Fragment merging – and flipping – on the leucine zipper of MITF

Transcription factors can be difficult drug targets, particularly those whose primary structure is a “leucine zipper” in which two α-helices gently coil around each other. Their three-dimensional structure provides few pockets suitable for binding small molecules. In a new (open-access) paper in Nat. Commun., Deborah Castelletti, Wolfgang Jahnke, and a large group of multinational collaborators at Novartis and elsewhere present progress toward one of these, microphthalmia-associated transcription factor (MITF), which has been implicated in melanoma.

 

Most of MITF is believed to be disordered, but the DNA-binding domain (DBD) homodimerizes as a basic helix-loop-helix leucine zipper. Unlike related transcription factors, the helices in MITF contain a small kink that keeps them from heterodimerizing and also creates a small “kink pocket.”

 

The researchers expressed the DNA-binding domain of MITF and screened it using ¹⁹F NMR against the LEF4000 library, which we described here. This yielded just 9 hits that confirmed in protein-observed NMR, a hit rate the researchers note “is amongst the lowest that we have observed across multiple FBS campaigns,” consistent with expectations for a difficult target. Two chemical series, represented by compounds 1 and 2, were prioritized, and analogs from the Novartis compound collection were screened to find more-potent compounds 3 and 4.

 

Crystallography revealed that compounds 3 and 4 both bound in the kink pocket. Excitingly, the binding modes are similar and overlapping, inviting fragment merging. This proved successful, yielding a compound that bound 100-fold more tightly than either fragment. Further optimization ultimately led to compounds 7 and 8, with low or sub-micromolar affinity as assessed by isothermal titration calorimetry (ITC).

 

The bound structures of compounds 7 and 8 were determined by crystallography. Compound 7 (gray, left) superimposes nicely onto compounds 3 (cyan) and 4 (magenta), showing successful fragment merging. Compound 8 (green, right), however, is flipped 180 degrees compared to compound 7, despite having similar structure and affinity. Although surprising, this is not too uncommon; we’ve written about previous flippers here, here, and here.

The MITF homodimer is asymmetric, with one helix kinked and the other straight. NMR experiments and molecular dynamics show that both compounds 7 and 8 slow the interconversion between kinked and straight forms, though it is unclear whether this has functional implications. The compounds do not seem to affect DNA binding, and with at best high nanomolar affinity towards MITF no cell data are reported with the molecules.

 

Nonetheless, the successful identification of ligands against a leucine zipper is exciting. The binding pocket is small; as shown in the figure above, the best compounds already stick out on either side of the helices. Further affinity improvements may be difficult, though perhaps covalent approaches could help. Alternatively, perhaps these molecules could be starting points for induced proximity strategies such as PROTACs. It will be fun to watch this story develop.

Fragments vs KEAP1: Fragment growing this time

Kelch-like ECH-associated protein 1 (KEAP1) binds to nuclear factor erythroid 2-related factor 2 (NRF2), targeting it for degradation. Blocking this interaction has anti-inflammatory effects, and indeed the approved drugs dimethyl fumarate and omaveloxolone are believed to act in part through this mechanism. But those drugs hit a lot of other targets, and more specific molecules have long been sought; we wrote about one in 2016 and another in 2021. In an open-access paper just published in Angew. Chem. Int. Ed., Anders Bach and an international team of collaborators at University of Copenhagen and elsewhere describe a new chemical series.

 

As in the 2016 paper, the researchers started with a crystallographic screen, in this case using the 768-member DSI-poised library, which we wrote about here. This resulted in 80 hits, all binding in the so-called Kelch pocket, which has previously been targeted. Thirteen of these bound in the central region, and compound 1 showed modest but measurable affinity by SPR.

 

All previously reported non-covalent high-affinity KEAP1 ligands contain at least one acidic moiety to interact with arginine residues in the protein, so the researchers used structure-based design to add carboxylic acids, resulting in compound 4, with low micromolar affinity. This molecule, unlike the initial fragment, could also block the KEAP1-NRF2 interaction in a fluorescence polarization assay.

 

Building into a hydrophobic sub-pocket yielded compound 12, and adding strategically placed hydrogen-bond acceptors led to further improvements in affinity, ultimately leading to compound 28, with low nanomolar activity. Crystallography revealed that these molecules bound in a similar fashion as the initial fragment.

 

Compound 28 and related molecules were tested in a variety of assays. They were selective for KEAP1 over 15 other human Kelch domains in a thermal shift assay. Compound 28 activated NRF-2 regulated cytoprotective genes and decreased inflammatory markers in multiple cell lines. It also displayed RNA expression profiles similar to those of other reported non-covalent KEAP1 inhibitors. Cellular potency in some of these assays was as good as 60 nM.

 

This is a nice fragment-to-lead story, though no ADME or DMPK data are reported, and the combination of relatively high molecular weight, negative charge, and lipophilicity suggest that permeability and oral bioavailability may be challenging. Indeed, the researchers note that no non-covalent KEAP1-NRF2 inhibitors have entered the clinic. Perhaps this target is better suited for covalent inhibitors, preferably ones more selective than dimethyl fumarate. More on those later.

