24 August 2015

Fragment-Based Drug Discovery

This is the straight-to-the-point title of a new book published by the Royal Society of Chemistry, edited by Steven Howard (Astex) and Chris Abell (University of Cambridge). It is the second book on the topic published so far this year, and it is a testimony to the fecundity of the field that the two volumes have very little overlap.

After a brief forward by Harren Jhoti (Astex) and a preface by the editors, the book opens with two personal essays. The first, by me, is something of an apologia for Practical Fragments and the growing role of social media in science (and vice versa). If you’ve ever wondered how this blog got started or why it keeps going, this is where to find out. The second essay is by Martin Drysdale (Beatson Institute). Martin is a long-time practitioner of FBDD, dating back to his early days at Vernalis (when it was RiboTargets) and he tells a fun tale of “adventures and experiences.”

Chapter 1, by Chris Abell and Claudio Dagostin, is entitled “Different Flavours of Fragments.” With a broad overview of the field it makes a good introduction to the book. There are sections on fragment identification, including the idea of a screening cascade, as well as several case studies, some of which we’ve covered on Practical Fragments, including pantothenate synthetase, CYPs, RAD51, and riboswitches.

The next two chapters deal with two of the key fragment-finding methods. Chapter 2, by Tony Giannetti and collaborators at Genentech, GlaxoSmithKline, and SensiQ, covers surface plasmon resonance (SPR). This includes an extensive discussion of data processing and analysis, which is critical for improving the efficiency of the technique. Competition studies are also described, as are advances in hardware, notably those from SensiQ. This is a good complement to Tony's 2011 chapter.

Chapter 3, by Isabelle Krimm (Université de Lyon), provides a thorough description of NMR methods, both ligand-based (STD, WaterLOGSY, ILOE, etc) and protein-based (mostly HSQC). The chapter does a nice job of describing techniques in terms a non-specialist can understand while also providing practical tips on matters such as optimal protein size and concentration.

Chapter 4, by Ian Wall and colleagues at GlaxoSmithKline, provides an overview of FBLD from the viewpoint of computational chemists. The chapter includes some interesting tidbits, such as the observation that fragment hits that yield crystal structures tend to be less lipophilic but also contain a smaller fraction of sp3 atoms and more aromatic rings. The researchers note that the current fashion for “3D” fragments is yet to be experimentally validated. They also include accessible sections on modeling, druggability, and integrating fragment information into a broader medicinal chemistry program.

The remaining chapters focus on specific types of targets. Chapter 5, by Miles Congreve and Robert Cooke (both at Heptares) is devoted to G protein-coupled receptors (GPCRs). This includes descriptions of how to screen fragments against these membrane proteins using SPR, TINS, CE, thermal melts, and competition binding. It also includes a detailed case study of their β1 adrenergic receptor work (summarized here). Congreve and Cooke assert that, although many of the GPCR targets screened to date have been highly ligandable, technical challenges only now being addressed have caused this area of research to lag about a decade behind other targets. They predict a bright future.

Rod Hubbard (Vernalis and University of York) turns to protein-protein interactions in Chapter 6. After describing why these tend to be more challenging than most enzymes and covering some of the methods for finding and advancing fragments, he then presents several case studies, including FKBP (one of the first targets screened using SAR by NMR), Bcl-2 family members (including Bcl-xL and Mcl-1), Ras, and BRCA2/RAD51. He concludes with a nice section on “general lessons,” which boils down to “patience, pragmatism, and integration.” As Teddy recently noted, this can lead to substantial rewards.

Allosteric ligands have potential advantages in terms of selectivity and addressing otherwise challenging targets, and in Chapter 7 Steven Howard (Astex) describes how fragments can play a role here. This includes how to establish functionality of putative allosteric binders, as well as case studies such as HIV-1 RT, FPPS, and HCV NS3. Astex researchers have recently stated that they find on average more than two ligand binding sites per protein, and this chapter includes a table listing these (including 5 binding sites each on bPKA-PKB and PKM2).

The longest chapter, by Christina Spry (Australian National University) and Anthony Coyne (University of Cambridge) describes fragment-based discovery of antibacterial compounds. After discussing some of the challenges, the authors report several in depth case studies including DNA gyrase, DNA ligase, CTX-M, AmpC, CYP121, and pantothenate synthetase, among others. At least one fragment-derived antibacterial agent entered the clinic; hopefully more will follow.

Chapter 9, by Iwan de Esch and colleagues at VU University Amsterdam, focuses on acetylcholine-binding proteins (AChBPs), both as surrogates for membrane-bound acetylcholine receptors and as well-behaved model proteins on which to hone techniques (see for example here, here, and here). Since AChBPs have evolved to bind fragment-sized acetylcholine, these proteins can bind tightly to small ligands; 14-atom epibatidine binds with picomolar affinity, for example, with a ligand efficiency approaching 1 kcal mol-1 atom-1.

And Chapter 10, by Chun-wa Chung and Paul Bamborough at GlaxoSmithKline, concisely covers epigenetics. Bromodomains are well-represented, including a table of ten examples (see for example here, here, here, here, here, and here). Happily, although some of these projects started from similar or identical fragments, the final molecules are quite divergent. However, the authors note that much less has been published on histone-modifying enzymes, such as demethylases and deacetylases, perhaps reflecting the challenges of achieving specificity with what are often metalloenzymes.

Finally, this is the 500th post since Teddy founded Practical Fragments way back in the summer of 2008. Thanks for reading, and special thanks for commenting!

19 August 2015

Caveat Emptor...or marketing does not always tell you whats really in the package.

In case you missed it, I spoke at the ACS on Sunday.  It was in a computational session looking at designing libraries and I am pretty sure I was the only non compchemist.  It was about all the problem compchemists have caused in library design.  My talk was even live tweeted by Ash (@curiouswavefn) and was well received.  So, looking at the next paper in my queue, its a computational-focused paper.  So, After spending several hours on a Sunday listening to compchemists, have I softened?  

