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.

Seventeenth Annual Discovery on Target

Just as April brings CHI’s Drug Discovery Chemistry in San Diego, September brings CHI’s Discovery on Target in Boston, and last week saw more than 1100 attendees attend 14 tracks over three days, along with associated training seminars and short courses. Fragments made appearances throughout.

Perhaps the most notable new development was an entire session devoted to covalent fragments. We spent three blog posts in June covering five papers on this topic, so this was a timely addition.

Eranthie Weerapana (Boston College) described proteomics methods for analyzing cysteine modification in cells. (Some of this is similar to Ben Cravatt’s work, which we discussed here.) She noted that many mitochondrial proteins have low abundance and are thus hard to detect, but by isolating the mitochondria she has been able to observe about 1500 cysteines on 500 proteins. Selenocysteine-containing proteins are even less common and can thus be lost in the noise, but by lowering the pH these rare beasts can be labeled selectively.

Alexander Statsyuk (University of Houston) gave two wide-ranging talks, and he noted that (beyond acrylamides) there are about 50 warheads that have not been widely explored. We’ve previously covered his work with fragments containing the 4-aminobut-2-enoate methyl ester, and Katrin Rittinger (Francis Crick Institute) discussed her recent use of a small library of just 104 of these to find a covalent inhibitor of LUBAC. And on the subject of very recent papers, I presented work from Carmot and Amgen on the discovery of covalent KRAS^G12C inhibitors.

You know a field is becoming popular when suppliers start selling reagents, and this is certainly the case for covalent fragments: Enamine and Life Chemicals both sell covalent fragment libraries. If you’ve had experience with them or others, please leave comments!

Natalia Kozlyuk (Vanderbilt) gave a nice presentation that emphasized some of the challenges that can arise in FBLD. Screening 14,000 fragments against the protein RAGE by NMR resulted in a number of hits, and crystallography revealed that a couple of these bind just 4 Å apart. Linking these together with variable-length linkers led to one molecule that bound as expected, while in another one of the linked fragments bound in a third site. Unfortunately, even the best dimeric molecules are quite weak, and they also cause the protein to precipitate. This appears to be a different mechanism from “classic” small molecule aggregation, in which the small molecules first form aggregates that block protein activity, though it is not unprecedented.

In a similar vein, Stijn Gremmen and Jan Schultz (ZoBio) described work done with Gotham Therapeutics to discover inhibitors of the methyltransferase complex METTL3/METTL14. HTS-derived compounds caused aggregation of protein as assessed by size exclusion chromatography and multiangle light scattering, but a fragment screen followed by optimization led to potent, well-characterized molecules.

Continuing the theme, Beth Knapp-Reed (GSK) described an HTS assay against the anti-cancer target LDHA in which 1.9 million compounds yielded 560 hits, almost all of which turned out to be false positives due to oxalic acid contamination. Fragment screening was more productive, yielding 16 crystallographically validated hits, and the researchers were able to improve the affinity more than 10,000-fold. And Puja Pathuri discussed successful efforts at Astex to discover ERK1/2 inhibitors (see here for more details).

Finally, last year we noted the increasing number of talks on PROTACs and targeted protein degradation. This year for the first time the conference held a full day and a half long program on the topic. PROTACs typically consist of two-component molecules in which one piece binds to a target of interest and the other piece binds to a protein called an E3 ligase which ultimately causes proteolytic degradation of the target. For example, Michael Plewe described how he and his colleagues at Cullgen have modified the fragment-derived vemurafenib to target an oncolytic mutant form of BRAF.

One of the interesting features of targeted protein degradation is that a high affinity ligand is not always necessary: Craig Crews (Yale) described how an 11 µM p38α ligand was sufficient to degrade 99% of the protein in cells. And fragments can help not just with target proteins, but in identifying new E3 ligase ligands as well. Carles Galdeano (University of Barcelona) described how fragment screening has yielded a couple sub-micromolar affinity ligands of an E3 ligase called Fbw7.

In the interest of time I’ll stop here, but if you were particularly struck by anything please mention it in the comments. And if you missed the meeting, be sure to mark September 15-18 on your calendar for next year!

Fragments find flexibility in fascin 1

Protein flexibility can be both an opportunity and a barrier – quite literally, when a solid wall of protein seems to block opportunities for fragment growing. But like secret doorways, protein domains can yawn open to expose tunnels and cavities. An example of this was published earlier this year in Bioorg. Med. Chem. Lett. by Stuart Francis and collaborators at the CRUK Beatson Institute.

The researchers were interested in fascin 1, which increases the invasiveness of multiple cancers by helping pack filamentous actin into bundles important for cell migration. The team began their search for an inhibitor by performing a surface plasmon resonance (SPR) screen of 1050 fragments, generating an impressive 53 hits. Although a number of these were reported to bind to multiple sites on the protein, only one is discussed.

Compound 1 binds between two domains of the protein in a pocket that does not exist in unbound fascin. However, the fact that the pocket completely envelopes the fragment “hampered attempts to develop the series.” Fortunately, the researchers were following the patent literature, and when they characterized compound 2 (not a fragment, and reported by a different group), they discovered that while it binds in the same pocket as compound 1, additional conformational changes occur to accommodate the larger molecule.

