Fragments vs GPx4 – in reverse micelles

Membrane proteins account for more than half of drug targets, but the fraction is far smaller for fragment-derived drugs. In part this is because biophysical methods, the mainstay of FBLD, have been harder to apply to membrane proteins. A recent (open-access) paper in JACS Au by Courtney Labrecque and Brian Fuglestad at Virginia Commonwealth University tackles this challenge.

 

The researchers use an approach called membrane-mimicking reverse micelles, or mmRM: tiny water-filled bubbles surrounded by lipids and suspended in an organic solvent. We last wrote about reverse micelles back in 2019, where they were being used to study high local concentrations of water-soluble proteins and ligands. Here, the researchers turned to membrane proteins.

 

There are actually two types of membrane proteins: integral membrane proteins and peripheral membrane proteins. The former, as their name implies, have at least part of the protein anchored in the membrane at all times; GPCRs are a prominent class. Peripheral membrane proteins are water soluble but associate with the membrane, and this interaction is often required for folding or function. One example is glutathione peroxidase 4 (GPx4), which reduces oxidized lipids. It is an intriguing but  challenging cancer target, with the only ligands being fairly reactive covalent modifiers. Thus the researchers turned to mmRMs, hoping these could both stabilize the protein in a biologically relevant state and also present binding opportunities unavailable in standard screens.

 

A library of 1911 fragments from Life Chemicals was screened against mmRM-encapsulated GPx4 using ¹⁵N-¹H HSQC protein-detected NMR. Fragments were chosen to have high aqueous solubility (at least 1 mM in PBS) and were screened in mixtures of 10 at 400 µM per fragment. After deconvolution, 14 hits were identified, and dose-response titrations revealed that 9 had apparent dissociation constants < 1 mM, with the most potent having a K_d = 105 µM.

 

Three fragments were studied in greater detail, and these were chosen to have a range of hydrophobicities from clogD = -2.1 (most polar) to clogD = 2.1 (most hydrophobic). Chemical shift perturbation (CSP) analyses suggested that the two more lipophilic fragments bind to the membrane-interacting region of the protein, while the more polar fragment likely binds to a water-exposed site. SAR-by-catalog was applied to find analogs, some of which had increased affinity for the protein, with the best being around K_d = 15 µM.

 

Interestingly, the fragments showed minimal binding to GPx4 under normal aqueous conditions (ie, in the absence of the mmRMs), even at very high fragment concentrations. The researchers suggest this is because the fragments are binding to the membrane-bound state of the protein found in mmRMs, which may adopt a different conformation than that in the absence of membranes. Perhaps. But as prior work shows, it is possible to detect extraordinarily low affinity interactions inside reverse micelles, so maybe these are just very weak binders. Ultimately it remains to be seen whether these fragments will have practical applications. I hope so, and look forward to seeing how they progress.

Fragments finger the BPTF PHD Finger

Plant homeodomain (PHD) fingers, despite their name, are found in nearly 300 human proteins. They are small (50-80 amino acid) domains that typically recognize post-translational modifications such as trimethylated lysine residues in histones. The PHD finger in BPTF is implicated in certain types of acute myeloid leukemia. However, because of the large number of PHD fingers as well as their small binding sites, few attempts have been made to develop corresponding chemical probes. (Indeed, the only mention of them on Practical Fragments was in 2014.) In a just-published ACS Med. Chem. Lett. paper, William Pomerantz and collaborators at University of Minnesota and St. Jude Children’s Research Hospital report the first steps.

 

The researchers started by screening a library of 1056 fragments (from Life Chemicals) against the BPTF PHD finger using ligand-observed (¹H CPMG) NMR. Fragments were at 100 µM in pools of up to five. This gave a preliminary hit rate of 5.7%, but only ten compounds (<1%) reproduced when compounds were repurchased and retested individually.

 

These ten fragments were next tested by SPR (at 400 µM), which confirmed six of them. Also, all ten CPMG hits were tested in an AlphaScreen assay in which they competed with a known peptide binder. This confirmed nine, including the six that confirmed by SPR.