Throwing the kitchen sink at IL-1β

Last year we highlighted a paper out of Novartis describing a fragment-to-lead story for interleukin-1 beta (IL-1β), a pro-inflammatory cytokine implicated in numerous diseases. The approved antibody drug canakinumab targets IL-1β, but a small molecule would provide easier oral dosing as well as better access to tissues such as the central nervous system. A new paper in J. Med. Chem. by Anna Vulpetti, Konstanze Hurth, and their Novartis colleagues describes the multiple approaches they've taken. (Anna also presented this work at Fragments 2024.)

 

The paper starts by summarizing the fragment work we described here. Notably, of nearly 4000 fragments screened, only a single super-sized fragment was validated, and it was quite weak. The researchers were able to optimize this to a molecule that inhibits binding of IL-1β to its receptor with an IC₅₀ = 1.1 µM.

 

Starting from the initial fragment hit, the researchers performed virtual screens to find alternative binders. Of 281 selected for testing by ¹⁹F NMR or TR-FRET, two hits were obtained, one with an affinity of around 230 µM and the other worse than 1 mM. These molecules were similar to each other, and merging them led to a 43 µM binder. All molecules exceeded conventional fragment size, with the smallest containing 24 non-hydrogen atoms. We’ve previously discussed the possible need for larger fragments for difficult targets such as protein-protein interactions.

 

In addition to FBLD, the researchers also performed DNA-encoded library (DEL) screens using 15 libraries containing >1.6 billion molecules. This led to one family of hits, one member of which inhibited binding of IL-1β to its receptor with an IC₅₀ = 8.3 µM. This molecule contains an aldehyde moiety, a reversible covalent electrophile. Subsequent experiments confirmed that the aldehyde reacts with a lysine residue on IL-1β, and the researchers were able to improve the potency to 1.2 µM. This molecule is even larger than the hit derived from fragments, with >50 non-hydrogen atoms. Interestingly, the molecule binds at a different site on the protein from the initial fragment hit.

 

Finally, the researchers screened a library of macrocyclic peptides in an mRNA display system. The macrocycles consisted of 10-14 amino acid residues, and the library was impressively large, containing “<10¹³ unique cyclic peptides.” This effort yielded a 14 µM inhibitor. Strikingly, crystallography revealed that the molecule binds at a site distinct from either the fragment- or DEL-derived hits.

 

This paper is a tour de force addressing a difficult target. Although the researchers conclude that the protein is “ligandable,” the physicochemical properties of all the hits will need to be improved, along with the affinities, in order to make useful chemical probes, let alone drugs. On the other hand, the fact that the ligands bind to different sites and yet can all inhibit the protein-protein interaction is encouraging, offering multiple opportunities for optimization.

Fragments vs hIL-1β: Growing into a cryptic pocket to inhibit a protein-protein interaction

Protein-protein interactions have a well-deserved reputation for being difficult to drug with small molecules. This is particularly true for cytokine-receptor pairs, which are involved in a host of extracellular signaling functions. Human interleukin-1β (hIL-1β) plays a key role in inflammation by binding to its receptor IL-1R1. Biologics such as anakinra and canakinumab have been approved as drugs, but apart from some very low affinity fragments no small molecule inhibitors are known. In a new (open access) Nat. Commun. paper, Frédéric Bornancin, and collaborators at Novartis and University of Leicester report the first.

 

The researchers started by screening the 3452-compound LEF4000 library, which we described here, using ¹⁹F-NMR. After confirmation using protein-observed 2D NMR just a single super-sized fragment hit remained, consistent with the difficulty of the target. The individual enantiomers of this racemic compound were studied, and only (S)-1 was found to be active. Further characterization revealed that, despite weak affinity, this compound had both slow association and dissociation rates. More on that below.

 

Fragment growing in multiple directions led to mid-micromolar compounds such as 11 and 12. Combining elements from these molecules ultimately led to compound (S)-2, with low micromolar affinity as assessed by SPR. 

 

 

Compound (S)-2 specifically blocked the binding of hIL-1β with its receptor IL-1R1, but did not inhibit the binding of the related cytokine hIL-1α to IL-1R1. Even better, the compound blocked IL-1R-mediated signaling in cells at low micromolar concentrations in two different assays. The similar activity in biochemical and cell assays is likely due to the fact that the compound only needs to act at the cell surface, so permeability is not an issue, in contrast to our post last week.

 

A crystal structure of (S)-2 bound to hIL-1β revealed important interactions between the protein and both the phenol and lactam nitrogen, two contacts that were maintained during fragment optimization. The structure explains why only the (S)-enantiomer is active, as maintaining these contacts would cause clashes for the other enantiomer.

