Fragments vs β-glucocerebrosidase

The protein β-glucocerebrosidase, also called GCase and GBA, is a lysosomal enzyme that cleaves glucosylceramide. People with inactivating mutations in both copies of GCase develop Gaucher’s Disease, which can be treated with a recombinant form of GCase. Heterozygous mutations increase risk for Parkinson’s Disease and for dementia with Lewy bodies, and though the mechanism is unclear, stabilizing the enzyme and/or boosting activity of residual GCase might help. This approach is described in a recent J. Med. Chem. paper by Nick Palmer and colleagues at Astex Pharmaceuticals.

 

The researchers started with a crystallographic screen of 440 fragments, resulting in a whopping 91 hits. In parallel, 1800 fragments (including the aforementioned 440) were screened using ligand-observed NMR, SPR, and thermal shift assays, and hits were confirmed crystallographically to yield another 15 structures. Astex has previously reported that multiple ligand binding sites are common in proteins, and GCase is no exception, with the 106 ligands binding to 13 distinct sites.

 

With this embarrassment of riches, prioritization became critical. Sites formed by crystal packing and shallow solvent-exposed sites were deprioritized, along with those near the active site, since ligands binding there might inhibit the enzyme. SPR was not well-suited to measuring ligand affinities due to non-specific binding, and ligand-observed NMR was similarly complicated due to multiple binding sites. However, isothermal titration calorimetry (ITC) proved to be effective, and this technique was used to narrow in on two binding sites.

 

Site A was particularly attractive: it had 31 fragment hits, one of which has a respectable dissociation constant of 12 µM. Screening of analogs did not lead to anything better, but merging this fragment with another Site A fragment led to compound 15. Interestingly, crystallography revealed that this molecule binds not at Site A but at Site B. Although the affinity is low, the ligand efficiency is respectable. The fragment also makes several polar interactions and has multiple vectors for growing the molecule.

 

 

Testing analogs of compound 15 led to compound 16, and growing led to compound 17, with low micromolar affinity. Further structure-based design ultimately led to compound 22, with low nanomolar affinity. The molecule increased GCase activity in a cellular assay, albeit at a fairly high (mid-micromolar) concentration. The molecule was found to be cell permeable with no efflux, so the source of the disconnect between affinity and cell activity is unclear.

 

This lovely example of structure-guided fragment-based ligand design holds several lessons. First, as noted above, finding fragments is often the easy part; selecting among them and figuring out what to do next can be challenging. Second, especially at the earliest stages of optimization, fragments can change not just their binding mode but their binding site entirely.

 

Finally, figuring out which sites will be best for high-affinity allosteric ligands isn’t necessarily straightforward. Of the 105 fragment hits at 13 sites, only four bound in Site B, yet this site turned out to be more fruitful than Site A, which had many more bound fragments. The researchers note that Site B had previously been identified as ligandable by FTMap, supporting the utility of computational approaches.

 

The researchers conclude, “we hope that our findings will be of use to the wider community.” Certainly from a best practices perspective the paper succeeds. And although the most advanced molecules described do not meet all the criteria for robust chemical probes, and it is unclear whether they will work with mutant proteins, they could still be useful to better understand the complicated biology of GCase.

Chemical couplets inhibit the GAS41 YEATS domain

Practical Fragments has covered bromodomains extensively, most recently just a couple months ago. But these epigenetic readers are not the only proteins that recognize acetylated lysine residues. Two years ago we highlighted fragment hits against one of the four YEATS domain proteins. A paper recently published in Cell Chem. Biol. by Jolanta Grembecka, Tomasz Cierpicki, and colleagues at University of Michigan tackles another member of the family.

 

The researchers were interested in the protein GAS41, which is amplified in multiple forms of cancer. The YEATS domain within this protein binds to acylated lysine residues in histone H3 proteins. However, unlike the deep pockets found in bromodomains, the acyl-lysine binding site in the YEATS domain consists of a partially solvent-exposed channel, making it a more challenging site to drug.

 

Nonetheless, an NMR-based screen of fragments (in pools of 10, each at 500 µM) led to compound 1, which produced multiple chemical shifts in a ¹H-¹⁵N HSQC experiment. Two different competition assays, fluorescence polarization (FP) and AlphaScreen formats, confirmed that compound 1 could compete with H3-derived peptides. Fragment growing led to compounds 7 and 11. (All IC₅₀ values below are from the FP assay; these are somewhat weaker than those from the AlphaScreen, but they more closely track binding affinities determined by isothermal titration calorimetry.)

 

A crystal structure of a compound closely related to compound 11 revealed that the molecule nearly fills the small binding channel, suggesting that further gains in affinity would be difficult. Indeed, no GAS41 inhibitors have been previously reported. However, the protein is dimeric, so the researchers decided to dimerize their molecule to bind to two YEATS domains simultaneously. This led to nanomolar molecules such as compound 19.

 

Not only was compound 19 potent in biochemical assays, it also disrupted binding of GAS41 to acetylated histone proteins in cells. Moreover, the compound inhibited growth of cancer cell lines with amplified GAS41.

 

This is a nice case study in fragment dimerization, an uncommon but interesting approach. The linking in this case led to a 44-fold improvement in affinity, which though impressive is far from synergistic, and is associated with a considerable loss in ligand efficiency. And although the micromolar potency of compound 19 in cells needs to be improved to generate a chemical probe, let alone a drug lead, these results nonetheless support the notion that targeting GAS41 could be a useful strategy for certain cancers.

Deconstructing an HTS hit for GyrB inhibitors

COVID-19 is deservedly engaging most of our collective mindspace when it comes to infectious diseases. Unfortunately, plenty other threats are out there, including antibiotic-resistant bacteria. A paper recently published in ACS Omega by Fumihito Ushiyama and colleagues at Taisho reports progress in this area.

