17 February 2020

Fragments vs MNK1 and MNK2: take three

Mitogen-activating protein kinase-interacting kinases 1 and 2, or MNK1 and MNK2, are implicated in several cancers while seeming to be dispensable for normal cells, making them attractive oncology targets. Indeed, we’ve previously written about two FBDD-derived clinical compounds against these kinases, eFT508 and ETC-206. In a J. Med. Chem. paper published last month, Alvin Hung and colleagues at A*STAR describe a third series.

The researchers started by screening 1700 fragments in a biochemical assay, resulting in 11 molecules that inhibited both MNK1 and MNK2 with decent ligand efficiency. Four of these had a meta-substituted pyridine, as in compound 6. Making a few analogs led to compound 13, with low micromolar potency against both enzymes.


Crystallography proved unsuccessful, but making the reasonable assumption that the pyridyl nitrogen binds to the kinase hinge region led to two models, only one of which was consistent with the SAR. Decoration of the phenyl ring led to compound 21, with submicromolar activity. Although still early, the researchers collected both in vitro and in vivo ADME data on this molecule, which turned out to be quite promising.

Next the researchers turned to the pyridyl ring and found that appending small (5-membered) heterocycles could also boost potency, as in compound 36. At this point, after multiple attempts with previous compounds, crystallography finally yielded a structure that confirmed the proposed binding mode. Combining elements from compounds 21 and 36 directly led to only a slight boost in activity, but further tweaking ultimately led to compound 47, the most potent member of the series. Unfortunately this molecule was unstable in mouse liver microsomes, but a related compound showed good mouse pharmacokinetics as well as impressive selectivity in a panel of 104 kinases.

No pharmacodynamic studies are described, and perhaps this series was deprioritized to focus on ETC-206, which was also developed at A*STAR. Indeed, the later compounds in this paper reveal a frustrating struggle between potency and stability often seen in medicinal chemistry. This is captured in a nice timeline that shows a rapid improvement in potency over about 8 months, followed by a slight drop as the researchers tried to improve exposure. Although no Goldilocks molecule is reported, this paper is nonetheless a lovely example of fragment to lead optimization done for the most part without the aid of crystallography.

10 February 2020

Toward SAR by SFX

As our poll last year revealed, X-ray crystallography has inched out ligand-detected NMR to become the most popular fragment-finding method. One criticism often leveled at crystallography is biological relevance: very few proteins in nature are found in the crystalline state. Moreover, crystallography is a dish usually served cold, with crystals typically frozen in liquid nitrogen. The reason for this is that the powerful X-ray beams used to elucidate the structure of molecules also rip them apart, and freezing them slows the damage. But protein-ligand complexes at low temperature may not always reflect our more temperate world.

One approach to collecting crystallographic data at room temperature is to do so very quickly, before radiation damage can occur. This is done using serial femtosecond crystallography (SFX), in which many crystals are individually examined using brief, intense beams from X-ray free-electron lasers (XFELs). The X-ray pulses last less than 20 femtoseconds, a time so breathtakingly short that light only travels the width of a typical human cell. In a recent IUCrJ paper, Robin Owen, Michael Hough, and collaborators at the Diamond Light Source, the University of Essex, and elsewhere describe a high-throughput version.

The protein crystals themselves can be quite small, just 1-20 µm across, compared with the > 50 µm crystals typically used in crystallography. These microcrystals are mounted in silicon “chips” containing 25,600 little apertures; the X-ray beam can then be swept across each of the positions.

The researchers demonstrated that they could collect high-quality data for three proteins that are particularly sensitive to radiation damage: two heme peroxidases and a copper nitrite reductase. For all three proteins, they were able to determine high-quality structures of bound fragment-sized ligands. Indeed, some of the ligands were even smaller than all but the smallest fragments: imidazole (five non-hydrogen atoms) and nitrite (three non-hydrogen atoms). The latter case was particularly impressive given that the nitrite displaces a bound water molecule, so the difference between empty and liganded protein is even more subtle.

The first word of this blog is “Practical,” so how does this technique stack up? The researchers used 2-4 chips for each structure, and data collection took about 14 minutes per chip. Despite the miniaturization, sample consumption is not trivial: 1.4 to 6 mg of protein and 4-40 µmol of ligand for each data set. However, the researchers showed that could get by with less data – in some cases significantly so – and state that a 4-5-fold improvement in throughput would be straightforward. Using processing software such as PanDDa could further improve results. I suspect it is only a matter of time before we see the first FBLD by SFX screen. It will be fun to see how useful it turns out to be compared with established methods.

03 February 2020

Fragments vs RIP2: from flat fragment to shapely selectivity

Last week we highlighted the utility of shapely fragments. However, as the latest review of fragment-to-lead success stories again shows, starting with a “flat” fragment does not condemn a lead to flatland. This is illustrated in a recent J. Med. Chem. publication by Adam Charnley and colleagues at GlaxoSmithKline.

The researchers were interested in receptor interacting protein 2 kinase (RIP2), which is implicated in various inflammatory diseases. A fluorescence polarization screen of 1000 fragments at 400 µM yielded 49 hits with inhibition constants ranging from 5-500 µM. Thirty of these confirmed in a thermal shift assay, and 20 were characterized crystallographically bound to the enzyme. Hit-to-lead chemistry was pursued for five series; the most successful started with compound 1a.


The crystal structure revealed that the carboxamide of compound 1a makes interactions with the hinge region of the kinase, with the phenyl group in the back pocket. A search of related molecules available in-house led to compound 2a, with a satisfying boost in potency. Interestingly, the crystal structure of this molecule bound to RIP2 revealed that the binding mode of the pyrazole moiety had flipped to keep the phenyl ring in the back pocket (compound 1a in cyan, 2a in gray). Enlarging the phenyl group to better fill the pocket led to compound 2k.


This molecule had relatively poor selectivity against several other kinases, but introducing a ring as in compound 8 improved the situation. Crystallography suggested that installing a bridged ring would pick up further interactions with the protein, and although the resulting molecule did not have better affinity, selectivity improved. Finally, a hydroxyl group was introduced (compound 11) to try to pick up interactions with a non-conserved serine residue. This addition did not improve biochemical activity, and in fact a crystal structure revealed that the hydroxyl group was pointing towards solvent, but the activity in human whole blood improved. Importantly, compound 11 was remarkably selective for RIP2: just 1 of 366 other kinases tested at 1 µM showed >70% inhibition.

This is a lovely fragment-to-lead success story that reiterates several important lessons. First, a generic (in this case commercial) and nonselective fragment can lead to novel, selective series. Second, as has been seen multiple times, fragment binding modes can flip unexpectedly, especially during early optimization. Finally, despite the relative flatness of fragment 1a (Fsp3 = 0, though the two aromatic rings are slightly twisted), it could be optimized to a more shapely lead, and the increased complexity is likely responsible for the impressive selectivity. Left unreported is the stability and pharmacokinetics of compound 11: the hydroxyl and all those sp3-hybridized carbons are likely metabolic hotspots. As is so often the case in lead discovery, what solves one problem can too often create another.