Crude reaction screening by crystallography

Getting from a fragment to something more useful can be time consuming, and frustrating. Often it requires making lots of inactive analogs. It is easy to set up multiple chemical reactions in parallel, but purifying products is tedious. Thus, there has been a trend towards screening crude reaction mixtures. Off-rate screening (ORS) using SPR was published back in 2014, and other techniques have been used as well. Now, two recent papers describe using crystallography as the assay.

 

In a J. Med. Chem. paper, Bradley Doak, Martin Scanlon, and collaborators at Monash University, La Trobe University, University of Wollongong, and Queensland University of Technology describe Rapid Elaboration of Fragments into Leads using X-ray crystallography (REFiL_x; see also here). The researchers note that ORS is best for identifying reasonably potent compounds, with low (<10) micromolar potency or better. For some targets, this is a bar too high. Specifically, they were interested in E. coli disulfide bond forming protein A (EcDsbA), an anti-virulence target for which they had found – after considerable effort – a 490 µM fragment.

 

The fragment contained a carboxylic acid, which could be conveniently used for amide bond formation, and the researchers decided to make a bespoke library around it. A collection of 93 small (5-12 heavy atoms) amines was assembled and each member was reacted with 2 micromoles (about 0.5 mg) of the fragment in a 96 well plate. The reactions were then evaporated and dissolved in DMSO to 100 mM concentration (assuming the reactions went to completion). HPLC-MS analyses revealed likely product for 83 reactions with yields up to 90%, though the average was closer to 25%. These crude reaction mixtures were then soaked into previously grown crystals of EcDsbA and analyzed crystallographically using both manual and automatic processing (including PanDDA).

 

The result was four hits, all of which were resynthesized and characterized in detail. Crystallography of the pure compounds confirmed the binding modes found in the crude reaction mixtures. Gratifyingly, one of the compounds bound 8-fold more tightly than the initial fragment, as assessed by two dimensional NMR.

 

The four hits all had purities >50% in the crude reaction mixtures, so the researchers resynthesized a few other non-hits that had lower yields. One of these was about 2-fold better than the original fragment, suggesting a false negative in the first screen. Experiments in which the most active hit was spiked into faux reaction mixtures at increasingly lower concentrations revealed – as expected – that detection was more difficult at lower concentrations.

 

A similar approach is described in Communications Chemistry by Rod Hubbard and collaborators at Vernalis, University of York, Diamond Light Source, University of Oxford, and University of Johannesburg. However, whereas the first paper focuses on trying to find improved hits against a difficult target, this paper focuses more on methodology. The researchers used two of the same protein targets for which they had previously performed off-rate screening, HSP90 and PDHK2. Conveniently, both these ATPases share some inhibitors, so the same set of 83 crude reaction mixtures could be used for both enzymes. Also in contrast to the EcDsbA example, the starting fragments (found in prior screens) were considerably more potent, as were the final molecules.

 

The researchers note that “as with any high-throughput experimental approach, there are false positives and false negatives.” Because off-rates were measured for all the crude reaction mixtures, the researchers could assess false positives and false negatives in the crystallographic screens. In false positives, products were seen in the crystal structures even though their affinities were no better than the reactant. In false negatives, starting material was seen in the crystal structures even though the products had better affinities. Together, these accounted for about half the results. Digging into the details, poor reaction yields and low solubility for the products accounted for many of the false negatives. False positives are harder to explain, though the researchers note that the buffer used for crystal soaking is different from that used for SPR.

 

There were also some notable successes: for one racemic compound, crystals of PDHK2 “correctly” selected the more potent enantiomer (K_d = 0.14 µM) over the less potent one (K_d = 17 µM). As in the first paper, there is a wealth of experimental details, including improvements to PanDDA protocols. The researchers performed the soaks in triplicate, and although this did lead to an increased number of hits, they note that singleton screening would probably be sufficient for most applications.

 

At the CHI FBDD meeting last month Frank von Delft, who is an author of the second paper, noted that he is increasingly using crude reaction screening to progress fragments, including against SARS-CoV-2. I look forward to seeing this approach, and the leads that come from it, advance.