The researchers were specifically interested in the protein DNA Gyrase B (GyrB), which is essential for bacterial replication (see here for previous work on the same target). A high-throughput screen against the E. coli protein led to a few dozen hits that were validated using a variety of biophysical methods including SPR, isothermal titration calorimetry (ITC), and crystallography. Compound 1 binds in the ATP-binding site, which is also where the natural product inhibitor novobiocin binds. The latter molecule makes an interaction with an arginine residue in the protein, but introducing a carboxylic acid moiety onto compound 1 to make a similar interaction was not successful (compound 8e).

Taking a step back, the researchers stripped compound 1 down to the core fragment 2a, which makes both polar and hydrophobic interactions with GyrB. Unfortunately, this fragment was too weak to show any affinity by ITC, as were 120 related fragments.

Looking closer at the structure of compound 1 bound to the protein revealed a small unfilled hydrophobic pocket near the 2-quinolinone fragment. Making appropriately substituted fragments was “relatively complicated,” and most of them were inactive. However, compound 2d showed binding by ITC as well as excellent ligand efficiency. Growing from this fragment ultimately led to compound 13e, with low nanomolar affinity. In addition to binding, compound 13e is a potent inhibitor of GyrB and is selective against a panel of 96 human kinases. Unfortunately though, it displays only modest antibacterial activity, likely due to efflux.

Nonetheless, this is a nice example of thoughtful structure-based design. In particular, the dramatic boost in potency gained by filling a small pocket (nearly 400-fold from compound 8e to 13e) validates the willingness to explore difficult chemistry rather than sticking with available analogs. The paper ends by noting that optimization is continuing, and I wish them well. By my count only a single fragment-derived antibacterial agent has entered clinical development, and that program is no longer active. We could use more.

Is thermodynamic data useful for drug discovery?

Just over a decade ago Ernesto Freire suggested that small molecules whose binding energy is dominated by the enthalpic – rather than the entropic – term make superior drugs. He also suggested that such molecules may be more selective for their target. But the backlash came quickly, and a couple years ago we wrote that focusing on thermodynamics probably isn’t particularly practical. A new perspective in Drug Disc. Today by Gerhard Klebe (Philipps-University Marburg) revisits this topic.

Klebe suggests that enthalpy was initially embraced “because readily accessible and easily recordable parameters are much sought after for the support of the nontrivial decision over which molecules to take to the next level of development.” (I would be interested to know whether sales of isothermal titration calorimetry (ITC) instruments spiked around 2010.) Unfortunately, both theoretical and practical reasons make thermodynamic measurements less useful than hoped.

First, and as we noted previously, “in an ITC experiment… the balance sheet of the entire process is measured.” In particular, water molecules – which make up the bulk of the solution – can affect both enthalpic and entropic terms. Klebe describes an example in which the most flexible of a series of ligands binds with the most favorable entropy to the target protein; this is counterintuitive because the ligand adopts a more ordered state once bound to the protein. It turned out that in solution the ligand traps a water molecule that is released when the ligand binds to the protein, thus accounting for the favorable entropy.

Indeed, water turns out to be a major confounding factor. We’ve previously written about “high-energy” water; Klebe notes that an individual water molecule can easily contribute more than 2 kcal/mol to the overall thermodynamic signature. And of course, proteins in solution are literally bathed in water. The structure of this water network, which may change upon ligand binding, is rarely known experimentally, but optimizing for it can improve affinity of a ligand by as much as 50-fold. Conversely, attaching a polar substituent to a solvent-exposed portion of a molecule to improve solubility sometimes causes a loss in affinity, and Klebe suggests this can be due to disruption of the water sheath.

Beyond these theoretical considerations, experimental problems abound. We’ve previously discussed how spurious results can be obtained when testing mixtures of ligands in an ITC experiment, but even with single protein-ligand complexes things can get complicated. Klebe shows examples where the relative enthalpic and entropic components to free energy change dramatically simply because of changes in buffer or temperature. This means that the growing body of published thermodynamic data needs to be treated cautiously.

So what is to be done? First, thermodynamic data should always be treated relatively: “we should avoid classifying ligands as enthalpy- or entropy-driven binders; in fact, we can only differentiate them as enthalpically or entropically more favored binders relative to one another.”

Klebe argues that collecting data on a variety of ligands for a given target under carefully controlled conditions will be useful for developing computational binding models. This is important, but not the kind of work for which people usually win grants, let alone venture funding.

He also suggests that, by collecting thermodynamic data across a series of ligands, unexpected changes in thermodynamic profiles might reveal “changes in binding modes, protonation states, or water-mediated interactions.” Maybe. But it takes serious effort to collect high-quality ITC data. Are there examples where you’ve found it to be worthwhile?

Taking a step towards STEP activators

Most drugs – and small molecule modulators in general – inhibit something, often an enzyme. Enzyme activation, on the other hand, is rare; we’ve highlighted just a few cases on Practical Fragments over the past decade. A new example is described in J. Med. Chem. by Christofer Tautermann and collaborators at Boehringer Ingelheim and the Beckman Research Institute of the City of Hope.

The researchers were interested in the protein tyrosine phosphatase non-receptor type 5 (PTPN5), also known as striatal-enriched protein tyrosine phosphatase (STEP). As its name suggests, this enzyme is found in the brain, and has been implicated in multiple neuropsychiatric disorders. However, phosphatases are tough targets due to their small, polar active sites. The problem is exacerbated for CNS targets, because negatively charged molecules have a hard time crossing the blood-brain barrier. Thus, the researchers sought allosteric modulators.

They began with a screen of 3083 fragments using STD NMR, differential scanning fluorimetry, and microscale thermophoresis. Validation of the several hundred hits by 2-dimensional NMR confirmed just seven, and comparison of the protein chemical shifts with those caused by a non-specific active site binder (sodium vanadate) suggested that compound 2 bound outside the active site. Crystallography confirmed this, revealing that the compound binds on the “back side” of the protein, about 20 Å from the catalytic pocket. The affinity was extraordinarily weak, with no functional activity, so the researchers used NMR to drive the SAR. Ultimately this led to fragment-sized BI-0314, with measurable affinity by isothermal titration calorimetry (ITC). Crystallography revealed that it binds in the same pocket as compound 2.