 

The structure also explains the mechanism of inhibition. (S)-2 binds to a cryptic pocket that forms in a region of hIL-1β important for interacting with IL-1R1, and formation of the pocket involves a loop movement that would be incompatible with the protein-protein interaction. The researchers argue convincingly that that the compound stabilizes the cryptic pocket, which naturally exists as a minor population within solution. This also explains the slow kinetics, which would be expected if the compound essentially has to wait until the cryptic pocket opens before it can bind.

 

There is still a long way to go to a drug. Not only is the affinity of (S)-2 modest, the two carboxylic acid moieties and the phenol are likely to impede oral bioavailability. Nonetheless, this is a lovely paper, and the researchers point out that cryptic pockets frequently involve “large movements of secondary structural elements” that could block biological function. Indeed, this is the case for approved drugs such as sotorasib. Don’t give up just because your protein of interest appears like a featureless billiard ball: there may well be opportunities hidden just beneath the surface.

Stabilizing protein-protein interactions: part 3 (fragment linking)

Stabilizing protein-protein interactions is becoming increasingly popular, and not just for PROTACs. Nearly three years ago we highlighted the use of crystallographic screening to find fragments that could stabilize interactions between the adapter protein 14-3-3δ and peptides derived from p53, a prominent cancer target. After noting how much work lay ahead, we ended the post with, “expect a part 3!” This has now been published (open access) in Angew. Chem. by Adam Renslo, Luc Brunsveld, Michelle Arkin, Christian Ottmann, and collaborators at UCSF and Eindhoven University of Technology.

 

In addition to crystallographic fragment screening, the researchers had previously performed a disulfide Tethering screen on the 14-3-3δ protein, which we described here. The fragments from the two screens bound next to one another, so the researchers decided to link them. They started by solving the crystal structure of compound 1 disulfide-bonded to 14-3-3δ in the presence of fragments from the crystallographic screen as well as a peptide derived from estrogen receptor alpha (ERα, another anti-cancer target). These co-structures guided the synthesis of new linked molecules, and these were soaked into crystals of 14-3-3δ and the ERα peptide. Compound 6 gave strong electron density and overlayed nicely on the initial fragments.

 

 

To determine whether the linked molecule could stabilize the 14-3-3δ/ERα complex, the researchers developed a fluorescence anisotropy assay with a dye-labeled peptide from ERα. Some of the linked molecules produced an increase in anisotropy, suggesting stabilization of the 14-3-3δ/ERα complex, but when the researchers ran the important control of repeating the experiment in the absence of 14-3-3δ they found that several molecules still increased anisotropy, which could be due to aggregation. (Adam published a nice early paper on aggregation and is thus particularly attuned to the dangers.)

 

Fortunately, some of the molecules passed this control, and with a robust crystallography system the researchers were able to use structure-based design to improve them, ultimately arriving at compound 24, which increased the affinity of the 14-3-3δ/ERα complex by 25-fold. It was also quite specific towards ERα, and did not increase the affinity of nine peptides from from other proteins for 14-3-3δ. The researchers attribute this selectivity to the fact that most other peptides would sterically clash with compound 24. (Not reported was the peptide from p53, which would be interesting.)

 

This is a nice paper on several levels. In addition to selectively stabilizing a therapeutically relevant protein-protein interaction, this is a rare example of starting with a covalent fragment and developing a non-covalent binder. (For another, see here.) Also, this is a good example of fragment linking, which is often challenging.

 

There is still a long way to go. The most potent molecules all contain amidine moieties, whose high polarity is a liability for cell permeability, let alone oral bioavailability. Moreover, the affinity of compound 24 is still quite weak, with a low ligand efficiency.

 

That said, with a wealth of structural and biological understanding I am optimistic further progress can be made, perhaps by rebuilding the covalent linkage to the protein, as was the case of sotorasib or this more recent paper from the UCSF team. I look forward to part 4!

Fragments vs IL17A: merging and linking

One of the talks at the Discovery on Target meeting last month described the discovery of small molecule inhibitors of IL17A, a pro-inflammatory cytokine. Antibody-based drugs against this protein are useful for psoriasis and other diseases, but they require regular injections. Also, because the antibodies stick around for a long time and dampen the immune response, they could leave patients less able to combat an infection. An orally available small molecule could solve both these problems, but blocking protein-protein interactions is generally difficult. Early progress towards this goal has been published (open access) in Sci. Reports by Eric Goedken and collaborators at AbbVie.

 

The researchers started by ¹³C-labeling the methyl groups of isoleucine, leucine, valine, and methionine. They then performed a 2D NMR screen ([¹H, ¹³C]-HSQC) of ~4000 fragments in pools of 12, with each fragment at 1 mM. This yielded multiple hits, including compound 4. Interestingly, the chemical shift perturbations (CSPs) caused by this fragment were distinct from those caused by known previously disclosed binders, suggesting a different binding site. In particular, one of these CSPs could be traced to a methionine residue. The full NMR assignment of the protein was not conducted, but modeling and mutagenesis narrowed the possibility down to a methionine near the C-terminus.

