15 April 2011

Sixth Annual Fragment-Based Drug Discovery

The only US-based conference completely devoted to fragment-based drug discovery ended in San Diego this week. As with last year, I won’t attempt to summarize all of the talks – there was far more information presented than I have time to write (or that you probably have patience to read!) For those of you who were there, please feel free to mention some of the things I missed.

One of the points that Don Huddler (GlaxoSmithKline) and I (Carmot) made in the pre-conference short-course is that finding fragments is a solved problem. As Rod Hubbard (Vernalis, University of York) noted in his opening presentation, “it’s pretty simple to find fragments that bind; a graduate student can do it in a couple months.” Even membrane proteins are starting to yield to fragment-based screening, as Gregg Siegal (ZoBio, Leiden University) discussed in his closing session (see also here).

That’s not to say that new methods for finding fragments aren’t useful, particularly if they open new target space, are faster or more reliable, or provide new information. An example of the latter was the presentation by Denis Zeyer (NovAliX) on native mass-spectrometry (see also here). Because hydrophobic interactions are weaker in the gas phase than in water, it should be possible to select for molecules that bind predominantly through polar interactions. In fact, by gradually increasing the voltage in their MS instrument, Zeyer and colleagues generated “VC50” curves, the voltage at which half the compound dissociates from the protein. At least in one case, a higher VC50 correlated with the presence of an additional hydrogen bond to the protein compared with related molecules.

Polar contacts are generally associated with enthalpic rather than entropic interactions, and whether such fragments are preferable was the subject of some discussion, particularly at a breakfast round-table discussion. In contrast to a meeting just last year, several participants were actively collecting thermodynamic data, though there was some uncertainty as to what to do with it. This is a controversial subject; one person suggested that enthalpic binders are likely to be more hydrophilic than entropic binders, so just keeping an eye on lipophilicity is likely to be just as useful and far easier than actually measuring thermodynamic parameters. Charles Reynolds (Ansaris) provided an analysis that illustrates some of the difficulties in using thermodynamic data – I’ll follow up on this in a later post.

The shape of fragments has been previously discussed, and Ivan Efremov (Pfizer) gave a nice case study of a strikingly three-dimensional fragment: an X-ray screen of 340 molecules against BACE resulted in a single hit, a spirocyclic pyrrolidine. The electron density of this was so clear that it didn’t even need to be deconvoluted from the other three compounds in the pool, and medicinal chemistry ultimately led to low micromolar inhibitors.

There was general consensus that ligand efficiency (and various lipophilicity adjusted versions) is a helpful metric. One practitioner said that his company had sometimes pursued more chemically tractable but less ligand efficient fragments and generally came to regret those decisions. But a fragment with lower ligand efficiency could still be interesting: with fragments, even small changes could have huge effects on binding (see for example AT13387, which was discussed by Chris Murray of Astex). Thus, a bit of initial fragment optimization could be a good investment before pursuing more intensive chemistry, particularly if commercial or in-house analogs are available. Interestingly, I couldn’t find anyone who uses either fit quality or %LE.

In the early days of fragment-based lead discovery a common selling point was that it sped up drug discovery, but a theme in this meeting was that it is not necessarily faster but can provide leads against more difficult targets or better leads against “normal” targets. Of course, one has to be wary of taking a good fragment, slapping a bunch of grease on it, and turning it into a lipophilic monster.

Indeed, an analysis of fragment-derived leads published a couple years ago was not flattering. Taking up the thrown gauntlet on behalf of fragments, Chris Murray presented a retrospective analysis of all 42 fragment to lead programs at Astex (including 21 kinases and 9 proteases). The average parameters of these leads were considerably more attractive in terms of both molecular weight and ClogP that the published values of the HTS hits. At least according to this analysis, fragment approaches have the potential to deliver superior molecules, as long as one is disciplined and creative in how these approaches are applied.


stowstepp said...

Thanks for the update Dan. I couldn't make this conference this year so it's good to hear an overview. I look forward to your post on Charles Reynolds' work.

Pete said...

The hydrophobic effect shouldn't operate at all in gas phase (sicne it's driven by cohesive forces between water molecules) and electrostatic interactions will be exaggerated. I'd be very concerned about the relevance of gas phase measurements to their solution phase equivalents.

Dan Erlanson said...

Thanks Pete,

The same thought occurred to me and in fact was brought up in the Q&A, but apparently NovAliX gets reasonably good correlations between IC50 values determined by mass-spectrometry and those determined using standard methods. One possibility is that at low voltage you still have a halo of loosely associated water molecules around the protein-ligand complex, but that these get blown away at higher voltage. It's an important point to consider, and it will be interesting to see more data.

Valerie Vivat (Novalix) said...

Hi everyone,
When analyzing non-covalent complexes by mass spectrometry under non-denaturing conditions, one must keep in mind indeed how interactions stabilizing the complex in solution will be affected by the ion transfer in the gas phase. Basically, interaction based on hydrophobic effect is lost while the electrostatic-based interactions (Van der Waals, H-bonds, ionic interaction) are preserved (and are even strengthened due to the absence of solvent shielding). Regarding water molecules now, during the ESI process, biomolecules are transferred in the gas phase as partially hydrated ions and complete ion desolvation is subsequently achieved through low energy collisions with the residual gas molecules in the mass spectrometer interface. This complete desolvation is required for accurate mass measurements. Consequently, in most cases, molecular mass measurement of the complexes is accurate enough to rule out the possibility that water molecules remain after ions are transferred from the atmospheric pressure to the deep vacuum of the TOF analyzer, even in the case of crystallographic water molecules.
Regarding gas phase stability : Reports in the literature mention that collision-induced dissociation experiments and the determination of Vc50s can be used to monitor gain or loss in polar interaction. Indeed, we have now accumulated a variety of in house examples showing that increase in the gas phase stability of a complex is correlated with a gain in polar interactions as shown by X-ray or ITC. Monitoring the complex gas phase stability is thus an attractive feature to quickly evaluate, for example, gain or loss in polar interaction of analog series targeting the same protein binding site.
Importantly, the gas phase stability is not an indication of the complex binding affinity. Evaluation of complex affinity (after complexes are formed in solution) is done under instrumental conditions showing no dissociation of the non-covalent complexes. For complexes stabilized by electrostatic-based interactions (and thus rather stable in the gas phase), it is indeed much easier to find optimal instrument settings compared to complexes stabilized by hydrophobic effect. However, this does not preclude complexes mainly stabilized in solution by hydrophobic effect to be detected at all. For example, nuclear hormone receptors in complex with fatty acids or phospholipids are readily detected with appropriate settings. In this case, the polar head of the ligands is likely to act as a stable anchor in the gas phase, resulting in non-covalent complexes which remain intact during the ca. 1 millisecond flight of the ion from the ionization source to the detector.
To sum up, native MS can be run under two different modes:
1. Low energy conditions where no complex dissociation occurs are used to assess the binding affinity of protein / ligand complexes,
2. High energy conditions inducing complex dissociation provide insight into the extent of polar interaction involved between the protein and the ligand. This type of experiment makes sense to compare a series of molecules binding to the same protein binding site.

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