Fragments in the clinic: AG-270

A promising oncology approach is to target “synthetic lethal” proteins that are required for cancer cells but not for ordinary cells. Methionine adenosyltransferase 2A (MAT2A) is a metabolic enzyme that produces S-adenosyl methionine (SAM). Cancer cells lacking another gene, methylthioadenosine phosphorylase (MTAP), appear particularly dependent on MAT2A. In a recent J. Med. Chem. paper, Zenon Konteatis and colleagues at Agios, Viva, and ChemPartner describe the first clinical inhibitor of MAT2A.

 

The researchers began by screening >2000 fragments in pools of 20, each at 50 µM, using ultrafiltration. The 31 hits were tested in enzymatic and SPR assays, and compound 1 confirmed in both. Testing 54 commercially available analogs led to compound 2, with low micromolar activity, and this molecule was profiled intensively.

 

Kinetic studies revealed compound 2 to be non-competitive with respect to substrates ATP and L-methionine, and crystallography confirmed that compound 2 binds in a previously discovered allosteric pocket. The molecule makes polar and hydrophobic contacts to MAT2A and also displaces several water molecules. SAR studies found a preference for aromatic moieties at the 2-position of the central core, but the phenyl off the 3-position could be substituted with more shapely moieties such as the piperidine in compound 9.

 

Closer examination of the structure of compound 2 bound to MAT2A revealed a protein-bound water molecule, and displacing this with the phenol in AGI-24512 led to a satisfying boost in biochemical potency as well as cell activity. However, the molecule has poor oral absorption and a short half-life in rats. Metabolite identification studies pinned the blame on the piperidine, with the phenol no doubt doing no favors. Medicinal chemistry ultimately led to AGI-25696, which despite its lower biochemical activity was active in cells, metabolically stable, and showed efficacy in a mouse xenograft model when dosed orally.

 

Despite these favorable properties, AGI-25696 has very high protein binding in human plasma (>99.9%) as well as high efflux, which would likely necessitate a high clinical dose. The researchers proposed that, due to the weakly acidic nature of the molecule, it could tautomerize, and each tautomer could bind to different plasma proteins. Simply methylating the N-H led to decreased plasma protein binding but also lower binding to MAT2A. Thus, the researchers sought to shield the N-H by forming an intramolecular hydrogen bond. After appending more than 70 heterocycles they eventually arrived at AG-270, which has a more respectable plasma protein binding of around 98.5%.

 

Extensive characterization of AG-270 revealed it to be potent with good pharmacokinetics and oral bioavailability. It is relatively clean in a panel of 95 potential off-targets and showed tumor growth reduction in xenograft models. But it is not without warts: low solubility necessitated a spray-dried dispersion for animal dosing, not surprising giving its high lipophilicity. Nonetheless, the molecule has entered a phase 1 clinical trial in patients with MTAP loss.

 

This is a lovely fragment-to-lead success story with several lessons. First, although enzymes are sometimes considered “easy,” this is not necessarily true. Indeed, at an ACS meeting in 2018 Anil Padyana mentioned that metabolic enzymes in particular often have shallow, polar active sites. Targeting allosteric sites, as done here, can be a useful alternative.

 

Second, it is striking how the core of the initial fragment remains intact in the clinical compound, a reminder of the power of fragments to efficiently explore chemical space.

 

Finally, this story is another important reminder that affinity is often just the beginning of a long journey. It took considerable effort to optimize the pharmaceutical properties from AGI-24512 to AG-270, including a decrease in ligand efficiency. In the end the team has succeeded, and Practical Fragments wishes them – and the patients – luck in the trials.

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.

Fragment growing and merging: an inverse agonist of RORγt2

The nuclear receptor transcription factor RORγt2 is involved in the differentiation of Th17 cells, and is thus a target for inflammatory diseases. The protein contains a large, hydrophobic ligand binding site, and as a result most known inverse agonists have less than ideal physicochemical properties. In a paper recently published in J. Med. Chem., Samuel Hintermann and colleagues at Novartis have taken a fragment-based approach.

The researchers screened a library of 1408 fragments using a “differential static light scattering (DSLS)” assay, which is a type of thermal shift assay that measures denaturation and aggregation of the protein. A few dozen molecules that stabilized RORγt2 were tested in dose-response curves prior to crystallization trials, ultimately yielding 13 structures. Compound 1 was particularly interesting because it binds in the center of the cavity, providing growth vectors in two directions. It also makes a couple hydrogen bonds with the protein, as opposed to purely hydrophobic interactions.

