Fragments vs TEAD: noncovalent this time

Last week we described a fragment-derived covalent probe that targets the four closely related TEAD transcription factors, which are part of the Hippo signaling pathway implicated in some cancers. A new paper in J. Med. Chem. by Timo Heinrich and collaborators at Merck KGa, iBET, and Cancer Research Horizons brings us another fragment-derived probe, this one noncovalent.

 

The researchers started by screening 1930 fragments, each at 2 mM, against TEAD1 and TEAD3 using SPR. Perhaps not surprisingly given the high concentration used, this led to a whopping 560 hits. These were then tested in dose-response format against TEAD1 with or without the coactivator YAP; 254 compounds showed differential affinity, among them compound 1. This molecule was crystallized bound to TEAD3, which revealed that it binds to the hydrophobic pocket normally occupied by a covalently-bound palmitoyl group required for activity. Despite being a fragment, compound 1 was active in a cell reporter assay, and the researchers state that further optimization was done using cellular assays rather than biophysical or biochemical experiments.

 

Analysis of the crystal structure suggested that enlarging the cyclopentyl moiety could fit more snugly into a hydrophobic pocket, while adding a small propyl moiety could extend into a separate pocket, leading to compound 6, with a 10-fold boost in activity. Replacing the propyl with an additional ring led to sub-micromolar compound 9. Finally, replacing the saturated ring with a substituted phenyl moiety led to MSC-4106, with low nanomolar activity in the cell reporter assay.

 

Thermal stabilization (specifically, nanoDSF) assays showed that MSC-4106 stabilized TEAD1 and TEAD3 but not TEAD2 or TEAD4. Palmitoylation assays confirmed this selectivity profile. The paper also includes a nice table comparing experimental selectivities of seven other non-covalent TEAD inhibitors, which vary from having activity only against TEAD1 to activity against all four homologs.

 

MSC-4106 was clean when tested at 10 µM against a panel of 58 receptors and 1 µM against nearly 400 kinases. It did not inhibit hERG or any of the common CYP450s. Finally, PK studies in mice, rats, and dogs showed that the compound is orally bioavailable with a long half-life. Given these favorable properties it was taken into xenograft studies, where it showed tumor growth inhibition at 5 mg/kg and tumor regression at 100 mg/kg. Analysis of tumor tissue showed downregulation of a TEAD-regulated gene, Cyr61.

 

Can we draw any lessons from comparing covalent MYF-03-176 (discussed last week) with non-covalent MSC-4106? Probably not, given that the former hits all TEAD homologs while the latter is selective for TEAD1 and TEAD3. Both molecules look to be excellent chemical probes for further dissecting Hippo signaling. I look forward to seeing how TEAD inhibitors ultimately fare in the clinic.

Twenty seven hits against four tuberculosis targets

1.2 million deaths. If you did not read the title of this post carefully you may assume this statistic refers to COVID-19. In fact, it is the number of people who died of tuberculosis in 2019. Worse, drug resistant forms of Mycobacterium tuberculosis, the organism that causes TB, are spreading far faster than new treatments are being developed. Initial efforts at addressing this problem are reported (open access) in Comp. Struct. Biotech. J. by Sangeeta Tiwari (University of Texas El Paso), Vitor Mendes (University of Cambridge) and a multinational team of collaborators.

 

M. tuberculosis is capable of making all 20 amino acids. The bug can also scavenge arginine from its host, but only inefficiently: knocking out the biosynthetic pathway abolishes virulence. Thus, targeting this pathway might lead to new drugs.

 

In total eight enzymes are needed to synthesize L-arginine from L-glutamate, and the researchers targeted four of them. The proteins were screened against a library of 960 fragments (each at 5 mM) using differential scanning fluorimetry (DSF). Depending on the specific target some of the hits were validated by SPR or ligand-based NMR before being taken into crystallography, which yielded structures of all the enzymes. In total 13 fragments were found to bind to ArgB, 4 bound to ArgC, 2 bound to ArgD, and 8 bound to ArgF. All the coordinates have been deposited in the protein data bank, though they don’t seem to have been released as of June 28.

