From covalent to noncovalent 14-3-3 modulator, unintentionally

The seven members of the 14-3-3 family act as “hub proteins” that bind to other proteins, bringing them together or affecting their subcellular localization. Some of the client proteins, such as estrogen receptor alpha (ERα), are associated with diseases such as cancer, and stabilizing the interaction with 14-3-3 could thus be useful therapeutically. The idea is that a small “molecular glue” could bind at the interface between 14-3-3 and a client protein, enhancing the interaction. We’ve written here, here, and here about successful examples, both covalent and noncovalent. An open-access paper in ACS Med. Chem. Lett. earlier this year by Richard Doveston and colleagues at University of Leiscester starts with a similar strategy, but ends up somewhere else entirely.

 

The researchers had previously found that WR-1065, the active metabolite of the approved drug amifostine, can covalently bind to C38 of 14-3-3σ. (This is the same cysteine residue that had been targeted by Michelle Arkin and coworkers in 2019 using tethering.) Disulfide bond formation between WR-1065 and 14-3-3σ enhances the affinity of ERα for 14-3-3σ by a modest 2.8-fold.

 

In the new paper, the Doveston group made a small set of analogs in which the thiol was replaced by other warheads. Compound 7, containing an acrylamide, improved the affinity of ERα for 14-3-3σ by nearly two orders of magnitude, from 206 to 2.8 nM. But surprisingly, mass spectrometry revealed no covalent modification. Indeed, compound 7 was also active at low micromolar concentrations against the C38A mutant form of 14-3-3σ, while WR-1065 was inactive.

An examination of the structure-activity relationships (SAR) revealed that the acrylamide was essential; reducing the double bond abolished activity, while replacing the amide nitrogen with an oxygen made it significantly less active. Removing the primary amine or replacing it with a hydroxyl or carboxylic acid was also not tolerated.

 

If compound 7 does not bind covalently to C38, how does it work? Surprisingly, the molecule does not even bind at the interface between 14-3-3σ and ERα. Indeed, it shows additivity with a well-characterized molecular glue (fusicoccin A) known to bind at the interface. Where exactly compound 7 does bind is uncertain. A series of biophysical experiments suggests that it may stabilize the dimeric form of 14-3-3σ, though the mechanism remains to be determined.

 

This paper is a useful reminder that, as the authors conclude, “even rational design approaches can lead to unexpected outcomes.” Sometimes a warhead does not behave as one.

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!

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!

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.

Covalent Inhibitor of KRas

So, Ras is big.  We keep on talking about it.  And sometimes we talk about the same work repeatedly.  This recent paper from AZ is a publication of work we have talked about here and here.  This follows on closely to work done by Vanderbilt and Genentech.  Those two papers were done using NMR and this one took a X-ray approach.  The AZ folks were taking a different approach to this PPI: stabilization of the interface.  They took 1160 fragments in pools of 4 and screened against HRas (homolog)-catalytic domain of SOS stable complex.  There were able to identify 3 bindings sites on HRas-SOS (Figure 1):

Figure 1.  HRas-SOS Complex.  HRas (Green), SOS (Blue), A: SOS binding site  (gold) (same as Vanderbilt), B SOS-Hras Interface binding site (Red) (same as Genentech), and C HRas covalent binding site (black).

 

Site A was the same site identified by the Vanderbilt group  Site B was the same as identified as Genentech.  However, the AZ compounds bound to both proteins at the interface.  Their initial hope was to use this site to stabilize the Ras-SOS interface.  Both of the fragments binding to these sites had their affinity determined by TROSY-HSQC NMR.    However, they were not potent enough to elicit a biological effect, which was not unexpected.  After several rounds of chemistry, they were not able to improve these fragments significantly, or even show that they actually stabilized the interface.  

Looking at the growing covalent literature, they hypothesized that an irreversible inhibitor may be the only way to inhibit GTPase activity, especially considering the pM affinity of GTP for Ras.  They identified Cys118R (conserved between HRas and KRas) as a potentially reactive sidechain proximal to the GDP binding site on Ras.  To go after this site covalently, AZ assembled a 400 fragment covalent library (Figure 2) and screened it by mass spectrometry.

Figure 2.  Chemotypes represented in AZ 400 fragment covalent library.

They chose the N-substituted maleimide was deemed "ideal"; other warheads were either insufficiently reactive or overly reactive.  Covalent modification of Cys118R by a fragment partially occludes the nucleotide binding site and potentially prevents the reorganization of the Cys118R loop, thus locking it into the catalytically inactive Ras-SOS complex.  Interestingly, their covalent compounds only inhibited catalytically activity when pre-incubated with Ras-GDP-SOS.  This supports the hypothesis that Cys118R becomes more accessible during SOS-mediated nucleotide exchange.  

This paper brings together several topics which I think are becoming hot: covalent fragments, mass spectrometry, and K-Ras.