Fragments in the clinic: Asciminib

Imatinib is the early poster child of personalized medicine. The drug famously works by binding to the mutant kinase BCR-ABL1, and its approval by the FDA for chronic myelogenous leukemia in 2001 arguably launched hundreds of programs targeting kinases. Although imatininb is remarkably effective, resistance sometimes develops, usually caused by mutations that lead to loss of affinity for the drug. Imatinib and other approved drugs that target BCR-ABL1 all bind in the active (ATP-binding) site of the kinase, and they all have various off-targets that can lead to toxicity. To sidestep these issues, researchers at Novartis have developed an allosteric inhibitor, as described by Andreas Marzinzik and colleagues in a new paper in J. Med. Chem.

The ABL1 kinase is naturally autoinhibited by the binding of a myristoyl group to an allosteric pocket. Although the pocket exists in BCR-ABL1, the site that is normally myristoylated is lost. The researchers wanted to create a molecule that would mimic the function of the myristoyl group and exert its inhibitory effect within the allosteric pocket.

An NMR-based screen of 500 fragments yielded 30 hits – perhaps not a surprisingly high hit rate given the lipophilicity of the pocket. Compound 2, with low micromolar affinity, had a high ligand efficiency. Unfortunately, it and similarly high-affinity fragments showed no cell-based activity. A crystal structure of compound 2 bound to the protein revealed that, although the fragment binds in the myristate pocket, its binding mode would actually prevent the conformational change necessary for allosteric inhibition. Tweaking and growing the fragment led to molecules such as compound 4, which were still inactive.

To determine why, the researchers developed a clever NMR assay based on a specific valine residue located in a disordered region of the protein that becomes helical in the allosterically inhibited state of the protein. This assay allowed them to distinguish which protein conformation molecules bound and revealed that, contrary to design, compound 4 did not in fact bind to the inhibited form of the protein. Other researchers had found a different series of molecules that also bind in the myristate pocket, and these all contained a trifluoromethoxy group. When this moiety was grafted onto compound 4, the resulting compound 5 showed cell-based activity.

Now the medicinal chemistry began in earnest. Crystallography revealed a lipophilic cleft in the allosterically inhibited form of the protein which could be filled with a pyrimidine, and the cationic solubilizing group in compound 5 was replaced by the neutral moiety in compound 7. This compound showed some hERG channel inhibition, which could be fixed by replacing the pyrimidine with a pyrazole. Also, crystallography revealed that there was a little extra space near one of the fluorine atoms, which could be replaced with a chlorine in the clinical compound asciminib (ABL001). A crystal structure of this molecule shows it binding to the inactive conformation of the protein (the helix that forms is in the upper right).

Asciminib effectively inhibits proliferation of cells containing either wild-type or T315I BCR-ABL1, the latter being one of the more pernicious resistance mutations. The compound is also highly selective against > 60 other kinases, and is only active against CML cell lines in a panel of 546 cancer cell lines, suggesting that it should be well tolerated. Mouse xenograft models were also impressive, and the compound is currently in a phase 3 clinical trial.

This is a thorough, clearly written account combining biophysics, modeling, chemistry, and biology to discover a first-in-class drug. It is also a useful reminder that binding alone may not be sufficient to cause desired effects. As with all the clinical-stage programs, Practical Fragments wishes everyone involved the best of luck!

Fragments in the clinic: ASTX660

Three years ago we highlighted a paper from Astex describing the discovery of an extraordinarily weak fragment and its advancement to a dual inhibitor of the anti-cancer targets cIAP1 and XIAP. We ended that post by writing, “whether or not this leads to a drug, it does look like another candidate for a useful chemical probe.” As three papers now make clear, the program has indeed led to an experimental drug.

The first paper, by Emiliano Tamanini and colleagues, was published in J. Med. Chem. last year and describes the optimization of Compound 21, one of the best compounds from the 2015 report. The researchers noticed that compounds in the series were chemically unstable: the amide bond was subject to hydrolysis. Fortunately this was readily fixed by repositioning the pyridyl nitrogen.

Optimization of the benzyl group was complicated by the fact that it binds in the P4 pocket, which differs between cIAP1 and XIAP. In the end, adding a fluorine gave a slight potency improvement against both proteins. The bulk of the work was focused on elaborating the methoxy group of compound 21. Detailed modeling experiments were used to choose moieties that would fold back on the core of the molecules in solution, thus pre-orienting them for binding as well as shielding a critical hydrogen bond. These efforts led to AT-IAP, with low nanomolar cell activity against both proteins as well as activity in mouse xenograft models.

Although AT-IAP is orally bioavailable in mice and rats, the bioavailability is much lower in monkeys, and it also inhibits the hERG channel, which can lead to cardiac toxicity. Fixing these problems is the focus of a paper published last month in J. Med. Chem. by Christopher Johnson and colleagues.

Metabolite identification studies revealed that the morpholine ring of AT-IAP is cleaved by CYP enzymes, so this was one area the researchers tried to modify. Although somewhat successful, hERG was still a problem, and this correlated with lipophilicity. Knowing how the molecules bound allowed the researchers to introduce small hydrophilic substituents without disrupting critical interactions, ultimately leading to ASTX660. Not only did the added hydroxymethyl group decrease hERG binding, it also improved bioavailability – a reminder that decreasing lipophilicity can have useful effects even on distant parts of the molecule.