 

The paper details the binding interactions for all the hits. Most of them are quite weak, though two hits against ArgB have low micromolar dissociation constants as assessed by ITC. Tantalizingly, these inhibit the growth of M. tuberculosis, and one of them seems to be on-target (adding arginine to the media rescues the inhibition). All the ArgB fragments bind not at the active site but rather at an interface between protein subunits. Unfortunately this site is quite hydrophobic, as are the fragments, suggesting an uphill battle in optimization.

 

A good antibiotic should not hit human proteins, and neither ArgB nor ArgC have human orthologs. ArgF does, but the region where the fragments bind is quite different. ArgD, with only two crystallographically-confirmed hits and 36% identity to the human enzyme, is probably the least attractive.

 

A year before the COVID-19 Moonshot launched we highlighted the Open Source Antibiotics initiative. I don’t think that team was involved with this research, but they would seem to be a natural fit. If you have spare bandwidth and are looking to do some fragment to lead optimization, this paper provides more than two dozen starting points.

Failing honorably and openly on PrP

Many of the posts on Practical Fragments – and indeed much of what appears in the literature – describe successes. This is obvious in the list of fragment-derived clinical compounds, and discoveries of high-affinity tool molecules or even advanceable fragment hits make up a large share of the 750+ posts on this blog. But of course, most of what we do in science fails, and such failures can also be informative. Eric Minikel and 25 collaborators from the Broad Institute and multiple other organizations have just published an illustrative case study on bioRxiv. (Eric also has a detailed and eloquent blog post of his own about the work.)

The researchers describe a five-year effort to find small-molecule binders of the prion protein, PrP, which misfolds and forms aggregates that lead to neurodegeneration. The hope was that binders could be turned into PrP stabilizers or perhaps even degraders. PrP has been studied for decades and there are plenty of literature reports of small molecules that seem to interact with the target, but none of these have been convincingly validated. Moreover, the crystallographic structure of the protein does not reveal attractive binding pockets.

Fragment-based methods have succeeded for other difficult targets, so the researchers performed STD and ¹⁹F NMR screens. Of 6630 pooled fragments, 238 initial hits were retested by STD NMR, leading to 80 hits that were then assessed by two-dimensional (TROSY) NMR. This led to a single hit, a substituted benzimidazole. Unfortunately the binding site could not be determined: chemical shift perturbations were spread across the protein. Differential scanning fluorimetry (DSF) showed the molecule caused a slight decrease in thermal stability. Both of these results suggest some sort of pathological mechanism, but the researchers did multiple experiments to rule out aggregation. A dose-response suggested a dissociation constant well above 1 mM, and none of 54 analogs tested proved any better. Soaking crystals of PrP with 20 of these was also unsuccessful.

Next, the researchers performed a thermal shift assay of just over 30,000 compounds (not necessarily fragments), which yielded both stabilizers and destabilizers. Unfortunately, none of 93 tested by two-dimensional NMR (HSQC) revealed any sign of binding. A DNA-encoded library (DEL) screen of 256,000 macrocycles also didn’t yield any confirmed hits (though more on that below), nor did a computational screen of just under 7 million molecules.

Given this experience, the researchers cautiously conclude that their “results may hint toward relative rarity of PrP binders in chemical space.” They do suggest several alternative approaches, such as screening the more biologically-relevant membrane-bound form of the protein. It may also be worth doing a high-concentration crystallographic screen. Finally, the researchers note that one of the DEL scaffolds “was judged to be a likely covalent binder and was not pursued further.” This decision may be worth revisiting. Indeed, covalent approaches have led to clinical compounds against another formerly undruggable target, KRAS.

According to the blog post, the researchers have pivoted to oligonucleotide therapeutics, where they seem to be making good progress. This makes sense, and I wish them luck. But I hope someone returns to PrP itself with new tools. If they do, the assays established and described here will prove invaluable.

