Fragments vs BET Bromodomains: FORMA’s story

Five years ago Teddy highlighted a paper from GlaxoSmithKline that reported the discovery and characterization of several different fragments that bind to members of the BET family of bromodomains, epigenetic readers that recognize acetylated lysine residues in histones. Researchers at FORMA were among those paying attention to these developments. David Millan and his colleagues have now published in ACS Med. Chem. Lett. their account of how they were able to advance one of these fragments to a chemical probe.

As the researchers note, many different BET inhibitors have been reported; we discussed two separate series just a few months ago. Chemical novelty was thus a challenge, particularly as they were starting with a fragment (compound 1) reported by a large company. They thus chose to tweak the fragment slightly to intermediate 2. Importantly, introduction of the second nitrogen also introduces another synthetic vector with potential to pick up interactions with the so-called “WPF shelf”. This explicit consideration of synthetic tractability in fragment design enables rapid progress.

Parallel chemistry led to compound 6, with measurable biochemical activity against BRD4. Further growing from the phenyl ring led to compound 8, with sub-micromolar biochemical and antiproliferative activity. A crystal structure revealed that the newly introduced amide functionality was pointed toward solvent, which would allow modulation of the physicochemical properties.

More medicinal chemistry followed, with considerable effort on improving the plasma and liver microsome stability. This campaign involved a combination of rational design and parallel synthesis along with a keen focus on minimizing lipophilicity. Ultimately the researchers arrived at FT001, with good activity and stability. This compound was also selective for BET family members over other bromodomains and displayed reasonable pharmacokinetics and impressive activity in a mouse xenograft model.

Last week we highlighted a paper that also started from a previously disclosed fragment to generate novel chemical matter. This paper provides another example of how useful public fragments can be in the hands of creative scientists.

Fragments vs histone KDM4 lysine demethylases: Celgene’s story

Last year we highlighted a paper from academia in which modeling was used to discover potent inhibitors of the lysine demethylase KDM4C, a potential anti-cancer target. In a recent paper in ACS Med. Chem. Lett., Michael Wallace and collaborators at Celgene, the European Institute of Oncology, and the University of Chicago report a chemical probe for KDM4 family members.

The researchers started with a literature screen of fragments known to bind to KDM4, leading them to compound 1, which previous work had shown binds to the catalytic iron through the pyridine ring nitrogen. Researchers from GlaxoSmithKline had also reported that growing off compound 1 could lead to more potent compounds, a strategy that proved successful here in the case of chiral compound 2a, which improved affinity by more than two orders of magnitude. Interestingly, the enantiomer had dramatically lower activity.

Armed with this information but no crystal structure, modeling suggested that further growing off the tetrahydronaphthalene would be productive, which turned out to be the case for compound 3, with similar affinity but improved activity in an antiproliferative cell assay. Further experiments showed that the compound increased levels of trimethyl-lysine on lysines 9 and 36 of histone 3, known substrates of KDM4 family members.

A crystal structure of compound 3 bound to KDM4A, which is closely related to KDM4C, suggested further room to grow. Compound 3 contains a carboxylic acid and has a low cLogD, traits that tend to reduce cell permeability. The researchers thus focused on increasing the lipophilicity of the molecules, leading to QC6352. Despite the fact that this molecule is less potent in the enzymatic assay, it has significantly improved cellular potency. It also has reasonable pharmacokinetics and oral bioavailability, and showed activity in a mouse xenograft model. QC6352 hits KDM4A, 4B, 4C, and 4D, but is quite selective against most other KDMs.

This paper illustrates three important points. First, as discussed previously, you don’t need a novel fragment to get to novel leads – you just need creative scientists. Indeed, the increasing number of fragment hits reported for various targets provides a wealth of starting points even for organizations that don’t do in-house fragment screening. Second, you don’t necessarily need a crystal structure as long as you have good modelers. And finally, while excess lipophilicity is rightly avoided, it is important to remember that compounds can also be too polar. As Oscar Wilde noted, “everything in moderation, including moderation.”

Fragments distinguish allosteric from active site binders

As discussed last year, secondary binding sites on proteins appear to be quite common. Some of these sites have no functional relevance, but others are allosteric sites, which can modulate the activity of proteins. Allosteric ligands can be useful for several reasons. First, unlike molecules that bind at the active (that is, catalytic) site of an enzyme, which usually inhibit activity, allosteric site binders can increase activity. Second, allosteric sites are usually less conserved than active sites, allowing greater selectivity. Finally, combining an allosteric inhibitor with an active site inhibitor can lead to synergy as well as lower the incidence of resistance mutations for cancer and anti-infectives. In a recent ACS Med. Chem. Lett. paper, Lukasz Skora and Wolfgang Jahnke at Novartis describe a simple NMR approach to differentiate these two classes of ligands.

The researchers used ¹⁹F NMR to screen 540 fragments containing a CF₃ group, each at 25 µM, in pools of 30 against the kinase ABL1 (at 4 µM); the BCR-ABL1 mutant form of this protein is a key driver for chronic myelogenous leukemia. Several approved drugs target the active site of ABL1, and Novartis researchers have recently launched clinical studies of a compound called ABL001, which binds to an allosteric pocket.

