How to avoid metal artifacts

Back in 2017 we observed with characteristic subtlety that “heavy metals suck.” That post described a hit-finding campaign which foundered when the apparent activity of the fragments turned out to be due to contaminating zinc. A new paper in J. Med. Chem. by Thomas Gerstberger, Peter Ettmayer, and colleagues at Boehringer Ingelheim (BI) describes a similar story, along with suggestions of how to avoid being misled.

 

BI had a collaboration with FORMA Therapeutics that entailed screening roughly 1.7 million compounds against ten targets using biochemical and cell-based assays. The effort resulted in chemical probes against BCL6 and SOS1 and a clinical compound against the latter. Another target was the activated (GTP-loaded) form of KRAS^G12D. Of the 6917 hits from the primary AlphaScreen assay, 1535 gave dose-response curves and passed various counter screens. Of these, 87 representative compounds were tested in STD NMR and thermal shift assays. Only seven confirmed by STD NMR, but these did not confirm by SPR or crystallography.

 

In parallel, the researchers were successfully using FBLD to develop inhibitors of KRAS^G12D, which we wrote about here. Some of the fragment hits were structurally similar to those from the HTS screen, and further searching of the FORMA library led to fairly potent (high nanomolar or low micromolar) hits in the AlphaScreen assay. Two of these even yielded crystal structures, though despite their chemical similarity to one another they bound to the protein in completely different orientations.

 

Unfortunately, follow-up work “revealed erratic structure-activity relationships,” and upon resynthesis the compounds were much less active. At this point the researchers became suspicious, and analyses of the original samples showed they contained >20,000 ppm of palladium contamination. Furthermore, PdCl₂ itself turned out to be a low micromolar inhibitor in the assay.

 

Metals are frequently used as catalysts or reagents in organic synthesis and can be difficult to completely remove during purification. Worse, their presence is often not detectable using standard purity assessments such as HPLC and NMR. Particularly in the case of fragments, which are expected to have low affinities, a small amount of metal contaminant could give a reasonable-looking but misleading signal in an assay.

 

To avoid this problem in the future the researchers developed a Metal Ion Interference Set, or MIIS, consisting of a dozen different metal ions and other salts, all soluble in DMSO so as to be compatible with typical screens. The MIIS is now routinely screened before initiating HTS campaigns, and the results of 74 assays are summarized in the paper. Pd²⁺, Au³⁺, and Ag¹⁺ are particularly nasty, often giving IC₅₀ values < 1 µM, but every metal gave IC₅₀ values < 10 µM in at least two assays. Biochemical assays such as AlphaScreen or TR-FRET were more susceptible to artifacts, with 20.9% showing IC₅₀ < 10 µM, while biophysics assays such as mass spectrometry were better behaved, with only 2.3% showing IC₅₀ < 10 µM. Cellular assays were also surprisingly robust, with 6.3% showing IC₅₀ < 10 µM.

 

This is a nice paper showing that even a massive screen may produce no useful chemical matter. Soberingly, the fact that some of the fragments gave reasonable-looking crystal structures even though the functional activity came from metal contaminants is a salutary reminder that just because you have a crystal structure of a bound ligand doesn’t mean you have a viable starting point.

 

Forewarned is forearmed, and the MIIS appears to be a valuable tool for assessing assay sensitivity to metal ions, which are all too often lurking invisibly in compound samples.

Multiplexing (native) mass spectrometry

Native mass spectrometry (nMS) is one of the less commonly used fragment-finding methods. The approach entails mixing proteins and ligands and gently ionizing them under non-denaturing conditions to look for complexes. As with many other methods, multiple fragments can be screened in a single sample. In a new ACS Med. Chem. paper, Ray Norton and collaborators at Monash University and CSIRO report screening multiple proteins in a single sample.

 

The researchers were interested in fatty acid-binding proteins, or FABPs. As their name suggests, these transporter proteins shuttle lipophilic molecules such as fatty acids around cells. The ten human isoforms are expressed in different tissues and have different functions in metabolic signaling, but their similarity to one another has made finding selective chemical probes difficult. Enter nMS.

