From fragment linking to covalent RAD52 inhibitor

Targeting proteins involved in DNA repair is a promising approach against cancer. However, finding effective small molecule inhibitors can be difficult because the protein-DNA interface tends to be very polar, with few classically ligandable pockets. In a new J. Am. Chem. Soc. paper, Nicolas Bocquet, Olivier Querolle, Ulrich Lücking, and collaborators at FoRx Therapeutics, WuXi AppTec, Diamond Light Source, University of Bern, Sandexis, and Weizmann Institute of Science provide a proof of concept.

 

The researchers were interested in RAD52, which mediates several DNA repair functions. The N-terminus of the protein forms an oligomeric ring structure, and each subunit contains a solvent-exposed groove that binds to single-stranded DNA. Since DNA is negatively charged, the researchers conducted a crystallographic screen against a library of 320 carboxylic acid containing fragments. This identified three related molecules, of which compound 3 had the highest occupancy. Because the compounds bind at the DNA-binding interface, the researchers developed a fluorescence polarization (FP) DNA-displacement assay and found that the fragments disrupt DNA binding at high micromolar concentrations.

 

Interestingly, the crystal structure revealed two copies of compound 3 bound to each subunit of RAD52: each positioned where a single nucleotide would normally bind, with the carboxylic acids making similar contacts as the phosphates. In other words, the fragments mimic two consecutive bases of a single-stranded oligonucleotide. Naturally, this suggested a linking approach.

Linking two close analogs of compound 3 led to compound 5, with a satisfying two orders of magnitude improvement in activity in the FP assay, which the researchers suggest is largely due to lowering the entropic penalty. Linking directly off the thiazole moieties produced similar results, and a macrocycle had slightly better activity, though not so much better as to justify the synthetic hassle. A crystal structure of compound 5 revealed a small hydrophobic cleft, and this was exploited with a phenyl moiety in compound 11, a high nanomolar inhibitor in the FP assay.

 

A crystal structure of compound 11 bound to RAD52 confirmed that the compound binds as designed. The structure also revealed a cysteine residue (C64) in close proximity to the phenyl moiety. Adding an acrylamide warhead yielded a covalent inhibitor, and further optimization ultimately led to FORX-864, which has low nanomolar activity in the FP assay after 4 hours. The researchers appropriately measured the k_inact/K_I, which came in at a respectable 230,000 M^-1s^-1, better than some covalent clinical compounds. Removing the warhead led to a dramatic loss in potency (IC₅₀ = 27 µM in the FP assay).

 

In a cellular assay to measure single-stranded annealing (SSA), FORX-864 showed roughly 1 micromolar activity, while a compound lacking the warhead was inactive. FORX-864 was also inactive in a cell line in which RAD52 had been knocked out, suggesting on-target activity. The researchers profiled the reactivity of FORX-864 in an activity-based proteomic profiling (ABPP) assay and found that it was reasonably selective. Intravenous dosing in mice showed encouraging pharmacokinetic properties, though with two carboxylic acid moieties oral dosing might be challenging.

 

Overall this is a nice example of liganding a difficult target. Given that FORX-864 mimics a dinucleotide, it may bind to other DNA- or RNA-binding proteins. However, covalent binding is required for high potency, and it is unlikely these other proteins will have cysteine residues in the appropriate position, which should help with selectivity. FORX-864 certainly appears to be the best chemical probe for RAD52 to date.

Focal molography for assessing protein-ligand interactions

The list of technologies used to find and validate fragments is long, with more than a dozen in our 2024 poll. But it’s always fun to learn about new approaches. At the Novalix conference earlier this year I learned of “focal molography,” and this is the topic of a recent paper in J. Med. Chem. by Nicholas Favalli, Andreas Frutiger, Sebastian Oehler, and colleagues at Philochem and lino Biotech.

 

The approach is described in detail in this Wikipedia article. Briefly, it is a biophysical technique based on changes in diffraction. A sensor chip contains a matrix of “molograms,” each of which is a lens constructed of micropatterned ridges and grooves. The ridges of each mologram are functionalized with unique single-stranded oligonucleotides. These capture complementary strands, each attached to a molecule to be screened. The grooves are coated with oligonucleotides without small molecules to act as a control in the subsequent readout. Proteins are then flowed over the chip. Protein binding to the small molecules of a specific mologram changes the topography of the ridge, and so the diffraction. This change can be measured in units of “coherent mass density” (pg/mm²). Because only specific binding to the ridges is detected (the grooves provide background controls for each mologram), the technique is suitable for even crude samples such as cell lysates.