 

Interestingly, the most potent fragment in the AlphaScreen assay was the starting point for the KRAS inhibitor we highlighted last year. However, this fragment did not show binding to the BPTF PHD finger by SPR, and the researchers had identified the 2-aminothophene substructure as a hit against an unrelated protein. Whether this fragment is privileged or pathological may be context dependent.

 

This and the top three fragments that confirmed in all assays were used as starting points for SAR by catalog, and a handful of analogs were purchased. The researchers also resynthesized two of the compounds. Oddly, resynthesized F2 turned out to be three-fold more active in the AlphaScreen assay than the commercial material. One analog, compound F2.7, showed mid-micromolar activity.

 

 

Docking and two-dimensional protein-observed (¹H,¹⁵N HSQC) NMR experiments suggest that most of the fragments bind in the “aromatic cage” which normally recognizes methylated lysine residues, but F2 may bind in an adjacent region. Both subpockets were also identified as being ligandable using the program FTMap.

This paper is a nice example of using orthogonal methods to find and carefully validate fragments against an underexplored class of targets. The researchers conclude by stating that “these hits are suitable for further SAR optimization and development into future methyl lysine reader chemical probes.” I look forward to seeing more publications.

Virtually screening 11 billion compounds – no problem!

Three years ago we highlighted virtual screens of roughly 100 million molecules which led to numerous high-affinity ligands against two targets. Those efforts made use of the Enamine “readily available for synthesis” (REAL) library, a virtual catalog of molecules that can be rapidly made and delivered. Enamine is continuing to grow this resource, which as of last year stood at 11 billion compounds. This is an impressive number, but how do you make use of it? In a just-published paper in Nature, Vsevolod Katritch (University of Southern California, Los Angeles) and a large group of collaborators provide a promising fragment-based solution.

 

Molecules in the Enamine REAL collection can be made using one-pot parallel synthesis from two or three reagents; for example, an amide could be made from an amine and a carboxylic acid. Enamine built a set of 75,000 reagents and 121 different reactions which collectively could produce 11 billion molecules (it’s even larger now). However, docking all of these could take thousands of years on a single CPU or cost hundreds of thousands of dollars on a computing cloud.

 

Rather than docking all the Enamine REAL compounds, the researchers developed an approach called virtual synthon hierarchical enumeration screening, or V-SYNTHES. The first step is to create a library of scaffolds with molecular weights in the 250-350 Da range. Taking the amide example above, imagine linking a set of 1000 amines to benzoic acid and a set of 1000 carboxylic acids to methylamine. This 2000 compound minimal enumeration library, or MEL, could be considered a subset of the full 1000 x 1000 = 1,000,000 virtual amide library. The numbers are even more dramatic for a three-component reaction: a MEL of just 1500 compounds could represent 125,000,000 fully elaborated molecules.

 

The MEL is docked against a protein of interest, and a diverse set of the top-scoring compounds chosen for fragment growing. In our example, the benzoic acid “cap” on the best compounds would be replaced by the full set of 1000 carboxylic acids. These would then be virtually screened, and the top compounds synthesized and tested.

 

The researchers applied V-SYNTHES to two targets. The first was a cannabinoid receptor bound to an antagonist. A total of 1.5 million molecules were docked against CB₂, representing 11 billion fully enumerated compounds. After filtering the best hits to remove PAINS and molecules similar to known CB₂ ligands, 80 diverse compounds were chosen for actual synthesis and testing, of which Enamine was able to deliver 60 in less than 5 weeks. One-third of these turned out to be antagonists with K_i values < 10 µM in biological assays.

 

How does this compare to a brute-force approach? Screening all 11 billion molecules wasn’t feasible, so the researchers screened a representative subset of the Enamine REAL library consisting of 115 million molecules – two orders of magnitude larger than the libraries screened in V-SYNTHES. Of 97 compounds synthesized and tested, only 5 turned out to be antagonists of CB₂ with K_i values < 10 µM.

