OK, time (finally) for the latest chapter in the GSK-Sirtris saga. (This is going to get fairly geeky, so feel free to skip ahead if you're not into enzymology). You'll recall from previous installments that Amgen and Pfizer, among others, had disputed whether the reported sirtuin compounds worked the way that had originally been reported. GSK has now published a paper in the Journal of Biological Chemistry to address those questions. How well does this clear things up? Let's take things in order:
Claim 1: Resveratrol is not a direct activator of SIRT1 activity (Amgen). Building on two 2005 papers, the Amgen team said that resveratrol, the prototype SIRT1 ligand, only works in that manner when the fluorescent peptide (Fluor de Lys) was used in the assay. This is due, they found, exclusively to the fluorophore on the peptide - it's an artifact of the assay conditions. Without it, no activation was seen with protein assays in vitro, nor in cell assays. Native substrates (p53-derived peptide and PGC-1alpha) show nothing.
GSK's response: This is true. They too, found that activation of SIRT1 depends on the structure of the substrate. Without the fluorescent label, no activation is seen.
Claim 2: Not only is this true for resveratrol, it's true for SRT1720, SRT2183, and SRT 1460 (Pfizer). The Pfizer team did a similar breakdown of the assay conditions, and found (through several biophysical methods) that the fluorophore is indeed the crucial element in the activity seen in these assays. And again, since that's an artificial tag, the Fluor de Lys-based assays can have nothing to do with real in vivo activity. Native substrates (p53-derived peptide, full-length p53, and acetyl CoA synthase 1) show nothing.
GSK's response: As above, activation of SIRT1 depends on the structure of the substrate. Without the fluorescent label, no activation is seen. SRT1460 and SRT1720 do indeed bind to the fluorescent peptide, but not to the unlabeled versions. Looking over a broader range of structures, some of them interact with the fluorophore, and some don't. There's no correlation between this affinity and a compound's ability to activate SIRT1.
A screen of 5,000 compounds in this class turned up three that actually do work with nonfluorescent peptide substrates (compounds 22, 23, and 24 in the paper). None of these have been previously disclosed. They, however, that even these still don't work when the peptide substrate lacks both the fluorescent tag and a biotin tag.
What's more, when these three compounds are tested on a p53-derived 20-mer peptide substrate, they actually inhibit acetylation, instead of enhancing it. Looking closer at a range of peptide substrates, SRT1460 and other compounds can also inhibit or enhance acetylation, depending on what peptide is being used. An allosteric mechanism could explain these results. It seems more likely that there are at least two specific sites on SIRT1 that can bind these compounds - the active site and an allosteric one. Thus there are several species in equilibrium, depending on whether these sites have substrate or small molecule bound to them, and on how this binding stabilizes or destabilizes particular pathways. In the real cell, this may all be part of various protein-protein interactions.
Claim 3: SRT1720 does not lower glucose in a high-fat-fed mouse model (Pfizer). Even though exposure of the drug was as reported previously, they saw no evidence (at 30 mg/kilo) of glucose lowering or of any increased mitochondrial function. These animals showed increased food intake and weight gain. The 100 mpk dose was not well tolerated, and killed some animals.
GSK's response: not addressed in this paper. It's an enzymology study only.
Claim 4: Resveratrol, SRT 1460, SRT1720, and SRT2183 are not selective (Pfizer). A screen of over 100 targets showed all of these compounds hitting multiple targets, with resvertrol itself showing the closest thing to a clean profile. None of them, say the Pfizer team, are suitable pharmacological tools.
GSK's response: not addressed in this paper. None of the newly disclosed compounds have selectivity data of this sort attached to them, either. I'd be very curious to know how they look, and I'd be very leery of attaching much importance to their behavior in living systems until that's been done.
The take-home: On the enzymology level, this new paper seems to be solid work. But it's the sort of solid work that should have been done around the time that GSK bought Sirtris, and not something appearing in 2010 in response to major attacks in the literature. The first main claim of those attacking papers is, in fact, absolutely true: the original Fluor de Lys assay is worthless for characterizing these compounds. What we learn from this paper is that the assay is worthless for even more complicated reasons than originally thought, and that the whole series of SRT compounds behaves in ways that were not apparent from the published work, to put it lightly.
As to the selectivity and in vivo effects of these compounds, Pfizer's gauntlet is still thrown down right where they left it. The fact that these compounds are so much harder to understand than was originally thought, even in well-controlled enzyme assays, makes me wonder how easy it will be to figure out the rest of the story. . .