 

An obvious application for focal molography is to confirm the binding of hits identified from DNA-encoded libraries (DEL), since the small molecules already have an oligonucleotide tag. This is the focus of the new paper.

 

The researchers started by assessing three known binders to the model protein carbonic anhydrase IX (CAIX). Surface plasmon resonance (SPR), the most frequently-used chip-based biophysical method, was used as a benchmark in two formats, one in which the small molecule was bound to the SPR chip, which is most comparable to focal molography, and one in which the protein was bound to the SPR chip.

 

SPR and focal molography agreed in rank order for the three compounds, but focal molography sometimes suggested tighter binding. For the highest-affinity compound, SPR gave a K_d of 28 nM for compound bound to the chip and 450-750 nM for protein bound to the chip, while focal molography gave a K_d of 0.82 nM. Agreement was better for the weaker binders, one of which was mid-nanomolar and the other low micromolar. The kinetic constants k_on and k_off also differed between focal molography and SPR by as much as two orders of magnitude.

 

Next, the researchers examined hits from a DEL screen against CAIX, some of which were selective against the related protein CAII. A total of two dozen molecules were tested by both focal molography and fluorescence polarization (FP) as an orthogonal technique. Again, the rank orders of affinities for the compounds to CAIX and CAII agreed while the absolute values varied, with focal molography usually suggesting ten-fold tighter binding. (Though the FP assay often bottomed out above the K_d values measured by focal molography.)

 

Finally, the researchers examined the binding of a known high-affinity binder to the oncology target fibroblast activation protein (FAP). In this case the measured affinities were close to the reported value.

 

This is an interesting approach, and according to the researchers “focal molography can support up to ~500 duplicate kinetic measurements per day on a single regenerable chip.” The observation that affinity measurements seem to be tighter than other methods does raise concerns. I wonder if the density of the molecules on the molograms leads to rebinding, as is sometimes seen when SPR chips are too heavily functionalized. Alternatively, since CAIX is a homodimer, perhaps avidity effects played a role. Also, the researchers suggest that the technique is best used for compounds with affinities better than 10 µM, which reduces utility for many fragment programs.

 

If you’ve used focal molography, how has it worked for you?

Twentieth-Fourth Annual Discovery on Target Meeting

Cambridge Healthtech Institute held its annual Discovery on Target (DoT) meeting in Boston last week. Attendance may have set a record, with over 1000 participants. There were sessions on antibodies, conjugates, and radioligands, but I’ll just touch on some of the small molecule themes most relevant to this blog.

 

Covalent approaches

As with last year, covalent approaches to drug discovery were well-represented. Dhirendra Simanshu (Frederick National Laboratory) described several drugs birthed in the NCI RAS Initiative, including ones targeting the G12C mutant of KRAS and C242 on the RAS-binding domain of PI3Kα. He also described how various KRAS mutants cause resistance to drugs, finding that most of them affect the K_I term (binding) more than the k_inact term (bonding). Continuing on the KRAS theme, Jack Sloan discussed efforts at BMS to discover inhibitors targeting both the on- and off-states of KRAS G12C.

 

Kasia Handing (Tango Therapeutics) presented both covalent and non-covalent approaches against VRK1, a synthetic lethal target for glioblastoma. A biochemical screen yielded covalent inhibitors that were characterized crystallographically, but they turned out to be too non-selective to progress. In the meantime, an in-cell DNA-encoded library (DEL) screen led to more advanceable molecules.

 

DEL was indeed a major topic of the conference, and here too covalent approaches are being explored. Elizabeth D’ambrosio described how GSK is identifying covalent binders from DEL screens, a process which includes testing for time-dependence as well as aggressive washing steps under denaturing conditions to quench unreacted molecules and wash away any sticky, non-covalently bound compounds. And Xiaojie Bruce Lu (Chinese Academy of Sciences) described applying DEL to a variety of targets, including some covalently targeting non-cysteine residues.