 

A nice feature of V-SYNTHES is that it is well-suited to SAR-by-catalog. This was demonstrated by looking for analogs of the three best hits within Enamine REAL space. Of 104 compounds synthesized and tested, more than half had K_i values < 10 µM, and 23 were submicromolar antagonists. In fact, several turned out to be low nanomolar and selective not just against the related CB₁ receptor but against a panel of 300 other GPCRs.

 

V-SYNTHES was also applied to the kinase ROCK1 and achieved similarly impressive results: six of 21 compounds synthesized and tested had K_d < 10 µM in a binding assay, and one was a low nanomolar inhibitor.

 

This is a lovely and practical application of fragment concepts. Importantly, because the computational cost only increases linearly with the number of synthetic components while the library size increases with the square (for two-component molecules), it is very scalable; the researchers suggest that “terascale and petascale libraries” should be “easily” accommodated. These are numbers beyond even what DNA-encoded libraries can promise.

 

Currently V-SYNTHES relies on a good structural model for docking, but as computational predictions of protein structures become ever more accurate, perhaps even this will cease to be a limitation. Our SkyFragNet post from 2019 is looking ever more prophetic, in a good way.

Fragments in the clinic: AG-270

A promising oncology approach is to target “synthetic lethal” proteins that are required for cancer cells but not for ordinary cells. Methionine adenosyltransferase 2A (MAT2A) is a metabolic enzyme that produces S-adenosyl methionine (SAM). Cancer cells lacking another gene, methylthioadenosine phosphorylase (MTAP), appear particularly dependent on MAT2A. In a recent J. Med. Chem. paper, Zenon Konteatis and colleagues at Agios, Viva, and ChemPartner describe the first clinical inhibitor of MAT2A.

 

The researchers began by screening >2000 fragments in pools of 20, each at 50 µM, using ultrafiltration. The 31 hits were tested in enzymatic and SPR assays, and compound 1 confirmed in both. Testing 54 commercially available analogs led to compound 2, with low micromolar activity, and this molecule was profiled intensively.

 

Kinetic studies revealed compound 2 to be non-competitive with respect to substrates ATP and L-methionine, and crystallography confirmed that compound 2 binds in a previously discovered allosteric pocket. The molecule makes polar and hydrophobic contacts to MAT2A and also displaces several water molecules. SAR studies found a preference for aromatic moieties at the 2-position of the central core, but the phenyl off the 3-position could be substituted with more shapely moieties such as the piperidine in compound 9.

 

Closer examination of the structure of compound 2 bound to MAT2A revealed a protein-bound water molecule, and displacing this with the phenol in AGI-24512 led to a satisfying boost in biochemical potency as well as cell activity. However, the molecule has poor oral absorption and a short half-life in rats. Metabolite identification studies pinned the blame on the piperidine, with the phenol no doubt doing no favors. Medicinal chemistry ultimately led to AGI-25696, which despite its lower biochemical activity was active in cells, metabolically stable, and showed efficacy in a mouse xenograft model when dosed orally.

 

Despite these favorable properties, AGI-25696 has very high protein binding in human plasma (>99.9%) as well as high efflux, which would likely necessitate a high clinical dose. The researchers proposed that, due to the weakly acidic nature of the molecule, it could tautomerize, and each tautomer could bind to different plasma proteins. Simply methylating the N-H led to decreased plasma protein binding but also lower binding to MAT2A. Thus, the researchers sought to shield the N-H by forming an intramolecular hydrogen bond. After appending more than 70 heterocycles they eventually arrived at AG-270, which has a more respectable plasma protein binding of around 98.5%.

 

Extensive characterization of AG-270 revealed it to be potent with good pharmacokinetics and oral bioavailability. It is relatively clean in a panel of 95 potential off-targets and showed tumor growth reduction in xenograft models. But it is not without warts: low solubility necessitated a spray-dried dispersion for animal dosing, not surprising giving its high lipophilicity. Nonetheless, the molecule has entered a phase 1 clinical trial in patients with MTAP loss.