Surprisingly, far from being an inhibitor, BI-0314 actually showed activation of the enzyme in functional assays, increasing the activity by up to 60% at 0.5 mM. Careful mechanistic analysis revealed that this was due to an increase of k_cat, while the K_M for substrate was mostly unchanged. Molecular dynamics simulations suggested that BI-0314 increases the rigidity of the enzyme, and also stabilizes the active conformation. As expected of an allosteric modulator, the molecule was selective for STEP, with no activity (activating or inhibitory) for a couple other phosphatases.

As it turns out, the researchers were actually interested in STEP inhibitors, so they didn’t pursue BI-0314 further. As they note, there is still much to be done to generate a useful chemical probe, in particular improving potency. Laudably, the researchers are making BI-0314 available to other researchers free of charge. Perhaps someone else will be able to take this forward, as we’ve seen for other published fragments. And indeed, as researchers at Novartis have shown with asciminib, the transition from an allosteric binder with no functional activity to an inhibitor is possible – perhaps the same will hold true for an activator. If you are interested in STEP, you now have a new site to explore, and even a well-characterized starting point.

Readers beyond bromodomains: Fragments vs YEATS

Epigenetic readers recognize modified amino acids in histone proteins to cause changes in gene expression. Readers containing bromodomains, which recognize acetylated lysine residues, have received particular attention, and fragment-based approaches have led to at least a couple bromodomain inhibitors entering clinical development. But the numerous bromodomains are not the only epigenetic readers to recognize acetylated lysine residues. In a recent paper in J. Med. Chem., Apirat Chaikuad, Stefan Knapp, and collaborators at Goethe-University Frankfurt and University of Oxford describe their efforts targeting a different family.

YEATS domains are present in four human proteins, three of which have been linked to cancer. Unlike bromodomains, YEATS domains recognize lysine residues modified with acyl derivatives beyond acetyl, such as propionyl, butyryl, and crotonyl. The biological significance of these modifications is not clear, and no inhibitors of these proteins had been reported when the work began.

The researchers focused on the oncogenic eleven-nineteen-leukemia protein (ENL). They solved the first apo crystal structure of ENL (ie, without a bound ligand), which revealed that although the binding pocket was pre-formed, there was some flexibility in the side chain residues. They also noted distinct differences in how the acylated lysine is recognized, including the absence of an asparagine residue that is conserved in all bromodomains, and a more-open pocket that can accommodate larger acyl chains.

Next, the researchers chose a set of nineteen fragments containing a central amide bond to mimic acetylated lysine. None of these showed activity in a thermal shift assay, but when the ligands were soaked (at 5-40 mM) into crystals of ENL, electron density consistent with binding was observed for ten of them, and two could be modeled with some confidence. (For the other nine compounds, the crystals no longer diffracted.) These two fragments also showed binding by isothermal titration calorimetry (ITC). This is a useful reminder of the need for orthogonal assays, and the power of crystallography to detect weak hits. Compound 19, a rather super-sized fragment, was similar to compounds identified in a high-throughput screen that the researchers reported here and here.

Using this information, the researchers made a handful of analogs and found that compound 20 had high nanomolar affinity as assessed by ITC. Like last week’s story, this effort could probably be considered more fragment-assisted than fragment-based. But whatever the precise genealogy, hopefully molecular descendants of compound 20 will help to elucidate the biological poetry of the YEATS domains.

Review of 2016 reviews

This year is finally coming to an end, and as we've done for the past four years, Practical Fragments will highlight some of the reviews that we didn't cover previously.

In terms of what we did cover, there were several excellent events, including the eleventh annual CHI FBDD Conference in San Diego, an inaugural meeting in Houston, and of course the first-ever major fragment event in Boston, FBLD 2016.

The twentieth anniversary of SAR by NMR was also commemorated by the eighth book devoted to FBLD, as well as a massive two volume work on lead generation. We also covered a special issue of Molecules and reviews on clinical candidates and library design.

Another review on library design was published recently in Drug Disc. Today by Ian Gilbert, Paul Wyatt, and colleagues at the University of Dundee. The researchers have built a set of 356 diverse compounds consisting of “capped” scaffolds, such that any hits could be rapidly expanded. Undergraduates did much of the actual library assembly, learning skills such as parallel chemistry and how to work with polar compounds. There is lots of nice detail in this paper, including on library storage conditions.

Targets

Practical Fragments often highlights successful fragment to lead programs, and these were the focus of a Perspective in J. Med. Chem. by Christopher Johnson (Astex) and collaborators: all 27 cases published in 2015 in which the affinity of a fragment was improved at least 100-fold to a 2 µM or better lead. Many of these were covered in Practical Fragments, including BTK, DDR1/2, ERK2, MELK, Mtb TMK, PKCθ, RET, FactorXIa, MMP-13, BCATm, PDE10A, soluble epoxide hydrolase, tankyrase, ATAD2, MCL-1, RAD51, XIAP/cIAP, and mGluR5. The paper also draws general conclusions about target types, molecular weights, cLogP values, and LE.

Targeting tuberculosis (TB) is the subject of two reviews from University of Cambridge researchers, one in Drug Discov. Today by Vitor Mendes and Tom Blundell and one in Parasitology by Anthony Coyne, Chris Abell, and colleagues. Fragment-based approaches have been more or less successful against several TB proteins, including pantothenate synthetase, CYP121, BioA, EthR, and thymidylate kinase, while other targets – such as shikimate kinase and CYP144 – have proven more difficult.

July was bromodomain month at Practical Fragments, and this target class is the subject of a review in Drug Discov. Today: Technol. by Dimitrios Spiliotopoulos and Amedeo Caflisch at the University of Zurich. The focus is on computational fragment screening methods, with examples for BRD4 and CREBBP. And while we’re on the topic of computational methods, Olgun Guvench of SilcsBio has a brief review in Drug Discov. Today on computational functional group mapping.