This paper is the subject of today's post.  It is an extension of this paper which describes their virtual screen.  From a 2 million compound virtual screen, they tested 17 compounds in vitro leading to 2 micromolar compounds.  This paper is the story of the most potent of the two micromolar compounds.  The target is CREBBP, which is another in the long line of epigenetic targets.  Compound A was one of the original in silico actives that was tested.  Three analogs were obtained and tested (B-D).
Figure 1.  Original active, A.  Analogs B-D.  Common structural motif is shown in blue.
Compound B was the most potent and become the focus of their optimization efforts. Of course, my eyes are drawn to that potential michael acceptor, but the authors dismiss it based upon their docking results: the only alkylatable residue in the area of its putative binding is well buried.  It is a moot point anyway because they were able to replace it with a isopthalate group and increase potency by 5x, 0.9uM (Compound 6).  Interestingly,the potency of 6 is different depending on the assay used: 0.8 um in a competition binding assay and 8.7uM in a TR-FRET assay.
Figure 2.  Compound 6
This compound was crystallized and showed that the predicted binding mode was correct. 

They then performed some gobbledy-gook MD calculations (finite-difference Poisson, warning PDF) in order to evaluate the electrostatic contribution of the polar contribution to binding of 6 and 7.  Compound 6 had more favorable electrostatic interactions (0.8 kcal/mol) than 7, which had more favorable van der Waals interactions (1.4 kcal/mol).  With this crucial information AND the crystal structure in hand, they then explored additional chemical space.  

Despite the authors' claim, I don't think they actually improved the potency significantly.  Compound 6 is 8 uM in the TR-FRET assay and the best compounds they claim are 1 or 2 uM.  I really have to call monkeyshines here.  They use the different assays interchangeably, yet never explain the one is used for what purpose.  Its cherry picking values.  When talking about selectivity, they switch to using thermal shift values.  And we all know the value of that.  So, I find it hard to believe their "most potent" this or "selectivity" that. The title of the paper includes "nanomolar", but that is only in one assay.  That's like saying I can run a 6 minute mile, since I did it once under optimum conditions.  Honestly, my typical times (WAY back when) were more 8:30 miles.  That honesty in data reporting.  Since they obviously had access to different assays, why weren't all compounds run in one, or optimally both.  I don't see that the MD calculations had any positive impact.  Maybe its the heat, but this paper is a not a sham, but definitely full of deceptive advertising.

17 August 2015

Fragments vs IAPs: resisting the affinity Siren

In 2011, Mike Hann decried “addiction to potency”. Indeed, newcomers to fragment-based methods often have to undergo a psychological shift to work with low affinity binders. But, as we asked last year, how weak is too weak? In a paper just published in J. Med. Chem., Gianni Chessari and colleagues at Astex may have set a new bar.

The researchers wanted to develop leads against inhibitor of apoptosis proteins (IAPs). The BIR3 domains of proteins such as cIAP1 and XIAP bind to and block the action of caspases and other proteins, allowing cancer cells to survive. Several groups have developed molecules that block these protein-protein interactions, usually by starting with an endogenous peptide inhibitor. However, most of these bind preferentially to cIAP1, and some evidence suggests that a balanced antagonist may have advantages.

The BIR3 domain is quite small, just 11.8 kDa, making standard ligand-observed NMR screening difficult. Instead, the researchers looked at line broadening and chemical shift changes of protein protons with δ < 0.4 ppm or 9.8 – 10.4 ppm on addition of fragments. Anticipating very weak binders, they used 0.2 mM protein and 10 mM fragments (in mixtures of two). A total of 1151 fragments were screened, of which 100 had been computationally preselected.

Among the best hits were those containing an alanine residue; one of these had mid-micromolar affinity and reasonable ligand efficiency. However, these bound preferentially to the BIR3 domain of cIAP1, in common with other reported inhibitors that also contain an alanine. In contrast, compound 1 appeared to have more balanced activity. Its affinity was risibly weak, but crystal soaking led to interpretable electron density, and also suggested that adding a suitably positioned methyl group could fill the small hydrophobic pocket normally occupied by the alanine side chain. The resulting compound 5 showed measurable – and balanced – activity against both proteins.

Next, a small virtual library was constructed in which the pyrrolidine was replaced with substituents to both improve affinity and create a scaffold for reaching into the P4 pocket. Thirty compounds were made, but disappointingly none of these had significantly improved affinity. Undeterred, the researchers obtained crystal structures of some of them bound to XIAP-BIR3 and performed careful modeling. The phenyl ring of compound 7 binds in a region of the protein with an electronegative potential, and by simply adding electron withdrawing substituents the affinity could be improved by >50-fold. Further growth ultimately led to compound 21, with nanomolar potency against both XIAP and cIAP1, though with a preference for the latter. This compound also showed on-target activity in cell-based assays as well as activity in mouse xenograft models.
It would have been easy to overlook compound 1; indeed, it took rather strenuous efforts to find it. Yet, comparing the structures of compound 1 and 21 bound to XIAP-BIR3 reveals that the initial fragment maintains its position and binding interactions in the elaborated molecule. This is a clear example that, with persistence and creativity, it is possible to advance even the weakest of fragments. The researchers note in the conclusion (and have reported at conferences) that they were able to optimize this series to low nanomolar inhibitors against both targets. Whether or not this leads to a drug, it does look like another candidate for a useful chemical probe.

12 August 2015

Silver Ain't Bad

For those of you who have been reading this blog for a while, you are familiar with the "Fragments in the Clinic" posts, Jan2015, Jan2013, and Jan2010.  Two of  those slowly making its way through the pipeline is navitoclax, ABT-263, and venetoclax, ABT-199.  Today Abbvie and Genentech announced that it had met its end point in phase II trials.  The companies plan to file for approval by the end of this year.  At that point we will then have TWO compounds approved from fragments.  Congratulations to all of those folks who have worked on this over the years!!!