Fragments that bind to ABL1 showed a decreased ¹⁹F NMR signal due to line broadening. Adding ABL001 displaced fragments that bind to the allosteric site, thereby increasing their NMR signals, while adding the active-site binding drug imatinib displaced fragments that bind to the catalytic site. Follow-up experiments with individual fragments identified a selective catalytic-site binder (CAT-1) and a selective allosteric site binder (ALLO-1). Both fragments are commercially available and quite weak (K_d = 43 µM for ALLO-1 and IC₅₀ = 380 µM for CAT-1), which in this case is a feature because they can easily be displaced.

Mixing these two fluorine-containing probes with ABL1, adding test compounds, and performing ¹⁹F NMR thus provides a simple means to determine whether a ligand binds to the allosteric site, the active site, or both sites. The researchers confirmed that the approved catalytic-site binding drugs nilotinib, dasatinib, and ponatinib displace CAT-1 but not ALLO-1, while allosteric-site binders such as ABL001 displaced ALLO-1 but not CAT-1.

Interestingly, a crystal structure of imatinib with the highly related protein ABL2 shows the compound binding to both the catalytic and allosteric sites, yet although imatinib clearly displaced CAT-1 it could not displace ALLO-1. This is a useful reminder that crystal structures say nothing about affinity.

The drug crizotinib, which binds to the active site of multiple kinases, has been reported by other researchers to bind to the allosteric pocket of BCR-ABL1, but this was not borne out in the competition assays. Similarly, the drug fingolimod has also been reported as an allosteric inhibitor of ABL1. This molecule did indeed displace ALLO-1, but only at concentrations so high as to be biologically irrelevant.

This is a nice paper, and a good reminder that fragments can make useful biophysical probes in and of themselves, even without the need for optimization.

Assessing ligandability by thermal scanning

Ligandability refers to the ability to find small-molecule leads against a target. A protein might be ligandable but not druggable if, for example, potent inhibitors of the target do not affect a disease state. But knowing in advance whether a target is ligandable can be useful, both to decide whether to embark on a campaign and to plan the resources it will likely require. Fragment screens by NMR have been shown to be good predictors of ligandability, but not everyone has access to this technology. Computational methods (such as FTMap) are also useful, but require a structure of the target. In a recent paper in J. Med. Chem., Stefan Geschwindner and colleagues at AstraZeneca describe high-throughput thermal scanning (HTTS) for assessing ligandability.

Thermal scanning (alternately called, as the researchers note, thermal shift, differential scanning fluorimetry (DSF), or thermofluor) relies on the preferential binding of a fluorescent dye to protein that is heat-denatured. Since ligands generally stabilize a protein against denaturation, an increase in melting temperature (Tm) is taken as an indication of binding. The assays can be plate-based and thus very fast.

The researchers chose 16 diverse targets (mostly enzymes) and screened their 763-ligandability fragment set (described here) at 1 mM by HTTS. Hits were defined as compounds that increased  thermal stability at least 3-fold above the standard deviation of controls. Targets were then categorized as follows:

Low ligandability: hit rate < 1.5%

Medium ligandability: hit rate between 1.5 and 4.5%

High ligandability: hit rate > 4.5%

Nine targets ranked low, and all of these failed high throughput screening (HTS), while 5 out of the 7 targets ranked medium or high by HTTS yielded useful HTS hits. Of course, failure in an HTS does not preclude target advancement by other means – including FBLD. Ultimately all but three targets (including all of those ranked medium or high and 6 of 9 ranked low) went on to enter hit-to-lead optimization programs.

Encouragingly, HTTS and NMR agreed perfectly for low and high ligandability targets, but NMR assigned three targets as medium where HTTS assigned them as low. The researchers thus set out to increase the sensitivity of HTTS.

It turns out that entropically-driven binders tend to cause greater thermal shifts than enthalpically driven binders. The observation that most fragments bind largely enthalpically, and with low affinity too, makes them particularly challenging to detect. To try to shift the balance, the researchers repeated the HTTS assay for three of the low-scoring targets in D₂O instead of H₂O, which enhances entropic interactions at the expense of enthalpic interactions. Indeed, all three targets showed enhanced hit rates, and two moved from low to medium ligandability.

Another way to improve sensitivity of a thermal shift assay is to add urea, which destabilizes proteins by lowering the unfolding enthalpy. Adding non-denaturing amounts of urea (0.8 to 2.4 M concentration) to the three low-scoring targets above did indeed increase the hit rate for two of them.

One interesting tidbit is the observation that particularly stable targets, with unfolding temperatures >70 °C, tend to produce lower hit rates in HTTS than less stable targets. This could account for the very different experiences people have had with the technique.

This is a nice paper, and the approach may be worth implementing, as the researchers note has already happened at AstraZeneca. Although HTTS is unlikely to ever be as robust as SPR, NMR, or crystallography, it is hard to beat the low cost and high speed.