 

Surface plasmon resonance (SPR) experiments revealed that compound 4 had very low affinity, but two rounds of optimization led to compounds 5 and 6. At this point the researchers were able to solve the crystal structures of these molecules bound to IL17A. The protein exists as a homodimer, and the molecules bind symmetrically, with two copies per homodimer. Moreover, the two copies bind close to one another.

 

 

In addition to these fragments, the researchers had also identified compound 7, and a crystal structure revealed that this binds in a similar fashion. Merging compound 7 with compound 6 and linking two copies of the resulting monomer led ultimately to dimeric compound 10, with nanomolar affinity by both SPR and isothermal titration calorimetry (ITC). This molecule inhibited the binding of IL17A with its receptor in a biochemical assay and also showed low micromolar activity in a cellular assay.

 

This is a nice paper that bears some similarity to previous work we highlighted from AbbVie on a different cytokine, TNFα. There too fragment linking was used initially. As in the present case, that effort led to a drop in ligand efficiency and a significant increase in the size of the molecules, resulting in suboptimal pharmaceutical properties. Identifying drug-like small molecules has been an ongoing challenge for IL17A; peptide-based inhibitors and macrocycles have been found that bind to other sites on the protein, but many of these are also well beyond rule-of-five space. As the researchers conclude, “we look forward to seeing which of these sites prove to be the most amenable to producing optimized drug candidates.”

Fragments vs KEAP1: deconstruction and merging

One of the more challenging protein-protein interactions targeted by drug hunters is the interface between the transcription factor NRF2 and its repressor KEAP1. This is part of the cellular defense against reactive oxygen species; increasing NRF2 activity may be useful for treating a variety of diseases. Unfortunately, the binding site on KEAP1 that interacts with NRF2 is large and has a predilection for carboxylic acids. Thus, many of the molecules reported as inhibitors tend not to be druglike. Anders Bach (University of Copenhagen) and a multinational team of collaborators sought to do better, and have just published some of their journey in J. Med. Chem.

 

The researchers had previously tested 19 reported small-molecule KEAP1 inhibitors, of which only nine confirmed. (This is a salutary reminder to take any individual publication with a large grain of salt.) The nine fell into six chemical series (two shown below), and the researchers decided to fragment some of these molecules into 77 fragments. The fragments were then tested in four assays: fluorescence polarization (FP), a thermal shift assay (TSA), saturation transfer difference (STD) NMR, and surface plasmon resonance (SPR).

 

Primary hit rates were generally high, from 25%-64%, but long-time readers will not be surprised that the overlap was not great: no fragments hit in all four assays, and only eight hit in three. As the researchers point out, this could reflect differences in sensitivity, conditions (from 3-8% DMSO and from 0.5 to 8 mM fragment), and different types of false positives and false negatives. Interestingly, and in contrast to previous work, overlap was good between STD NMR and SPR.

 

Crystal structures of seven hits were solved bound to the protein, and compounds 4c and 1m (from different precursor molecules) were merged to provide compound 8, with low micromolar affinity. Compound 8 was the subject of considerable medicinal chemistry, with five different vectors chosen for growing. Despite being structurally enabled, the researchers struggled; changes that improved affinity in one context did not do so in another. After considerable effort, the researchers obtained compound 77o, with mid-nanomolar activity.

 

Compound 77o is stable in human plasma and mouse liver microsomes. Unfortunately, and unsurprisingly given the two carboxylic acids, it has poor permeability. Indeed, a fragment-derived KEAP1 inhibitor we described previously has only a single carboxylic acid, as does precursor compound 7. As the researchers themselves acknowledge, “the physicochemical properties of our compounds are not favorable for membrane permeability.”

 

Nonetheless, this paper is a lovely example of fragment-based deconstruction reconstruction (FBDR) and is well worth studying for the thorough descriptions of fragment screening in orthogonal assays and structure-based design. Another lesson may be that despite considerable effort, the final molecule is far from a chemical probe, let alone a drug. Perhaps some targets truly are undruggable. Or maybe – as for other seemingly undruggable targets – a change in strategy is needed.

Fragments vs TNFα advanced with biophysics, linking, and growing

The cytokine tumor necrosis factor α (TNFα) is a key mediator of inflammation and has long been a target for rheumatoid arthritis, Crohn’s disease, psoriasis, and a host of other inflammatory diseases. Several biologic drugs, such as adalimumab, are approved but these monoclonal antibodies and fusion proteins can be immunogenic or induce neutralizing antibodies. Small molecules could avoid these pitfalls and also reach organs, such as the brain, less accessible to biologic agents. Impressive efforts towards this goal have just been reported in J. Med. Chem. by Justin Dietrich, Chaohong Sun, and colleagues at AbbVie. (Andrew Petros presented this work at the CHI DoT meeting last September.)

 

The researchers began by screening 18,000 fragments using two-dimensional (¹³C-HSQC) NMR against TNFα in which the methyl groups of isoleucine, valine, leucine, and methionine were isotopically labeled. Only 11 fragments caused significant perturbations, an 0.06% hit rate reflecting the difficulty of finding hits against this target. All the fragments were characterized by SPR, and compound 1 turned out to have reasonable affinity and ligand efficiency. Synthesis of a few dozen analogs led to compound 2, with improved activity.