Fragments vs Mycobacterium TrmD – and native ESI-MS revisited

The Mycobacterium genus of bacteria contains more than 190 species, including the pathogens that cause leprosy and tuberculosis. Mycobacterium abscessus (Mab) is not as famous, though it is a growing concern for people living with cystic fibrosis. The enzyme tRNA (m¹G37) methyltransferase (TrmD) is essential for Mycobacterium growth. In a recent open-access paper in J. Med. Chem., Chris Abell, Tom Blundell, Anthony Coyne, and collaborators at University of Cambridge, Royal Papworth Hospital, and the US NIH describe potent inhibitors of this target.

The researchers screened 960 fragments using differential scanning fluorimetry (DSF). The 53 hits were soaked into crystals of TrmD, and 27 yielded electron density – all in the cofactor (S-adenosyl methionine, or SAM) binding site. One weak but particularly ligand-efficient fragment was advanced through fragment growing to low micromolar affinity, but further improvements proved challenging.

A powerful feature of FBLD is that screens often yield multiple starting points, and this proved useful here. Specifically, compound 16 bound in the pocket where the adenine of SAM normally sits, while compound 20 bound nearby in the ribose pocket. Merging these led to compound 23,  and a crystal structure suggested that the indole nitrogen would be a good vector for fragment growing, resulting in compound 24c. Addition of a positively charged moiety to engage a couple glutamic acid residues led to compound 29a, a mid-nanomolar compound as assessed by isothermal titration calorimetry (ITC).

Several of the molecules were characterized by native electrospray ionization mass spectrometry (ESI-MS) – an interesting but somewhat controversial technique in which protein-ligand complexes are gently ionized and detected in the gas phase. For large molecules such as proteins, even the gentlest ionization will generate highly charged species; in this case the dimeric TrmD protein had between 13 and 17 positive charges. Each of these charge states represents a different species, or perhaps several since the positive charges could be on different regions of the protein.

Compound 24c has modest affinity, but encouragingly, native ESI-MS at 100 µM ligand revealed two bound ligands, as expected for a protein dimer. However, for the much more potent compound 29a, native ESI-MS at 100 µM ligand revealed two bound ligands for a high charge state but largely unbound protein for a lower charge state. The researchers speculate this could be due to dissociation of the positively charged ligand in the gas phase.

Overall this paper is a nice exercise in fragment merging and growing, guided by crystallographic data. Some of the molecules elaborated from 24c (including 29a) inhibited growth of Mycobacteria species, though further improvements in affinity will be needed, and no DMPK properties are provided. Still, it's good to see solid work being done on new antibacterial agents.

The disconnect between observed affinity by native ESI-MS and that observed by ITC does make one cautious about the utility of the former technique. We’d welcome comments on your experiences with it.

Fragments vs the PWWP1 domain of NSD3: a chemical probe

Epigenetics is a topic we’ve covered frequently at Practical Fragments. Much attention has been focused on bromodomains, which recognize acetylated lysine residues. However, lysine side chains are also methylated to affect gene expression. The PWWP1 domain of the protein NSD3 (NSD3-PWWP1) recognizes these modified lysines. This protein is amplified in several tumour types, and so makes an intriguing cancer target. At the CHI DDC conference last year Jark Böttcher presented how Boehringer Ingelheim and a large multinational group of collaborators developed a chemical probe for NSD3. The story now appears in Nat. Chem. Biol. (and see here for a fun animated short set to music).

The researchers started by screening a library of 1899 fragments against NSD3-PWWP1. STD NMR (at 0.25 mM of each fragment, in pools of four) as well as differential scanning fluorimetry (at 0.5 mM of each fragment) resulted in 285 and 20 hits, respectively. Two-dimensional NMR was used to confirm hits. Interestingly, only three fragments were identified from both STD-NMR and DSF, and these did not confirm – a cautionary reminder that screening orthogonal methods is not necessarily the best path.

Fortunately, 15 fragments not only confirmed, but also caused the same changes to the 2D-NMR spectra as a histone-derived peptide containing a dimethyl-lysine residue, suggesting that the fragments bind at the recognition site for modified lysines. Those fragments with dissociation constants better than 2 mM were pursued crystallographically, and some of the successes included compound 4. This molecule was used in a virtual SAR-by-catalog screen of internal compounds. Of the 601 fragments experimentally tested, compound 8 was the most potent. Crystallography confirmed that the compound binds in the expected site, and further structure-based design ultimately led to BI-9321.