Next, the researchers looked for analogs of compound 2 and also performed a virtual screen against the enlarged pocket. Of 110 commercial compounds tested, three gave dissociation constants better than 100 µM, including compound 3. The researchers recognized that compound 3 lacks the halogens found on both the original fragment and compound 2, and by adding these they were able to improve the affinity more than ten-fold. Further optimization ultimately led to BDP-13176, with mid-nanomolar affinity by SPR and ITC as well as activity in a functional assay. Although the molecule has reasonable solubility and stability against liver microsomes, it has low permeability and high efflux.

This is a nice structure-based design story, and while the fragment did provide some information about the binding site, one could argue that the real breakthrough came with determining the binding mode of compound 2. Indeed, without this information, it would have been all too easy to assume that the pocket was not ligandable. This is an important reminder that crystal structures usually only reveal one form of a protein. The system is also a good test case for modelers who want to see how their algorithms perform against a dynamic protein. Breakthroughs are often unexpected, and it is always worth making a few compounds that don’t look like they’ll fit.

Fragments vs sepiapterin reductase, via 19F NMR

It has been two years since we’ve had a post devoted to fluorine NMR. Though I don't share Teddy’s “fetish” for ¹⁹F-based screening, I do think the technique can be quite powerful, as demonstrated in a recent J. Med. Chem. paper by Jo Alen, Markus Schade, and their colleagues at Grünenthal GmbH.

The researchers were interested in sepiapterin reductase, which is abbreviated as SPR but which I’ll spell out to avoid confusion with surface plasmon resonance. This enzyme performs the last step in the production of tetrahydrobiopterin, an essential cofactor for multiple enzymes, including some that synthesize neurotransmitters and produce nitric oxide. Sepiapterin reductase has been proposed as a target for non-opioid-based pain medications.

The primary assay involved displacement of a fluorine-containing inhibitor that binds in the substrate site of the enzyme; thus, the researchers could use ¹⁹F NMR without requiring fluorinated fragments. A total of 4750 fragments were screened at 250 µM, initially in pools of 12. The 26 hits were then tested in an enzymatic assay, and 21 showed activity better than 75 µM. The best, compound 3, was sub-micromolar.

Crystal structures were obtained for six compounds, including compound 3, and all bound in the substrate pocket as predicted from the original displacement assay. The phenolate of compound 3 makes hydrogen bonds to two critical catalytic residues. Not surprisingly, capping this moiety with a methyl group led to an inactive compound. The researchers made dozens of variants, but aside from compound 26, most of these were disappointingly less active. Compound 26 does show good solubility and permeability, though no cell data are provided, and the phenol will likely be glucuronidated in vivo.

This is a nice story that illustrates a not-infrequent frustration: after identifying the initial nanomolar hit from a small library, the researchers likely thought improving potency still further would be easy. Instead, it took more than 60 analogs just to gain another order of magnitude. That said, 57 nM is nothing to sneeze at. And this situation is certainly preferable to the more common alternative of starting with a weak fragment that remains weak no matter what you do to it!

Fragment vs hematopoietic prostaglandin D2 synthase: a chemical probe

Six years ago we highlighted work out of GlaxoSmithKline and Astex describing some of their efforts to find inhibitors of hematopoietic prostaglandin D2 synthase (H-PGDS), an enzyme implicated in asthma, lupus, and multiple other inflammatory diseases. A recent paper in Bioorg. Med. Chem. by David Deaton and collaborators describes another chemical series from that program.

As noted in the earlier publication, the researchers were graced with 76 crystallographic fragment hits, of which compound 1a was a weak but ligand-efficient member. Several other fragments that bound in the same region contained a methoxy group, and a quick survey of commercially available analogs led to compound 1b, with a nice bump in potency. Replacing the nitrile with an amide (compound 1d) improved activity further.

What do you do when you’ve got an amide? Make lots of them! This was effective, and the researchers show more than 60 analogs leading to low nanomolar inhibitors such as compound 1bg. One problem with amides is that they can be enzymatically cleaved in vivo, but this challenge was surmounted by tweaking the substituents.

The researchers also noted that the compounds are quite electron-rich, potentially leading to phototoxicity, and in fact some of the molecules degraded upon exposure to UV light. Also, methoxy substituents are prone to dealkylation in vivo. The researchers solved both problems by replacing the methyl with a difluoromethyl group, leading to GSK2894631A.

This molecule was put through a battery of tests and found to be orally bioavailable (at least in mice) with good pharmacokinetics. It is selective against related enzymes as well as a larger panel of receptors and transporters. Encouragingly, the compound showed potent activity in a mouse model of acute inflammation. In other words, this looks to be a useful chemical probe to explore the biology of prostaglandin signaling.

This is a nice story on several levels, and it also illustrates an important point that younger researchers and folks in academia sometimes overlook: it can take ages before work done in industry sees the light of day. Indeed, one of the authors on the paper left Astex more than five years ago, so the work described is likely several years older than that. Still, better late than never. Good science is always worth publishing, even if – like another paper we recently highlighted – it happened some time ago.