10 August 2015

Fragments vs PAK1 – allosterically

Some of our recent posts have discussed the use of fragment-based approaches to discover selective new chemical probes. Continuing this theme, a paper from Alexei Karpov and colleagues at Novartis in ACS Med. Chem. Lett. describes a success story against the kinase PAK1.

The six p21-activated kinases (PAKs) have been implicated in a variety of indications, from cancer to neurodegenerative diseases. Unfortunately, most reported inhibitors are not sufficiently selective to elucidate the biology. The researchers were particularly interested in PAK1, against which they performed a fragment screen (no details of methods or libraries are provided). One of the more interesting hits was fragment 1. Although a bit chunky (24 heavy atoms, MW = 328 Da) with only modest ligand efficiency, it is structurally unusual for a kinase binder.

In fact, crystallography revealed that the fragment binds not in the active site at all, but rather in an allosteric site adjacent to the ATP-binding site, with the so-called DFG-loop in the “out” conformation. Not surprisingly, this molecule was more active against the inactive form of the enzyme. Despite its binding mode, it did appear to be ATP-competitive, perhaps because the DFG-out conformation of the protein is not able to bind ATP.

Aficionados of GPCRs will not be surprised to learn that fragment 1 – a dibenzodiazepine – bound several with high affinity, but the researchers used structural information both to ablate this off-target activity as well as improve affinity for PAK1, resulting ultimately in compound 3. This molecule was completely selective for PAK1 when tested at 10 µM concentration against a panel of 442 kinases, as well as against a panel of 22 other potential off-targets. Unexpectedly, it was even >50-fold selective against the closely related PAK2. It also displayed good permeability and acceptable solubility, though the stability could be improved.

Compound 3 blocked PAK1 autophosphorylation in cells, but was only modestly effective at blocking proliferation of a pancreatic cancer cell line with high levels of PAK1. As it turns out, all known cancer cell lines that are dependent on PAK1 also seem to use PAK2, so perhaps this chemical probe is too selective. Nevertheless, it will be useful to help disentangle the overlapping roles of these two kinases.

More broadly, this is a nice illustration of the selectivity achievable with allosteric kinase inhibitors. Indeed, as we recently noted in a comment to a post earlier this year, Novartis has put at least one allosteric kinase inhibitor into the clinic.

05 August 2015

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? 

03 August 2015

Fragments and HTS vs BCATm

One of the themes throughout this blog is that fragments are useful not just in and of themselves, but as part of a broader tool kit, what Mark Whittaker referred to as fragment-assisted drug discovery, or FADD. A nice example of this has just been published in J. Med. Chem. by Sophie Bertrand and colleagues at GlaxoSmithKline and the University of Strathclyde.

The researchers were interested in mitochondrial branched-chain aminotransferase (BCATm), an enzyme that transforms leucine, isoleucine, and valine into their corresponding α-keto acids. Knockout mouse studies had suggested that this might be an attractive target for obesity and dyslipidemia, but there’s nothing like a chemical probe to really (in)validate a target. To find one, the researchers performed both fragment and high-throughput screens (HTS).

The full results from the fragment screen have not yet been published, but the current paper notes that the researchers screened 1056 fragments using biochemical, STD-NMR, and thermal shift assays. Compound 1 came up as a hit in all three assays, and despite modest potency and ligand efficiency, it did have impressive LLEAT. The researchers were unable to determine a crystal structure of this fragment bound to the protein, but STD-NMR screens of related fragments yielded very similar hits that could be successfully soaked into crystals of BCATm.

The HTS also produced hits, notably compound 4, which is clearly similar to compound 1. In addition to its increased biochemical potency, it also displayed good cell activity. Moreover, a crystal structure revealed that the bromobenzyl substituent bound in an induced pocket that did not appear in the structure with the fragment, or indeed in any other structures of BCATm.

The researchers merged the fragment hits with the HTS hits to get molecules such as compound 7, with a satisfying boost in potency. Interestingly, the fragment-derived core consistently gave a roughly 10-fold boost in potency compared to the triazolo compounds from HTS. Comparison of crystal structures suggested that this was due to the displacement of a high-energy water molecule by the nitrile.

Extensive SAR studies revealed that the propyl group could be extended slightly but most other changes at that position were deleterious. The bromobenzyl substituent was more tolerant of substitutions, including an aliphatic replacement, though this abolished cell activity. Compound 61 turned out to be among the best molecules in terms of potency and pharmaceutical properties, including an impressive 100% oral bioavailability and a 9.2 hour half-life in mice. Moreover, this compound led to higher levels of leucine, isoleucine, and valine when mice were fed these amino acids.

This is a lovely case study of using information from a variety of sources to enable medicinal chemistry. Like other examples of FADD, one could argue as to whether the final molecule would have been discovered without the fragment information, but it probably at least accelerated the process. More importantly, molecules such as compound 61 will help to answer the question of whether BCATm will be a viable drug target. 

29 July 2015

Novel Assay (SPR) Leads to Novel Allosteric Binding Site (nAChR)

I love this blog.  But sometimes it drags.  Finding new articles to blog about can be hard, there seems to be waves.  So, I love it when someone points one out to me.  This article was sent to me by someone associated with it.  We don't always blog those articles people point out to us, but this one has all the hallmarks: high visibility journal, good target, and "innovative" in the approach.  So, let's dive in and see what we have here.