 

One of the advantages of DEL is being able to screen large libraries, and increasing throughput is also of interest to Harvard’s Steve Gygi, who is screening covalent fragments against cells and cell lysates. In 2024 we described how he was using tandem mass tag (TMT) multiplexing to analyze 18 samples in a single mass-spectrometry run, and he has now extended this to 35 samples. What’s more, Steve is combining 16 different fragments in each sample and introducing clever deconvolution approaches to further accelerate the process.

 

Not everyone is targeting cysteine residues. Mark Tye described the discovery of Revolution Medicines’ zoldonrasib, which uses an aziridine warhead to target the G12D mutant of KRAS. And Balyn Zaro (University of California San Francisco) described using ninhydrin derivatives to target arginine. Most organic chemists are familiar with ninhydrin’s reaction with primary amines such as the N-termini of peptides, but Balyn has been able to optimize selectivity for arginine and has used the resulting chemical probes to identify more than 7000 arginine residues in cells, about 5% of which are hyperreactive. Interestingly, some cell lines seem to have a greater proportion of reactive arginine residues than others.

 

Direct-to-Biology (D2B) approaches

Another recurring theme was direct-to-biology, in which crude reaction mixtures are screened directly in various assays without purification. Indeed, Nicholas Bland of Domainex said that after looking at the agenda of the meeting he considered skipping the introductory slides – and this was on the first official day of the conference. Nicholas described using D2B for optimizing PROTACs as well as for improving fragment hits against the GPCR A_2A receptor.

 

Jack Sadowsky, who also presented at DDC this past spring, described how he and his colleagues at Kimia are using D2B in combination with machine learning to develop selective inhibitors of kinases while also improving their in vitro ADME properties. This effort entailed making and testing tens of thousands of compounds from a virtual space of some 80 million molecules.

 

But even small-scale D2B can be valuable. Trevor Sherwood described how he and his colleagues at BMS used D2B to make and test 663 molecules to improve the potency of an EP4 agonist identified from a high-throughput screen. This involved multiple reaction types, including photoredox chemistry, for which they built a custom-designed reactor to evenly heat and illuminate the microtiter plates. The D2B work was done in parallel with medicinal chemistry, but interestingly the development candidate BMS-986526 came directly from the D2B work and has entered a phase 1/2a trial for rheumatoid arthritis.

 

There were other talks and posters on D2B that I missed, but I did attend two breakfast roundtables on the topic that were quite lively despite happening at 7:30 – a testament to the interest in the topic.

 

“Traditional” fragment-based drug discovery

At a plenary panel moderated by Drug Hunter’s Dennis Hu on drugging difficult targets, Biogen’s Klaus Urbahns asked the other panelists if they were still doing fragment-based lead discovery. John Tallarico of Novartis and Ian Storer of AstraZeneca immediately said yes. And in a separate presentation, Kris Borzilleri described how she and her Pfizer colleagues used fluorine-detected NMR and fragment optimization to identify ligands for an orphan GPCR.

 

Debanu Das described using high-throughput crystallography screening at XPose Therapeutics, now part of Mid-Atlantic BioTherapeutics, to identify inhibitors of DNA damage response pathway proteins. Although the initial hits had no detectable activity, SAR by catalog was able to get to nanomolar inhibitors of the protein APE1, which is overexpressed in ovarian cancer, in a matter of weeks.

 

But perhaps the most robust validation of FBDD was provided by Steve Fesik of Vanderbilt, who described a decades-long effort to drug the MYC pathway, which is probably dysregulated in almost all cancers. We described some of his published work on WDR5 here, but he is now making progress against MYC itself. This has not been easy: MYC is intrinsically disordered, and SAR-by-NMR screens against both the transactivation domain and the DNA-binding domain failed. However, MYC forms a structured leucine-zipper heterodimer with the protein MAX, and screening this complex yielded hits at the DNA binding interface. (Earlier this year we described work from Novartis on a different leucine zipper.) The hits have been optimized to low nanomolar binders that disrupt DNA binding, unlike a slide full of other (alleged) MYC inhibitors, including high-profile commercially available compounds such as MYCi361 and MYCi975.

 

I’ll close – hopefully before you stop reading. If you attended, what talks or posters stood out? And if you wish you had attended, mark your calendar for Oct. 18-21 next year, when DoT returns to Boston in time for peak leaf-peeping season.