Rounding out target-focused reviews, Paramjit Arora and colleagues at New York University focus on protein-protein interactions (PPIs) in a Trends Pharm. Sci. paper. This covers multiple approaches to finding PPI inhibitors, including fragment-based, and also touches on hotspots and structure-based design.

Biophysics

It is impossible to imagine FBLD without biophysics, and this is the topic of an authoritative review in Nat. Rev. Drug Disc. by Jean-Paul Renaud (NovAliX), Chun-wa Chung (GlaxoSmithKline), U. Helena Danielson (Uppsala University), Ursula Egner (Bayer), Michael Hennig (leadXpro), Rod Hubbard (University of York) and Herbert Nar (Boehringer Ingelheim). In addition to covering all the major techniques, the paper does a great job of delving into some of the more obscure and emerging methods, providing an excellent discussion of the throughput and requirements for each technique as well as the kinds of information obtained. Although the review is broader than FBLD, the application of biophysical techniques to fragments is a major theme. The researchers also remind us that, “contrary to the belief that all drug discovery challenges are best solved through the introduction of new technologies, substantial advances can also be driven by innovative application.”

Individual biophysical techniques also received plenty of attention over the year, including three on NMR. The first, by Alvar Gossert and Wolfgang Jahnke (Novartis) in Prog. Nucl. Magn. Reson. Spectrosc., is a 44-page practical guide to identifying and validating protein ligands. This contains a wealth of information on most of the NMR methods you will ever likely encounter; it includes a handy chart summarizing the molecular weight and concentration limits for each technique, suggested workflows, and thorough discussions of potential pitfalls. The review may appear daunting to the novitiate – it is replete with equations and pulse sequences – but the writing is clear. In the end, much comes down to the concept of the “validation cross”, a rubric for assessing the integrity of both ligand and protein, and evaluating binding effects on both ligand and protein.

Two additional reviews, both from William Pomerantz and colleauges at the University of Minnesota, focus specifically on protein-observed ¹⁹F NMR. The first, a Perspective in J. Med. Chem., is a good general introduction. Despite being the 13^th most abundant element on our planet, only five natural products are confirmed to contain fluorine. Introducing this element into proteins – as has been done in more than 70 cases – can be a useful approach for discovering new ligands. And if you want to start doing this yourself, a paper in Nature Protocols provides practical details.

Turning to other biophysical techniques, surface plasmon resonance (SPR) continues to be very popular, and is reviewed by Alain Chavanieu and Partine Pugnière in Expert Opin. Drug Discov. The paper provides a good general overview on using SPR for FBLD, covering the theory, history, various screening strategies, comparison to other methods, recent applications to a variety of different targets, and a suggested workflow.

Calorimetry is less commonly used for fragment screening, even though it can provide thermodynamic data. Michael Recht and collaborators at the Palo Alto Research Center and Zenobia discuss both enthalpy arrays as well as more conventional isothermal titration calorimetry (ITC) in a Methods Enzymol. chapter.

Chemistry

But while biophysics is important, FBLD would be nowhere without chemistry. In MedChemComm, Stefan Kathman and Alexander Statsyuk (then Northwestern, now University of Houston) review one chemical approach, covalent tethering. This touches on the original reversible (thermodynamically-controlled) disulfide tethering approach developed back at Sunesis but is primarily focused on irreversible (kinetically-controlled) methods. The paper does an excellent job summarizing challenges, potential pitfalls, design rules, and recent successes. As of early this year the Statsyuk lab had sent their 100-member covalent fragment library to nine different research groups, three of which had already identified hits. The review ends with some provocative questions, and it will be fun for practitioners to answer them as covalent approaches garner increasing attention.

Another chemical technique we’ve touched on is substrate activity screening (SAS), and this is reviewed in ChemMedChem by Pieter Van der Veken and collaborators at the University of Antwerp. All published examples are summarized, including the modified approach developed by the Van der Veken lab; some unpublished data are also discussed. The paper also includes a good general section on the subtleties and complexities of transforming substrates into inhibitors.

Finally, if all this is a bit too much, a good general review on FBLD was published in Pharmacol. Ther. by Martin Scanlon and colleagues at Monash University. This concise but thorough paper covers theory, history, library design, hit finding and characterization, and select clinical success stories. The longest section is devoted to chemical strategies for elaborating fragments, and includes some of the less commonly used methods such as target-guided synthesis, Tethering, and off-rate screening.

And that’s it for this year. Thanks for reading, and especially for commenting. Take care, do important work, and may 2017 be better than we can reasonably hope.

CYP121 revisited: fragmentation approaches

Three years ago we highlighted work out of Chris Abell’s lab at the University of Cambridge targeting CYP121, an important enzyme for the pathogen Mycobacterium tuberculosis (Mtb). Two new papers from his group discuss progress on this target using conceptually similar approaches.

A previous fragment screen had identified some very weak fragments, and merging had led to low-micromolar compound 2 – the starting point for a (free access) J. Med. Chem. paper by researchers at Cambridge, the University of Manchester, the Francis Crick Institute, and São Paulo State University. The researchers used a “retrofragmentation” or deconstruction approach: systematically dissecting the molecule into component fragments (such as compounds 4 and 5) to see which bits were most important. Group efficiency analyses revealed that the two lower aromatic rings were important, while the upper one was much less so.

Crystallography revealed that compound 2 did not make direct interactions with the active-site heme molecule in CYP121, so the researchers sought to create some by growing out from compound 4. This led to a nice increase in affinity (compound 19a). Incorporating the other ring led to compound 25a, with sub-micromolar affinity as measured by isothermal titration calorimetry (ITC). Of course, heme is common to every CYP – including those found in humans – raising the question of selectivity. Happily, compound 25a turned out to be reasonably selective for CYP121 compared with a panel of Mtb and human enzymes.

There’s lots more in this (30 page!) paper, including extensive SAR supported by crystallography, ITC, native mass spectrometry, and an interesting spectroscopic binding assay. But unfortunately, the compounds are not active in a cellular assay, and the researchers are trying to figure out why.

