Tiny fragments at high concentrations give massive hit rates

Screening fragments crystallographically is becoming more common, especially as the process becomes increasingly automated. Not only does crystallography reveal detailed molecular contacts, it is unmatched in sensitivity. At the FBLD 2018 meeting last year we highlighted work out of Astex taking this approach to extremes, screening very small fragments at very high concentrations. Harren Jhoti and colleagues have now published details (open access) in Drug Discovery Today.

The researchers assembled a library of 81 diminutive fragments, or “MiniFrags”, each with just 5 to 7 non-hydrogen atoms. Indeed, the fragments adhere more closely to the “rule of 1” than the “rule of 3.” Because the fragments are so small, they are likely to have especially low affinities: a 5 atom fragment with an impressive ligand efficiency of 0.5 kcal mol^-1 per heavy atom would have a risibly weak dissociation constant of 14 mM. In order to detect such weak binders, the researchers screen at 1 M fragment concentrations, almost twice the molarity of sugar in soda! Achieving these concentrations is done by dissolving fragments directly in the crystallographic soaking solution and adjusting the pH when necessary. Although this might mean preparing custom fragment stocks for each protein, it avoids organic solvents such as DMSO, which can both damage crystals and compete for ligand binding sites.

As proof of concept, the researchers chose five internal targets they had previously screened crystallographically under more conventional conditions (50-100 mM of larger fragments). All targets diffracted to high resolution, at least 2 Å, and represented a range of protein classes from kinases to protein-protein interactions. The hit rates were enormous, from just under 40% to 60%, compared to an average of 12% using standard conditions.

Astex has previously described how crystallography often identifies secondary binding sites away from the active site, and this turned out to be the case with MiniFrags: an average of 10 ligand binding sites per protein. In some cases protein conformational changes occurred, which is surprising given the small size and (presumably) weak affinities of the MiniFrags.

All this is fascinating from a molecular recognition standpoint, but the question is whether it is useful for drug discovery. The researchers go into some detail around the kinase ERK2, which we previously wrote about here. MiniFrags identified 11 ligand-binding sites, several of which consist of subsites within the active site. Some of the MiniFrags show features previously seen in larger molecules, such as an aromatic ring or a positively charged group, but the MiniFrags also identified new pockets where ligands had not previously been observed. The researchers argue that these “warm spots” could be targeted during lead optimization.

One laudable feature of the paper is that the chemical structures of all library members are provided in the supplementary material. Although it would be easy to recreate by purchasing compounds individually, hopefully one or more library vendors will start selling the set. If MiniFrag screening is standardized across multiple labs, the resulting experimental data could provide useful inputs for further improving computational approaches, as well as providing more information for lead discovery.

Better properties from fragments: c-Abl kinase activators

Last year we described the discovery of asciminib, an allosteric inhibitor of the kinase BCR-Abl that binds in the enzyme’s myristoyl-binding pocket. As we also highlighted nearly a decade ago, molecules that bind in this pocket can either inhibit or activate the enzyme. Although inhibitors have the most obvious therapeutic potential as anti-cancer agents, activators of the ubiquitously expressed c-Abl protein could potentially treat chemotherapy-induced neutropenia. In a recent J. Med. Chem. paper, Sophie Bertrand and coworkers at GlaxoSmithKline describe their efforts in this area.

The researchers started with a high-throughput screen of 1.3 million compounds. Among the hits was fragment-sized compound 2, which showed good binding and activation in biochemical assays but only modest activity in cells. Building off the left side of the molecule improved biochemical potency, but cell activity still lagged. SAR studies on the dichlorophenyl moiety suggested that this hydrophobic group was probably optimal, and a crystal structure of an analog bound to the enzyme confirmed this. Replacing the central thiazole with other aromatic rings also did little to improve cell activity.

The researchers acknowledge “that the chemistry strategy was largely pursuing compounds with rather poor physical properties,” notably low solubility, high lipophilicity, and high aromatic character. As co-author Robert Young has noted previously, physical properties matter. Happily, a fragment screen identified compound 28.

Adding the acetyl group from the HTS hit generated compound 29, with improved activity compared to the fragment. Moreover, this molecule had better solubility and permeability compared to the more lipohilic, thiazole-containing compound 2. Compound 29 also showed significantly improved activation of c-Abl in a cellular assay. Crystallography revealed that it bound in a similar fashion as compound 2, but with a twisted, more “three-dimensional” shape.

Further optimization, in part informed by previous work done on the thiazole series, ultimately led to compound 52, the most active compound synthesized. Another molecule in the pyrazoline series showed good pharmacokinetic properties in mice. Unfortunately, in vivo efficacy studies had to be halted early due to unexpected (and not clearly understood) toxicity.

This paper nicely illustrates several points. First, the power of fragment-assisted drug discovery, in which information from both HTS and FBLD is combined for lead optimization. Second, the inherently fuzzy line between FBLD and other discovery approaches: had compound 28 been tested in the HTS collection, it likely would have been a hit. Third, the importance of physicochemical properties. And finally, the inadequacy of potency and physicochemical properties alone to produce a developable compound. You can optimize your molecule to the best of your ability but still be sideswiped by nasty surprises such as toxicity. It is helpful to be clever in drug discovery, but you need to be lucky too.