Eighteenth Annual Discovery on Target Meeting

Last week CHI held Discovery on Target – virtually of course. There were 20 tracks over three days and more than 650 attendees, down from 1100+ last year. Because the event was more fragmented (pun intended) than the recent DDC, which had at most four parallel tracks, the Q&As and discussions seemed smaller, though that could have just been the ones I attended. On the other hand, one of the huge advantages of the format is being able to watch concurrently scheduled talks later. More thoughts on virtual conferences are here, and if you have not already done so please take our poll on the right side of the page.

 

This conference has always been more biology-focused than DDC, with tracks on antibodies, immunology, NASH, gene therapy, disease modeling, and fibrosis, among others. But there were also plenty of talks on targets and methodology, which is where I’ll focus most of this post. Please add your highlights and thoughts in the comments.

 

Julien Orts (ETH Zurich) presented an update on his NMR² method, which uses information from intermolecular NOEs to computationally determine protein-ligand structures without requiring full NMR assignment of the protein. We wrote about this technique in 2017 and at the time we questioned how applicable it would be to fragments due to low affinities, multiple binding modes, and fewer contacts. As it turns out, very: Julien described successes with proteins including HDM2, DsbA, bromodomains, and Pin1. Even with as few as 10-12 intermolecular NOEs he has been able to get good agreement with crystal structures. Currently he is applying this approach to SARS-CoV-2 proteins as part of the COVID-19-NMR project.

 

William Pomerantz (University of Minnesota Twin Cities) also presented NMR techniques. He is particularly known for his protein-observed ¹⁹F (PrOF) NMR screening, in which fluorinated tyrosine and tryptophan residues are introduced into proteins. Ligand binding changes the chemical shifts of the fluorine atoms, and by varying the concentration of the fragment, accurate dissociation constants can be determined. In early work, a screen of 930 fragments in pools of 5 against BRD4 took 11 hours (and another 11 hours for deconvolution) and provided multiple hits. We’ve covered some of his more recent work using shapely fragments here, and in unpublished work he has been screening the dual-domain construct of BRD4 and finding fragments that are ten-fold selective for one over the other bromodomain. He is further improving throughput by screening two proteins simultaneously.

 

Rounding out NMR, Andrew Petros (AbbVie) presented a beautiful fragment-to-lead success story on TNFα, a trimeric cytokine that has been the subject of numerous (often unsuccessful) lead-discovery efforts. A 2-dimensional NMR screen of 18,000 fragments gave just 11 hits. Crystallography of one showed two copies binding in close proximity, and linking these ultimately led to a low nanomolar binder. The series showed high clearance and no oral bioavailability, so they performed additional screens to identify different fragments that were ultimately advanced to potent compounds with animal efficacy. I look forward to reading the paper when it is published.

 

Finding fragments is important, but so is avoiding artifacts, the subject of a talk by Samantha Allen (Janssen). Around 2% of screening compounds can form small molecule aggregates that can interfere with assays, and if these aren’t weeded out they can quickly overwhelm an assay. Samantha described the use of resonance waveguide grating (RWG) technology, as used in the Corning Epic BT. This label-free technology is similar to SPR, but RWG can be run in 384 or 1536 well plates. Samantha showed that RWG compares favorably to dynamic light scattering for detecting aggregates. It is also 4-5 times faster and less prone to false-positives.

 

Covalent fragments were a theme of last month’s DDC, and they were prominent here as well. Four years ago we highlighted work out of Ben Cravatt’s lab doing covalent fragment screening in cells, but this was a rather time-consuming process. Steve Gygi (Harvard) has streamlined activity-based protein profiling and was able to screen 288 fragments in just 7 days and identify more than 1500 modified cysteine residues.

 

Dan Nomura (UC Berkeley) continued the theme with a wide-ranging presentation using chemoproteomics to discover covalent ligands for a variety of targets, including new E3 ligases, which can be used for developing targeted protein degraders. (Shameless plug/disclosure: Dan Nomura is a founder of my company, Frontier Medicines, and we are actively hiring across multiple positions and levels.)

 

Targeted protein degraders such as PROTACs were the subject of one track at last year’s DoT meeting, and this year two sequential tracks were devoted to the topic. As I suggested in 2018, fragments could be ideal starting points given that high affinity is not always necessary. This year, Stewart Fisher confirmed that he and his colleagues at C4 Therapeutics often “detune” chemical matter, lowering the binding affinity to get efficient degraders. That doing so can improve physicochemical properties is a nice bonus.