 

TNFα forms a homotrimer, and a crystal structure of compound 2 bound to TNFα revealed that two copies of the fragment bind within a large hydrophobic cavity at the interface of the three protein monomers. Not present in the apo-form of the protein, this central pocket is formed by the movement of tyrosine side chains, causing desymmetrization of the protein trimer. The researchers linked the two nearby fragments to produce compound 3 with improved affinity but decreased ligand efficiency. Further optimization led to compound 4, which was active in cells. But perhaps not surprisingly given its size and lipophilicity, this molecule had high clearance and poor oral bioavailability in mice. 

 

 

A second fragment, compound 6, had lower affinity than fragment 1, and parallel chemistry efforts generated only flat SAR. A crystal structure revealed that compound 6 bound in a similar manner as compound 1, with two copies in the central cavity. Surprisingly, a crystal structure of compound 8, which differs from compound 6 only by a single methyl group and actually has slightly lower affinity, revealed a singly copy bound in the central void. Scaffold hopping led to compound 9, which was ultimately optimized through structure-based design and careful attention to drug-like properties to compound 12. This molecule is orally bioavailable and showed activity in a mouse arthritis model.

 

This is a lovely paper that illustrates several important lessons. First, as the researchers note, “we have learned from multiple programs, including this one, aimed at developing small-molecule inhibitors of protein-protein interactions, that biophysical methods, when used to drive a fragment-based approach, offer the greatest chance of success.” NMR was essential for finding the initial fragments, SPR provided necessary thermodynamic and kinetic information, and crystallography led to the breakthrough discovery of the binding mode of compound 8.

 

Second, although the initial series generated by fragment linking ultimately did not advance, it proved critical for developing chemical tools, validating assays, and providing structural insights.

 

And finally, this paper is a paean to persistence for difficult targets. As the researchers note, scientists have been seeking small molecule inhibitors of TNFα for decades, and although compounds were reported as early as 2005, most of these have had poor physicochemical properties. Seemingly undruggable targets can sometimes be unlocked. But it usually takes time.

Fragment mixtures vs protein mixtures

In FBLD – as in most areas of research – speed and efficiency are prized. The faster you can find quality fragments, the faster you can advance them. NMR-based screening remains one of the most popular fragment-finding methods, and in a recent Molecules paper William Pomerantz and collaborators at the University of Minnesota and Gustavus Adolphus College provide an accelerated workflow.

 

The Pomerantz lab is well known for protein-observed ¹⁹F (PrOF) NMR, in which fluorine-labeled residues are incorporated into proteins. This is easily accomplished by supplementing the media with fluorine-containing amino acids during protein expression. To date more than 15 fluorinated amino acids have been tested in more than 70 proteins, ranging from 7 to 180 kDa in size. Because the chemical shift of fluorine is so sensitive to its environment, a fragment binding nearby can be readily detected by PrOF NMR.

 

When a single type of amino acid is fluorinated, the resulting protein spectrum is considerably simpler than in traditional protein-observed NMR methods. Taking advantage of this, the researchers mixed two different bromodomain proteins: the human oncology target BPTF and PfGCN5 from the malarial parasite Plasmodium falciparum. Both of these bromodomains contain a tryptophan in their N-acetyl-lysine binding sites, so each protein was labeled with 5-flurotryptophan. The proteins were then screened (at 50 µM each) against 467 fragments from Life Chemicals in pools of 4-5 (at 400 µM each). Chemical shift perturbations of the binding-site tryptophan were seen for half of the 98 pools. To determine which fragments were responsible for these shifts, the researchers tested their fragment mixtures against the relevant proteins using (ligand-detected) CPMG NMR. Since they had previously determined the ¹H NMR spectra of all their fragments, it was easy to pick out the binders.

 

Hit rates were similar for both BPTF (9.8%) and PfGCN5 (9.2%), and 4.1% of fragments hit both bromodomains. The researchers had previously screened this library, which is enriched for shapely fragments, against the bromodomain BRD4 D1 (see here) and obtained a similar hit rate. Statistical analyses revealed that the 3D-character for PfGCN5 hits is similar to the library as a whole, as had also been seen for BRD4 D1, while the BPTF hits tended to be flatter.

 

The researchers also followed up on several  fragments individually. One in particular had low micromolar affinity for PfGCN5 as assessed with both PrOF NMR and ¹H-¹⁵N HSQC NMR titrations. Interestingly, this fragment also caused a chemical shift in a different 5-fluorotryptophan residue some 22 Å away from the canonical binding site. Binding at this site could not be competed by a known high-affinity ligand, and a computational screen using FTMap suggested that this does appear to be a secondary binding site.

 

Overall this approach appears to be an appealing workflow as judged by comparing required time, protein, and ligand amounts to other NMR-based screening cascades. As the researchers note, it is advantageous to assess both protein and ligand behavior, as done here. Have you tried using PrOF, and if so how has it performed for you?