Nicotinic receptors (nAChR) are pentameric, ligand-gated ion channels.  nAChR mutations are implicated in a wide range of neurological disorders and are the targets for a wide range of current drugs.  It is important to note that most of these drugs work through an allosteric site distant from the agonist binding site.  Most high resolution, structural data is from homologous molluscan Acetylcholine binding proteins.  The orthosteric site is located at an interface of a "principal" and "Complementary" subunit.  Ligand-binding induces conformational changes in the ligand binding site which are coupled to the ion opening.  There is little information on structural implications at allosteric sites.  The authors decided to address this unmet need with a chimeric human α7 ligand binding domain and AChBP which has 71% sequence similarity to the native protein, compared to 33% for the most commonly used target (Aplysia).  

The authors used SPR against a target with a blocked orthosteric site.  They did this one of two ways: 1. pre-incubation with a high affinity orthosteric ligand or 2. mixing each fragment with an orthosteric ligand of lower affinity.  This second approach (Figure 1) allows detection of fragments not competing for binding to the orthosteric site and were therefore potential allosteric ligands.  
Figure 1.  To distinguish allosteric binders from competitive binders using SPR spectroscopy we perfused each fragment alone (green triangle) or in combination with the competitive antagonist d-tubocurarine (black circle). In the case of an allosteric binder, the response units observed for the mixture of fragment + d-tubocurarine is close to the sum of fragment alone + d-tubocurarine alone (blue dashed line). No competition exists because the fragment and d-tubocurarine bind at distinct sites. (C) In the case of a competitive binder, the response units for the mixture of fragment + d-tubocurarine is lower than the sum of fragment alone + d-tubocurarine alone because both compounds compete for binding at the same site. (D) Example traces for fragment 4, which was identified as one of the allosteric binders in this study.
 In screening 3000 fragments, they found 300 putative allosteric binders.  Follow up, including dose-response, led to 24 fragments being selected for co-crystallization.  Crystal trials were set up using blocked nAChR and fragments that were soluble at 5-10mM; yielding in the end 5 crystal structures.  All five proved to be allosteric binders in three separate locations, including one never observed before (top pocket).  The most potent of these fragments had a IC50 of 34 uM, while the least potent was 400 uM. 
Figure 2.  Allosteric Binding Sites
This is a really nice piece of work.  I think the SPR assay is really clever and I would expect that many people will now be taking a similar approach to discovering allosteric sites in their targets.

27 July 2015

Fragments vs DDR1/2: a chemical probe

Our last post was about the utility of chemical probes: small molecules with sufficient potency and selectivity to be able to address specific biological questions. A recent paper in ACS Med. Chem. Lett. by Chris Murray and colleagues at Astex describes an excellent example of finding a new probe for the discoidin domain receptors (DDR1 and DDR2). Previous publications had suggested a role for these receptor tyrosine kinases in certain types of lung cancer, but some of this work had relied on non-selective inhibitors.

The researchers started with a thermal shift assay of their 1500 fragment library against DDR1, followed by crystallographic screening, resulting in around 50 fragment-protein complexes. Not surprisingly most of the fragments bound in the hinge-binding region of the kinase, but around 10 bound in the so-called “back pocket”, with the protein in the inactive DFG-out conformation. As the researchers point out, it is rare to see fragments binding here.

Compound 1 was of particular interest. At first glance, it's not impressive: with 18 heavy atoms and MW > 250 Da, it is on the large end for an Astex fragment, and it had low activity in a biochemical assay. However, its binding mode revealed potential areas for improvements, and the methylene was an unusual feature in a back-pocket binder.

The first step in improving affinity was to add a hinge binder. This was done with the aid of an in-house program called AstexMerge, based on the program BREED, which superimposes a set of ligands. The user chooses a starting molecule, and the program tries to merge that with other molecules while taking account of bond angles and distances. This process led to the design of compound 2, and a few tweaks quickly led to compound 4.

Although compound 4 was potent against DDR1 and 2 and showed good cell-based activity in a phosphorylation assay, it did potently inhibit some other kinases too, most notably c-kit. That problem was fixed with further medicinal chemistry, notably addition of a methyl group to the previously mentioned methylene and replacement of the urea, leading to compound 9, which was potent, selective, and showed good pharmacokinetics in mice.

Although compound 4 was not completely selective, it was more so than some of the previously described molecules, so the researchers tested it in lung cancer cells and found that, despite the fact that it inhibited DDR2 phosphorylation, it showed no effect on cell proliferation. Thus, “the project was halted in favor of more attractive targets.”

Clearly the researchers didn't start out trying to disprove the role of DDR1/2 in squamous cell lung cancer, but their efforts will save others from pursuing the same course. The publication introduces some attractive chemical probes for interrogating the biology of these receptors; hopefully one of these molecules will be added to the Chemical Probes Portal as an alternative to the less-selective probes that have been used in previous studies. Who knows, perhaps someone will find another indication for which DDR1/2 inhibitors are just the ticket.

22 July 2015

Introducing the Chemical Probes Portal

Chemical probes can be incredibly powerful reagents for understanding biology. A potent, selective, and cell-active modulator of a specific protein can be invaluable for figuring out what that protein actually does. Fragment-based methods can be effective at identifying these tool compounds, as we've described here and here.

Unfortunately, good chemical probes are difficult to discover, and scientists are left struggling with suboptimal reagents that hit multiple targets, often through pathological mechanisms. This leads to "pollution of the scientific literature," in Jonathan Baell's memorable phrasing. Despite our occasional PAINS Shaming, high-profile articles in C&EN and Nature, and even a dedicated blog, the problem continues. What is to be done?

Yesterday, a team of 53 authors from 46 academic and industrial organizations published a Commentary in Nature Chemical Biology entitled "The promise and peril of chemical probes" (see here for excellent coverage in Nature, here for Science's take, and here for In the Pipeline). This provides a good working definition for a chemical probe. According to the Structural Genomics Consortium, a chemical probe for epigenetics targets must have:

  • Potency < 100 nM against the desired target
  • >30-fold selectivity vs related targets
  • On-target cell activity < 1 µM

It should also be profiled against a larger panel of potential off-targets, and a related inactive compound (such as a stereoisomer) should be available as a control.

