Flipping fragments in CDK8

The cyclin-dependent kinases (CDKs) are targets for a variety of diseases, particularly cancers. One of the earliest posts at Practical Fragments discussed the clinical-stage AT7519, which inhibits several CDKs. A new paper in Bioorg. Med. Chem. Lett. by Xingchun Han, Song Yang, and their colleagues at Roche Innovation Center Shanghai describes the discovery of a selective CDK8 inhibitor.

The researchers started with a biochemical screen (at 100 µM) of ~6500 fragments, all with less than 19 non-hydrogen atoms. A whopping 403 compounds showed >70% inhibition, and of 227 tested in full dose-response curves, 48 had IC₅₀ < 50 µM with ligand efficiency > 0.3 kcal/mol/atom. Compound 1 was both potent and structurally interesting.

SAR by catalog led to several more active analogs, including compound 4, which was crystallographically characterized bound to CDK8 (blue). The pyridine nitrogen makes a hydrogen bond with the hinge-region of the kinase, while the pyrrole nitrogen makes a water-mediated bond to the protein. Interestingly though, benzylation of the pyrrole slightly improved affinity, suggesting that the molecule can bind in a flipped orientation, with the pyrrole nitrogen pointing out towards solvent. This binding mode would provide easy access to a small hydrophobic pocket, a hypothesis that was supported when compound 17 showed a dramatic increase in affinity. A crystal structure of compound 17 bound to CDK8 confirmed the flipped binding mode.

A closely related molecule (replacing the chlorine atom with a trifluoromethyl group) showed oral bioavailability and good pharmacokinetics in mice. And another closely related compound (methyl instead of chlorine) showed excellent selectivity against a panel of 43 kinases.

There are several practical lessons in this brief paper. First, very minor changes can lead to massive improvements in affinity. Indeed, compound 17 has the same number of non-hydrogen atoms as the initial fragment, yet binds almost 1000-fold more tightly. Second, it is possible to discover selective kinase inhibitors while staying well within the ATP-binding pocket, and doing so may cut down on molecular obesity too (compare this paper with the CK2 story highlighted last week.) And finally, while structural information can be enabling, it is always important to remember that molecules – even reasonably potent ones – can dramatically change binding modes with the slightest modification. Remaining alert to this possibility can open new opportunities.

Fragment linking to a selective CK2 inhibitor

The kinase CK2 is an intriguing anti-cancer target, but most of the reported inhibitors bind in the conserved hinge region and so also hit other kinases, complicating interpretation of the biology. A team based at the University of Cambridge has taken a fragment-linking approach to discover more selective inhibitors. The first report was published last year by Marko Hyvönen, David Spring, and colleagues in Chem. Sci., and they have now published a more complete account in Bioorg. Med. Chem.

A crystallographic screen identified compound 1, which bound to six different sites! One of these sites was particularly interesting as it appeared to be a previously undiscovered “αD” pocket near the ATP-binding site. A couple cycles of SAR by catalog, informed by computational screening, led to compound 7, which binds in the desired pocket but not at other sites.

Although compound 7 has measureable affinity for CK2α as judged by ITC, it does not inhibit the enzyme, which is not surprising because it does not bind in the ATP-binding site. Thus, the researchers screened 352 fragments from Zenobia in cocktails of 4, each at 5 mM, and found 23 that bound in the ATP site. Reasoning that the hinge region is the most conserved portion of the ATP-binding site, the researchers avoided fragments that bound there. This led them to focus on compound 8, which has a synthetic handle pointing towards the αD pocket.

Next, modeling was used to generate a series of appendages from compound 7 to try to reach compound 8. Compound 19 looked like it could bridge the gap, a hypothesis which was confirmed when linking led to a low micromolar binder. Tweaking the linker led to CAM4066, which showed nanomolar binding as well as inhibition of CK2. Crystallography revealed that the linked molecule bound as expected.

CAM4066 was tested against 52 other kinases at 2 µM and showed at most only 20% inhibition, suggesting that it is indeed quite selective for CK2. Unfortunately, perhaps because of its carboxylic acid, it did not show any cell activity. This was addressed by making a methyl ester prodrug – a strategy that was also taken for another fragment linking campaign on a very different target.

As the researchers point out, CAM4066 follows the Evotec model of a largely lipophilic fragment linked to a more polar fragment. There is still much more to do: no pharmacokinetic data are provided, and the potency still falls short of what is needed for a chemical probe. Still, this is a nice illustration of the power of fragment linking, guided by both modeling and crystallography, to generate molecules with interesting properties.

Chiral fragments – and poll!

Chirality underpins all life. Nineteen of the twenty amino acids contain at least one stereocenter, as do all nucelosides, sugars, and most metabolites. The very first fragment I ever found was chiral, but that is not typical, at least judged by those that show up in publications. Only 5 of the 27 fragment to lead success stories published in 2015 started with a fragment containing a chiral center. This probably reflects what people choose to screen and pursue. Chiral centers can lead to challenging chemistry, and chiral centers also add to molecular complexity.