 

FABP isoforms 1-5 are the most heavily studied, and these were first assessed individually. They ionized well, though in some cases peaks corresponding to both the native protein and a complex with acetic acid was observed, not surprising given that the buffer contained 50 mM ammonium acetate.

 

Next, all five proteins were mixed together at 10 µM each. All the proteins could still be observed (with or without bound acetate), though some proteins did give stronger signals than others due to differences in ionization efficiency.

 

Adding small molecule WY14643, which the researchers had previously found to bind to FABPs in a fluorescence polarization (FP) assay, led to a more complex spectrum, with peaks corresponding to unbound proteins, proteins bound to WY14643, proteins bound to acetate, and proteins bound to both acetate and WY14643. When WY14643 was added at 10 µM, the selectivity profile was consistent with the FP data. Interestingly though, when ligand was added at the total concentration of all protein isoforms (50 µM), the selectivity profile changed. The researchers suggest this may be due to nonspecific binding at higher ligand concentrations, as has been seen previously for nMS.

 

To explore the generalizability of multiplexing nMS, the researchers turned to more potent (nanomolar) ligands. As with WY14643, these molecules showed good agreement with published selectivity rankings at lower ligand concentrations with some non-specific binding at higher concentrations.

 

When I first wrote about nMS back in 2010, I noted that “the stability of protein-small molecule complexes in native mass spectrometry assays does not necessarily correlate with the (more relevant) solution-phase affinity,” and this fact is investigated in the paper. Careful optimization of the experimental conditions, including ionization voltage and temperature, led to good relative selectivity rankings for a given ligand across the different FABP isoforms but differences in absolute values from those measured by ITC.

 

Another challenge is the fact that the five FABP isoforms tested have similar molecular weights; in one case a ligand complexed with FABP3 was difficult to distinguish from free FABP2. The researchers could solve this by using different protein constructs, such as a hexa-histidine-tagged version of FABP3.

 

Overall this is an interesting approach, and the paper does an excellent job describing the technical details and limitations. Along with protein-observed ¹⁹F NMR, mass spectrometry is a rare experimental technique suitable for screening mixtures of proteins in solution. Indeed, this becomes even easier when screening covalent binders, as seen in this paper from 2003, since there is no need to worry about ligand dissociation during ionization. And with the increasing interest in covalent drugs, the use of MS is only likely to increase.

SAR by TR-HT-SAXS

Well that’s an acronym soup! SAR by NMR was the first practical fragment-finding method, and over the years Practical Fragments has covered lots of other techniques. Small-angle X-ray scattering, or SAXS, has not been among them. As the name suggests, this technique uses X-rays, typically produced at a synchrotron. However, unlike conventional crystallography, it doesn’t require crystalline material. Instead, proteins in solution are analyzed to provide information on their size and shape. The resolution is too low to assess small molecule binding, but suitable for observing dimerization or changes in conformation.

 

Time-resolved SAXS, or TR-SAXS, examines SAXS over time in response to a trigger. For example, you can rapidly add a ligand to a protein and watch for changes in conformation. And HT simply means high throughput. A recent Nature Chemical Biology paper from Chris Brosey, John Tainer, and collaborators at the University of Texas MD Anderson Center, Lawrence Berkeley National Laboratory, University of California Santa Cruz, and University of Arkansas for Medical Sciences Little Rock describes structure-activity relationships by time-resolved high throughput small-angle X-ray scattering (TR-HT-SAXS).

 

The researchers were interested in apoptosis-inducing factor (AIF), a mitochondrial protein with potential implications for cancer and other diseases. AIF normally exists as a monomer in complex with an FAD cofactor. Binding of NADH causes reduction of FAD to FADH^- and concomitant dimerization of the protein. Could fragments do the same, allowing dimerization on demand?

 

A library of 2500 fragments purchased from Life Chemicals was screened at 0.75-1.5 mM against the AIF-FAD complex using differential scanning fluorimetry (DSF), and those that raised or lowered the temperature by more than 1.7 ºC were further characterized by microscale thermophoresis (MST). This led to 32 binders and 7 negative controls, or molecules that did not confirm either by DSF or MST. (Side note: although many people discount compounds that give negative thermal shifts, the natural ligand NADH lowers the melting temperature of AIF by a whopping 10.8 ºC.)

