PhABits: photoaffinity-based fragment screening

Three years ago we highlighted work out of Ben Cravatt’s lab describing “fully-functionalized fragments” that – in addition to a variable portion – contain a photoreactive diazirine moiety and an alkyne moiety. These were incubated with cells and irradiated with UV light to crosslink the fragments to bound proteins. The alkyne was then used in click chemistry to isolate and identify the bound proteins. Cell-based screening is not for the faint of heart, but as demonstrated in a paper recently posted on ChemRxiv by Jacob Bush and collaborators at GlaxoSmithKline and University of Strathclyde, the functionalized fragments can also be used in biophysical screening. (Emma Grant presented a nice poster on some of this work at FBLD 2018.)

A small library of 556 fragments, rebranded as PhotoAffinity Bits (or PhABits), was synthesized by coupling the alkyne- and diazirine-containing carboxylic acid with a diverse set of amines (each with < 16 heavy atoms). These were then screened at 200 µM against six pure recombinant proteins, irradiated with UV light, and analyzed using intact protein mass spectrometry as in Tethering and other forms of covalent FBLD. Hit rates varied tremendously, from less than 3% for myoglobin to 47% for lysozyme. It would be interesting to see whether this approach, like other fragment finding methods, is able to assess protein ligandability.

Most of the PhABits did not react with the proteins tested, though 58 crosslinked to at least four, and 10 crosslinked to all six. For one of the proteins screened, the bromodomain BRD4-BD1, a known high-affinity ligand could compete 68 of the 89 fragment hits, suggesting a specific interaction at the acetyl lysine pocket. Of the 21 fragments that were not competed, 19 bound to at least three other proteins. Interestingly, the physicochemical properties and solubilities of these fragments were not notably different from the rest, and the researchers speculate that their non-specificity may be due to a longer-lived reactive intermediate generated after UV irradiation.

Several of the BRD4-BD1 fragments were confirmed as binders using a TR-FRET assay, some with low micromolar affinities, though the tighter ones tended to contain known bromodomain binding motifs such as isoxazoles. A couple of these were successfully used to generate PROTACs, as suggested here. Protein digestion and LC-MS/MS sequencing revealed that the fragments crosslinked residues near the acetyl lysine binding site, and this binding mode was confirmed using X-ray crystallography for one of the fragments.

In addition to BRD4-BD1, another target the researchers highlight is KRAS4B^G12D. Of the 11 unique hits, some resembled previously reported molecules, and LC-MS/MS studies suggested that they do in fact bind in the same pocket. Competition studies confirmed this, and the resulting IC₅₀ values were similar to those previously determined using HSQC NMR.

As the researchers point out, this photoaffinity-based screening approach is limited to homogenous proteins that are suitable for mass spectrometry. Also, the crosslinking efficiency is not necessarily related to the affinity of the fragment. Still, this is an interesting approach to both find fragments and identify their binding sites. It will be fun to see how it develops.

Back to the Future: HIV protease offers lessons for SARS-CoV-2

Today’s guest post is by Glyn Williams (University of Cambridge). Fragment aficionados will recognize Glyn as the former VP of Biophysics at Astex, but before that he worked at Roche. His experiences there in the 1990s have lessons for today. -Dan Erlanson

In two recent Practical Fragments posts (here and here), Dan Erlanson noted efforts which will allow the scientific community to contribute to drug design efforts against the SARS-CoV-2 main protease (M^pro). Leading the charge at the moment is the COVID Moonshot consortium who have already received design proposals for covalent inhibitors, based on the structures of fragments bound to M^pro that have been generated by researchers at the Diamond Light Source. At the same time, more information about M^pro, including its substrate preferences, is being published. Soon there will be an urgent need to define a selection procedure which will allow valuable drug candidates to be progressed.

A similar situation was faced in 1985 when HIV protease was being considered as a drug target for AIDS. An excellent description of a pragmatic, and ultimately successful, procedure was published in 1993 by Noel Roberts and Sally Redshaw of Roche in The Search for Antiviral Drugs:Case Histories from Concept to Clinic.

When the project began there was no definitive proof that this aspartyl protease was essential for viral replication in human cells and that it could not be substituted by a cellular protease. However, its in vitro ability to cleave a Phe-Pro or Tyr-Pro peptide bond (amongst others) marked it out as unusual, and that was sufficient encouragement for Roche to initiate a discovery programme. Inhibitor design then took advantage of this feature to build in selectivity over human aspartyl proteases, ultimately giving a high therapeutic index while also improving inhibitor absorption after oral administration. 
 