BI-9321 was put through a battery of tests. Affinity was confirmed in biochemical, SPR, and ITC assays, and crystallography revealed the binding mode to be similar to the initial fragment. BI-9321 was selective for NSD3-PWWP1 when tested against 14 other PWWP domains, and showed no activity against 35 protein methyltransferases, 31 kinases, and 48 bromodomains. Solubility, in vitro metabolic stability, permeability, and plasma protein binding all look good.

Multiple assays also demonstrated selective target engagement in cells at a concentration of around 1 µM. BI-9321 showed downregulation of MYC mRNA levels, though the effect was both modest and transient. Antiproliferative activity was also observed in cells, and the effects were synergistic with a bromodomain inhibitor. Moreover, these effects were only seen in NSD3-dependent cells, suggesting that the activity is on-target and that the compound is not generally cytotoxic.

All of this makes BI-9321 an attractive chemical probe, at least for cell-based assays. More work will need to be done to improve potency and further understand the biology. Laudably, to this end, the researchers have made the molecule publicly available.

Fragments in China

The 2017 International Symposium on Fragment Based Lead Discovery (pdf here) was held in Shanghai, China last week. I was fortunate to be able to attend what I believe was the first significant FBLD meeting in Asia. Antimicrobials were a major theme, particularly against drug-resistant pathogens. The two days were filled with nearly 20 talks, so I’ll just try to capture a few impressions.

Ian Gilbert discussed the fragment-based efforts underway at the University of Dundee, focusing especially on library design. Among initially purchased commercial compounds, only 56% passed quality control, with 26% insufficiently soluble (at least 2 mM in water) and most of the rest either unstable or impure, similar to what has been seen by others. Ian has also enlisted undergraduate students to make “capped” fragments ready for optimization, as well as novel heterocycles.

Biophysics was a major theme of the conference, and Ian made a strong case for biolayer interferometry (BLI), one of the lesser-used fragment finding techniques. A screen can be completed in just a few days with less than a milligram of protein. In particular, BLI may be useful for assessing ligandability: Ian tested 31 targets, 13 known to be ligandable and 5 known to be not ligandable, and found good agreement with previous research. Ligandable targets generally gave primary hit rates >4.5%.

Ismail Moarefi (Crelux, now part of WuXi AppTec) highlighted microscale thermophoresis (MST) and differential scanning fluorimetry (DSF). NMR had identified ten hits against Pim1, but only six had yielded crystal structures, despite considerable effort. Of the four that didn’t, three had no activity by MST, while the fourth was very weak. Ismail also discussed the Prometheus nanoDSF instrument, which is sufficiently sensitive that it can resolve two-stage melting curves for a two-domain protein.

Another lesser used fragment-finding technique, affinity mass spectrometry, was described by Wenqing Shui (ShanghaiTech University). This uses ultrafiltration to separate protein-bound ligands from unbound molecules and mass spectrometry to identify hits; up to 1000 molecules can be screened in a single assay! Wenqing provided several success stories, including fragment hits with very weak (millimolar) affinity. She also demonstrated that the technique works against a membrane preparation of a GPCR.

Among more common biophysical methods, NMR was represented by Ke Ruan (University of Science and Technology of China). The challenge was characterizing a low-solubility ligand which caused extensive line-broadening of the protein due to intermediate exchange rates. This was solved by examining the distance between a fluorinated ligand and a paramagnetic label on the protein and using this to model the binding mode.

But by far the star of the show was crystallography. We’ve previously mentioned the high-throughput capabilities developed at the Diamond Light Source, and part of the impetus for this conference was to bring these technologies to China. Frank von Delft (Diamond and University of Oxford) noted that since the XChem platform launched in late 2015 more than 50,000 crystals have been screened against more than 40 targets, resulting in more than 1000 fragment structures. The group is committed to removing barriers and bottlenecks and today can process 1000 crystals per week through compound soaking, harvesting, data collection, and processing (using specially developed programs such as PanDDA). More than 30 external groups have used the facility, and every target has yielded at least one hit.

