Review of 2020 reviews

An old curse runs, "may you live in interesting times." And 2020 has been interesting indeed. Amid all the tumult, Practical Fragments will maintain its tradition of ending the year with a post highlighting conferences and reviews.

 

Despite the travel restrictions caused by COVID-19, some conferences did go ahead, adapted to online formats: I highlighted CHI’s Fifteenth Annual Fragment-based Drug Discovery and their Eighteenth Annual Discovery on Target. Although these were quite successful, I think most of us are looking forward to returning to in-person events sometime in the coming year.

 

Perhaps because so many people were stuck working from home, the number of reviews of potential interest to fragment fans has soared to a record number of more than twenty. I’ve tried to group these thematically.

 

General

If you’re looking for a concise yet thorough review, Harren Jhoti and colleagues at Astex provide one in Biochem. Soc. Trans. Harren is one of the pioneers of FBDD, and the review touches on library design, detection of fragment binding, and fragment to lead strategies. A review in Front. Mol. Biosci. by Qingxin Li (Guangzhou Sugarcane Industry Research Institute) goes into more detail on fragment screening, optimization, and biological targets.

 

For the past five years a few fragment fanciers (myself included) have been writing annual reviews in J. Med. Chem. covering fragment-to-lead success stories from the previous year, each with a handy table showing fragment, lead, and key parameters. The 2018 edition, led by yours truly (Frontier Medicines), was published at the beginning of the year, while the 2019 edition, led by Wolfgang Jahnke (Novartis), just came out a few weeks ago. At the risk of self-promotion, both are well worth perusing to see the growing diversity of targets and emerging trends, such as covalent fragments.

 

Biophysics

Biophysical methods are by far the most commonly used for finding fragments, and an excellent overview of thermal shift, SPR, and NMR by Joe Coyle and Reto Walser (Astex) appears in SLAS Discovery. The goal is “to help the anxious biophysicist withstand the relentless unforeseen,” and the paper provides loads of practical advice. For example, over more than 50 thermal shift screens, “we have never derived anything useful from negative Tm shifts.” The researchers note that “SPR is particularly user-friendly and particularly prone to artifact, overinterpretation, and varying degrees of frustration.” As for validating ligand-observed NMR hits crystallographically, rates range from 5% to 80%.

 

As we noted earlier this year, crystallography is becoming increasingly dominant in fragment screening, and in Molecules Laurent Maveyraud and Lionel Mourey (Université de Toulouse) provide an overview of the process, covering theory, workflow, practical aspects, pitfalls, examples, and other emerging methods. David Stuart and colleagues at Diamond Light Source discuss structural efforts on SARS-CoV-2 proteins in an open-access paper in Biochem. Biophys. Res. Commun. As of late October this included more than 500 released structures of 16 different proteins. Efforts against the main protease (which I reviewed in Nat. Commun.) have led to molecules with mid-nanomolar activity, and the researchers rightly highlight the worldwide collaboration that has led to such rapid progress.

 

NMR

NMR is of course a biophysical technique, but there are so many papers this year that it makes sense to group them into their own section. Ray Norton (Monash Institute of Pharmaceutical Sciences) and Wolfgang Jahnke (Novartis) introduce a special issue of J. Biomol. NMR focused on “NMR in pharmaceutical discovery and development” by briefly summarizing the state of the art and introducing 13 articles, one of which we covered previously and three of which are highlighted below.

 

“NMR in target driven drug discovery, why not?” ask Gregg Siegal and collaborators at ZoBio and Gotham in an (open access) J. Biomol. NMR review. In addition to characterizing small molecules, proteins, and their interactions, the researchers present cases studies in which NMR data has helped clarify a crystallographic protein-ligand structure, or even suggested that the crystal structure represented at most a minor conformation in solution.

 

In other words, NMR is “the swiss army knife of drug discovery,” as Reto Horst and colleagues at Pfizer put it in another J. Biomol. NMR review. The researchers describe successful NMR fragment screens against difficult targets such as an ion channel and a large (145 kDa) trimeric enzyme. They also make a good case for using NMR to determine the solution conformations of small molecules early in a project, a strategy that has paid off in more than 15 Pfizer projects over the past six years.

 

Benjamin Diethelm-Varela (University of Maryland) focuses on using NMR for “fragment-based drug discovery of small-molecule anti-cancer targeted therapies” in ChemMedChem. This is a thorough yet accessible overview of FBDD, ligand- and protein-based NMR methods, plus ten case studies. “A practical perspective on the roles of solution NMR spectroscopy in drug discovery” is provided by Qinxin Li and CongBao Kang (A*STAR) in Molecules. As the title suggests, this review is fairly broad, and includes an interesting section on NMR screening in cells.

