Fragments towards the clinic: ERK1/2

The first fragment-based drug to reach the market, vemurafenib, targets a mutant form of the kinase BRAF. Initial responses can be miraculous, but metastatic melanoma is an implacable foe, and patients often relapse. One mechanism of resistance involves upregulation of the kinases ERK1 and EKR2, which are downstream of BRAF. These are the subject of a paper just published in J. Med. Chem. by Tom Heightman and collaborators at Astex and Sygnature.

Most kinase drugs bind to the so-called hinge region of the protein, where the adenine moiety of ATP binds. Previously reported ERK1/2 inhibitors do indeed bind here, but one molecule from Merck (Schering) also binds in a second pocket some distance away. This molecule both inhibits the kinase and also blocks it from becoming phosphorylated itself, thereby preventing it from becoming activated.

Unfortunately this molecule did not have good pharmacokinetic properties, so the researchers sought a new series. They began with virtual, crystallographic, and thermal-shift fragment screens against ERK2. Compound 5 was active in a biochemical assay with impressive ligand efficiency. The bound structure showed multiple interactions with the protein as well as good vectors for further growth. Recognizing that spanning from the hinge region to the second pocket would require a large molecule, the researchers first sought to increase the sp³ character of the fragment to maximize solubility by replacing the pyrazole with a tetrahydropyran (compound 7), which also provided a nice boost in potency. 

Next, the researchers started growing towards the second pocket, guided by docking. This led to another large jump in potency, to the low nanomolar compound 11. Further growth to compound 16 led to marginal improvements in biochemical potency but did show antiproliferative activity in the Colo205 cell line, which contains the oncogenic BRAF mutation. Building into the second pocket, again guided by both modeling and crystallography, significantly improved the cell activity, ultimately leading to compound 27. Consistent with the design, the molecule blocks ERK activity as well as phosphorylation of ERK. It is also orally bioavailable, has good pharmacokinetics, and causes tumor regression in mouse xenograft models. Moreover, Compound 27 is quite selective for ERK1/2 in a panel of 429 kinases.

This is a lovely example of fragment-based and structure-based design. Although the final molecule is on the large side, careful attention to molecular properties maintained acceptable pharmacokinetics. The paper ends by noting that “further pharmacological characterization of 27 will be published elsewhere.” Indeed, Astex has taken an ERK1/2 inhibitor called ASTX029 into the clinic. Practical Fragments wishes everyone involved the best of luck.

Fifth NovAliX Biophysics in Drug Discovery Conference

Last week NovAliX held its biophysics meeting outside of Strasbourg for the first time. Naturally they chose Boston, one of the most European of US cities and a major hub of drug discovery. The event brought together 118 participants from 15 countries, roughly 80% from industry. Although the food and drink could not compare to France, the science and discussion were every bit as satisfying. With 30 talks and 22 posters I won’t attempt to be comprehensive, but as with last year just try to capture a few themes. 

One particularly noteworthy session was devoted to single particle cryo-electron microscopy (cryo-EM), which was recently reviewed in Nat. Rev. Drug Discov. by conference chairman Jean-Paul Renaud and a multinational team of experts. The approach involves flash-freezing a thin film of sample and using transmission electron microscopy to capture two-dimensional “projection” images of your target. If the protein is randomly oriented you can computationally combine thousands of individual images into a three dimensional structure. Although the technique has been around for decades, until recently the resolution was too low to be useful for structure-based drug design. Recent advances in hardware and computation have led to what’s come to be known as the “resolution revolution,” explained Gabe Lander (Scripps).

One advance is the 300 keV Titan Krios – a massive (and massively expensive) instrument that is so widely coveted that Gabe showed pictures of happy scientists hugging newly delivered crates. Indeed, of the ~1000 structures solved to < 4 Å resolution, the vast majority of them were solved on one of more than 130 Krios instruments throughout the world. But Gabe showed that high resolution structures can be obtained with more common 200 keV instruments, including a 2.6 Å resolution structure of aldolase (150 kD), a 2.9 Å structure of hemoglobin (64 kD), and a 2.9 Å resolution structure of alcohol dehydrogenase (81 kD) with bound NAD⁺ cofactor. Although only a handful of sub-2 Å structures have been reported, he thought these would become routine in the next few years.

