Combining fragments and HTS hits to target PHGDH

Boehringer Ingelheim has been on something of a tear reporting new chemical probes for difficult targets – see here for their NSD3 inhibitor and here for their RAS inhibitor. This is part of an ambitious effort to develop probes for the entire human proteome by 2035. In a new paper published in J. Med. Chem., Harald Weinstabl and collaborators at BI and Shanghai ChemPartner describe the discovery of BI-4924, a potent inhibitor of phosphoglycerate dehydrogenase (PHGDH).

The enzyme is the rate-limiting step in serine synthesis, and has been implicated in multiple types of cancers. However, metabolic enzymes such as PHDGH are particularly challenging drug targets for several reasons: cofactors such as NADH are present at high concentrations in cells, the substrate binding pocket is both shallow and polar, and one often needs near complete inhibition to see an effect. Thus, the researchers chose multiple approaches.

An STD NMR fragment screen was conducted against the apo form of the protein (250 µM fragment and 20 µM protein) to find compounds that would bind in the NAD⁺-binding site. Of 1860 fragments screened, 60 hits were identified, and 19 of these gave measurable dissociation constants in an SPR assay and were selective against two other proteins. Compound 9 was found crystallographically to bind in the adenine pocket of the NAD⁺-binding site. Fragment growing was challenging due to the “kinked shape” of this pocket: elaborated molecules tended to point out of the pocket into solvent. Careful design led to modest improvements in potency (compound 11), and adding a negatively charged moiety led to potent molecules such as compound 43. To avoid problems with permeability, the researchers tried various uncharged bioisosteres, but these were not tolerated. Interestingly, crystallography revealed that the carboxylic acid does not seem to make specific interactions with the protein; its necessity may be due to long-range electrostatic interactions with multiple nearby basic residues.

In parallel, a biochemical HTS screen of more than a million molecules yielded 27,000 hits, which were whittled down to 11,250 that confirmed and didn’t interfere with the assay. Removing PAINS and large, lipophilic molecules narrowed the set to 4750 compounds. Further rigorous assessment included biophysical methods, as recently recommended. Aware of the potential for metal contaminants to give false positives, the researchers examined select samples with inductively coupled plasma mass spectrometry and found that some contained mercury or copper, which inhibited the enzyme. Ultimately 77 hits were validated with dissociation constants better than 300 µM, including compound 8, which crystallography revealed binds in a similar manner to fragment 9.

Combining information from both campaigns and growing to engage an aspartic acid side chain ultimately led to BI-4924. This compound is soluble, stable, and selective against other dehydrogenase enzymes. Unfortunately, the carboxylic acid moiety does indeed impart low permeability, and perhaps because of this the molecule has only low micromolar activity in cells. However, the ethyl ester (BI-4916) transiently accumulates in cells and modulates serine levels.

Unfortunately, the researchers appear to have been scooped; as they politely note, “subsequently, these findings were independently confirmed….” As it stands BI-4916 is too unstable for use in vivo. Still, it could be useful for further unraveling the biology around serine biosynthesis and its role in cancer cells, and the paper itself stands as a nice example of structure-based lead design combining information from multiple sources.

Biophysics beyond fragments: a case study with ATAD2

Three years ago we highlighted a paper from AstraZeneca arguing for close cooperation of biophysics with high-throughput screening (HTS) to effectively find genuine hits. A lovely case study just published in J. Med. Chem. shows just how beneficial this can be.

Paul Bamborough, Chun-wa Chung, and colleagues at GlaxoSmithKline and Cellzome were interested in the bromodomain ATAD2, which is implicated in cancer. (Chun-wa presented some of this story at the FragNet meeting last year.) Among epigenetic readers, bromodomains are usually quite ligandable, but ATAD2 is an exception, and when this work began there were no known ligands.

Recognizing that they might face challenges, the researchers started by carefully optimizing their protein construct to be stable and robust to assay conditions. This included screening 1408 diverse compounds, none of which were expected to bind. Disturbingly, a TR-FRET screen at 10 µM yielded a 4.1% hit rate, suggesting many false positives. Indeed, when an apparently 30 nM hit from this screen was tested by two-dimensional ¹⁵N-¹H HSQC NMR, it showed no binding. The researchers thus made further refinements to the protein construct to improve stability and reduce the hit rate against this “robustness set.”

This exercise illustrates an important point: make sure your protein is the highest quality possible!

Having done this, the researchers screened 1.7 million compounds and obtained a relatively modest 0.6% hit rate. Of these 9441 molecules, 428 showed dose response curves and were tested using SPR and HSQC NMR. In the case of SPR, the researchers also tested a mutant form of the enzyme that was not expected to bind to the acetyl-lysine mimics being sought. Most of the hits did not reproduce in either the SPR or the NMR assays, and at the end of the process just 16 closely related molecules confirmed – a true hit rate of just 0.001%!