Stabilizing protein-protein interactions: part 2

Stabilizing – rather than disrupting – protein-protein interactions is increasingly popular, particularly in the context of PROTACs and molecular glues. Last year we highlighted research in which covalent Tethering was used to identify fragments that could stabilize a protein-protein interaction, but only when the fragments were disulfide-bonded to one of the proteins. In a new (open access) J. Med. Chem. paper some of the same researchers, including Christian Ottmann and a large group of collaborators at Eindhoven University of Technology, University of Lille, AstraZeneca, and UCSF, have extended the approach to non-covalent fragments.

 

As before the researchers were interested in the adapter protein 14-3-3; the various isoforms of this “hub” protein each interact with hundreds of other proteins, many of which are implicated in disease. Natural products such as fusicoccin A stabilize interactions between 14-3-3 and some protein partners, suggesting ligandability, but the 13 chiral centers make analoging somewhat daunting. Two 14-3-3 partners implicated in oncology include the transcriptional coactivator TAZ and the tumor suppressor p53. Stabilizing the interactions of either of these proteins with 14-3-3 could have anti-cancer effects.

 

To find fragments that could stabilize these interactions, the researchers first grew crystals of 14-3-3σ in complex with peptides derived from either TAZ or p53. These crystals were then soaked in 100 pools of five fragments, each at 10 mM. Electron density was seen for several fragments; all fell into one of two scaffolds. Interestingly, both scaffolds contained an amidine moiety that forms a salt bridge with a glutamic acid side chain in 14-3-3σ.

 

One fragment, AZ-003, made interactions both with 14-3-3σ as well as with the TAZ-derived peptide. The peptide itself actually changed conformation so that a carbonyl oxygen could form a hydrogen-bond to the fragment, and three additional peptide residues could be resolved in the electron density that were not seen in the absence of fragment.

 

In the case of the other complex, none of the fragments interacted directly with the p53-derived peptide, though they bound nearby. Fragment growing and crystallography revealed that some of these larger molecules made water-mediated interactions to the peptide and were specific for p53 over TAZ, demonstrating that selectivity can be achieved. In the case of one molecule, AZ-008, crystallography was unsuccessful but modeling and protein-observed NMR experiments suggested direct interactions with the p53-derived peptide. Fluorescence polarization and SPR experiments revealed that 1 mM AZ-008 improved the affinity of 14-3-3σ for the peptide by an unambiguous but modest two-fold.

 

As with the previous paper, there is still much to do, and it will likely not be easy. The Supporting Information contains more than 30 crystal structures of small molecules bound to 14-3-3, both covalently and non, hinting at the effort required. However, the fact that both AstraZeneca and Novartis are working on 14-3-3 proteins – along with at least one startup – bodes well. Expect a part 3!

Fragments vs RAS family proteins: A chemical probe

RAS family proteins are considered a holy grail of oncology research. Way back in 2012 we discussed a couple papers disclosing low affinity fragments that bind in a small, shallow, polar pocket found in KRAS, NRAS, and HRAS. At the time we wondered “whether this is a ligandable site on the protein.” Last year we highlighted a paper proving that the site is, in fact, ligandable, as exemplified by the mid-nanomolar molecule Abd-7. A paper just published in Proc. Nat. Acad. Sci. USA by Darryl McConnell and collaborators from Boehringer Ingelheim and Vanderbilt University (including Steve Fesik, who published one of the 2012 reports) describes successful development of another ligand. (See here for a fun animated description set to music.)

Consistent with the “undruggable” reputation of RAS family proteins, a high-throughput screen of 1.7 million compounds failed to find anything useful. In contrast, a library of just 1800 fragments screened using STD NMR and MST identified 16 fragments that bind to an oncogenic mutant form of KRAS, as confirmed by 2-dimensional (HSQC) NMR. A separate HSQC NMR screen of 13,800 fragments identified several dozen more, though all the fragments from both screens have dissociation constants weaker than 1 mM. SAR by catalog led to amine-substituted indoles such as compound 11, which modeling suggested could form a salt bridge to an aspartic acid side chain.

The pocket in which all of these molecules bind, between the so-called switch I and switch II regions of KRAS, is much smaller than typical drug-binding sites, but modeling suggested that fragment growing could pick up an additional hydrogen bond, leading to compound 15. Crystallography confirmed the predicted binding mode of this molecule, and informed additional structure-based design, leading first to compound 18 and ultimately to BI-2852, with low or sub-micromolar affinity for wild-type and mutant KRAS, NRAS, and HRAS as assessed by ITC. The researchers also confirmed that the enantiomer is about 10-fold less potent, thereby providing a control compound. Commendably, the researchers have made BI-2852 and the enantiomer available (for free!) to the research community as a chemical probe.

A crystal structure of KRAS^G12D bound to BI-2852 (cyan) compared with Abd-7 (magenta) reveals how shallow the pocket is; both molecules are largely surface-exposed. The conformational flexibility of the protein is also interesting: Abd-7 would not be accommodated by the protein conformation bound by BI-2852.