Growing from the ethoxy position quickly led to improvements in affinity. To avoid the possibility of toxic iminoquinone metabolites, the researchers replaced the central phenyl ring with a pyridine, resulting in the low micromolar inverse agonist compound 8.

To further improve affinity, the researchers merged an element from a previously reported GlaxoSmithKline molecule (compound 2) onto compound 8, resulting in the potent compound 9, which was characterized in a battery of assays.

The crystal structure of compound 9 bound to the protein revealed that the core fragment moiety binds in the same manner as the original compound 1, though the added benzyl ether binds in a subpocket that had not previously been observed to bind ligands.

Kinetic studies using a reporter displacement assay revealed that compound 9 has both a slow on-rate as well as a slow off-rate, consistent with the fact that it is fully enclosed by the protein. The researchers performed molecular dynamics simulations to try to determine how the ligand could enter or leave, which suggested large conformational changes in a flexible region of the protein. Isothermal titration calorimetry (ITC) showed that the binding of compound 9 is enthalpically driven, with an unfavorable entropy. Although interpreting thermodynamics is fraught, this result makes intuitive sense given the hydrogen bonds formed and the fact that the molecule seems to rigidify the protein.

Biophysics is interesting, but of course biology is what was driving the program. Compound 9 is potent in a variety of cell assays and is also selective for RORγt2 over other nuclear hormone receptors. However, it is also mostly insoluble, and although it did show efficacy in a rodent inflammation model, plasma concentrations of compound 9 were highly variable between individual rats, which the authors attribute to poor physicochemical properties.

This is a nice application of fragment growing and merging that demonstrates how difficult it is to find useful leads for lipophilic sites: even with favorable biochemistry and biophysics, the pharmacokinetics are a slog. That said, others have made progress against similarly hydrophobic targets, so it will be fun to watch this story progress.

Fragments score a win against WDR5-WIN

Protein-protein interactions (PPIs) can be difficult targets for multiple reasons. First, the contacts often cover large, flattish areas with few “ligandable” pockets. Second, they can involve multiple proteins; imagine trying to disrupt a huge multicomponent machine with a little widget. The protein WDR5 falls into the second category. It serves as a scaffold around which other proteins assemble to regulate epigenetics. One of these proteins, MLL1, is implicated in certain leukemias and binds to WDR5 through the WDR5 INteraction (WIN) motif, making this protein-protein interaction an intriguing anti-cancer target. In a recent paper in J. Med. Chem., Stephen Fesik and colleagues at Vanderbilt University describe their efforts towards this target.

Unlike some PPIs, the WIN motif does contain a nice little pocket which normally recognizes arginine residues. However, since the highly basic guanidine moiety of arginine is undesirable in drugs, the researchers conducted a fragment screen to find new WIN-site binders. A two-dimensional (¹H-¹⁵N HMQC) NMR screen of a large fragment library (>13,800 fragments, more than the majority of respondents in the poll to the right) identified 47 hits that produced similar spectral changes as a peptide that binds in the WIN site. Compound F-1 was the most potent.

A crystal structure of compound F-1 bound to WDR5 revealed that the imidazole moiety binds in the same deep pocket normally occupied by the arginine side chain, with the phenyl ring pointing up out of the pocket. Initial growing off the phenyl ring into nearby hydrophobic pockets produced more potent compounds, but at best these were still micromolar binders. The researchers had more success by targeting a slightly more distant pocket with compounds such as 4a and subsequently compound 4i. A crystal structure of compound 4a bound to WDR5 suggested that the biologically active conformation might not be the lowest energy conformation of the free molecule. Introducing a ring to restrict the conformation led to more potent molecules such as 6e, with sub-nanomolar affinity.

Unfortunately, though potent in biochemical assays, compound 6e and related molecules were about 2800-fold less potent in cell-based assays. The compound is cell permeable and not effluxed, so the disconnect must be due to something else – perhaps the multiple other proteins in the cellular environment. Anyone who has spent much time doing medicinal chemistry will have encountered frustrating situations like this. Perhaps a new chemotype is needed, or perhaps the compounds need to be made even more potent. Indeed, several years ago the Fesik group reported nanomolar binders of MCL-1, but it was not until they improved affinity to picomolar that they saw good cell potency. Stay tuned!