 

Finally, although not directly fragment-related, William Kaelin (Dana Farber Cancer Institute) gave an inspiring talk on the discovery and development of MK-6482, an allosteric HIF2α inhibitor in late-stage clinical trials for cancer linked to Von Hippel-Lindau disease; data released just last week shows durable responses in patients with kidney cancer. The science itself was lovely, but he reminded us of the ultimate stakes: “It’s not about what journal your paper is published in or whether you can fool reviewer 3, it’s about whether you publish things that are true and robust and can be built upon by others.”

 

Words to live by.

Poll: Conferences in the age of COVID-19

The last fragment-relevant conference of 2020 is happening this week – virtually of course. I attended my first virtual conference last month and wrote up my thoughts here. As I noted, I look forward to the resumption of in person events. However, despite rapid progress, we do not know when a safe, effective COVID-19 vaccine will be widely available.

 

In the meantime, conference organizers need to plan – usually months ahead. If you would like to help, please answer the brief (six question) survey on the right-side of the page. Also, please leave comments below – anonymously if you’d prefer – on what virtual conferences you’ve attended, what worked, and what didn’t. We'll post results early next month.

 

Hope to see you next year – ideally in-person!

From noncovalent to covalent fragment for NSD1

As evident at the CHI Drug Discovery Chemistry meeting a couple weeks ago, covalent fragment-based lead discovery is becoming increasingly popular. Normally this entails screening a library of electrophile-containing fragments. However, it is also possible to start with a noncovalent fragment and add the “warhead” later. This is the approach taken by Jolanta Grembecka, Tomasz Cierpicki, and collaborators at University of Michigan, Memorial Sloan Kettering Cancer Center, and Columbia University in a paper just published in Nat. Chem. Biol.

 

The researchers were interested in NSD1, one of three related histone methyltransferases whose potential role in cancer is uncertain – in part due to the lack of good chemical probes. They started with a two-dimensional (¹H-¹⁵N HSQC) NMR screen of 1600 fragments in pools of 20, with each fragment present at 250 µM. BT1 was one of the hits, and synthesis of several analogs led to BT2, which showed low micromolar affinity as assessed by ITC as well as an IC₅₀ of 66 µM in a functional assay. Crystallography was unsuccessful, but NMR experiments suggested considerable changes in an autoinhibitory loop that blocks the substrate-binding region of the so-called SET domain.

For whatever reason, the researchers tested thiocyanate analog BT3 and found that this binds covalently as a disulfide to a cysteine in the autoinhibitory loop. They were able to get a crystal structure of BT3 bound to the protein, which revealed significant rearrangements that allow BT3 to bind deep in the SET domain, where it makes multiple polar and hydrophobic contacts. Prudently they chose to replace the unstable thiocyanate warhead, and while acrylamide derivatives were inactive, aziridine BT5 modified the protein as assessed by mass spectrometry.

 

BT5 inhibited NSD1 with an IC₅₀ of 5.8 µM after a four hour incubation and was somewhat selective against the related proteins NSD2 and NSD3 as assessed both by mass spectrometry and activity assays. It also showed good selectivity at 50 µM against 20 other epigenetic enzymes and 291 kinases. Interestingly though, at this concentration BTK was inhibited by 41% and EGFR was inhibited by 49%; both these kinases are targeted by approved covalent drugs.

 

Next, the researchers conducted multiple cell assays. A cellular thermal shift assay (CETSA) revealed that BT5 stabilized NSD1 but not NSD2 or NSD3. Growth of an NSD1-dependent cancer cell line was inhibited with GC₅₀ = 1.3 µM after 3 days, NSD1-mediated histone methylation was suppressed, and several target genes showed reduced expression. BT5 also inhibited growth of non-NSD1-dependent cell lines, though at 6-8 higher concentrations, and did not alter methylation or target gene expression. Finally, the compound impaired colony formation of cells from a primary patient-derived sample with an NUP98-NSD1 translocation.

 

This is a nice, carefully conducted study. Refreshingly, the researchers do not attempt to oversell their results, and acknowledge that “further optimization is needed to develop NSD1 chemical probes.” But they’re off to a good start, and it will be fun to see what they – and others – will come up with.