The biology is also quite interesting – and complicated. RAS family proteins behave as molecular switches, cycling between the “on” (GTP-bound) state and the “off” (GDP-bound) state, with these transitions assisted by other proteins. On-state RAS drives cell-proliferation and survival. Molecules that bind at the switch I/II pocket block the transition from off to on, but they also block the transition from on to off. Thus, cellular effects are modest. Moreover, BI-2852 hits all RAS isoforms, which could lead to unacceptable toxicity in animals.

This is a lovely paper, but I do quibble that the promise of the title – “drugging an undruggable pocket on KRAS” – remains to be demonstrated. First, both the biochemical and cell-based potency need to be further improved. As the molecule is already large, gaining this needed potency could come at the cost of physicochemical properties. Indeed, the researchers do not discuss the pharmacokinetics of BI-2852. And finally, as the authors themselves note, they will probably need to improve selectivity to spare one or more wild-type RAS isoforms.

What this work does establish indisputably is that the switch I/II pocket is ligandable, though not without effort, as indicated by the 42 authors. Whether or not the site is actually druggable may require another seven years to determine.

Fragments in the clinic: AZD5991

Venetoclax, the second fragment-based drug to reach the market, binds to and blocks the activity of the anti-apoptotic protein Bcl-2, allowing cancer cells to undergo programmed cell death. The drug is effective in certain cancers such as chronic lymphocytic leukemia and small lymphocytic lymphoma. However, a related protein called Mcl-1 is more important in other types of cancers. Like Bcl-2, it binds and blocks the activity of pro-apoptotic proteins, allowing cancer cells to survive even when Bcl-2 is inactivated. A paper in Nat. Comm. by Alexander Hird and a large group of collaborators (mostly at AstraZeneca) describes a successful effort to target Mcl-1.

Given that the researchers were targeting a protein-protein interaction, they took multiple approaches, including their own fragment-based efforts. They also characterized previously reported molecules, such as those the Fesik group identified using SAR by NMR (which we wrote about in 2013). A crystal structure of one of these revealed a surprise: two copies of compound 1 bound to Mcl-1, which had undergone conformational changes to accommodate the second molecule in an enlarged hydrophobic pocket.

Recognizing the potential synergies of linking these together, the researchers prepared a dimer of a related molecule, but unfortunately the affinity of this much larger molecule was actually worse. However, they wisely isolated and tested a side product, compound 4, and found that this had improved potency. A crystal structure of this molecule bound to Mcl-1 revealed that the pocket had expanded to accommodate the added pyrazole moiety. Since compound 4 adopted a “U-shaped” conformation, the researchers decided to try a macrocyclization strategy to lock this conformation and reduce the entropic penalty of binding. This produced compound 5, and adding a couple more judiciously placed atoms led to AZD5991, with a nearly 300-fold improved affinity. The molecule binds rapidly to Mcl-1 and has a relatively long residence time of about 30 minutes. A crystal structure reveals a close overlay with the initial compound 1 (in cyan).

In addition to picomolar affinity, AZD5991 showed excellent activity in a variety of cancer cell lines dependent on Mcl-1. The compound was tested in mouse and rat xenograft models of multiple myeloma and acute myeloid leukemia and showed complete tumor regression after a single dose. This is all the more remarkable given that AZD5991 is about 25-fold less potent against the mouse version of Mcl-1 than the human version. The molecule was also effective in cell lines resistant to venetoclax, and combining the two molecules caused rapid apoptosis in resistant cell lines. AZD5991 is currently being tested in a phase 1 clinical trial.

This paper holds several lessons. First, the researchers did extensive mechanistic work (beyond the scope of this post to describe) to demonstrate on-target activity. Second, although the initial dimerization strategy was unsuccessful, the researchers turned lemons into lemonade by pursuing a byproduct; we’ve written previously about how even synthetic intermediates are worth testing. Third, the macrocyclization and subsequent optimization is a lovely example of structure-based design and medicinal chemistry. And finally, the fact that the researchers started with a fragment-derived molecule reported by a different group is a testimony to the community nature of science. Last week we highlighted the Open Source Antibiotics initiative, which is actively encouraging others to participate in advancing their early discoveries. Good ideas can come from anywhere, and it takes a lot of them to make a drug.

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.

Stabilizing protein-protein interactions

Despite the fact that the second FDA-approved fragment-derived drug targets a protein-protein interaction (PPI), these types of targets have a well-earned reputation for being difficult. Most researchers try to disrupt PPIs. An alternative is to stabilize PPIs. This is not as crazy as it sounds: rapamycin, tafamidis, and PROTACs all stabilize PPIs. In a paper just published in J. Am. Chem. Soc., Michelle Arkin, Christian Ottmann, and collaborators at UCSF, Eindhoven University of Technology, Novartis, and the University of Duisburg-Essen bring fragments to bear on the problem.