 

Next, the fragment binders and negative controls were screened at 0.5-1 mM by TR-SAXS. Intense X-rays cause reduction of the FAD cofactor, but in the absence of NADH or other ligands the AIF protein remains monomeric. However, some fragments did cause dimerization of the protein during TR-SAXS. Interestingly, these fragments were structurally related to one another. Subsequent crystallography revealed that they bind where NADH normally binds and make some of the same interactions to induce protein dimerization. The paper includes much more detailed characterization, including mutagenesis, spectroscopic, and protein crosslinking experiments to further understand the mechanism.

 

TR-SAXS is an interesting addition to our toolbox of biophysical methods suitable for fragment screening. It does have some disadvantages, such as the need for large amounts of protein at high concentrations: 67 µM in this case. Also, the “HT” may be somewhat aspirational, with a current throughput of 100-200 compounds per synchrotron shift. Finally, the technique is probably best suited to well-characterized proteins where SAXS data can be carefully modeled. With these limitations in mind, it will be fun to see how generally TR-SAXS finds fragments that alter the conformation and multimerization of proteins.

Fragment-based Drug Discovery Down Under (FBDD-DU) 2024

The end of June brought me to Brisbane for the fifth FBDD-DU Conference, which was meeting for the first time outside Melbourne. This was also my first FBDD-DU conference since 2019, and it was nice to see a wide range of talks from around Australia and beyond. As always, I won’t attempt to be comprehensive, so if you attended, please feel free to add your observations.

 

Techniques

Experimental techniques received considerable attention. Félix Torres (NexMR) described using an inexpensive benchtop NMR that doesn’t require liquid helium. Fragments were screened using photochemically induced dynamic nuclear hyperpolarization (photo-CIDNP). The method is so rapid that it is limited more by sample handling than data collection, and the Torres team is speeding things up using flow technology. Right now photo-CIDNP is still very much DIY, but rumor has it that Bruker may soon launch a photochemical module for their benchtop instrument.

 

We’ve written about high-throughput crystallographic screening at the Diamond Light Source, and synchrotrons around the world are building similar platforms. Kate Smith described integrated systems at the Swiss Light Source which automate crystallization, fragment screening, data collection, and data processing. She also described increasing automation of fragment screening using the free-electron laser (FEL), which we wrote about here. Current throughput is around 40 compounds per day and requires large amounts of protein, but these are still early days.

 

Australia is building their own high-throughput crystallography platform, and various components were described by Roxanne Smith (University of Melbourne), Gautham Balaji (Monash Univesrity), and Yogesh Khandokar (ANSTO-Australian Synchrotron). Watch this space!

 

Speaking of Australia, Nyssa Drinkwater described Compounds Australia, a national repository of more than 2.5 million molecules, including several fragment collections. Members, who can be from outside Australia, can store their own libraries within the facility to ease collaborations with other groups, and they can also access public libraries of compounds, including unusual Antipodean natural product extracts. I was fortunate to be able to visit the facility at Griffith University and can attest that it is easily the equal of those in large pharma.

 

Turning to mass spectrometry, Sally-Ann Poulsen (Griffith University) described covalent library screening against PRMT5, a target we’ve written about here. Sally-Ann is also a pioneer of (conventionally non-covalent) native mass spectrometry, and she described applying this methodology to screen small molecules against RNA.

 

But the star of the conference was SPR, appearing in multiple talks. Long-time readers may recall an instrument made by SensiQ, with its gradient injection capability to accelerate data collection. This is now marketed by Sartorius, and Lauren Hartley-Tassell (Griffith University) described using it to screen a glycoprotein. The larger plumbing in the instrument is less prone to clogging, and Lauren said it can even accommodate screening of whole cells.

 

Anything to accelerate the (sometimes painful) process of advancing fragments is always welcome. As Jason Pun (Monash University) noted, eight of nine targets screened in Martin Scanlon’s group started with fragments having affinities worse than 100 µM. Off-rate screening, an SPR technique we wrote about here, can rapidly identify more potent molecules from crude reaction mixtures, but data processing can be tedious. Jason described new software tools to automate this process, and hopefully he will publish the methodology and code. (An aside: over coffee Yun Shi of Griffith University noted that off-rate screening, or ORS, should really be called off-rate constant screening, which would give the more amusing acronym ORCS.)