The second (open access) paper also takes a deconstruction approach, this time starting from the substrate cYW. Fragmentation of this and related cyclic dipeptide substrates into amino acid derivatives and analogs led to the testing of 65 commercial compounds in a thermal shift assay, resulting in seven hits that increased the denaturation temperature by more than 1 °C. Compound 1a was the most stabilizing, and a spectroscopic assay suggested interaction with the heme group.

The spectroscopic assay also revealed a high micromolar affinity for the fragment. Attempts to improve this ultimately led to compound 31, with comparable affinity as cYW but with improved ligand efficiency. The thioester could be replaced with only a modest loss in potency, and interestingly the stereochemistry of these molecules did not seem to make a difference. Compound 31 was also reasonably selective for CYP121 in a panel of other CYPs.

Both papers cover lots of ground. Reading some publications you can be lulled into thinking that FBLD is an easy progression of increasingly potent compounds. These examples are useful reminders that many compounds turn out to be dead ends, and that even potent and selective molecules may not have the desired biological effects. Sometimes doing everything right can still leave you short of the goal – at least for a while.

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.

Fragments deliver a chemical probe for Family VIII bromodomains

Today’s post continues the theme of July as bromodomain month at Practical Fragments. The 61 human bromodomains (found in 46 proteins – some proteins have more than one) have been divided into eight families based on their sequences. Family VIII contains ten members, some of which are involved in keeping stem cells from differentiating. Two papers describe chemical probes that target some or most members of this family.

The first paper, which actually came out last year in Science Advances, is from a multinational group including Thomas Günther (Universität Freiburg), Stefan Knapp and Susanne Müller (both University of Oxford) and collaborators at Pfizer. The researchers started by screening libraries of acetyl lysine mimetics that had yielded inhibitors against other bromodomains. These came up empty; even promiscuous bromodomain inhibitors failed to hit Family VIII members. As is so often the case, when all else fails, the researchers turned to fragments. A thermal shift assay revealed that salicylic acid – the polypharmacological metabolite of aspirin – binds to the bromodomain PB1(5). Isothermal titration calorimetry (ITC) confirmed this result, providing a dissociation constant of 250 µM.

The researchers were also able to obtain a crystal structure of PB1(5) bound to salicylic acid in the acetyl lysine binding site common to all bromodomains, with the carbonyl making the usual hydrogen bond with a conserved asparagine. But whereas most other bromodomain binders make a water-mediated bridge to a conserved tyrosine, the phenol makes a direct hydrogen bond. The benzene ring also binds deeper in the pocket, displacing four highly conserved water molecules.

The subsequent medicinal chemistry optimization of this fragment is described in a paper published earlier this year in J. Med. Chem. by Dafydd Owen and colleagues at Pfizer, along with collaborators at the University of Oxford, DiscoveRx, Eurofins, the University of Massachusetts Worcester, and Johann Wolfgang Goethe University. Testing commercial and proprietary analogs of salicylic acid quickly revealed that uncharged enamides such as compound 2 were more effective at stabilizing PB1(5) against thermal denaturation than salicylic acid, and crystallography confirmed a similar binding mode.

Two rounds of library synthesis were conducted, first with 130 amines and then with 320 amines, with physicochemical properties of target compounds chosen in advance such that cLogP would range between 1 and 4. Seven family VIII bromodomains were screened in parallel, and compounds were identified with differing specificities. Some of the compounds were unstable in water, but introducing steric hindrance around the amine improved stability and led to compounds such as PFI-3. This is potent against the family VIII bromodomains PB1(5), SMARCA2A, and SMARCA4 and did not hit at least 40 other bromodomains tested. A related compound is active against more of the family VIII bromodomains while still maintaining good selectivity against other bromodomains.

Both of these probes are able to bind to family VIII bromodomains in cells and were used to explore the proteins’ biological roles. A variety of cellular phenotypic assays showed minimal changes, and the compounds do not appear to be toxic. They did attenuate myocyte or adipocyte differentiation, while PFI-3 caused embryonic stem cells to differentiate. One gets the impression that the researchers were hoping for more profound effects, but that’s why you make chemical probes in the first place. Whether or not these compounds will ultimately prove useful as drug leads, they should help to unravel some fiendishly complex biology.

Fragments deliver a chemical probe for CBP and EP300

As we mentioned last week, July is bromodomain month at Practical Fragments. Today we’ll start by looking at two closely related bromodomains, one found in cyclic-AMP response element binding protein (CBP) and another from adenoviral E1A binding protein of 300 kDa (EP300). Both proteins have been implicated in a variety of diseases, particularly cancer, so a chemical probe would be very valuable.

Alexander Taylor and collaborators at Constellation Pharmaceuticals, Genentech, and WuXi, describe such a probe in a recent paper in ACS Med. Chem. Lett. The researchers screened about 2000 fragments in a thermal shift assay using 0.8 mM of each fragment. Compounds that increased the melting temperature of the CBP bromodomain by at least 1° C were validated first by time-resolved fluorescence resonance energy transfer and then by ¹⁵N HSQC NMR, ITC, and X-ray crystallography. Compound 1 was one of the more attractive hits, in particular because it was considerably less active against BRD4, whose inhibition causes all sorts of changes to cells.

Crystallography of the racemic compound clearly showed that only one of the enantiomers bound, and this was confirmed in functional assays when both enantiomers were tested separately. The active enantiomer makes some of the same interactions typical of all bromodomains with the natural ligand (N-acetylated lysine). Fragment growing was attempted off the aromatic ring, and although several vectors were tolerated, most decreased selectivity against BRD4. However, close examination of the structures revealed a promising vector that led to compound 14, with good selectivity against BRD4. Further optimization ultimately led to CPI-637, with low nanomolar activity against both CBP and EP300 as well as good cell-based activity. Crystallography revealed that this compound binds in a similar manner as the initial fragment.

The selectivity of CPI-637 against other bromodomains is also good (> 700-fold less active against BRD4), though it does hit BRD9 with sub-micromolar activity. Just as with the initial fragment, the opposite enantiomer of CPI-637 is considerably less active. Although no pharmacokinetic data are provided, at the very least this should be a useful probe for cell-based studies.