 

All these papers might have you thinking that NMR is a “Gold Standard,” and that phrase does indeed appear in the title of another Molecules review by Abdul-Hamid Emwas (King Abdullah University of Science and Technology) and a multinational group of collaborators. This is a large (66 page) monograph with 455 references and is particularly detailed on various NMR techniques; if you want to see the pulse sequence of the HSQC experiment or review the Einstein-Stokes equation this is the place to turn.

 

In addition to the six reviews on NMR above, two specifically cover ¹⁹F NMR. The first, from the J. Biomol. NMR special issue by Claudio Dalvit (Lavis) and colleagues, focuses on fluorine NMR functional screening, or n-FABS. This paper provides an excellent theoretical and practical overview of the technique, and includes a handy table of 17 published case studies. And in Prog. Nuc. Mag. Res. Spect. Peter Howe (Syngenta) reviews “recent developments in the use of fluorine NMR in synthesis and characterization.” As the title suggests, much ground is covered, from spectrometer technology to quantum chemistry calculations, and there is a short section on fragment-based screening.

 

Computational

Turning to in silico techniques, Floriano Paes Silva Jr. and collaborators at LaBECFar and several other (mostly) Brazilian institutes provide an open-access overview in Front. Chem. After summarizing FBDD they describe how computational techniques can help along the way, from druggability prediction to docking, de novo design, and assessment of ADMET properties and synthetic accessibility. The review ends with several case studies.

 

In an open-access article in Drug Disc. Today, Stefano Moro and colleagues at University of Padova focus on “the rise of molecular simulations in fragment-based drug design.” This accessible overview covers hotspot identification, hit identification and characterization, and hit to lead optimization, and includes a nice section on free energy perturbation.

 

Other topics

Molecular properties are critical for developing good drugs, and in J. Med. Chem. Christopher Tinworth (GlaxoSmithKline) and Robert Young (Blue Burgundy) “appraise the rule of 5 with measured physicochemical data.” This is packed full of good stuff including a supplementary table with calculated and measured data for hundreds of compounds. The summary is that molecular weight is much less important than (measured) lipophilicity and hydrogen bond donors. “Good practice is all about compromise, aiming to maximize efficacy and efficiency while navigating many potential pitfalls in molecular optimization.” People sometimes obsess over rules vs guidelines, and the researchers close by stating that “rules are for the obedience of fools and guidance of the wise.”

 

As a poll from several years ago suggested, fragment linking tends to be less common than fragment growing, though it can work spectacularly. In J. Med. Chem. Isabelle Krimm and colleagues mostly at Université de Lyon review 45 successful fragment linking case studies (though it would have been appropriate for them to acknowledge Practical Fragments for the clearly borrowed table of clinical compounds). While by no means exhaustive, this is a useful resource. Interestingly, only 20% of the examples display superadditivity.

 

Target-guided synthesis (TGS) can be thought of as a special case of fragment linking. In J. Med. Chem., Rebecca Deprez-Poulain and colleagues at Université de Lille review kinetic TGS, in which two components react irreversibly with one another in the context of a protein to form a higher-affinity binder. Kinetic TGS may have some practical advantages over reversible TGS (or dynamic combinatorial chemistry), but as the researchers note most examples start with compounds larger than fragments, and thus only 38% of examples lead to products with a molecular weight less than 500 Da. This could partly explain why only 6 of the 50 reported examples have gone into animal studies.

 

Finally, György Keserű and collaborators at the Hungarian Research Centre for Natural Sciences review covalent fragment-based drug discovery in Drug Discovery Today (open access). Library design and validation is well-covered, as are various methods for screening covalent fragments, and there is a handy table of some four-dozen published examples. Given the increasing popularity of covalent FBLD, this contribution should be of wide interest.

 

When I wrote my concluding post for 2019, COVID-19 was an obscure and nameless disease, and SARS-CoV-2 had not even been identified. I ended with, "may 2020 bring wisdom, and progress." We've gained both, though the cost has been incalculable. So I'll just close this post by thanking you for reading and commenting.

Benchmarking docking methods: a new public resource

Despite advances in crystallography, obtaining structures of fragments bound to proteins is still often elusive. Computational docking is likely to forever be faster than experimental methods, but how good is it? A new paper in J. Chem. Inf. Mod. by Laura Chachulski (Jacobs University Bremen) and Björn Windshügel (Universität Hamburg) assess four popular methods and also provide a public validation set for others to use.

 

When evaluating fragment docking methods, it is essential to have a well-curated set of experimental structures. To this end, the researchers started by combing the PDB for high quality, high resolution (< 2 Å) structures of protein-fragment complexes. They used automated methods to remove structures with poor electron density, close contacts with other ligands, and various other complications. Further manual curation yielded 93 protein-ligand complex structures. The fragments span a relaxed rule-of-three, with 7 to 22 non-hydrogen atoms (averaging 13) and ClogP ranging from -4.1to 3.5 (averaging 1.1). I confess that some choices are rather odd, including oxidized dithiothreitol, benzaldehyde, and γ-aminobutyric acid. The researchers might have saved themselves some effort, and obtained a more pharmaceutically attractive set, by starting with previous efforts such as this one.