Bridget Carragher (New York Structural Biology Center) described challenges and how to overcome them. Currently it takes at best eight hours to go from data to structure, but she thought getting this to under one hour would be achievable. Moreover, cryo-EM can be used to characterize different conformational or oligomeric states present in a single sample, as Giovanna Scapin (Merck) demonstrated with insulin binding to its receptor. Indeed, even simple visualization – without fancy computational processing – can provide useful information about protein aggregation, as demonstrated by Wen-ti Liu (NovAliX).

Although primary fragment screening still looks a long way off for cryo-EM, it should start to provide useful structural information for fragments bound to targets less amenable to conventional biophysical techniques, such as membrane proteins – the topic of another session.

Miles Congreve (Heptares) discussed how their stabilized “StaR” GPCRs can provide high-resolution crystal structures suitable for FBDD (see for example here). This has allowed them to discover less lipophilic, more ligand-efficient drug candidates against a variety of targets.

According to Anass Jawhari, it isn’t even necessary to make mutant GPCRs: Calixar has developed proprietary detergents that can stabilize full length adenosine A_2A receptor for a week – more than enough time to perform STD NMR screens of 100 fragments and identify 19 hits, some of which turned out to be functional antagonists. Matthew Eddy (University of Southern California) used two-dimensional NMR on this same protein to reveal dramatic differences in conformational dynamics when bound to agonists vs antagonists.

Indeed, conformational changes and dynamics were a running theme throughout the conference. Keynote speaker and Nobel-laureate Martin Karplus (Harvard) quoted fellow Nobelist Richard Feynman: “everything that living things do can be understood in terms of the jiggling and wiggling of atoms.” (As an aside, Martin’s MCSS method pioneered computational FBDD approaches, predating SAR by NMR.) Göran Dahl (AstraZeneca) described how large scale conformation changes well outside of the active site of PI3Kgamma were responsible for freakishly high selectivity of a class of inhibitors.

But how do you detect conformational changes? We’ve previously mentioned Biodesy’s SHG approach, and Parag Sahasrabudhe (Pfizer) described how this proved useful for classifying ligands for IL-17A. Gerrit Sitters (Lumicks) described a completely different “dynamic single-molecule” (DSM) approach, which involves trapping a single fluorescently labeled protein between DNA strands tethered to two microspheres. Changes in protein conformation caused by ligand binding change the distance between microspheres, and these can be detected to within 1 Å.

Kinetics is intimately linked to dynamics, but the factors responsible for slow binding and dissociation are still poorly understood. Chaohong Sun (AbbVie) examined an archive of 8000 data points and found that on-rates and off-rates each varied by more than five orders of magnitude. There was no correlation with ClogP of the ligands, though larger ligands were more likely to have slower kinetics. There were also significant target effects; on-rates were consistently slow for one target.

As we’ve previously discussed, off-rate screening (ORS) can be used to identify hits in crude reaction mixtures, and Menachem Gunzburg (Monash University) described how this technique is being used in hit-to-lead efforts. Lowering the temperature to 4 °C and adding 5% glycerol further slows dissociation, allowing weaker hits to be discovered.

At the extreme, irreversible inhibitors have an off-rate of 0, and Gregory Craven (Imperial College London) described quantitative irreversible tethering of electrophilic fragments to cysteine residues in proteins using a fluorimetric plate-based assay. As we’ve noted, one challenge with irreversible tethering is deconvoluting intrinsic reactivity from proximity-directed reactivity, which Gregory addresses using a reference thiol such as glutathione.

There is much more to say but in the interest of time I’ll stop here. If you missed the conference you have two chances next year: June 4-7 when it returns to Strasbourg, and November 20-22 when it will be held in Kyoto. And there are still excellent events coming up this year – hope to see you at one!

The origins and development of FBDD

Most of the papers Practical Fragments cover are limited in scope: a new chemical probe, say, or an NMR method. Even in our annual review of reviews, most of the publications have a focus, such as a particular technique. But a paper just published in Drug Discovery Today, by Iwan de Esch (VU University Amsterdam) and an international group of collaborators (including FragNet scholar Angelo Romasanta), is a rather different beast.

The (open access) paper is a bibliometric analysis of FBDD. The researchers first assembled all papers in Thomson-Reuter’s Web of Science which had “fragment” and one of several other terms as a keyword. If you try this at home you will find all sorts of irrelevant topics (such as antibody fragments), so these were manually removed, leaving 2781 publications. But many early papers did not refer to fragments, so all references that had been cited at least ten times were added, resulting in a total of 3642 papers published between 1953 and 2016. What can be learned with such a data set?