Switching gears to another aspect of CBP, the multidomain protein p300/CBP-associated factor (PCAF) has a bromodomain that may bind to CBP, though the biology is not entirely clear. PCAF is known to bind an acetylated HIV protein, and has been proposed as a target for AIDS. Obviously this is another opportunity for a chemical probe! The first steps are reported in a paper by Stefan Knapp and collaborators at Goethe University Frankfurt, University of Oxford, Leiden University, ZoBio, and University of Cambridge, published in J. Med. Chem (and open-access).

The researchers screened two separate fragment libraries using either thermal shift assays (at 1 mM fragment) or TINS. Hits were confirmed using SPR and crystallography, resulting in seven structures. As expected, all the fragments bound at the site where N-acetylated lysine normally binds. The PCAF bromodomain appears to be quite rigid, with little movement in structures with the different bound fragments. A few elaborated molecules were tested, with the best showing low micromolar affinity as assessed by ITC; crystal structures with these molecules are also reported and deposited in the protein data bank. It will be fun to see whether their potency can be improved.

We’ll have another post on bromodomains next week, but first stay tuned later this week for an updated list of fragment-derived drugs that have entered the clinic.

Biophysics: not just for fragments

Biophysics and fragment-based drug discovery go together like Nutella and strawberries. Indeed, SAR by NMR ushered in the dawn of fragment-based methods two decades ago, and most fragment-based programs today make use of NMR, SPR, and/or ITC – not to mention X-ray crystallography. Interestingly, the same is not necessarily true for high-throughput screening (HTS) programs. In a recent paper in Drug Discovery Today, Rutger Folmer makes a strong case for engaging biophysics early and often in HTS. He bolsters his argument with more than 20 examples from internal programs at AstraZeneca.

The first descriptions of using NMR to profile HTS hits were not published until several years after SAR by NMR, but they were rather shocking, with up to 98% of hits failing to confirm. Nor is this merely a historical problem, as discussed here. Aggregators, redox cyclers, generically reactive covalent modifiers – all of these are problems not just in fragment screening but in HTS as well. Sometimes the most potent hits are artifacts, particularly for more difficult targets. The key to triaging out pathological actors is to assess binding and not rely solely on inhibition.

That means bringing biophysics into hit profiling at the earliest stages, before trying to optimize fruitless hits. As Rutger points out, it is often difficult to rally colleagues to look at less active molecules after they have wasted months pursuing more potent dead ends.

And biophysics can make an impact even before running screens. Profiling published tool compounds or in-licensing opportunities with biophysical techniques can reveal unwelcome surprises. Testing the output of early HTS pre-screens (7000-10,000 compounds) before a full HTS (2 million compounds at AstraZeneca) can reveal whether an assay is particularly susceptible to false positives. In some cases this can result in reconfiguring the assay, for example by choosing a different detection technology or modifying the protein construct.

A key element to gaining such benefits is organizational commitment. At AstraZeneca, a biophysicist is assigned to a project team immediately after target selection – well before any screens are run. This seems like prudent practice. How many other organizations are doing this?

EthR revisited again: fragment merging this time

Fragment linking, growing, and merging: these are the main methods for enhancing affinity. Two years ago we highlighted a fragment screening effort against the tuberculosis target EthR, which involved fragment linking. A few months later we discussed fragment growing against the same target by a different group. Now the first group, led by Chris Abell at the University of Cambridge, has published a new paper in Org. Biomol. Chem. describing fragment merging.

In the original paper, a thermal shift assay had led to the discovery of a few dozen fragments, several of which were characterized crystallographically bound to EthR. In some cases, two molecules of the same fragment could bind in the large lipophilic cavity of the protein and block binding to DNA, as assessed by SPR. Capitalizing on this, two copies of compound 1 were linked together to generate a micromolar binder.

In the new paper, the researchers tried merging compound 1 with another fragment, compound 2, which also binds at two positions within the protein. Several merging strategies were attempted, and although they all stabilized the protein against thermal denaturation and could be characterized crystallographically bound to the protein, most were no better at blocking DNA binding than the original fragments. Compound 5, however, did show enhanced activity, and was the subject of additional SAR. This led to compound 15, which showed low micromolar binding by isothermal titration calorimetry (ITC) and functional activity. (Oddly, compound 1 appeared to bind considerably more tightly by ITC than suggested by its functional activity, perhaps a result of having two binding sites.) The crystal structure of the optimized, merged compound bound to EthR revealed that compound 15 binds as expected (gray), overlaying with one copy each of compound 2 (magenta) and compound 1 (cyan). 

Unfortunately, aside from compound 1, none of the molecules showed activity in a cell-based assay. The researchers propose that this is due to poor permeability across the notoriously impenetrable envelope of the mycobacterial envelope. All in all this is a nice story, as well as a sobering reminder that while potency is important, it is just one of many properties that need to be optimized.

As to the question of whether one should apply growing. linking, or merging, a single case study does not really permit generalization. However, it is satisfying that all three techniques can lead to early leads.

The Value of DSF

Science is based upon incremental advances of previous work.  A year ago, Dan blogged about worked on BioA.  The key take home from that work was that a hydrazine fragment ended up destabilizing the target by 18C.  It ended up being, as expected, a reversible, SAM-competitive inhibitor with modest potency.  As Dan concluded:

This is a very nice paper, and it will be fascinating to try to understand how the fragments so effectively destabilize the protein despite binding tightly, and how this translates into inhibition. The researchers suggest that finding ligands that destabilize proteins could be generally useful for turning off proteins.

In this paper, the same group is back (This work was also presented at DDC in San Diego in April). Interestingly, they seemed to have abandoned the hydrazine.  Taking the same approach (DSF-Xray-ITC) they identify different fragments (2% hit rate from a 1000 screened).  9 were stabilizers (average of +3.8C) and 12 were destabilizers (average of -13.8C(!)).  5 fragments were able to be crystallized by soaking, co-crystallization was able to add one more structure (Figure 1).  Interestingly, the calorimetry showed that only F5's binding is strongly, enthalpically driven.

