Review of 2019 reviews

The year ends, and with it the awkward teenage phase of the twenty-first century. As we have done since 2012, we're using this last post of the year to highlight conferences and reviews over the previous twelve months.

There were some good events, including CHI’s Fourteenth Annual Fragment-based Drug Discovery meeting in San Diego in April, their Discovery on Target meeting in Boston in September, and the third Fragment-based Drug Design Down Under 2019 in Melbourne in November, which also saw the launch of the Centre for Fragment-Based Design. Our updated schedule of 2020 events will publish next week.

Turning to FBLD reviews, Martin Empting (Helmholtz-Institute for Pharmaceutical Research Saarland) and collaborators published a general overview in Molecules. This is a nice up-to-date summary, covering library design, methods to find, confirm, and rank fragments, and optimization approaches. It’s also open access so you can read it anywhere.

Targets

Protein-protein interactions can be particularly challenging drug targets, and these are covered in a Eur. J. Med. Chem. review by Dimitrios Tzalis (Taros Chemicals), Christian Ottmann (Technische Universiteit Eindhoven) and colleagues. The focus is on clinical compounds, and several of these – including venetoclax, ASTX660, mivebresib, onalespib – are discussed in detail. The article is particularly useful in discussing late-stage optimization of pharmacokinetic and pharmacodynamic properties. It also provides a nice summary of physicochemical properties for fragment hits and derived candidates.

Target selectivity is always important, and this is the focus of a review in Exp. Opin. Drug Disc. by Rainer Riedl and collaborators at the Zurich University of Applied Sciences and the Università degli Studi dell’Insubria. Although the broader topic is de novo drug design, fragment-based methods are prominent, and include case studies we’ve discussed on nNOS, pantothenate synthetase, and MMP-13.

In terms of specific targets, Fubao Huang, Kai Wang, and Jianhua Shen at the Shanghai Institute of Materia Medica provide an extensive review of lipoprotein-associated phospholipase A2 (Lp-PLA₂) in Med. Res. Rev. This serine hydrolase has been studied for four decades but – as the researchers note – “divergence seems to be ubiquitous among Lp-PLA₂ studies.” At least this is not for lack of good chemical tools, fragment-derived (see here, here, and here) and otherwise.

Methods

Although NMR has fallen behind crystallography in our latest poll, that is certainly not reflected in terms of reviews. In particular, ¹⁹F NMR is covered in three papers. CongBao Kang (A*STAR) manages to pack a lot (including 261 references!) into a concise review in Curr. Med. Chem. Topics include protein-observed ¹⁹F NMR, in which one or more fluorine atoms are introduced into a protein genetically, enzymatically, or chemically, as well as ligand-observed methods, in which fluorine-containing small molecules are directly observed or used as probes that are displaced by non-fluorine-containing molecules.

Protein-observed ¹⁹F NMR (PrOF NMR) is covered in Acc. Chem. Res. by William Pomerantz and colleagues at the University of Minnesota. Although the first example was published 45 years ago, only in the past few years has the technique been used for studying protein-ligand interactions. The researchers note that introducing fluorines into aromatic residues is ideal because they are relatively rare, simplifying interpretation, and overrepresented at protein-protein interactions, maximizing utility. Several case studies are described, and even proteins as large as 180 kDa are amenable to the technique.

Ligand-based fluorine NMR screening is simpler and more common than techniques that focus on proteins, and this topic is thoroughly reviewed by Claudio Dalvit (Lavis) and Anna Vulpetti (Novartis) in J. Med. Chem. After a section on theory, the researchers discuss library design, including a long section on quality control (which involves assessing solubility, purity, and aggregation of the molecule in a SPAM filter). Direct and competition-based screening approaches are covered in detail; for the latter, a new method for determining binding constants is provided. The paper concludes with more than a dozen case studies. Clearly much has changed in the ten years since I wondered “why fluorine-labeled fragments are not used more widely.” This perspective is a definitive guide to the topic.

Moving to less common methods for characterizing fragments, György Ferenczy and György Keserű (Research Center for Natural Sciences, Budapest) cover thermodynamic profiling in Expert Opin. Drug Disc. After discussing several case studies, they conclude that “thermodynamic quantities are not suitable endpoints for medicinal chemistry optimizations” due to the complexity of contributing factors. This is consistent with another recent paper on the subject (see here), though the information provided is still interesting for understanding molecular interactions.