Of course, to collect data on 1000 crystals requires you to reproducibly grow lots of well-diffracting crystals that can handle the rigors of soaking, and Diamond has released a handy list of tips and tricks. Getting the right crystals was also the theme of two talks, one by Sheng Ye (Chinese Academy of Sciences) and the other by Carien Dekker (Novartis). Sheng emphasized the importance of optimizing the protein construct, which could include trimming flexible termini or disordered loops, mutating flexible surface residues, or considering different species. He also noted that adding heavy metal ions can actually improve the quality of the crystals as well as making the structures easier to solve. Carien also emphasized the importance of getting the construct right and discussed how seeding (crushing a hard-won crystal and using this to seed new drops) can be very useful. As we’ve noted, screening fragments at extremely high concentrations seems to be the current state of the art, with Novartis moving to 50 mM in the final soak and Diamond going beyond 200 mM! (In contrast to other types of screens at high concentrations, crystallography should not yield false positives, though hits might bind so weakly as to be undetectable by any other method.)

Such a wealth of structures can be daunting, and Anthony Bradley (Diamond) described the construction and use of a “poised library” for follow-up studies. The 768 fragments are (mostly) soluble to 500 mM in DMSO and are designed such that simple chemistry could generate 1.4 million analogs based on reagents currently in stock at Enamine. Potential analogs can be searched using the Fragment Network approach described here, and I was happy to see that Diamond has released their own open-source version (updated link as of 3 Jan 2018).

Jianhua He (Chinese Academy of Sciences) described the facilities at the Shanghai Synchrotron Radiation Facility (SSRF). This is the first third-generation synchrotron in China and has hosted more than 200 research groups since it opened in 2009. Feng Ye, who works at SSRF, gave a talk (in Mandarin) about screening a bacterial protein at XChem; the movies showing liquid handling and robotics would be impressive in any language. Renjie Zhang (Diamond), who also spoke in Mandarin, gave a talk describing (I’m told) not just XChem but how outside users can apply for access. Although there is currently a long waiting list, this should be addressed within the next year or so when SSRF gains Diamond status.

At the 2015 Pacifichem meeting there were only a few speakers from China. Given the level of interest and expertise I saw last week, I predict that the 2020 meeting will see many more.

Assessing ligandability by thermal scanning

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

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

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

Low ligandability: hit rate < 1.5%

Medium ligandability: hit rate between 1.5 and 4.5%

High ligandability: hit rate > 4.5%

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

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

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

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

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

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

Native mass spectrometry revisited

Native electrospray ionization mass spectrometry (ESI-MS) is one of the less-commonly used fragment finding methods. The technique relies on gently ionizing a protein-fragment complex without causing denaturation; bound fragments reveal themselves as shifts in mass. The technique is truly label-free, and can use very small amounts of protein and fragments. In practice the technique can work really well, reasonably well, or quite poorly. Two new papers shed light on factors that influence success.

The first paper, by Kevin Pagel (Freie Universität Berlin), Benno Kuropka (Bayer), and collaborators, examines four different cancer-related proteins. Let me say up-front that that the paper is remiss in not disclosing the chemical structures of any of the fragments, so in a very real sense this work is not reproducible. It is a shame the editors of ChemMedChem were not more demanding. That said, there is some useful information here.

Most of the focus is on the protein MTH1, screened at 10 µM concentration with 100 µM of each fragment. This was not a naïve screen; the fragments were previously identified from a thermal shift assay (TSA): 24 stabilized the protein, 4 destabilized it, and 5 had no effect. Remarkably, all of the fragments showed complexes in ESI-MS ranging between 6 – 66%, even those that had no effect in the TSA! Choosing an (admittedly arbitrary) 20% cutoff weeded out most of the false positives: 16 of the 24 stabilizers passed, while none of the destabilizers or neutral molecules did.

The best hit by ESI-MS also gave the strongest thermal shift, and a titration curve revealed an impressive dissociation constant of 1.7 µM. However, even at high concentrations of fragment the amount of bound complex did not exceed 70%, meaning that interpretation of single-dose experiments (for example, from a primary screen) could be problematic.