More characterization of ASTX660 is provided in a paper by George Ward and colleagues in Mol. Canc. Ther. This reports the crystal structure of the molecule bound to XIAP. As Johnson et al. note, the polar interactions made by the molecule are conserved from the original fragment – the additional protein interactions that improve affinity by more than a million-fold are all hydrophobic.

Ward et al. also provide more detailed mechanistic cell biology, pharmacokinetics, and xenograft data. In particular, ASTX660 is a much more potent antagonist of XIAP activity in vivo than other clinical-stage compounds, which will hopefully translate to better efficacy. The compound is currently in a phase 1-2 study.

Collectively these papers provide a valuable lesson in structure- and property-based drug design and illustrate just how much effort can be required to go from fragment to clinical compound. I’ll end this post with an echo of the original: whether or not this leads to an approved drug, it is a lovely story of perseverance combined with creative chemistry and biology. Practical Fragments wishes everyone involved the best of luck.

Fragment flipping during optimization

Last month we highlighted a study that asked how often the binding mode of a fragment changed during optimization. A new paper in J. Med. Chem., by Swen Hoelder and collaborators at Institute of Cancer Research, University of Oxford, and Universitat de Barcelona provides an interesting case study.

The researchers were interested in the kinase ALK2, which is implicated in an aggressive and universally fatal childhood cancer called diffuse intrinsic pontine glioma. They started by screening a library of fragments designed to target kinases, which yielded compound 1. This compound actually contains two moieties that are known kinase hinge binders, a quinazolinone and a pyrazole. Unfortunately the researchers could not obtain a crystal structure of the fragment bound to ALK2, but SAR suggested that the pyrazole was not essential, and indeed replacing this with a quinoline led to compound 7, with sub-micromolar activity.

Next the researchers introduced methyl groups at various positions around the quinazolinone and found that these neither significantly improved nor decreased binding. Modeling based on similar reported molecules led them to grow the molecule towards solvent, ultimately leading to the mid-nanomolar compound 16 (blue in figure below), which they characterized crystallographically bound to ALK2. The molecule bound as expected, with the unsubstituted nitrogen of the quinazolinone forming a hydrogen bond to the hinge region of the kinase.

So far so good, but the researchers were still curious about some of their earlier SAR. In particular, the methyl groups added to some of the molecules should have been incompatible with the observed binding mode of compound 16, suggesting an alternative binding mode for these molecules. This insight proved correct, and in fact adding two methyl groups to compound 7 led to compound 21, which is more potent than compound 7 and binds such that the amide of the quinazolinone core forms hydrogen bonds with the hinge region, as confirmed by crystallography (green in figure).

Compounds 16 and 21 have similar affinities yet different binding modes, so what about selectivity? Testing them in a panel of ~110 kinases revealed both to be quite selective for ALK family kinases, though they had different off-targets. The selectivity of compound 21 is particularly impressive given its small size – it teeters on the edge of being rule of three compliant. A related molecule also showed activity in a cell-based assay.

An interesting unanswered question is the binding mode of the initial fragment. Perhaps it binds in multiple orientations, which could explain why crystallography was unsuccessful. Regardless, this is a nice study that illustrates how close attention to confusing SAR can lead to attractive new series.

From generic fragment to selective BET-family BD2 inhibitor

Fragments have been a rich source of leads against bromodomain-containing proteins, epigenetic readers that recognize acetylated lysine residues and are implicated in a variety of diseases. The four members of the BET family in particular have been heavily explored. Each of these proteins actually contains two separate bromodomains, called BD1 and BD2, and most reported inhibitors hit both of them more or less equally. To follow up on some intriguing biological hints that this may not be necessary, Robert Law and collaborators at GlaxoSmithKline and University of Strathclyde pursued selective BD2 inhibitors, which they describe in J. Med. Chem.

The researchers started with a fragment they first reported six years ago, and which has been used by Forma as the starting point for one of their own programs. Although fragment 10 is equipotent against BD1 and BD2 of BRD4, growing led to compound 12, with an encouraging 60-fold selectivity for BD2 (all values shown below are for BRD4 BD2). A crystal structure of a close analog suggested several opportunities for further growth to improve potency.

Changing the methyl group to a cyclopropyl group improved selectivity, and introducing a hydroxymethyl substituent off the phenyl ring (compound 44a) improved potency for BD2. This molecule was fairly lipophilic, so the researchers explored adding a variety of polar substituents to improve solubility, ultimately resulting in GSK340.

GSK340 was profiled against 35 bromodomains and found to be at least 40-fold selective for the BD2 domain compared to the BD1 domain of the four BET family members. It showed the highest affinity for BRD4 but also bound tightly to the BD2 domains of BRD2, BRD3, and BRDT and was selective against non-BET family bromodomains. The compound was cell permeable and inhibited the release of the inflammatory cytokine MCP-1, supporting the notion that BD2 domain inhibition alone could have useful anti-inflammatory effects. Unfortunately GSK340 shows sufficiently high clearance in rat and human hepatocytes that the researchers suggest its utility will be limited to in vitro assays. Still, this paper provides another illustration that – with the help of creative medicinal chemistry – a generic, non-specific fragment can lead to a novel and selective chemical probe.