Figure 1.  Crystallographically Confirmed Fragment Hits

The authors make several interesting observations:

• Little correlation between magnitude of Tm shift and confirmation by crystallization
• Stabilizing and destabilizing compounds were confirmed by Xray
• No correlation between magnitude of the Tm shift and calorimetry determined Kd.
• Conformational flexibility in the target active site need to be taken into account.

This is not surprising to me; I have seen/heard this many times.  What does this mean for DSF in general? 

Fragments vs 53BP1

As we’ve noted (repeatedly), epigenetics is big. However, much of the focus has been on bromodomains, which recognize acetylated lysine residues. In a paper published earlier this year in ACS Chem. Biol., Lindsey James, Stephen Frye and collaborators at the University of North Carolina, the University of Texas, the Mayo Clinic, and the University of Toronto describe their efforts on a protein that recognizes methylated lysine residues (a Kme reader).

The protein 53BP1 is involved in DNA repair and could have anticancer potential. It recognizes a dimethylated lysine sidechain within a histone protein, so the researchers screened a set of molecules containing amines to mimic this moiety. They used an AlphaScreen assay, with each compound at 100 µM. This does not appear to have been a library of fragments (and unfortunately the number of compounds screened was not stated), but the most notable hit was the fragment-like UNC2170.

Although the affinity was modest, it was quite selective for 53BP1, showing no activity up to 500 µM against 9 other Kme readers. Since AlphaScreen assays can be prone to false positives (the original PAINS compounds were identified in this assay), the researchers tested their compound using ITC, which gave a dissociation constant of 22 µM, in good agreement with the AlphaScreen assay, though with unusual stoichiometry (more on that later).

Thus encouraged, the researchers set off to optimize their hit. Initially they tried modifications around the amine, but even changes as subtle as adding or removing a methyl group killed activity. Attempts to rigidify the propyl linker were also unsuccessful, and shortening it or lengthening it failed too. Replacing the amide with a sulfonamide or amine abolished activity. Most substitutions around the phenyl ring also gave dead compounds, though the bromine atom could be replaced with similarly hydrophobic moieties such as iodine, isopropyl, or trifluoromethyl. Many other analogs were made too, all to no avail. Though the text is measured, the frustration is palpable.

Ultimately the researchers were able to solve the crystal structure of the compound bound to 53BP1, which produced a surprise: one molecule of UNC2170 binds to two molecules of protein, making interactions with each. This explains the stoichiometry seen in the ITC data. It also explains the intolerance to substitutions, as “the ligand is encircled by both proteins,” with no room for modifications.

Happily, UNC2170 is highly cell permeable and non-toxic, and does show some modest activity in cell-based assays. Hopefully the researchers will ultimately find more potent compounds, though this may require a different approach. Indeed, another Kme reader also proved to be quite challenging, but was amenable to fragments. It would be fun to see whether an explicit fragment screen produces more tractable starting points against 53BP1.

Rad fragments revisited

Two years ago we highlighted a paper in which Cambridge University researchers identified fragments that bind to the protein RAD51, which in turn binds to the protein BRCA2 to protect tumor cells from radiation and chemotherapeutics. In a new paper in ChemMedChem, Marko Hyvönen and colleagues describe how they have grown these fragments into low micromolar binders.

One of the best fragments identified in the previous work was L-tryptophan methyl ester (compound 1), so the researchers naturally tried substituting the methyl group. A phenethyl ester (compound 5c) gave a satisfying 10-fold boost in potency, but this turned out to be the best they could get: shorter or longer linkers were both less active, and modifications around the phenyl ring gave marginal improvements at best. Also, changing the ester to an amide decreased affinity. They were, however, able to improve potency another order of magnitude by acylating the nitrogen (compound 6a).

At the same time, the researchers made a more radical change to the initial fragment by keeping the indole and replacing the rest with a sulfonamide (compound 7a). This also boosted potency. Further optimization of the sulfonamide substituent improved the affinity to low micromolar (compound 7m) and increased ligand efficiency as well.

The original fragments had been characterized crystallographically bound to the protein, but the researchers were unable to obtain structures of the more potent molecules, though they did sometimes see tantalizing hints of electron density. Competition studies with known peptide inhibitors also suggested that the molecules do bind in the same site as the initial fragments.

The thermodynamics of binding were characterized using isothermal titration calorimetry (ITC). Although the initial fragments owed their affinity largely to enthalpic interactions, the more potent molecules were more entropically driven. This, the researchers suggest, could partially account for the failure of crystallography despite extensive efforts: the lipophlic molecules can bind in a variety of conformations.

Some have argued that enthalpic binders should be prioritized, but this study illustrates one of several problems: even if you start with an enthalpic binder, there’s no guarantee it will stay that way during optimization.

This is a nice paper, but I do wonder how much affinity there is to be had at this site on RAD51. Given the micromolar affinity of the natural peptides, nanomolar small-molecule inhibitors may not be possible. Then again, like other difficult PPIs such as MCL-1, perhaps the right molecule just hasn’t been made. How long – and how hard – should you try?

Fragments as enzyme activators

About half of all approved drugs are small molecules that inhibit enzymes. This makes sense intuitively: an enzyme is like a complicated little machine, and there are lots of ways you can wreck a machine. But there are times when you might want to activate an enzyme, and this is conceptually more difficult. In a new paper in Angew. Chem. Int. Ed., Rod Hubbard and colleagues at the University of York show how fragments can help.

The researchers were interested in the enzyme O-GlcNAc hydrolase (OGA), which removes N-acetylglucosamine from proteins. They were looking for inhibitors of a bacterial version of this enzyme, so they screened it against 100 fragments using three ligand-observed NMR methods (STD NMR, PO-WaterLOGSY, and T_1ρ-filter). This resulted in a very high hit rate: 22 fragments showed binding in all three assays, and 18 of these were competitive with a known substrate-like inhibitor, PUGNAc. Some of these were also active in an enzymatic assay.