The results were similar for the protein KDM5B: 8 of 9 stabilizing fragments were hits by ESI-MS, as were two of 7 destabilizing fragments. Note that fragments that destabilize proteins can still be tight binders, as illustrated here.

For two additional proteins, however, ESI-MS was disappointing. For BRPF1, ESI-MS didn’t find any of the 11 hits from TSA, while for UHRF1 it found only a single hit – though this hit was not one of the 10 stabilizers identified by TSA. One could argue that the TSA hits were false positives were it not for the fact that, in the case of BRPF1, 6 of them were confirmed by crystallography.

The second paper, in Angew. Chem., comes from Chris Abell and coworkers at the University of Cambridge, and focuses on the protein EthR, a potential target for tuberculosis that we’ve previously discussed.

EthR binds to DNA, so rather than look for direct binding of fragments to EthR the researchers instead looked for fragments that could disrupt the EthR-DNA complex. A small library of 73 fragments was tested (at 0.5 mM each, in 2% DMSO), yielding 8 hits. The same library was screened under the same conditions using differential scanning fluorimetry (DSF), yielding 7 hits, 4 of which had also been identified using ESI-MS. All 11 of these molecules were then tested under the same conditions in an SPR assay to see if they could disrupt the interaction between EthR and chip-bound DNA. The 7 best SPR hits were all fragments that had been identified by ESI-MS. Moreover, two fragments – one identified solely by ESI-MS and one identified by both ESI-MS and DSF – were characterized bound to EthR crystallographically, and these represent new chemotypes for this target.

So what are we to make of all this? In common with other techniques, ESI-MS works well for some targets and less well for others. The problem is that it is not clear what distinguishes the two classes of targets. If you have access to the equipment and expertise you might consider adding ESI-MS to your screening cascade. But if you can only afford to buy one instrument for fragment screening, you’d probably be better off investing in NMR or SPR.

Disrupting constitutive protein-protein interfaces

Protein-protein disruptions are notoriously difficult because the interfaces between proteins tend to be large and flat, with few of the deep pockets where small molecules prefer to bind. That's not to say they're impossible: the second approved fragment-derived drug targets a protein-protein interaction. This interaction, as with most others studied (see here, here, and here, for example), is transient: two proteins come together to transmit a biological signal, then dissociate. But many proteins form constitutive dimers or oligomers, and these tend to be even more challenging to disrupt. This is the class of targets discussed in a paper just published in J. Am. Chem. Soc.

Wei-Guang Seetoh and Chris Abell (University of Cambridge) were interested in the protein kinase CK2, a potential anti-cancer target. The enzyme is a tetramer containing two identical catalytic subunits (CK2α) and two identical regulatory units (CK2β). Previous experiments had shown that introducing mutations into CK2β that disrupted dimer formation decreased enzymatic activity and increased protein degradation. Would it be possible to find small molecules that did this?

Chris Abell is a major proponent of the thermal shift assay, in which a protein is heated in the presence of a dye whose fluorescence changes when it binds to denatured protein. The way this assay is normally conducted, small molecules are added, and if they bind to the protein they stabilize it, thus increasing the melting temperature (see here for an interesting counterexample).For oligomeric proteins, one might expect that anything that disrupts the oligomers would destabilize the proteins, thus lowering the thermal stability, and indeed this turned out to be the case in a couple model systems. Thus, the researchers screened dimeric CK2β against 800 fragments, each at the (very high) concentration of 5 mM. No fragments significantly increased the melting temperature, but 60 decreased the stability by at least 1.5 °C.

Best practice for finding fragments includes using multiple orthogonal methods, so all 60 hits were tested (at 2 mM each) in three different ligand-detected NMR assays: STD, waterLOGSY, and CPMG. Impressively, 40 of these showed binding in all three assays. There was no correlation between the binding affinity and the magnitude of thermal denaturation, which is not surprising because the thermal shift incorporates not just the enthalpy change of ligand binding but also the enthalpy change of protein unfolding. Thus, as the researchers note, “the extent of thermal destabilization cannot be used as a measure of its binding affinity.”