All of which brings us to the topic of our new poll: do you include chiral fragments in your primary screening collection? If so, do you include both enantiomers? Please vote in the poll to the right.

If you do include chiral fragments, do you screen racemic mixtures? Crystallography can sometimes reveal which enantiomer is active if the quality of the structure is good enough, but woe betide anyone screening racemic mixtures by ITC! In a new paper in Magn. Res. Chem., Claudio Dalvit (University of Neuchatel) and Stefan Knapp (Goethe University Frankfurt) show that fluorine NMR can also be used to screen racemic mixtures.

As Teddy wrote more than five years ago, ¹⁹F NMR is “just like ¹H NMR”. Most applications of ¹⁹F rely on detecting the line broadening that occurs when a fluorine-containing fragment binds to a protein. However, the chemical shift of the fluorine atom(s) can also change, particularly if the ligand forms hydrogen bonds to the protein. This “chemical shift perturbation” can be large enough to be detectable.

In the absence of protein, ¹⁹F NMR shows the same signal for different enantiomers, so a racemic ligand containing a single trifluoromethyl group gives a single sharp peak. However, upon addition of a protein that binds one enantiomer, the signal splits into two; one remains sharp and retains essentially the same chemical shift, while the other becomes broader and moves. The researchers show this both theoretically and experimentally with a racemic fragment that binds to the bromodomain BRD4. Adding a high-affinity ligand that binds to the same site displaces the fragment, causing the two signals to again converge.

Unfortunately there is no X-ray structure of the ligand bound to the protein, and the two pure enantiomers were not tested individually. And of course, unlike crystallography, ¹⁹F NMR does not reveal which enantiomer in a racemic mixture binds. Still, enantioselective binding can itself be indicative of specific binding, as opposed to various artifacts, and the researchers recommend that “racemates should always be included in the generation of the fluorinated fragment libraries.” What do you think?

Efficiently searching for fragments

What do you do when you find a fragment? After checking for artifacts and getting as much structural information as possible, the next step is usually to test analogs for improved potency. But how do you go about that? Richard Hall and his colleagues at Astex provide their approach in a recent paper in J. Med. Chem.

Readily available analogs can come from two sources. Larger organizations generally have massive libraries of compounds, and it’s easy enough to order these for testing. There are also plenty of commercial vendors, enabling SAR by catalog. But how do you sort through the millions of possibilities to find those that are most likely to improve potency?

Sub-structure searches are generally the first approach: look for fragments containing a central core, perhaps differently decorated. A nice example of this is described here, where a search for related pyrimidines led to an increase in potency by replacing one atom. Sometimes more dramatic changes are necessary though. Searching for similar molecules that do not share the same core can be successful, as in this case, but often requires multiple searches. Also, particularly for smaller fragments, “similarity” can encompass significant differences.

The Astex researchers have created a computational tool to streamline this search procedure. It is called the Fragment Network, which is a “graph database,” a type of database in which information is stored as nodes and edges – like the webpages (nodes) and links (edges) used in Google searches. In the Fragment Network, each fragment is computationally dissected into component parts (such as a phenyl ring or a hydroxyl), with edges representing the connections between the parts (such as carbon-carbon bonds). The database contains about 5 million compounds of up to 24 non-hydrogen atoms, and these are further annotated as to whether they are available in-house or from more or less reliable vendors.

A search of the Fragment Network – which takes just a fraction of a second – can be customized depending on the goal. A default search returns compounds that are up to two edges away from the query, which can yield quite a large number of compounds, many of which would not come up in a substructure query, as shown for the simple but useful 4-hydroxybiphenyl.

Plodding through lists of compounds can be tedious, and one nice feature of the Fragment Network is that it groups compounds by type – so for example the ring substitutions are grouped separately from the linker replacements. Compounds are also sorted by commonality of replacement: for example, published data reveals that the most common replacement of a methyl group is a chlorine atom, followed by a methoxy group, with an amine way down the list.

The researchers applied the Fragment Network retrospectively to two previously disclosed programs, campaigns against protein kinase B and HCV NS3. In both cases the program identified most of the changes explored by the medicinal chemists on the project, as well as some that were not tested. Of course, often times the best fragments are not available and need to be synthesized, and the grouping of results returned by the Fragment Network quickly highlights these regions of less-populated chemical space.

Those of you who have seen Astex researchers present at conferences will be familiar with AstexViewer, a powerful open-source molecular visualization program. Hopefully the code for the Fragment Network will also be publically released. If not, it might be worth talking to your computationally gifted colleagues to see if they can create something similar. In the meantime, how many of you are using something similar?