182601382..comments2024-03-27T06:45:59.174-07:00Comments on Practical Fragments: Substrate activity screening revisited: substrates as inhibitorsDr. Teddy Zhttp://www.blogger.com/profile/07288045760981372367noreply@blogger.comBlogger3125tag:blogger.com,1999:blog-1136153439451224584.post-83986254024928129162014-10-09T08:00:29.421-07:002014-10-09T08:00:29.421-07:00To date, not too many teams have provided accounts...To date, not too many teams have provided accounts of their experience with SAS in the literature and there are I think still a number of open questions. Please note that as the authors of the publication mentioned in the post, we do support the rationales behind the original SAS methodology, and share the opinion that screening a SAS-library is a good approach to obtain “hit” fragments and inhibitors of enzymatic targets. The main points we try to put forward in the publication are related to designing a screening experiment that renders a maximal amount of information with a minimal amount of resources. This experiment is what we provisionally call MSAS. Our manuscript covers only one target (urokinase), but we have meanwhile significantly expanded our SAS-library and garnered additional experience with two other (cysteine) proteases. In all of these cases, our findings have been fundamentally identical: when screening only for substrates, you overlook a significant number of valuable fragments.<br />Our latest experiments with the cysteine proteases also did not change our view regarding the issue brought up by Matt Soellner. We do need rather large amounts of enzyme to generate a reliable fluorescence signal during a substrate screen, even for the “best” substrates in the library. Examples from the publication include the alkylguanidines that were inspired by the side chain of arginine: one could reasonably argue that urokinase has evolved to specifically accomodate this functionality and to efficiently process substrates containing it. You would therefore expect the corresponding SAS-members to be good substrates. This was not the case. Evidently, the proteases we selected might have lower intrinsic catalytic turnover efficiencies than the targets that Matt Soellner worked on. Soellner’s targets might also be less dependent on processing assistance by other structural features present in the target’s natural substrates, but not in the fragments. Once more, it would be good to learn about other researchers’ experiences. <br />Finally, we are still a bit puzzled by the inhibition experiments mentioned by Soellner. It seems that these were carried out on a subset of the substrates/library (?) to assess the possibility of false negatives, and not on the full library for the sake of identifying inhibitors. All in all, the publications do not go in much depth on these experiments and provide little experimental details for the experiments (substrate/enzyme) concentrations used. It is therefore not evident to compare whether Soellner and our results are unisonous for these experiments.<br />Pieter Van der Vekennoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-79446855183879271302014-09-29T05:07:26.623-07:002014-09-29T05:07:26.623-07:00We tried this with a iso-peptidase and ended up wi...We tried this with a iso-peptidase and ended up with an expensive library with no hits.In the end it turned out the enzyme had an odd biding mechanism where it first bound close to the N-terminal of the ~10 kDa artificial substrate we had been using initially, before the catalytic pocket opened allowing entry of substrate. Our fragment based substrates stood no chance of making this interaction and thus we only got a few false positives.<br /><br />Just goes to show that if the enzymology and crystallography are done well before you start your proposed screen you can avoid slip ups like the above. Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-44405310696499612322014-09-22T07:30:39.763-07:002014-09-22T07:30:39.763-07:00I'm biased because I worked on the phosphatase...I'm biased because I worked on the phosphatase/SAS methodology in Jon Ellman's lab and my laboratory recently reported SAS methodology for kinases (Angewandte 2014), but:<br /><br />The idea that you need to use a lot of enzyme, but that low levels of a contaminating enzyme pose a problem is more than a bit contradictory. If you are looking for substrates for any enzyme, you are going to want to use pure enzyme. But SAS can clearly use low enzyme concentrations, as found with the low amount of contaminating protease giving "false" posititives.<br /><br />In my experience with SAS, the same concentrations used to screening for enzyme inhibitors are sufficient to screen substrate libraries (for example: we use 30 nM kinase with both SAS and traditional inhibitor screening methods). If you cannot find substrates with a reasonable amount of enzyme, then you need to improve your substrate library.<br /><br />Screening the substrate libraries as inhibitors should always be done to avoid false negatives that can occur SAS. In fact, we did this with both phosphatase and kinase libraries. <br />Matt Soellnernoreply@blogger.com