Critical issues, such as the decision to target the HIV-1 viral strain, access to suitable protease constructs and clear criteria for project progression, were defined early on. Novel protease and anti-viral assays were then developed in parallel with transition-state mimetic leads. From the start, it was recognised that the low aqueous solubility of the optimal peptide substrates could imply that peptidomimetic inhibitors were also likely to have poor physico-chemical properties. At the time there was no structural information on the enzyme or its complexes, so there was little opportunity to avoid these shortcomings.

As with COVID-19, the worldwide health implications of HIV were obvious and scientific interactions between different research groups were driven by a spirit of cooperation. Public laboratories contributed clinical data and provided access to assays for viral activity. In 2020 the ability to share data has improved beyond recognition but the ability to interpret and act on it is still subject to political and commercial pressures. At Roche, a series of hydroxyethylene inhibitors was not pursued due to its prior inclusion in multiple patents for renin inhibitors. In addition, sensitivity to criticism from AIDS activist groups during the project discouraged Roche from developing follow-up candidates later.

Many current predictions and public expectations about COVID-19 now depend on the availability of vaccines in 2021. After more than three decades of research, no preventative vaccine is yet available for HIV. However, the ability to treat a viral infection, even with a drug that contains and controls the infection rather than eliminates it, should not be undervalued. In 1993 the Roche HIV protease clinical candidate, Ro 31-8959, was in Phase 2 evaluation. Roberts and Redshaw pointed out then that lowering a patient’s viral load would reduce the risk of further infections amongst health-care workers and social contacts, while the persistence of immature and non-infectious viral material in cells could stimulate the patient’s own immune system to eliminate the virus.

Roberts and Redshaw concluded their 1993 analysis with the statement that "although there is still much work to be done, we remain very hopeful that Ro 31-8959 will make a positive contribution to the therapy of AIDS". Two years later Ro-31-8959, as Saquinavir, was approved by the FDA and, with Ritonavir, a second protease inhibitor from Abbott Labs, led to a 64% reduction in deaths from AIDS in the US over the next 2 years. Let us now hope for the same degree of success from new COVID-19 treatments.

Fragment chemistry roundup part 3

Last week’s post discussed three papers describing new chemistries for building fragment libraries. The theme continues this week with three more.

The first, in ACS Med. Chem. Lett. from Philip Garner (Washington State University Pullman), Philip Cox (AbbVie), and colleagues describes the synthesis of a library of pyrrolidine-based fragments in just three steps. A chiral auxiliary, which is subsequently removed, enables an asymmetric cycloaddition reaction to generate pyrrolidine rings containing three defined stereocenters. Using this method, the researchers made 48 fragments from simple starting materials.

As one might predict looking at the structures, the fragments have low lipophilicity (average AlogP = 0.12) and high levels of saturation (Fsp³ = 0.47), though with an average MW = 225 they are a bit portly.

The fragments are also quite shapely, as assessed both by principal moments of inertia (PMI) or plane of best fit (PBF). The researchers acknowledge that this shapeliness increases the fragments’ molecular complexity, and they also note the difficulty of quantifying this, “as current estimates do not take into consideration 3D, let alone the multidimensional descriptors of chemical space.” Thus, they may have lower hit rates. Hopefully we’ll see screening data from this set at some point in future.

Diversity oriented synthesis (DOS) has only been occasionally applied to fragments, perhaps in part due to issues Teddy raised in his Safran Zunft Challenge. In an (open access) Bioorg. Med. Chem. Lett. paper, Nicola Luise and Paul Wyatt (University of Dundee) describe a set of 22 fragments in 12 scaffolds starting from just 3 precursors; a few examples are shown.

Although the embedded pyrazine, pyridine, and pyrimidine moieties are found in many drugs, some of the bicyclic cores are novel or rarely found in commercial sets.

In both these papers, the chemistry is sufficiently straightforward that a hit could rapidly lead to numerous analogs, which is a selling point for including them in a library. But in advancing other fragments a common problem is that the analog you most want to make is synthetically difficult. A crystal structure may reveal that an otherwise useful synthetic handle is making intimate contacts with the protein, while a hard-to-functionalize aliphatic ring is situated next to an attractive subpocket. A clear example of this is the phase 2 IAP inhibitor ASTX660 from Astex, whose fragment starting point consisted of a piperidine linked to a piperazine.

Perhaps building on this experience, Rachel Grainger, Chris Johnson, and collaborators from Astex, University of Cambridge, and Novartis have published in Chem. Sci. a high-throughput experimentation method to functionalize cyclic amines. The researchers used nanomole-scale reactions run in 1536-well plates to explore and optimize photoredox-mediated cross-dehydrogenative heteroarylation.