The researchers were interested in the protein 14-3-3δ, a “hub” protein that binds to more than 300 other proteins (not all at the same time). One of these is estrogen receptor α (ERα): binding prevents the transcription factor from dimerizing and binding to DNA. The natural product fusicoccin A (FC-A) binds at the interface of 14-3-3δ and ERα and stabilizes that interaction, thereby inhibiting the growth of breast cancer cells. Because FC-A is a structurally complex natural product, the researchers sought fragments that would have a similar effect. They used Tethering, in which reversible disulfide bond formation stabilizes a protein-ligand complex, allowing its identification (see here and here). Specifically, fragments that bind near a cysteine residue are resistant to reduction, and the extent of binding can be detected by mass spectrometry.

The 14-3-3δ protein conveniently contains a cysteine residue in the vicinity of the ERα binding groove; the researchers used this native protein and also created two additional mutant proteins in which the native C38 cysteine was removed and new cysteine residues were introduced nearby. These three proteins were then screened against a library of 1600 disulfide-containing fragments under mildly reducing conditions in the presence or absence of a phosphopeptide derived from ERα. Most of the hits against the native protein were weak, but several hits against the N42C mutant were both resistant to reduction and also bound preferentially to the 14-3-3δ/ERα peptide complex compared to 14-3-3δ alone. Thus, ERα could enhance the binding of fragments to 14-3-3δ.

Next, the researchers used a fluorescently labeled peptide derived from ERα to show that one fragment could improve the apparent dissociation constant for the peptide and 14-3-3δ about 40-fold, from 1.3 µM 32 nM. Crystallography revealed that the cooperative fragments bound at the PPI interface, as expected given the location of the cysteine residues. The cooperative fragments placed a phenyl group in close proximity to a valine residue from the ERα peptide.

The researchers then examined the selectivity of one of their stabilizing fragments for other 14-3-3δ client proteins. In the case of a phosphopeptide derived from TASK3, which has a similar sequence to that of the ERα peptide, the fragment also showed cooperative binding. However, two peptides from other client proteins competed with the fragment for binding, and crystal structures revealed that the binding modes would be incompatible.

This is a nice illustration of site-directed fragment discovery to identify fragments that can modulate protein function in a more sophisticated manner than simple inhibition. One of the nice features of Tethering is that – like crystallography – it is able to identify extraordinarily weak binders. Unfortunately, this sometimes makes the hits challenging to advance: NMR experiments do show binding between a non-disulfide-containing derivative of one of the fragments and the 14-3-3δ/ERα peptide complex, but at high concentrations. It will be interesting to see whether this can be built into a potent non-covalent binder, and/or whether other types of covalent modifiers will be able to produce useful chemical probes for this target.

Fragments vs Ras – part 3

Six years ago we highlighted papers from Genentech and Steve Fesik’s group reporting fragments that bind to Ras-family proteins, which are among the best validated but most difficult anti-cancer targets. The fragments bind some distance from the GTP-binding site, but can block Ras signaling by interfering with important protein-protein interactions. However, the most potent molecules reported bound at this site with just ~200 µM affinity, and we concluded by musing that “it still remains to be determined whether this is a ligandable site on the protein.” As reported recently in Nature Communications by Terence Rabbitts and collaborators at the University of Oxford, St. James University, Domainex, and the University of Aberystwyth, the answer appears to be yes.

The researchers screened HRas against 656 fragments, each at 200 µM, using SPR, resulting in 26 initial hits. These were tested again by SPR against active-form protein (bound to the GTP mimetic GTPγS) or inactive protein (bound to GDP). A single compound, Abd-1, was selective for the activated form of the protein, and did not bind when the protein was complexed to an antibody the researchers had previously generated that binds at the same PPI site.

Abd-1 had low affinity and was not particularly soluble, so the researchers looked for analogs with better properties, resulting in Abd-2, which binds to both HRas and KRas. Further growing in the direction taken by the Fesik group did not lead to significant improvements, but a breakthrough occurred when the researchers grew off a different region of the fragment, towards what looked to be the wall of the small pocket. As Trevor Perrior mentioned at the DOT meeting last month, this led to the opening up of a new channel and a substantial boost in affinity for Abd-5. Further growing allowed the researchers to trim off the right-hand portion entirely, leading to Abd-7, with mid-nanomolar activity and good ligand efficiency. Crystallography revealed that, despite the conformational changes, the core of Abd-7 still binds in the same location as Abd-2.

Not only did Abd-7 bind tightly to KRas, it also inhibited the pathway in cell-based assays (albeit at 100-fold higher concentrations), presumably by blocking interactions with Ras-effector proteins. The compound also showed low micromolar activity against cancer lines with different Ras mutations in cell viability assays. The researchers note that “the observed discrepancy between affinity (in vitro Kd) and efficacy (IC₅₀ in cells) is a known challenge that can be addressed through chemistry.” Other possible challenges include metabolic stability and oral bioavailability, neither of which is discussed. Nonetheless, the paper reveals that this site in Ras family proteins is ligandable. It is also a useful reminder that proteins can be remarkably plastic, and sometimes the best route forward really is by slamming into what appears to be a solid wall.

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.

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!