Targeting RAS via PDEδ: another protein-protein interaction

Last week we highlighted molecules that inhibit the interaction between oncogenic RAS proteins and an activator protein, SOS1. This week continues the subject of fragments and RAS, but with a different protein-protein interaction, described in a recent paper in Eur. J. Med. Chem. by Min Huang, Naixia Zhang, Bing Ciong, and colleagues at Shanghai Institute of Materia Medica.

The researchers were interested in the protein PDEδ, which binds to lipidated RAS proteins and helps shuttle them to the plasma membrane. Blocking this protein-protein interaction could interfere with RAS signaling. PDEδ was screened against just 535 fragments using two ligand-observed NMR techniques (STD and CPMG), yielding five hits. Crystallography revealed that compound 1-H9 bound at the site where RAS normally binds. Other groups had previously identified molecules that bind in this same region, and the researchers used this information to grow their fragment to compound 16, with low micromolar activity.

Interestingly, a crystal structure of compound 16 showed that the binding mode had flipped relative to the initial fragment: the isobutyl group, which had been designed to replace the isopropylthio group, was binding in a region of the protein previously unoccupied by the fragment. Further growing led to compound 40, with mid-nanomolar potency in a biochemical assay.

Unfortunately, compound 40 and other molecules in the series had at best only modest activity in a viability assay of cells dependent on PDEδ. This result is in contrast to a previously reported PDEδ inhibitor, and the researchers suggest that the difference could be due to off-target activity of that molecule. Indeed, a third group has reported that inhibition of PDEδ would need to be nearly complete to be pharmacologically useful. As the researchers conclude somewhat optimistically, “all these complexities of PDEδ-associated proteins may impose a challenge and opportunity for PDEδ-targeted anticancer drug discovery.” While it is easier to see the challenges than the opportunities, this is nonetheless a nice example of using fragments to target a protein-protein interaction.

Stabilizing and destabilizing SOS1-RAS interactions

Last week we highlighted an example of fragments stabilizing a protein-protein interaction. This week continues the theme, with a paper published in Proc. Nat. Acad. Sci. USA by Roman Hillig, Benjamin Bader, and colleagues at Bayer.

The protein of interest was KRAS, inhibitors of which have long been sought as anti-cancer agents (see here and here for previous fragment efforts). KRAS binding to GTP activates cell survival and proliferation pathways. Guanine nucleotide exchange factors (GEFs) such as the proteins SOS1 and SOS2 facilitate the exchange of GDP for GTP. While inhibitors of this interaction would seem an obvious goal, other researchers had discovered molecules that stabilize the interaction, so the team looked for these too.

An STD-NMR screen of 3000 fragments (in pools of 8, each at 200 µM) yielded 310 hits, of which 97 bound to the complex of a mutant form of KRAS (G12C) and SOS1, but not to either isolated protein. Crystallography was attempted on 42 of these molecules, resulting in 13 structures. All compounds bound in a small hydrophobic pocket on SOS1, near where KRAS binds. Interestingly, two of these, including compound F1, stabilized the interaction between KRAS and SOS1, as assessed by 2-dimensional protein-observed NMR, SPR, and a biochemical assay. The remaining fragments bound to the complex but neither stabilized nor destabilized it. Unfortunately, efforts to improve the affinity of F1 proved unsuccessful.

Meanwhile, the researchers conducted an HTS screen of more than 3 million molecules, which they validated in a variety of biochemical and biophysical assays. Compound 1 passed all of them, and crystallography revealed that the naphthyl moiety binds in the same hydrophobic pocket of SOS1 as compound F1. Unlike the fragment, however, compound 1 inhibits the interaction of KRAS^G12C and SOS1. Structural analysis suggests that this is in part steric: one of the methoxy groups would clash with KRAS. Also, binding of compound 1 causes a conformation change in a critical tyrosine side chain of SOS1 that normally interacts with KRAS. Interestingly, the fragment F1 also interacts with this residue, but enforces a conformation similar to what it adopts when bound to KRAS, thus explaining the stabilization of the complex caused by F1.

Those of you who have worked on kinases will immediately recognize the quinazoline core of compound 1, and indeed this molecule inhibits kinases such as EGFR with nanomolar potency. This activity would make cell assays difficult to interpret, so the researchers added a methyl group to prevent interaction with the hinge region of kinases. Other changes improved the solubility, but only marginally improved the affinity of the best molecule, compound 17.

With two separate series, both of which bind in the same region, the researchers tried merging F1 and compound 17, ultimately leading to BAY-293, with low nanomolar affinity as assessed by isothermal titration calorimetry and functional activity in disrupting the KRAS-SOS1 interaction. Crystallography confirmed that the molecule binds as designed, with the amine group from F1 making similar interactions. BAY-293 was also active in a variety of cell-based assays, and should be a good chemical probe for better understanding the complexities of KRAS signaling.

Superficially BAY-293 bears more resemblance to its HTS parent than its fragment parent, and perhaps this story is best described as an example of fragment-assisted drug discovery. It is also a nice reminder that sometimes subtle chemical changes can make the difference between activation, disruption, or simple binding with no functional activity.