After discussing examples of high-quality probes, the researchers turn their attention to what they term – rather charitably – "probes of lesser value:"
The continued use of these probes poses a major problem: tens of thousands of publications each year use them to generate research of suspect conclusions, at great cost to the taxpayer and other funders, to scientific careers and to the reliability of the scientific literature.
The authors then go on to describe best-practices. For example, even high-quality probes can give spurious results when used at high concentrations. As Paracelsus recognized five centuries ago, the dose makes the poison.

All of this is important, but as the authors acknowledge, it's been said before. What really differentiates the Commentary is the simultaneous launch of a companion web site, the Chemical Probes Portal. Its creators hope that this will lead to vigorous community discussion around questions such as:

Is there a probe for my target protein?
Which ones should I use?
How should I use this probe properly?
Is this probe suitable for use in animal models?

Currently the Portal lists just seven probes with links to references and descriptions of selectivity, solubility, and the like. All of these are “good probes,” but hopefully this will expand: the paper itself discusses the shortcomings of molecules such as staurosporine, chaetocin, obatoclax, and gossypol, and including them in the portal with detailed warnings would be valuable for the scientific community.

I hope this takes off. Understanding the natural world is hard enough even with well-behaved reagents and carefully controlled experiments. Practical Fragments will check back in a year or so to see how the site is doing. In the meantime, probe cautiously!

15 July 2015

Covalent Inhibitor of KRas

So, Ras is big.  We keep on talking about it.  And sometimes we talk about the same work repeatedly.  This recent paper from AZ is a publication of work we have talked about here and here.  This follows on closely to work done by Vanderbilt and Genentech.  Those two papers were done using NMR and this one took a X-ray approach.  The AZ folks were taking a different approach to this PPI: stabilization of the interface.  They took 1160 fragments in pools of 4 and screened against HRas (homolog)-catalytic domain of SOS stable complex.  There were able to identify 3 bindings sites on HRas-SOS (Figure 1):
Figure 1.  HRas-SOS Complex.  HRas (Green), SOS (Blue), A: SOS binding site  (gold) (same as Vanderbilt), B SOS-Hras Interface binding site (Red) (same as Genentech), and C HRas covalent binding site (black). 
Site A was the same site identified by the Vanderbilt group  Site B was the same as identified as Genentech.  However, the AZ compounds bound to both proteins at the interface.  Their initial hope was to use this site to stabilize the Ras-SOS interface.  Both of the fragments binding to these sites had their affinity determined by TROSY-HSQC NMR.    However, they were not potent enough to elicit a biological effect, which was not unexpected.  After several rounds of chemistry, they were not able to improve these fragments significantly, or even show that they actually stabilized the interface.  

Looking at the growing covalent literature, they hypothesized that an irreversible inhibitor may be the only way to inhibit GTPase activity, especially considering the pM affinity of GTP for Ras.  They identified Cys118R (conserved between HRas and KRas) as a potentially reactive sidechain proximal to the GDP binding site on Ras.  To go after this site covalently, AZ assembled a 400 fragment covalent library (Figure 2) and screened it by mass spectrometry.
Figure 2.  Chemotypes represented in AZ 400 fragment covalent library.
They chose the N-substituted maleimide was deemed "ideal"; other warheads were either insufficiently reactive or overly reactive.  Covalent modification of Cys118R by a fragment partially occludes the nucleotide binding site and potentially prevents the reorganization of the Cys118R loop, thus locking it into the catalytically inactive Ras-SOS complex.  Interestingly, their covalent compounds only inhibited catalytically activity when pre-incubated with Ras-GDP-SOS.  This supports the hypothesis that Cys118R becomes more accessible during SOS-mediated nucleotide exchange.  

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

13 July 2015

Fragments vs BTK: metrics in action

Sometimes the discussions over metrics, such as ligand efficiency, can devolve into exegesis: people get so worked up over details that they forget the big picture. A recent paper in J. Med. Chem. by Chris Smith and (former) colleagues at Takeda shows how metrics can be used productively in a fragment-to-lead program.

The researchers were interested in developing an inhibitor of Bruton’s Tyrosine Kinase (BTK) as a potential treatment for rheumatoid arthritis. This is the target of the approved anti-cancer drug ibrutinib, but ibrutinib is a covalent inhibitor, and the Takeda researchers were presumably concerned about the potential for toxicities to arise in a chronic, non-lethal indication. Many of the reported non-covalent BTK inhibitors are large and lipophilic, with consequently suboptimal pharmacokinetic properties. Thus, the team set out to design molecules with MW < 380 Da, < 29 non-hydrogen atoms (heavy atoms, or HA), and clogP ≤ 3.

The first step was a functional screen of Takeda's 11,098 fragment library, all with 11-19 HA, comfortably within the bounds of generally accepted fragment space. At 200 µM, 4.6% of the molecules gave at least 40% inhibition. Hits that confirmed by STD NMR were soaked into crystals of BTK, ultimately yielding 20 structures. Fragment 2 was chosen because of its high ligand efficiency, novelty, and the availability of suitable growth vectors.
Close examination of the structure suggested a fragment-growing approach. Throughout the process, the researchers kept a critical eye on molecular weight and lipophilicity. This effort led through a series of analogs to compound 11, with only 24 heavy atoms and clogP = 1.7. This molecule is potent in biochemical and cell-based assays and has excellent ligand efficiency as well as LLE (LipE). Moreover, it has good pharmacokinetic properties in mice, rats, and dogs, with measured oral bioavailability > 70% in all three species. Finally, compound 11 shows efficacy in a rat model of arthritis when dosed orally once per day.

Although compound 11 is selective over the closely related kinase LCK, unfortunately it is a double digit nanomolar inhibitor of oncology-related kinases such as TNK2, Aurora B, and SRC, which would probably be unacceptable in an arthritis drug. Nonetheless, this study is a lovely example of fragment-growing guided by a strict commitment to keeping molecular obesity at bay.