For one thing, the term “fragment-based drug discovery” didn’t appear until 2002. In the early 2000s “fragment-based lead discovery” was more common, though for roughly the past decade the former term and “fragment-based drug design” have co-dominated.

The researchers also examined the number of citations each paper has received to reveal interesting trends. For example, in the early years (1996-2001), industry dominated. Indeed, 9 of the 10 most cited papers of all time come from industry, and the sole outlier describes the protein data bank (PDB). In the past decade academics have become significant contributors, which is not surprising given their stronger incentive to publish.

Moving beyond raw citations, the researchers manually classified papers into scientific disciplines (methods, molecular basis, applications, and crystallography) to explore the diffusion of knowledge. This reveals the centrality of the 1996 “SAR by NMR” article, which was the first to cite theoretical and computational approaches and also bring in biophysics. Deservedly, this is the most highly-cited paper (454 citations within the set of articles, and currently >2100 total according to Google Scholar).

Our most recent poll of fragment-finding methods revealed a spike in crystallography, driven both by higher hit rates as well as technical advances, and this is also seen in the paper, where the 2011-2016 period shows a significant increase in crystallography over earlier five-year periods. As we’ve also noted, there has been a shift in content: while many earlier publications focused on techniques, medicinal chemistry has become a much more common subject in recent years.

There is plenty more here, and the paper is fun reading for anyone in FBDD, whether you have lived through the history or are new to the field. My one quibble is that the list of 3642 papers is not provided as supplementary material. Indeed, it is the open nature of the PDB that has made it such a valuable resource. Hopefully the authors will release their underlying data so others can build upon it.

Fragments in the clinic: ETC-206

A few weeks ago we highlighted the story of eFT508, a clinical MNK1/2 inhibitor derived from a previously published fragment. One of the comments to that post mentioned another example describing a clinical compound against the same targets – also derived from a previously published fragment! This work was recently published in J. Med. Chem. by Kassoum Nacro and a large, multinational group of collaborators from A*STAR and other institutes.

The kinases MNK1 and MNK2 are responsible for phosphorylating and thereby activating eIF4E, a protein that regulates messenger RNA translation. All three proteins are overexpressed in various cancers, particularly blast crisis chronic myeloid leukemia (CML), in which patients stop responding to drugs such as dasatinib. An inhibitor of MNK1/2 could thus potentially resensitize the cancer cells. Moreover, MNK knockout mice are healthy, suggesting that the therapy might be minimally toxic.

The researchers started with a 2010 paper which reported a virtual screen against MNK1; nearly three quarters of the hits were fragments. The A*STAR researchers were particularly attracted to molecules such as ETP-38766, and they used modeling along with a previously reported structure of MNK2 to scaffold-hop to compound 4, with sub-micromolar activity. (MNK1 and MNK2 are closely related, and most reported compounds show similar activity against both; values for MNK1 are given here.)

Building out the molecule further did not do much for biochemical potency but did yield molecules with improved solubility, permeability, and cell-based activity – such as compound 27. Further tweaking of the core and replacement of the metabolically labile methyl piperazine ultimately led to ETC-206, with nanomolar potency in biochemical and cell-based assays. It also shows good pharmacokinetics, is orally bioavailable, and is remarkably selective for MNK1/2: in a panel of 104 kinases screened at 1 µM compound, only one other kinase showed significant inhibition. As expected, the molecule showed little antitumor activity in a xenograft assay when dosed by itself, but significantly improved the activity of dasatinib. Indeed, the molecule has recently entered a phase 1 clinical study in combination with dasatinib.

Several lessons can be drawn from this paper. First, it appears that ETC-206 was derived solely with the aid of modeling, without recourse to experimental structural data for any molecules in the series. Second, both ETC-206 and eFT508 had their origins in fragments previously discovered by others – a reminder that, with the increasing number of publications, you don’t necessarily have to do your own fragment screen in order to do FBLD. (An important corollary is that a fragment does not itself need to be novel to generate patentable chemical matter.) Finally, ETC-206 and eFT508 are both selective MNK1/2 inhibitors but look very different from one another – a reminder that many roads can lead to different clinical candidates for the same target.