Fragment linking for specific Bcl-2 inhibitors

One of the most well-known examples of a fragment-based program that has yielded a clinical compound is Abbott’s Bcl-2 effort: ABT-263 is currently in over a dozen trials for various cancers. However, this molecule hits several proteins in the Bcl-2 family, and a more specific inhibitor of Bcl-2 alone may have lower toxicity. Phil Hajduk and colleagues have used SAR by NMR to do this, as they report in the latest issue of Bioorg. Med. Chem. Lett.

The researchers started with a protein-detected NMR screen of 17,000 compounds; compound 1 (see figure) was found to be fairly potent, and also roughly 20-fold selective for Bcl-2 over the related protein Bcl-xL. Hajduk’s team did not have a crystal structure at this point, but they were able to use NMR to determine that the compound lies in a large hydrophobic groove. This is the same groove found to bind biaryl acids in earlier work, so the researchers screened a set of 70 of these to see if they could bind in the presence of compound 1. Interestingly, compound 6 was equally potent in the presence or absence of compound 1, suggesting that both fragments could bind simultaneously.

NMR was then used to determine the ternary structure of fragments related to compound 1 and compound 6 bound to Bcl-2, and linking these led to compound 25, with high nanomolar potency. Although this represents a good boost in potency, the binding energies were not additive (let alone synergistic). An NMR structure of one of the linked molecules revealed that, although it binds in the same groove as its component fragments, its position is shifted, and also that one of the protein side chains moves to deepen a hydrophobic pocket. Additional chemistry to fill this pocket led to compound 29, with 40 nM biochemical potency and measurable cell activity, as well as greater than 1000-fold selectivity for Bcl-2 over Bcl-xL and at least 28-fold specificity over other Bcl-2 family members.

This is a nice example of starting with a modestly selective fragment (albeit a jumbo-sized one) and, through fragment linking, increasing both the potency and specificity towards the target protein.

Small but PAINful

We’ve written previously about the phenomenon of compound aggregation, and how this can lead to false positives in high-concentration screening (see also here). Unfortunately, aggregation is not the only thing that can trip you up. Earlier this year Jonathan Baell and Georgina Holloway published a description and systematic catalog of “Pan Assay Interference Compounds,” or PAINS, and Baell has now followed up on this with a Perspective in the October issue of Future Medicinal Chemistry. (For those of you without journal access, he has also summarized some of this material here.)

PAINS are compounds that frequently show up as screening hits, but that act through non-specific mechanisms such as covalent attachment to proteins or generation of hydrogen peroxide. Sometimes it is not the compound itself that is problematic but a breakdown product or leftover reactant; most readers with experience in hit-to-lead discovery will experience a painful sense of déjà vu reading about hits that did not confirm when the compound was resynthesized or repurified. This problem is exacerbated when trifluoracetic acid (TFA, which is toxic to cells and can accelerate compounds’ decomposition) is used in reverse-phase purification of compounds; apparently Pfizer has systematically repurified hundreds of thousands of their screening compounds to remove traces of TFA.

The problem with PAINS is that they may show convincing biochemical and even cell based activity, but mechanistically be useless for further advancement to drugs or even chemical probes. Unfortunately this does not prevent them from being published, where they are picked up by other researchers and identified as “privileged pharmacophores” by computational chemists. This leads to more (wasted) research and more (useless) publications in a vicious circle Baell terms “pollution of the scientific literature.”

Although the focus of the Perspective is high-throughput screening, fragments can be PAINS too, as the sampling below shows.

Compound 1 is obviously a Michael acceptor and compound 2 is unsurprisingly reactive with cysteine, but the problems with compound 3 are less obvious, and compound 4 generates hydrogen peroxide in the presence of reducing agents and oxygen – a mechanism it likely took some time to track down.

One of the fun aspects of reading this paper is that Baell is not shy about calling out high-profile publications that are likely to report false positives, though he readily admits that he too has been misled. The question is what to do: using computational filters to get rid of the worst offenders only eliminates 5-12% of commercial compounds, but more stringent screens can eliminate upwards of 95%!