08 July 2015

Merging Fragments for Matriptase

We often talk about methods here: how to screen, how to prosecute those actives, and everything in between.  This is one of those what you do with the actives posts.  In this paper, a group from Aurigene and Orion present their results on Matriptase.  There have been multiple reported matriptase inhibitors, small molecule and peptide based.  Previous work from this group showed compounds that were active in cell-based migration and invasion assays and in mice with tri-substituted pyridyls and benzene compounds.  For this work, they take a SBDD approach:  "structure divulges a trypsin-like S1 cavity, a small hydrophobic S2 subpocket, and a solvent exposed spacious S4 region."

In screening benzamidine fragments (MW less than 300) they found 2 actives, ~80uM (Figure 1).
Figure 1.  Benzamidine screening actives
These were modeled in to the active site and obviously the amidine moiety went into S1.  Cpd 1's benzene moiety went nicely into S4 while 2's piperidyl went into S1'.  S4 easily accepted the more hydrophobic napthyl instead of phenyl and then they decided to see if the napthyl compound and 2 could be "linked" and the beta carbon. [So, my first quibble here is that this is not really a linking approach; this is fragment merging. Linking involves modeling, SBDD, and discovery of different linkers.  Its very difficult to do without specialized methods.  What they did here was see huge spatial overlap of compounds and voila, "we can add something right here".]  Well, not surprisingly, this worked.  They describe their SAR around each pocket to pick the compounds to merge, go read it if that interests you. The did crystallize the merged compound and it confirmed the modeling.  The final compound showed activity in cell-based assays and in mice.  That's good.  

This work can be summarized as follows: if you have significant spatial overlap you have a very good chance of merging disparate moieties.  So, two things bother me here.  First, the actives 1 and 2 are mighty big for fragments (more than 22 HAC).  That's fine, tomato...to-mah-to.  The final compound ends up pretty honking big too (37 HAC).  What is really bothersome, at least to me, is the LE.  Both actives start well below 0.2 (for a protease!) and they never improve on it. Now, Pete may disagree, but metrics have a place in FBDD.  Does the LE metric in this case tell us anything? 

06 July 2015

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.

01 July 2015

Updated: fragment events in 2015 and 2016

It is hard to believe that the year is already half over, but there are still important events coming up, and 2016 is already starting to take shape!


August 11-13: The OMICS Group is holding a conference entitled Drug Discovery & Designing in Frankfurt, Germany, with FBDD listed as a conference highlight.

September 22-24: Newly added! CHI's Thirteenth Annual Discovery on Target will be held in Boston again, and fragments play an important role in several tracks, including epigenetics and kinases. Also, Teddy and I will be teaching a short course on Targeting Protein-Protein Interactions on Monday, September 21.

December 15-17: More than 40 presentations. 8 countries. 3 days. One event:
The first-ever Pacifichem Symposium devoted to fragments.

The Pacifichem conferences are held only once every 5 years in Honolulu, Hawaii to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, New Zealand, and the US. Registration is now open!


February 21-24: Zing conferences is holding its inaugural Structure Based Drug Design Conference in Carlsbad, California. This looks like a cousin of last year's Caribbean meeting, so it should be a lot of fun.

April 19-22: CHI’s Eleventh Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of this year's meeting here, here, and here; last year's meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

October 9-12: Finally, FBLD 2016 will be held in Boston, MA. This marks the sixth in an illustrious series of conferences organized by scientists for scientists, the last of which was in Basel in 2014. Surprisingly, this also seems to be the first dedicated fragment conference in Boston. You can read impressions of FBLD 2012FBLD 2010, and FBLD 2009.

Know of anything else? Add it to the comments or let us know!

29 June 2015

Crystallographic screening of soluble epoxide hydrolase

Last year we highlighted a paper from Yasushi Amano and colleagues at Astellas in which they performed fragment-screening on soluble epoxide hydrolase (sEH), a potential target for inflammation and hypertension. A new paper from the same group in Bioorg. Med. Chem. builds on that work and provides some interesting comparisons.

In the first paper, the researchers performed an enzymatic screen with fragments at high concentrations, resulting in a hit rate of around 7.3%, of which 126 of the 307 hits resulted in crystal structures. However, despite this bounty of hits, only 2 new scaffolds were found that bind to the catalytic triad of the enzyme.

Given the success rate with crystallography, the new paper focused on crystallographic screening as the primary fragment-finding method. The researchers chose 800 fragments from their in-house collection with molecular weights between 151-250 Da. To identify new scaffolds, fragments containing amide or urea moieties – known catalytic-site binders – were excluded. The fragments were then pooled into cocktails of 10 and soaked into crystals of sEH, with each fragment at a final concentration of 1 mM. X-ray diffraction data of the soaked crystals resulted in 8 hits. To ensure that nothing was missed, cocktails of the remaining 9 fragments from pools with a hit were retested, but nothing new came up. Although the researchers do not comment on the lower hit rate compared with the original screen, this could be because they were looking specifically for new scaffolds.

Despite the 1% hit rate, the fragments identified were quite interesting, with IC50 values ranging from 52 to 2200 µM. Most of the fragments formed hydrogen bonds to the catalytic triad, but the details differed from reported inhibitors. For example, several fragments contained secondary amines. Fragment 1 (cyan) in particular was well-positioned to reach into two sub-pockets on either side of the catalytic center, so 14 analogs were chosen for screening, resulting in molecules with significantly increased activity, such as compound 9 (magenta).
The crystal structure of compound 9 bound to sEH reveals that it binds in a similar manner as fragment 1. However, the added hydroxyl group is able to make new interactions that were unavailable to fragment 1, and the larger adamantyl group of compound 9 is able to make more hydrophobic interactions than the smaller phenyl ring.