 

Having built their benchmark data set, called LEADS-FRAG, the researchers next tested AutoDock, AutoDock Vina, FlexX, and GOLD to see how well they would be able to recapitulate reality. The results? Let’s just say that crystallographers look likely to have job security for some time.

 

Only 13 of the 93 protein-fragment complexes were correctly reproduced as the top hit using all four methods (even with a reasonably generous RMSD cutoff criterion of < 1.5 Å).There were 18 complexes that none of the methods predicted successfully. Across the four methods, the top-ranked poses were “correct” 33-54% of the time. Docking methods usually provide multiple different poses with different scores; up to 30 were considered here. Looking at lower-ranked poses increased the number of successes to 27 of the 93 fragments, while only three failed in all methods. Overall, the correct structure was present among the poses in 53-86% of cases. Changing the scoring function sometimes led to further improvements.

 

Why were some fragments more successfully docked than others? Fragments that were more buried within the protein (lower solvent-accessible surface area, or SASA) yielded better predictions than those that were more solvent-exposed. The researchers did not report on the effect of rotatable bonds; intuitively, one might think that a more flexible fragment would be harder to dock. A study we highlighted nearly ten years ago found that fragments with higher ligand efficiency also had higher docking scores, and it would be interesting to know if that reproduced with this larger data set.

 

The researchers conclude by noting that “these programs do not represent the optimal solution for fragment docking.” I think this is a fair assessment. And as the researchers acknowledge, the bar was set low: compounds were docked against the crystal structure of the protein with ligand computationally removed. In the real world, proteins often change conformation upon ligand-binding, which would make docking even more difficult.

 

In addition to trying to determine how a specific fragment binds, it can also be valuable to computationally screen large numbers of fragments. The programs used here took between 10 seconds and 42 minutes per ligand, but as we highlighted last year speed continues to increase.

 

Most importantly, the public availability of LEADS-FRAG will allow others to assess their own computational approaches. It will be fun to revisit this topic in a few years to see how much things have improved.

Fragments vs LpxC, two ways

Gram negative bacteria such as Pseudomonas aeruginosa are a continuing threat, and antibacterial drug discovery is not keeping pace. The enzyme UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is critical for the synthesis of the bacterial cell wall lipopolysaccharide. In a new J. Med. Chem. paper, Yousuke Yamada, Rod Hubbard, and collaborators at Taisho and Vernalis describe progress against this target.

 

LpxC is a zinc hydrolase, and although previous potent inhibitors have been reported against the metalloenzyme, these contained hydroxamate moieties. Unfortunately, hydroxamic acids are rather nonspecific zinc binders, and many of them hit human enzymes such as HDACs and MMPs. Thus, the researchers turned to fragments to find new metallophilic starting points.

 

The 1152 members of the Vernalis fragment library were screened against LpxC using three NMR experiments: STD, WaterLOGSY, and CPMG in pools of six. This yielded a remarkable 252 hits in at least one assay. These were retested individually and for competition with a substrate pocket-binding small molecule, resulting in 28 hits, two of which were advanced.

 

A crystal structure of compound 6 bound to LpxC suggested that adding a hydroxyl group could make additional interactions with the protein, and this was confirmed in the form of compound 10. Further fiddling in this region of the molecule was not successful, and the phenyl ring did not provide good vectors to a hydrophobic tunnel. However, replacing the phenyl with a more shapely piperidine yielded compound 17. Although this molecule had slightly lower affinity, it did provide a better starting point for further optimization, ultimately leading to compound 21, with low nanomolar potency against LpxC. Unfortunately, this and other members of the series showed only weak antibacterial activity.

 

Compound 9 was weaker than the other fragment starting point, but making and testing related compounds led to improved binders such as compound 27. This was the first molecule in this series to be structurally characterized, and crystallography revealed that the imidazole was making a single interaction with the zinc at the heart of the LpxC active site. Adding a hydroxyl led to bidentate chelator 29 (i.e. two interactions with the zinc) that had better activity, and further structure-based design ultimately led to low nanomolar inhibitors such as compound 43. In contrast to the other series, this one did show antibacterial activity, and the researchers eventually discovered molecules with in vivo efficacy. Both series were also selective against a small panel of human metalloproteases.

 

 

This is a nice fragment to lead story (expect it to be included in the next compilation). As the researchers note, it provides two important lessons. First, fragments can provide multiple different starting points for a target. Second, because fragment libraries tend to be small, it can be valuable to take some time to refine a fragment before launching into fragment growing or merging. Indeed, compound 38 (itself fragment-sized) contains only four more atoms than the initial fragment hit, yet has more than a thousand-fold higher affinity. During lead optimization you often need to add molecular weight, lipophilicity, and possibly polar atoms, so it is crucial to get the core binding elements as good as possible.