Baell calls for better stewardship of the scientific literature:

Journals therefore have a responsibility here because if misleading information is published, unintentional though it may be, it is more likely to propagate and be taken as fact by others who may then initiate flawed research projects. Research resources are too precious for this to be acceptable.

I agree whole-heartedly with this, though given what I see published on a weekly basis I despair of this happening any time soon. Ultimately of course the onus is on researchers to carefully follow up and fully understand the mechanism of their hits, particularly those with dubious structures: don’t contribute to the pollution yourself, and don’t let friends (or papers you review) pollute!

FBLD 2010

The last major fragment event of this year is over, but it ends on a high note: FBLD 2010 has remained true to its predecessors in bringing together a great group of fragment enthusiasts in a Gordon-Conference-like environment. With 30 talks and even more posters I won’t attempt to be comprehensive or even representative, but will instead just pick out a few themes. Those of you who were there, please chime in with your own observations.

One of the themes was the shape of chemical space, and what makes a good binder. Jean-Louis Reymond, who has been systematically enumerating all stable molecules containing carbon, nitrogen, oxygen, and a few other atoms, has already published up to 13 heavy atoms but is now expanding his analysis to molecules containing up to 17. The issue of whether more attention should be given to three-dimensional fragments was discussed, with Ken Brameld reporting that fragments in crystal structures at Roche and the protein data bank contain fewer “flat” compounds than does the ZINC database of commercial molecules. However, analyzing 150,000 molecules with MW < 300 that had been screened in 40-100 high-throughput screens at Roche did not show any shape differences between the 50,000 molecules that showed up in at least one screen and those that didn’t. Interestingly, this ratio also came up in a talk by Tony Giannetti of Genentech, who said that across 13 screens 36% of their fragments hit at least one protein, while the rest didn’t hit any. Vernalis has found similar results; is there any way to enrich for the productive binders?

While FBLD 2009 had a strong computational theme, a major thrust of this conference was using biophysics to detect and confirm fragment binding. Tony discussed best-practices in SPR, and noted that since small molecules are “brighter” in NMR assays than proteins it is possible to find even very tiny fragments, including a 6 heavy-atom compound with a Kd of 600 micromolar. Tony also described the use of SPR for weeding out badly behaved compounds. Spookily, he noted that promiscuity is a function of compound, protein, and buffer, so it is not possible to weed out bad actors in a library before screening: one compound that was promiscuous against 8 targets bound legitimately and gave a crystal structure with a ninth. Adam Renslo of the University of California San Francisco described how easy it is to be misled by such phenomena. Glyn Williams described how Astex uses biophysical techniques to detect problem compounds, and noted that oxidizers can be particularly insidious – a trend that will likely continue as people explore novel heterocycles.

Glyn also presented a fascinating if slightly depressing discussion of ligand efficiency. As many have found, it can be challenging to maintain ligand efficiency during the course of fragment optimization. Yet even this goal is too modest. A fragment pays about 4.2 kcal/mol in binding energy when it binds to a protein due to loss of rotational and translational entropy; since this enropy cost is only paid once, atoms added to this molecule do not have this liability . Thus, merely maintaining ligand efficiency means that the atoms being added are binding less efficiently. This point was also emphasized by Colin Groom of the Cambridge Crystallographic Data Centre.

Membrane proteins are increasingly being targeted by fragment-based methods, as recently discussed on this site, and both Gregg Siegal of ZoBio and Rebecca Rich of the University of Utah presented progress against GPCRs.

There was general agreement that many approaches can find fragments, and that using several orthogonal methods is a good way to separate the true binders from the chaff, but a continuing challenge is what to do next. The last two sessions were devoted to chemical follow-up strategies and success stories. Some of these have been at least partially covered on Practical Fragments (for example here, here, and here) but there were a number of unpublished examples too – we’ll try to discuss these individually as they emerge.