This is a lovely illustration of the gains in both affinity and ligand efficiency that can be had by scaffold-hopping. It is also a nice example of using fragments to explore new chemical space. Finally, it is laudable that all the structural information is deposited in the protein data bank.

24 June 2015

One Fragment to Rule them All

Recently, I have been riffing on the ontology of FBDD.  FBDD has become so popular that we are now seeing appropriation of the term in many papers that don't really mean it.  So, I came across this paper.  Now, don't be fooled by the title, this is about fragments, the abstract promises me so.  Let me skip the science, which to my eyes is actually quite boring, and get right to the heart of their fragment case.  
How is this paper fragments you ask?  Well, this is not about scaffold hopping or innovative uses of fragments to develop SAR.  This is not about interesting approaches to screening.  It is most certainly not about in silico approaches.  This is most certainly about fragment library design.  We often discuss here the sizes of fragment libraries and what they should look like.  One important concept we often tackle here is how big should the libraries be and what size should fragments be.  More importantly we often discuss how much of chemical space a fragment library should cover.  This paper takes an anti-Reymond approach to address that question. 
The Reymond approach tries to determine how big chemical space is, what it looks like, and what portion of it is available.  The Anti-Reymond approach identifies what is available and validates its inclusion in a fragment library.  Here is the last sentence of this paper:
"These findings...verify the value of the benzamide fragment in drug design."
Now, I was worried that benzamidine was not a valuable fragment.  This paper has removed all doubt in my mind.  Now that is settled, we can go on an validate the other 165, 999, 999,999 other possible fragments. 

22 June 2015

Fragments vs P2X1

Four years ago we highlighted a paper in which researchers performed a fragment screen against ion channels. There have been other occasional reports, but for the most part this has been a quiet area. A new open-access paper in Neuropharmacology by Andrew Thompson and collaborators at Cambridge University, University of Bern, VU University Amsterdam, and Washington State University provides another case study.

The researchers were interested in the P2X1 purinergic receptor, which allows calcium ions to pass into cells when ATP binds. An antagonist could be a safe anti-clotting agent as well as a potential male contraceptive. However, the only reported inhibitors are freakish molecules like suramin.

The paper is heavily focused on assay development and validation, in this case using cells stably transfected with P2X1. These were loaded with a voltage-sensitive fluorescent dye: when the channel opens, fluorescence increases. (Control cells not expressing P2X1 do not behave this way.) By adding potential ligands first and then adding ATP, both agonists and antagonists could be identified.

The researchers screened 1443 fragments (from IOTA) at 300 µM each. Cell-based fragment screens are rare but not unprecedented. In this case, 46 hits were obtained, and these were retested at multiple concentrations; 39 hits showed dose responses. These were both agonists and antagonists, with EC50 values ranging from low micromolar to above 1 millimolar.

For confirmation, the researchers used a fluorescently labeled analog of ATP that binds to the P2X1 on transfected cells but not to cells that don’t express P2X1; the increased fluorescence of the cells could be visualized using confocal microscopy. Most of the fragment hits reduced the fluorescent signal, suggesting that they block ATP binding.

A structural analysis suggested that the hits are quite diverse, though annoyingly only a single fragment structure is provided. Still, these do look like useful assays, and the paper provides another successful example of fragment screening in a complicated cellular system.

17 June 2015

Fragments vs HIV Reverse Transcriptase - again

Some targets are so heavily studied that you would think there is nothing left to discover. HIV-1 Reverse Transcriptase (HIV-1 RT) is one of these, with 13 marketed drugs against it: half of all anti-HIV drugs. But as Gilda Tachedjian and collaborators at Burnet Institute, Monash University, the University of Pittsburgh, and the University of Melbourne show in a recent (and open-access) paper in Proc. Nat. Acad. USA, there are still new insights to be learned about this target.

The researchers started with an STD NMR screen of 630 Maybridge fragments, each at ~350 µM in pools of up to five. This gave 84 hits – a healthy 13% hit rate. However, when these were tested in a functional assay (RNA-dependent DNA polymerase activity, or RDDP) only 12 showed significant inhibition, of which 6 were better than 1 mM. Testing 14 related compounds led to 2 more hits, for a total of 8 fragments with IC50s from ~70-750 µM. However, one showed signs of aggregation in dynamic light scattering and was not further pursued.

Since HIV-1 RT has been the object of such intensive research, the team looked at the similarity of their fragments to known binders, including those from previous fragment screening. Surprisingly, their hits turned out to be quite distinct.

Next, the researchers looked at the effect of their fragments on the DNA-dependent DNA polymerase activity of HIV-1 RT, and happily found results similar to the RDDP assay above. The 5 most potent fragments were also tested against three clinically important mutants of HIV-1 RT, and while two of them showed reduced activity, the other three were either as potent or even more so. Testing these against unrelated polymerases revealed that they are not merely promiscuous inhibitors.

Of course, functional activity at high concentrations can have all sorts of causes, so the researchers performed a battery of careful enzyme kinetics experiments to ascertain the mechanisms. One fragment turned out to be competitive with respect to deoxynucleotide triphosphate substrate, even though it looks nothing like a nucleotide. Another is competitive with the DNA substrate. In other words, both these fragments operate through different mechanisms of action from clinically approved HIV-1 RT inhibitors.

One of the most potent fragments is a p-hydroxyaniline, which the researchers recognized as a PAINS compound (it can form reactive quinones). However, freshly prepared samples of this fragment were just as active as samples that had been stored in DMSO for months. Also, an analog without the ability to form a quinone was still active, albeit less so.

The p-hydroxyaniline fragment also showed activity in a cell-based assay. Just as with biochemical assays, cell-based assays are also susceptible to false positives, but the kinetics of viral inhibition were consistent with inhibition of HIV-1 RT rather than other other mechanisms. Further work on the compound may be merited; these are exactly the kinds of investigations necessary to decide if an interesting PAINS molecule is worth pursuing.