 

Targets

Turning to targets, Ben Davis (Vernalis) described a collaboration with Servier to advance oncology target USP7 inhibitors from a literature fragment to a preclinical candidate. Crude reaction mixture screening was used extensively, not just by SPR but even in microsome stability studies. Unfortunately the project ended when on-target toxicology effects emerged, which were perversely more severe in higher animal species than they were in mice.

 

Yun Shi described finding tiny heterocyclic fragments that react with the NAD⁺ cofactor of neurodegenerative target SARM1 in situ to generate a potent inhibitor, as we wrote about here. Yun is using ¹⁹F NMR to follow the base-exchange reaction to identify inhibitors to other glycohydrolases too.

 

Deaths due to E. coli are – somewhat surprisingly – more common than those caused by any other pathogen, and Christina Spry described her work at the National Australian University to discover inhibitors of the essential dephosphocoenzyme A kinase (GPCK) enzyme, which catalyzes the final step in the synthesis of Coenzyme A (CoA). Fragment screening by DSF and NMR identified a weak (K_D=380 µM) binder, and fragment growing has led to a low nanomolar inhibitor that is selective against the human form of the enzyme.

 

Continuing the E. coli theme, several talks discussed efforts against the challenging bacterial virulence target DsbA, a twenty-year campaign in Martin Scanlon’s group at Monash as noted by Yildiz Tasdan. The enzyme has a shallow, hydrophobic active site, but the discovery of fragments binding to a cryptic site and crude-reaction screening by ORS (ORCS?) and affinity-selected mass spectrometry (ASMS) has finally led to molecules with dissociation constants around 1 µM.

 

Finally, in his closing keynote address Alvin Hung, who recently founded NeuroVanda, described a wide range of fragment success stories, many of them covered on Practical Fragments, against targets including pantothenate synthetase, GSK3β, PKC-ι, and MNK1/2. Although structural enablement helped in many cases, Alvin was not rigid about the need for atomic-level details: in response to the question whether he would advance a fragment in the absence of structure, he answered simply, “of course.” Perhaps it's time to redo my poll on this subject.

 

I’ll wrap up here, but if you missed this or earlier events this year there are still a couple more conferences in Boston, and 2025 is already starting to take shape.

Fragment events in 2024 and 2025

The year is half-way done, and we've seen some great events; I'll share my thoughts on FBDD Down Under 2024 next week.

Boston is where it's at in the second half of 2024, and it's not too soon to start planning for 2025.

September 22-25: After a six year hiatus, FBLD 2024 will be held in Boston. This will mark the eighth in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2018, FBLD 2016, FBLD 2014, FBLD 2012, FBLD 2010, and FBLD 2009. Early-bird registration ends August 12, so don't delay!

 

September 30 to Oct 3: Autumn is usually a nice time of year in Boston, so stick around to attend CHI’s Twenty-Second Annual Discovery on Target. As the name implies this event is more target-focused than chemistry-focused, but there are always plenty of FBDD-related talks. You can read my impressions of the 2023 meeting here, the 2022 meeting here, the 2021 event here, the 2020 virtual event here, the 2019 event here, and the 2018 event here.

 

Finally, from December 3-5 CHI holds its first-ever Drug Discovery Chemistry Europe in beautiful Barcelona. This will include tracks on lead generation, protein-protein interactions, degraders, and machine learning, with several fragment talks. (Updated July 8.)

 

2025

April 14-17: CHI’s Twentieth Annual Fragment-Based Drug Discovery, the longest-running fragment event, returns as always to San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of the 2024 meeting here, the 2023 meeting here, the 2022 event here, the 2021 virtual meeting here, the 2020 virtual meeting here, the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting here, here, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here. 

 

May 5-6: Returning after a four five year hiatus, Industrial Biostructures of America will be held in Cambridge, MA and includes a session on FBLD. (Updated Aug 21 and Oct 6.) 

  

Know of anything else? Please leave a comment or drop me a note.