If you missed this or the previous two conferences you’ll have another chance in 2012, when the meeting will be held in my fair city of San Francisco. And if you can’t wait that long, there are at least two fragment conferences scheduled for next year – details to come shortly.

Commercial fragments – how do they compare?

Earlier this year we updated our list of commercial fragment suppliers. Now Chris Swain at Cambridge MedChem Consulting has analyzed the structures and properties of eleven of these.

A major conclusion, which will be a disappointment to purchasers of fragments but a boon to suppliers, is that there is very little overlap in terms of exact molecules. Given the vastness of chemical space this shouldn’t be too much of a surprise, but it is striking that, of the 40,000+ molecules represented, less than a dozen are sold by four or more companies. Looking at molecular similarity rather than identity increases the amount of overlap, but for the most part each collection is quite differentiated from the others.

Where it gets really interesting is in the analysis of chemical properties, which Swain has calculated for each fragment set. These include cLogP, molecular weight, polar surface area, H-bond donors and acceptors, heavy atom count, and rotatable bond count. Here the collections are dramatically different, with some being strictly Rule-of-3 compliant while others are much less so. There are also interesting differences in distributions: some collections are distributed around a low molecular weight median, while others are biased towards larger molecules. Finally, an analysis of molecular diversity reveals some collections to be very diverse while others have clusters of closely related molecules.

Of course, people differ in how much weight to put on simple molecular properties. Also, an analysis such as this is necessarily a static snapshot: commercial offerings change over time, and new suppliers continue to enter the market. Moreover, some of the companies offer many more fragment-sized molecules as extension sets beyond their core fragment collections. Still, this is a valuable resource for anyone building or expanding a custom fragment collection. The only thing that would make it even more useful would be price per fragment!

TINS and STD and SPR – oh my!

Following up on our last post on the use of the NMR technique TINS applied to a membrane protein, the same research group has now compared TINS with other techniques on a more conventional target. In addition to TINS, they conducted fragment screens using another commonly used NMR technique as well as surface plasmon resonance (SPR) and high-concentration screening; the results appear in the Journal of Biomolecular Screening.

TINS involves immobilizing a target protein onto a resin, then flowing fragments across the resin and determining whether they bind to the target as assessed by a reduction in their NMR amplitudes. A reference protein is evaluated at the same time; only fragments that bind to the target protein and not the reference are considered hits. In this case, the researchers chose the viral protein RNA-dependent RNA Polymerase (RdRP) as the target and the PH domain from the human protein Akt1 as the reference. Using a total of 4 mg of RdRP, they screened a library of 1270 commercially available fragments in pools of 3 to 5 compounds each, resulting in 74 hits.

One of the most commonly used NMR techniques for fragment screening is saturation transfer difference (STD), in which the magnetization of the protein target is saturated, and so magnetization transfers to any ligands bound to the protein. The researchers tested 133 fragments (both hits and non-hits from the TINS experiments) and found a total of 49 hits, of which 40 had also been found by TINS.

The 83 fragment hits from both TINS and STD were tested for their ability to inhibit polymerase activity at concentrations up to 2 millimolar; 70 of them showed some activity, and a few of these seemed to actually activate the enzyme.

Finally, a selected set of 62 fragments (all of which were hits in at least one of the three assays) were tested in an SPR assay at concentrations up to 0.2 millimolar. Of these, around half showed binding, and these tended to be the fragments that showed the greatest activity in the enzymatic assay.

The authors conclude that TINS picks up more hits than the other assays, though high-concentration screening comes close. This may be true, but it would have been nice if they had run the entire set of 1270 fragments through each of the different methods; it is possible that there were false negatives in the TINS experiments that could have been picked up by the other techniques. Moreover, some of the TINS hits that didn’t confirm in other assays may well have been false positives. Still, there are lots of useful data in this paper, and it demonstrates yet again the importance of using multiple, orthogonal techniques to discover and properly validate fragments.