Unfortunately there is no crystallographic or detailed NMR structural information as to how these molecules actually bind. Previous work has identified multiple fragment binding sites on HIV-1 RT, so further work should eventually reveal how these molecules interact with the protein.

In the end this paper shows that, even in the absence of structure, it is possible to learn a great deal about how fragments inhibit an enzyme. It is also a useful reminder that fragment-based approaches can identify new types of inhibitors even for a target that has been intensively – and successfully – studied for decades.

15 June 2015

Natural Product Derived Fragments against MMP-13

I have been lucky to work on a lot of systems that very much interest me.  I, in particular, love metallo-proteins.  I worked on rubredoxin as a post-doc and when I moved into industry I worked on a slew of metalloproteins.  So, I love it now when I see papers on targets I used to work on.  This paper does exactly that while also letting me riff (later) on Natural-Product-Derived Fragments (NPDF). 

NPDF has a long history in FBDD having been discussed here, here, here, and so on.  Many vendors and some companies have NPDF libraries (whether they call them that or not).  However, these libraries have yet to be proven to be an efficient route for "discovering clinical drug candidates".  Lanz and Riedl set out to do this against MMP-13 (how many of your just said, yeah I worked on that target?).  All MMP-13 clinical candidates with strong ZBG (Zinc-binding groups) have failed.  They are aiming to develop a MMP-13 without a strong ZBG.  Of course, we have seen a LOT of work towards this goal: here, here, and here for example.  The authors propose that the use of NPDF prevents the problem of using fragments with "debatable biological properties".  This seems to the be the argument used by the NPDF people, since these fragments are found in nature they have desirable properties.  I have never bought this line of reasoning for a variety of reasons.  

To their end, the authors selected uracil as their starting NPDF for these reasons: good synthetic starting points, cis amide bonds, and its found in a variety of natural products (nucleic acids).  They docked it in the S1' non-zinc binding site and found a strongly conserved binding site. [For me, and I would imagine a whole lot of people, this fits in the "things you already knew" category.]  The uracil interacted with the NH an CO of Met232 via its cis amide bonds and "addresses" Lys228.  Several compounds were made from the uracil starting point (Figure 1):
Figure 1.  2: 5 uM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 20 uM. 3: 10 nM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 20 uM2: 5 nM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 10 uM
So, in the end, they have created a potent and selective compound.  They did use a NPDF as a starting point.  Making these compounds is not something that bowls me over either for a Technical Difficulty score or Artistic Merit.   However, I would not go so far as to say that they have validated the NPDF approach.  I think to show that a generic approach works you need more than one (relatively well known) target with more than one (relatively well known) fragment. 

08 June 2015

Benchmarking native mass spectrometry

Mass spectrometry (MS) is one of the less common tools to find fragments. In the conceptually simplest approach (native mass spectrometry), you incubate your protein with a putative ligand and ionize the mixture. Fragment binding is detected by an increased mass for the complex, and the strength of binding by the ratio of heavier bound complex peak to protein peak. However, the liquid to gas phase transition is a big step, and often the complex does not survive. Aside from more specialized applications of MS (such as herehere, and here) there aren’t many published examples. A recent paper from Federico Sirtori and colleagues at Nerviano and Università degli Studi di Milano in Eur. J. Pharm. Sci. describes fragment screening by native MS in detail.

The researchers used the reliable model protein Hsp90, which was also used in a previous MS study and in benchmarking other techniques. One of the many benefits of Hsp90 is a wealth of well-characterized inhibitors with a range of affinities, and these were used to calibrate the technique. This turned out to be critical: beyond sample preparation itself (beware non-volatile buffer components), all kinds of parameters can be adjusted including various voltages, temperatures, vacuum strength, and ion source. Get one of these wrong and your non-covalent complex either fails to ionize or blows apart.

In addition to using published data on known compounds, the researchers ran both fluorescence polarization (FP) and surface plasmon resonance (SPR) assays to independently determine dissociation constants. Initially the results from MS (a Q-TOF) were quite different, but after optimization the team was ultimately able to find conditions that gave qualitatively as well as quantitatively similar results for ligands with affinities ranging from picomolar to ~100 micromolar.

Thus encouraged, the team embarked on a fragment screening campaign. The Nerviano fragment library consists of 1914 molecules mostly following the rule of 3, though halogenated fragments up to 380 Da are allowed as are compounds with up to 6 hydrogen bond acceptors. The fragments were run in mixtures of 5, with protein at 2.5 µM and each compound at the low concentration of 10 µM. Sample injection and data processing were automated, and the entire screen took 2 days and 2 mg of protein.

Given the low concentration of fragments, the researchers lowered the bar for potential hits, yielding 282 compounds. These were retested individually, yielding 146 confirmed hits that gave signals of 5.2-29.7% bound protein. This is a high hit-rate, particularly given that these binding levels suggest affinities in the 20-179 µM range. Indeed, only 5 fragments could be competed by a high-affinity binder, suggesting either that the others bind outside the active site or are non-specific (false positives). Regarding false negatives, Nerviano reported the results of an NMR fragment screen against Hsp90 last year, and 12 of 14 hits identified there could also be detected by MS. The other two were likely below the detection limit of the MS assay.

Unfortunately, the researchers do not discuss thermodynamics. In theory enthalpic interactions dominate over entropic interactions in the gas phase, but it is unclear whether any of the observed binders were strongly entropy-driven.

In the end, it appears that fragment screening by native MS is workable, but the sensitivity is probably lower than other techniques. Of course, increasing the ligand concentration would increase the sensitivity to weaker binders, but at the cost of more non-specific binding – which is already considerable. Also, Hsp90 is about the friendliest protein one can imagine. I would be reluctant to try this with a more challenging target that lacks good tool ligands. But if you want to give it a go, this paper provides a wealth of information for getting started. And if you have experience with native MS, please share it in the comments.