Next, all 40 confirmed fragments were tested at 2 mM to see whether they caused CK2β dimer dissociation, as assessed by native state electrospray ionization mass spectrometry (ESI-MS). 18 fragments shifted the equilibrium to monomeric protein, though interestingly no protein-fragment complexes could be observed. These 18 fragments also decreased dimerization in an isothermal titration calorimetry (ITC) assay.

There is still a long way to go: all the fragments are very weak, and preliminary SAR studies were unable to find analogs with significantly improved activity. Indeed, it is unclear where the fragments bind, or whether the binding site(s) are even ligandable. Still, the combined use of biophysical techniques on a particularly gnarly target make this an interesting study on the frontiers of molecular recognition.

Microscale thermophoresis revisited

One of the less commonly used fragment-finding methods is microscale thermophoresis (MST). This measures the movement of proteins in a temperature gradient; ligand binding changes the movement. When we first described MST in 2012, we noted that the technique seemed relatively low throughput. In a paper recently published in J. Biomol. Screen., Alexey Rak and colleagues at Sanofi teamed up with Dennis Breitsprecher and researchers at NanoTemper (which makes MST instruments) to try to increase this.

The researchers chose the kinase MEK1 and carefully developed assay conditions; their detailed description is a useful resource for those who decide to give MST a try. Adding nonionic detergent to the assay proved to be essential for reproducibility and to prevent the protein from sticking to the capillary or aggregating. Also, rather than relying on the weak chromophores (such as tryptophan) in native proteins, MEK1 was labeled with a fluorescent dye. The substrate ATP was used as a positive control, and the measured affinity was in good agreement with previous results.

The screen itself was performed on a set of 193 fragments that had been computationally preselected as potential ligands for the kinase MEK1 (work we blogged about here). These were serially diluted using automated liquid handling and tested in 12-point dose-response curves to try to determine dissociation constants (Kd values) for each fragment. All together this run of more than 2000 capillary tubes required only 90 micrograms of protein and took less than 7 hours. Retrospective analysis suggested that a single-point screen at 150 µM of each fragment would have caught most of the best hits and cut analysis time to 70 minutes, so it looks like MST is becoming competitive with other biophysical screening methods in terms of time and reagent consumption.

What about results? The overall hit rate was nearly 38%, which is high, though not outrageously so given that the fragments were computationally pre-selected. Of these, the best 25 fragments showed well-defined dose-response curves with
Kd < 200 µM and competition with ATP. One nice feature of the method is that pathological behavior such as aggregation or denaturation could be observed directly in the form of irregular or bumpy MST traces, thus allowing false positives to be rapidly weeded out. Similarly, a loss in fluorescence signal was interpreted as the protein unfolding and sticking to the wells or pipette tips.

It is always useful to cross-check hits in orthogonal assays. As we noted previously, these fragments had previously been screened against MEK1 using surface plasmon resonance (SPR) and differential scanning fluorimetery (DSF). Most of the best hits from DSF were rediscovered by MST, though MST found many hits DSF had missed. In contrast, most of the SPR hits did not confirm in MST. The rank order of hits was also similar for MST and DSF but not for MST and SPR.

A picture is worth a thousand words, and some of the best hits were subjected to crystallography. In fact, 7 of the top 15 MST hits had previously been characterized by crystallography, and 7 new crystal structures could be determined out of 11 additional MST hits for which crystallography was attempted.

Overall then it appears that MST is coming into its own. If you’ve tried it, please share your experiences.

Review of 2015 reviews

In the Northern Hemisphere the winter solstice has passed but the days are still short, and 2015 is hurtling into history. As we did in 2014, 2013, and 2012, Practical Fragments will spend this last post of the year highlighting notable events as well as reviews we didn't previously cover.

Two major conferences this year were CHI’s Tenth Annual FBDD meeting in San Diego (discussed here and here) and Pacifichem 2015. If you missed these don’t worry – we’ll have an updated list of 2016 events soon.

After a three year drought, two new books published in 2015: Fragment-based methods in drug discovery and Fragment-based drug discovery. And the trend looks set to continue, with a new book edited by Wolfgang Jahnke and me set to publish in early 2016.