Compound 23 is the most potent molecule disclosed, but the researchers mention a manuscript in preparation that describes further optimization. The compound shows promising selectivity against other bromodomains; it certainly doesn’t look like a classic bromodomain binder. X-ray crystallography revealed that the binding mode is in fact different from other bromodomain ligands. Trimming down compound 23 produced compound 35, which shows reasonable activity and ligand efficiency.

This paper nicely demonstrates the power of biophysics to discern a still small signal in a sea of noise. As the researchers note, PAINS filters and computational approaches would not have worked due to the sheer diversity of the misbehaving compounds. (That said, if the library had been infested with PAINS, the false positive rate would have been even higher!)

The paper is also a good argument for FBLD. Compound 35 is probably too large to really qualify as a fragment, but perhaps related molecules could have led to this series. And GSK also discovered a different series of potent ATAD2 inhibitors from fragments, which Teddy wrote about.

Inhibition in Solution Assay (ISA) – on a surface

As illustrated by our poll, surface plasmon resonance (SPR) is one of the most widely used techniques for finding fragments. However, as commonly practiced, SPR – like all techniques – has drawbacks. For one thing, despite impressive recent advances, it is still not particularly high throughput. Also, the protein is typically immobilized on a sensor chip, and the detection of binders depends on the mass ratio of the ligand to the protein. With larger proteins and smaller fragments, this can quickly push the signal below the noise.

A seemingly simple solution is to reverse the experiment: immobilize the small molecule and add the (comparatively large) protein to get a whopping signal. Indeed, this is the approach that Graffinity (now part of NovAliX) takes, and is conceptually similar to work done at RIKEN. However, both these techniques require dedicated surfaces functionalized with fragments.

In a recent paper in J. Med. Chem., Stefan Geschwindner, Jeffrey Albert, and colleagues at AstraZeneca sought to simplify matters. Their idea is to immobilize a single high-affinity molecule to a chip. Protein in solution should give a good signal when the protein binds to the surface, and adding competitor to the solution should decrease protein binding to the immobilized target compound, thereby reducing the signal. They call this the “inhibition in solution assay”, or ISA.

The researchers used the protein PDE10A as a test case and attached a previously characterized small molecule to the surface; this modified small molecule has an IC₅₀ of 991 nM for the target. They then used two different approaches for detecting interactions, SPR (GE/Biacore) and a 384-well plate-based optical waveguide grating (OWG) from SRU Biosystems. Both formats work and give comparable results for a set of molecules ranging in affinities from 40 nM to 0.5 mM.

One nice feature of this approach is that, as a competition assay, it should only identify molecules that are competitive with a known binder. On the flip side, ISA does require a reasonably potent binder for your protein, and you must be able to modify this molecule such that it can be immobilized to the surface. And of course, there are still problems at high concentrations; the researchers mention that high loading of immobilized small molecule can cause other molecules to stick to the surface. Still, this is an interesting approach that should be easily applied to many systems. I’d be curious to know whether you’ve tried it or a variant, and how it compares to more conventional SPR methods.

Another (impractical) NMR Screening Method

NMR has a checkered history in drug discovery. In the 90s, it promised to deliver structures just like X-ray. Strike 1! After that, especially after the advent of SAR by NMR, it promised to deliver boatloads of hits from screening. Strike 2! After that, pharmaceutical NMR worked hard to make sure that it was impactful and value-added. It found niches in which it thrives, e.g. a variety of -omics. In drug discovery, NMR still needs to realize it is living with two strikes. How can NMR survive and even thrive? Quite simply. NMR needs to provide rapid, robust, and easily understandable data to medchemists that leads to decisions. Data that results in no action has no value.

In this paper, Salvia et al. present a ligand-based NMR screening method using "long-lived states (LLS)" of the ligand to boost the sensitivity of ligand-based screening. This new method provides 25x better signal-to-noise than established (T1rho) methods and uses less protein. One of the benefits of this method is it allows NMR to study interactions as tight as 100nM and up to 1 mM.

The graphical abstract (above) shows that while this method is very similar in concept to other ligand-based methods (TOP: equilibrium between NMR differentiated states) it requires much more work than these other methods (Bottom: titrations of ligands). The data (Below) does generate quite satisfying curves, and as noted by the authors, are in agreement with previously published values.
I think this work, while an interesting application of Long Lived States, has really no practical value to the screening world. The strength of the binding can be too strong, making the bound lifetime too long, and thus there is a practical floor for Kd. Of course, because it is based upon kinetics, it can be very different for every system.

If you want to determine Kds for a complex < 10uM there are better, far more robust methods (SPR, for one). The amount of time and effort required to generate Kds from this method seems to run contrary to the tenets I described above (rapid, robust, and (most importantly) easily understandable). To me, the title of the paper simply does not deliver. This method is NOT a screening application. A screening application is one experiment (NMR or otherwise) from which you can determine whether a compound is binding or not, ideally from a mixture of compounds.

