21 September 2020

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 We’ve covered some of his more recent work using shapely fragments here, and in unpublished work he has been screening the dual-domain construct of BRD4 and finding fragments that are ten-fold selective for one over the other bromodomain.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.

13 September 2020

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!

07 September 2020

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.