In addition to complete books, several book chapters may be of interest to readers, the first being “Fragment-based drug discovery” by Jean-Paul Renaud and NovAliX colleagues, published in Small molecule medicinal chemistry (Wiley). This is a general review of the topic, focused heavily on biophysical techniques, especially SPR, NMR, and native MS. It also includes a couple case studies – one on the clinical compound AT9283 and one on the bromodomain BRD2.

The next three chapters all come from Springer’s massive Methods in molecular biology series. Continuing the biophysical theme is “Biophysical methods for identifying fragment-based inhibitors of protein-protein interactions,” by Michelle Arkin and colleagues at UCSF. This provides background and step-by-step instructions for SPR, differential scanning fluorimetry (DSF), NMR (including STD, WaterLOGSY, and HSQC/HMQC), and X-ray crystallography. A more detailed guide to STD NMR is provided by Hai-Young Kim and Daniel Wyss (Merck) in “NMR screening in fragment-based drug design: a practical guide,” while Byeonggu Han and Hee-Chul Ahn (Dongguk University-Seoul) discuss STD NMR applied to kinases in “Recombinant Kinase Production and Fragment Screening by NMR Spectroscopy.”

Moving on to journals, two reviews focus on protein-protein interactions. The first, by Thomas Magee (Pfizer) in Bioorg. Med. Chem. Lett., briefly touches on challenges and solutions before focusing on several case studies, including navitoclax, Mcl-1, RPA, KRas, Rad, bromodomains, XIAP, HCV NS3, and more. The second, by Chunquan Sheng (Second Military Medical University, Shanghai), Wei Wang (University of New Mexico and East China University of Science and Technology) and colleagues is published in Chem. Soc. Rev. This is much broader, covering not just fragment-based approaches but others as well, and includes 229 references and 21 figures. There’s a lot of good stuff in this paper, but unfortunately the authors do not discuss the numerous false positives that can occur, such as aggregation and PAINS, and some of their examples are artifacts. Caveat lector.

The next two papers focus on specific therapeutic areas. Xinyong Liu and colleagues at Shandong University discuss the application of fragment approaches to HIV targets in Expert Opin. Drug Discov. In addition to recent examples, this covers some of the older literature, as well as less conventional topics such as dynamic combinatorial chemistry. And in Front. Neurol., Jeffry Madura and Christopher Surratt (Duquesne University) discuss the role fragment-based approaches can play in developing drugs that target the central nervous system (CNS). This review is particularly focused on computational methods.

The next three papers continue the computational theme. Dima Kozakov, Adrian Whitty, Sandor Vajda (Boston University) and co-workers have two reviews discussing work we highlighted earlier this year. The first, in Trends Pharmacol. Sci., is an excellent summary of how computational hot spot analysis can predict whether a protein will be ligandable, and includes a number of case studies. The second, a Perspective in J. Med. Chem., is a much more wide-ranging analysis of the approach. This paper also considers difficult targets, some of which may be tackled with larger molecules such as macrocycles, and others of which may simply not be druggable. And in Chem. Biol. Drug Des., Matthew Bartolowits and V. Jo Davisson (Purdue University), focus on “subpockets,” which are essentially the regions surrounding individual amino acid residues in proteins. This paper also includes an extensive list of software tools for analyzing binding sites.

Finally, Chris Murray and David Rees (Astex) have a brief but lively essay in Angew. Chem. Int. Ed. After providing essentially a target product profile for an ideal fragment, they challenge chemists to devise new routes to superior fragments. Although fragments may seem simple, the “precision synthesis” required to elaborate them “is often rate-limiting.” Diversity-oriented synthesis (DOS) is one potential solution, although there does not seem to have been as much activity here as might have been hoped. Some of the problems are prosaic but significant: as we’ve noted, highly water soluble fragments can be hard to isolate. The authors call for new synthetic methodology compatible with small fragments containing diverse hydrogen-bonding functional groups.

And with that, Practical Fragments says farewell for the year. Thanks for reading (and especially for commenting) and may 2016 bring brilliant breakthroughs!