More interestingly, the four hits that were not competitive with PUGNAc actually appeared to bind more strongly to the protein in the presence of that inhibitor. In this case, the inhibitor can be considered a stand-in for the natural substrate, and the results suggested that the fragments might enhance binding of the enzyme to its substrate. Indeed, when these fragments were tested in the enzymatic assay, one of them (compound 2) actually activated the enzyme, with an “AC₅₀” value of 3.5 mM.

The activator was further characterized by several orthogonal methods. NMR revealed that, in the presence of PUGNAc, compound 2 bound with a dissociation constant of 3.1 mM, very close to its AC₅₀ value. In the absence of PUGNAc, the dissociation constant was too weak to be determined. Isothermal titration calorimetry experiments revealed that compound 2 increased the affinity of the enzyme for PUGNAc by more than three-fold, while Michaelis-Menten kinetics revealed that, at a concentration of 8 mM, compound 2 nearly doubled the k_cat/K_M. A crystal structure of both compound 2 and PUGNAc bound to the enzyme revealed that the two molecules bind near one another in what appears to be a catalytically active conformation of the protein.

Next, the researchers took an “SAR by catalog” approach to find more potent molecules, leading to compound 4, with an AC₅₀ value roughly ten-fold better than compound 2. Detailed enzymatic characterization revealed that the molecule acts as a “nonessential reversible activator,” and improves substrate binding roughly 7-fold while improving k_cat by a factor of 1.7.

Interestingly, differential scanning fluorimetry (DSF) revealed that all the activators destabilize the enzyme, while PUGNAc stabilizes the enzyme. This is yet more evidence that compounds can decrease the melting temperature of a protein while still binding specifically.

This is an academic study in the best sense of the phrase. The immediate utility of these molecules is tenuous, as they do not work with human OGA (which has some important active site differences), though there may be industrial applications. The more important finding of this rigorous paper is that discovering enzyme activators might be easier than expected. There aren’t that many reported, but this may just be because people tend not to look for them: if you find an activator of an enzyme you are trying to shut down you probably won’t pursue it. That said, a few companies have been founded on enzyme activators. Perhaps fragments can help discover more.

Benchmark Your Process

So, not everybody agrees with me on what a fragment is.  As has been pointed out years ago, FBDD can be a FADD.  In this paper, from earlier this year, a group from AZ discusses how FBDD was implemented within the infectious disease group. Of course, because of the journal, it emphasizes how computational data is used, but you skim over that and still enjoy the paper :-). They break their process into several steps.

Hot Spots: This is a subject of much work, particularly from the in silico side.  In short, a small number of target residues provide the majority of energy for interaction with ligands.  Identifying these, especially for non-active site targets (read PPI), is highly enabling, for both FBDD and SBDD. To this end, the authors discuss various in silico approches to screening fragments.  They admit they are not as robust as would be desired (putting it kindly).  As I am wont to say, your computation is only as good as your experimental follow up.  The authors indicate that the results of virtual screens must be experimentally tested.  YAY!  They also state that NMR is the preferred method; 1D NMR in particular being the AZ preferred method.  [This is something (NMR as the first choice for screening) that I think has become true only recently.  Its something I have been saying for more than a decade, but I guarantee my cheerleading is not why.] They do note that of the two main ligand-based experiments, STD is far less sensitive than WaterLOGSY.  There is no citation, so I would like to put it out there, is this the general consensus of the community?  Has anyone presented data to this effect?  Specifically, they screen fragments 5-10 per pool with WaterLOGSY and relaxation-edited techniques.  2D screening is only done for small proteins (this is in Infection) and where a gram or more of protein is available.

Biophysics:  They have SPR, ITC, EPIC, MS, and X-ray.  They mention that SPR and MS require high protein concentrations to detect weak binders and thus are prone to artifacts.  They single out the EPIC instrument as being the highest throughput.  [As an aside, I have heard a lot of complaints about the EPIC and wonder if this machine is still the frontline machine at AZ.]  60% of targets they tried to immobilize were successful.  They also use "Inverse" SPR, putting the compounds down; the same technology NovAliX has in their Chemical Microarray SPR.  In their experience, 25% of these "Target Definition Compounds" still bind to their targets. 

They utilize a fragment-based crystallography proof of principle (fxPOP).  Substrate-like fragments (kinda like this?) are screened in the HTS, hits [not defined] are then soaked into the crystal system, and at least one structure of a fragment is solved.  This fragment is then used for in silico screening, pharmacophore models, and the like.  So, this would seem to indicate that crystals are required before FBDD starts.  They cite the Astex Pyramid where fragments of diverse shape are screened and the approach used at JnJ where they screen similar shaped fragments and use the electron density to design a second library to screen.

As I have always said, there are non-X-ray methods to obtain structural information.  AZ notes that SOS-NMR, INPHARMA, and iLOE are three ways.  These are three of the most resource intensive methods: SOS-NMR requires labeled protein (and not of the 15N kind), INPHARMA requires NOEs between weakly competitive ligands (and a boatload of computation), while iLOE requires NOEs of simultaneously binding ligands.  I think there are far better methods, read as requiring fewer resources, to give structural information more quickly (albeit at lower resolution).

The Library:  The describe in detail how they generated their fragment libraries.  They have a 20,000 fragment HCS library.  The only hard filter is to restrict HA less than 18.  I fully support that.  They also generated a 1200 fragment NMR library biased towards infection targets.

The Process:   The authors list three ways to tie these methods together:

1. Chemical Biology: Exploration of binding sites/development of pharmacophores.  I would add that this is also for target validation.  As shown by Hajduk et al. and Edfeldt et al., fragment binding is highly correlated to advancement of the project. 
2. Complementary to HTS.  At the conference I am at today, one speaker (from Pfizer) said that HTS was for selectivity, FBDD was for efficiency (or Lord, here comes Pete with that one).  I really like that approach.
3. Lastly, stand alone hit generation.  

I think this paper is a nice reference for those looking to see how one company put their FBDD process in place. Not every company will do it the same, nor should they.  But there is a FBDD process for every company.