 

This is a lovely fragment-to-lead success story with several lessons. First, although enzymes are sometimes considered “easy,” this is not necessarily true. Indeed, at an ACS meeting in 2018 Anil Padyana mentioned that metabolic enzymes in particular often have shallow, polar active sites. Targeting allosteric sites, as done here, can be a useful alternative.

 

Second, it is striking how the core of the initial fragment remains intact in the clinical compound, a reminder of the power of fragments to efficiently explore chemical space.

 

Finally, this story is another important reminder that affinity is often just the beginning of a long journey. It took considerable effort to optimize the pharmaceutical properties from AGI-24512 to AG-270, including a decrease in ligand efficiency. In the end the team has succeeded, and Practical Fragments wishes them – and the patients – luck in the trials.

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.

Fragments vs RAS family proteins: A chemical probe

RAS family proteins are considered a holy grail of oncology research. Way back in 2012 we discussed a couple papers disclosing low affinity fragments that bind in a small, shallow, polar pocket found in KRAS, NRAS, and HRAS. At the time we wondered “whether this is a ligandable site on the protein.” Last year we highlighted a paper proving that the site is, in fact, ligandable, as exemplified by the mid-nanomolar molecule Abd-7. A paper just published in Proc. Nat. Acad. Sci. USA by Darryl McConnell and collaborators from Boehringer Ingelheim and Vanderbilt University (including Steve Fesik, who published one of the 2012 reports) describes successful development of another ligand. (See here for a fun animated description set to music.)

Consistent with the “undruggable” reputation of RAS family proteins, a high-throughput screen of 1.7 million compounds failed to find anything useful. In contrast, a library of just 1800 fragments screened using STD NMR and MST identified 16 fragments that bind to an oncogenic mutant form of KRAS, as confirmed by 2-dimensional (HSQC) NMR. A separate HSQC NMR screen of 13,800 fragments identified several dozen more, though all the fragments from both screens have dissociation constants weaker than 1 mM. SAR by catalog led to amine-substituted indoles such as compound 11, which modeling suggested could form a salt bridge to an aspartic acid side chain.

The pocket in which all of these molecules bind, between the so-called switch I and switch II regions of KRAS, is much smaller than typical drug-binding sites, but modeling suggested that fragment growing could pick up an additional hydrogen bond, leading to compound 15. Crystallography confirmed the predicted binding mode of this molecule, and informed additional structure-based design, leading first to compound 18 and ultimately to BI-2852, with low or sub-micromolar affinity for wild-type and mutant KRAS, NRAS, and HRAS as assessed by ITC. The researchers also confirmed that the enantiomer is about 10-fold less potent, thereby providing a control compound. Commendably, the researchers have made BI-2852 and the enantiomer available (for free!) to the research community as a chemical probe.

A crystal structure of KRAS^G12D bound to BI-2852 (cyan) compared with Abd-7 (magenta) reveals how shallow the pocket is; both molecules are largely surface-exposed. The conformational flexibility of the protein is also interesting: Abd-7 would not be accommodated by the protein conformation bound by BI-2852.

The biology is also quite interesting – and complicated. RAS family proteins behave as molecular switches, cycling between the “on” (GTP-bound) state and the “off” (GDP-bound) state, with these transitions assisted by other proteins. On-state RAS drives cell-proliferation and survival. Molecules that bind at the switch I/II pocket block the transition from off to on, but they also block the transition from on to off. Thus, cellular effects are modest. Moreover, BI-2852 hits all RAS isoforms, which could lead to unacceptable toxicity in animals.

This is a lovely paper, but I do quibble that the promise of the title – “drugging an undruggable pocket on KRAS” – remains to be demonstrated. First, both the biochemical and cell-based potency need to be further improved. As the molecule is already large, gaining this needed potency could come at the cost of physicochemical properties. Indeed, the researchers do not discuss the pharmacokinetics of BI-2852. And finally, as the authors themselves note, they will probably need to improve selectivity to spare one or more wild-type RAS isoforms.

What this work does establish indisputably is that the switch I/II pocket is ligandable, though not without effort, as indicated by the 42 authors. Whether or not the site is actually druggable may require another seven years to determine.