And although you might have thought the 2017 VAPID publication was the last word on the limitations of ligand efficiency (LE), Pete Kenny has published a splenetic jeremiad on the topic in J. Cheminform. (see also his blog post on the topic, which includes a sea serpent). This is largely a retread of a 2014 article on the same topic (reviewed by Teddy in his inimitable manner here). Pete also describes a more complicated alternative to LE involving residuals, though unfortunately he provides no evidence that it provides more useful information. Pete is of course correct to remind us that metrics have limitations, but assertions that LE “should not even be considered to be a metric” are overwrought.

Chemistry

Two articles discuss virtual chemical libraries. In J. Med. Chem., W. Patrick Walters (Relay Therapeutics) describes efforts to measure, enumerate, and explore chemical space. He notes that false positives could quickly overwhelm a virtual screen of a hundred million molecules, but as we saw earlier this year, progress is being made. Indeed, Torsten Hoffmann (Taros Chemicals) and Marcus Gastreich (BioSolveIT) focus on navigating the vastness of chemical space in Drug Disc. Today. They note that the Enamine REAL Space is up to 3.8 billion commercially accessible compounds, more than double the number of stars in the Milky Way. But this pales in comparison to the 10²⁰ potential compounds in Merck’s MASSIV space. Just storing the chemical structures of these in compressed format would require 200,000 terabytes – and searching them exhaustively is beyond current technology.

Ratmir Derda and Simon Ng (University of Alberta) discuss “genetically encoded fragment-based discovery” in Curr. Opin. Chem. Biol. This involves starting with a known fragment that is then coupled to a library of peptides and screened to find tighter binders. The researchers provide a number of case studies, though adding even a small peptide to a fragment will generally have deleterious effects on ligand efficiency. And – Rybelsus not withstanding – oral delivery of peptides is challenging.

Finally, Vasanthanathan Poongavanam, Xinyong Liu, and Peng Zhang, and collaborators at Shandong University, University of Bonn, University of Southern Denmark, and K.U. Leuven review “recent strategic advances in medicinal chemistry” in J. Med. Chem. Among a wide range of topics from drug repurposing to antibody-recruiting molecules is a nice, up-to-date section on target-guided synthesis. As I opined a couple years ago, I still doubt whether this will ever be generally practical, but from an intellectual standpoint I’m happy to see work continue on the approach.

And with that, Practical Fragments says goodbye to the teens and wishes you all a happy new year. Thanks for reading and commenting. May 2020 bring wisdom, and progress.

Fragments in the clinic: S64315 / MIK665

Earlier this year we highlighted the discovery of AZD5991, a phase 1 compound from AstraZeneca that inhibits the anti-apoptotic cancer target Mcl-1. Those efforts made use of a fragment previously published by a different research group. Mcl-1 has been a popular target for some time; the first mention on Practical Fragments dates to 2010. The story behind another investigational drug is described in a couple papers from earlier this year.

The first, in ACS Omega by Rod Hubbard and colleagues at Vernalis, University of York, and Servier, describes fragment screening efforts against both Bcl-2 and Mcl-1. The proteins are related both structurally and functionally, and Bcl-2 is the target of venetoclax – the second fragment-derived drug approved. Some of the early fragment hits bound to both proteins, but selective and potent inhibitors were ultimately developed. In the interest of space only those against Mcl-1 will be discussed here.

Both proteins required considerable protein engineering, which is described in detail. Ultimately one form of human Mcl-1 was used for crystallography, while mouse protein was used for NMR screening due to its better stability. A total of 1064 fragments were screened at 0.5 mM each (in pools of eight) using ligand-observed NMR; 39 confirmed using STD NMR, WaterLOGSY, and CPMG. Additionally, fluorescence polarization, 2-dimensional (HSQC) NMR, ITC, and SPR were used to validate hits. Crystallography proved challenging in the beginning but ultimately helped drive optimization of more potent molecules. The large number of different assays employed is consistent with our recent poll results.

Protein-observed NMR was particularly useful in providing information on the quality of both the ligand and protein (reminiscent of the “validation cross” discussed here). Before crystallography was able to play a meaningful role, “NMR-guided models,” combining partial protein assignments with flexible docking, were used to drive SAR.