After optimizing conditions, the researchers moved to larger (milligram) scale to couple 64 different protected amines against heteroarene 3a and 48 heteroarenes against N-Boc-morpholine, thereby obtaining a variety of interesting molecules, many of which contain polar functionalities. Finally, they used flow chemistry to generate more than a gram of product 5g, demonstrating scalability. The paper ends with a half dozen examples of fragments taken from recent reviews, noting how the cross-dehydrogenative coupling could be used to elaborate them.

Progress often comes from expanded possibilities. By facilitating new chemistries, this paper lowers the barriers for drug hunters to make the most promising molecules. And taken together, all six of these papers advance the field of fragment chemistry.

Fragment chemistry roundup part 2

It has been more than a year since we devoted a post solely to fragment library synthesis (though see here for an example describing library synthesis and screening). Since you can’t screen fragments without a library, Practical Fragments will spend the next two posts focusing on recent library design papers.

The first, from David Spring (University of Cambridge) and collaborators at the Technical University of Denmark, California State Polytechnic University Pomona, and University of Leeds, was published earlier this year in Chem. Commun. David Spring has long been interested in fragments that resemble natural products (NPs), such as those with multiple sp³ stereocenters.

The researchers focus on 3-hydroxy-2,2-disubstituted-cyclopentan-1-ones, which are found in natural products and derived drugs. The two building blocks syn-1 and anti-1 were elaborated in fewer than six synthetic steps into a total of 38 small molecules in 20 scaffolds, a few of which are shown.
 

Close attention was paid to physicochemical properties, and consequently the library is rule-of-three compliant, with a mean molecular weight of just 208 Da. The library is also quite shapely, as judged either by a high (0.70) mean Fsp³ or by individual members' principal moments of inertia (PMI).

Another paper from David Spring’s lab was published last year in Eur. J. Org. Chem. In it, the researchers describe the synthesis of nine heterocyclic spirocycles, a couple of which are shown here.

As with the newer paper, the physicochemical properties conform to the rule of three, and the molecules are quite shapely as assessed by their Fsp³ values.

Wrapping up this week’s installment is a paper in Chem. Eur. J. from Richard Bayliss, Stuart Warriner, Adam Nelson (all at University of Leeds) along with collaborators at University of Leicester, Diamond Light Source, University of Oxford, and University of Johannesburg. The researchers set out to assemble a diverse set of 80 shapely fragments for general use. Several rounds of computational pruning arrived at 60 commercial compounds and 20 that were synthesized de novo. Both approaches ran into problems: some “commercially available” compounds proved “difficult to obtain in practice,” while several synthetic approaches that looked good on paper turned out to be anything but. The final library is quite shapely though: all the synthesized compounds have at least one stereocenter, and only two fragments in the entire set are “close to the rod-disk axis” of a PMI plot.

Usefully, this paper presents screening data, in this case a high-concentration (80-200 mM) crystallographic screen against Aurora A kinase. This yielded just four hits, a 5% hit rate much lower than some other crystallographic screens. Interestingly none of these bound at the kinase hinge region where fragments often bind but instead were found at an allosteric site. The authors do not speculate on the low hit rate, which could be due either to the shapeliness of the fragments or their portliness, with 18-22 heavy atoms, considerably above the optimum suggested by Astex. The fragments are available for screening at Diamond’s XChem, though they don’t seem to have been used in the recent SARS-CoV-2 main protease screen.

We’ll cover three more papers next week. In the meantime, stay safe and please leave comments!

Fragment screening in cells with cryo-EM

Of all the biophysical advances so far this century, cryogenic electron microscopy (cryo-EM) has probably made the most impressive strides. Frequently dismissed as “blobology” just a few years ago, the technique now regularly produces three-dimensional structural models that rival those from X-ray crystallography. Indeed, it is rare to pick up an issue of Science or Nature that doesn’t contain a cryo-EM structure. Earlier this year, researchers from Astex described the structures of fragment hits against two proteins determined using cryo-EM. Now, the boffins from DREADCO (who previously brought us universal crystallography) have begun fragment screening in cells using cryo-EM.

Fragment screening in cells is not new: we previously highlighted work using either covalent or non-covalent fragments. However, figuring out which proteins the fragments bind can be challenging, which is one of the reasons structural information is so useful.

The researchers from DREADCO incubate their fragment library against cells – human or otherwise – for varying lengths of time. They then flash-freeze the cells in liquid ethane, collect, and process the data, using standard cryo-EM workflows. Of course, given the complexity of cells, the computational processing power needed is enormous – but nothing their SkyFragNet platform can’t handle.

One of the advantages of cryo-EM is that larger structures are more easily solved, so the researchers are focusing on organelles such as mitochondria, as well as ribosomes. Already they’ve found dozens of hits that resolve to high resolution, and they are in active fragment-to-lead optimization. Surely it is only a matter of time before our list of fragment-derived drugs includes one discovered with the aid of cryo-EM.