I would be curious to see in the comments if anyone (especially our NMR savvy readers, you know who you are) think that this method has practical applications.

Fragments vs Abl: antagonists and agonists

A common concern with using biophysical techniques to identify fragments is that the functional implications of identified binders are not always clear, an issue we’ve discussed previously. In a new paper in J. Am. Chem. Soc., Wolfgang Jahnke (co-editor of the first book on FBDD) and colleagues at Novartis describe a clever NMR approach to address this problem and identify both agonists and antagonists that bind to an allosteric site on the protein tyrosine kinase Abl.

Abl is less well known than its famous cousin, Bcr-Abl, an oncogenic fusion protein in which the kinase activity is always turned on. Bcr-Abl is targeted by imatinib and a number of other kinase inhibitors; indeed, the success of imatinib against certain types of cancer has been largely responsible for the rush to develop drugs targeting kinases.

Most kinase-targeted drugs (including imatinib) bind in or near the conserved ATP-binding site. However, Abl offers another binding site, a pocket that can be filled by the fatty-acid myristic acid. This interaction causes conformational changes in the protein, stabilizing an inactive state. Indeed, previous research had identified molecules that bind in this pocket and block activity. Jahnke and colleagues used NMR screening of a 500-fragment library to try to identify new chemical scaffolds.

Several fragments were identified, some of which bound relatively tightly as judged by NMR and ITC. However, these fragments did not inhibit kinase activity. Crystallographic analysis of the fragments bound to Abl revealed that, although the fragments do bind in the myristate pocket, their binding modes are incompatible with the conformational changes needed to inhibit the kinase. Realizing that a specific valine residue is structurally disordered in the absence of myristate, the researchers established an NMR assay using Abl in which valine had been isotopically labeled to assess which molecules bind in a similar fashion to myristate (and thus block activity).

But what of the molecules that bind in the myristate pocket without causing conformational changes? Some of these can actually activate the kinase by competing with endogenous myristoyl groups. Fragment-based discovery of agonists is not unprecedented (see for example here and here), but it is rare. Assays such as the one described here to distinguish between different conformations of a protein could be practical complements to approaches that focus on binding alone. The paper is also a useful reminder that binders are not necessarily inhibitors, and can in fact be just the opposite.

Guest Blogger: Brian Stockman

[DrZ: Most of you probably know Brian and his excellent work in NMR and drug discovery, especially fragments. I have asked Brian to summarize his most recent paper for us. Below is his contribution. The Editors would welcome others to do the same if they are so inclined.]


A recent paper from Pfizer [Chemical Biology & Drug Design 73, 179-188 (2009)] described the concerted use of NMR screening, competition binding, TROSY-based binding site mapping, and NMR-based activity assays to identify allosteric fragment activators of 3-phosphoinositide-dependent kinase-1 (PDK1). This protein kinase presented an interesting challenge since, in addition to the ATP site typically targeted by structure-based drug design efforts, it was known to have an allosteric site that could activate (or potentially inhibit) activity.

An STD-based NMR screen resulted in 372 fragment hits out of 10,237 fragments screened. Testing the compounds in an activity assay would normally eliminate the many false-positive artifacts of the STD assay. A first pass of the hits through a Kinase-Glo assay revealed that many were in fact inhibitors. Fragments without activity in this assay, however, could not be discarded since this assay was not capable of monitoring events at the allosteric site and could not distinguish ‘non-inhibitors’ from activators. Thus fragments that did not inhibit in the Kinase-Glo assay were also run in a Caliper assay. This assay uses a shorter peptide substrate and is capable of detecting inhibition and activation. Ultimately, a subset of the original fragment hits that were either inhibitors with high ligand efficiencies, activators, and/or had very novel chemical structures were chosen for further studies.

STD competition binding experiments using the known ATP-site binder staurosporine or a short peptide known to bind in the allosteric pocket were very useful to distinguish these two binding sites. TROSY-based binding site mapping, using 15N-labeled PDK1 expressed in baculovirus, was used to confirm the binding site for several key compounds. Finally, the biochemical assay data was complemented with 19F NMR-based activity assays. These assays used the 2-fluoro-ATP method described in a previous paper from Pfizer [Journal of the American Chemical Society 130, 5870-5871 (2008)].

NMR-based activity assays proved very valuable since they could easily handle high fragment concentrations, and, since they directly monitor conversion of substrate to product, were capable of detecting both inhibition and activation. NMR-based activity assays are single-enzyme assays. As such, they are quite useful as both primary fragment screening assays and as orthogonal HTS-triage assays. NMR-based activity assays have been characterized as the ‘uncola’ of biochemical assays because, as opposed to many HTS and bench top assays, they do not rely on any coupling enzymes for their detection. NMR-based activity assays should prove very valuable for accurately evaluating compounds in the 10 uM to 1 mM dynamic range of activity typical of fragments.