While the first paper focuses on protein optimization and biophysics, the second (in J. Med. Chem.), by András Kotschy and collaborators, focuses on chemistry. Fragment 1a was one of several hits pursued, initially by looking for analogs, but most of these had comparable (weak) activity. In the absence of a crystal structure a systematic chemistry campaign was conducted, varying elements of the core and sidechains. Many of these molecules had comparable activity against both Mcl-1 and Bcl-2, but replacing the nitrogen linker with an oxygen led to selectivity against the former. The addition of hydrophobic substituents led to compound 10c, with submicromolar activity.

Anticipating poor cell permeability for a negatively charged, lipophilic molecule, the researchers introduced a positively charged methylpiperazine moiety at various positions around the molecule, ultimately leading to compound 18a. In addition to potent Mcl-1 binding, this molecule is active in cells and shows reasonable pharmacokinetic properties in mice. Further optimization to S64315 does not appear to have been published yet, though the structure was disclosed earlier this year, and the fragment origins remain clear.

Together these papers provide a thorough description of drugging a difficult target. They also provide insights into the investment required. The Mcl-1 project began around 2007, and it took a decade before S64315 entered the clinic. Enabling drug discovery against protein-protein interactions required multiple biophysical techniques in addition to all the standard components of pharmaceutical research. The researchers note that “establishing such a platform can take some time and resource – a tool compound is usually needed to validate the assays, but the assays are needed to identify the tool compound.” In the end they have succeeded, and Practical Fragments wishes them – and the patients being treated – the best of luck.

A new library of fluorinated Fsp3-rich fragments

Among fragment-finding methods, ligand-based NMR ranks near the top in terms of popularity. Of its many variations, fluorine (¹⁹F) NMR appears to be gaining in popularity. Fluorine NMR has several advantages, including high sensitivity and the fact that many fragments can be screened simultaneously because of the wide chemical shift range for fluorine. Although more commercial fluorine-enriched libraries are available now than when we first wrote about the approach a decade ago, the diversity of these libraries is still somewhat limited. This problem has been tackled by Mads Clausen at the Technical University of Denmark and an international team of collaborators in a new Angew. Chem. Int. Ed. paper.

The researchers wanted to create a fluorinated fragment library that would be not just diverse but also contain a high fraction of sp³-hybridized carbons (high Fsp³). Some of the early claims around “three dimensional” fragments have been questioned, and there seems to be little if any correlation between the shapeliness of fragments and that of derived leads, but if you’re going to make new fragments in academia it makes sense to explore interesting molecular architectures.

Starting from just six simple building blocks, each containing a trifluoromethyl group, the researchers generated nine different cores which were further derivatized at multiple positions to yield 115 diverse fragments. Consistent with diversity-oriented synthesis, no more than five synthetic steps were used for any molecule. All molecules were made as racemates in order to further increase the diversity of the library.

The resulting “3F Library” is mostly rule-of-three compliant, though given that the trifluoromethyl moiety alone adds 69 Da the fragments do tend to be larger, with an average molecular weight of 284 Da. They are, however, less lipophilic than two commercial fluorinated fragment libraries. And with an average Fsp³ = 0.7 and 3.3 chiral centers they are also quite shapely as assessed by principal moment of inertia.

Building a library is nice, but will it provide hits? To find out, the researchers screened the 102 fragments that passed quality control against four targets. They used a transverse (T₂) relaxation assay (specifically, CPMG) in which fragments bound to a protein tumble more slowly, causing a reduction in ¹⁹F signal intensity. Hit rates ranged from 3% to 11%, and about two thirds of these confirmed in STD or WaterLOGSY assays. As seen by the examples shown here, the fragments are quite diverse.

Whether these hits will lead to more potent molecules remains to be seen. Laudably the paper ends with the statement: “we hope that the 3F library will find use for other researchers and we encourage anyone interested in screening the fragments to contact us.” If you are looking for interesting new fragments that are tailored for follow-up chemistry, I encourage you to take the team up on their offer.

Poll results: affiliation and fragment-finding methods in 2019

The fourth iteration of our fragment-finding methods poll has just closed. If you want to jump right to the results feel free to skip the next paragraph, which focuses on methods.

The poll was run using Crowdsignal, the successor to Polldaddy, and ran from 20 October through 30 November. This free polling software tabulates total number of votes for a question but not the number of individual respondents. To determine individual respondents, we included a question on “workplace and practice.” Of the 137 individual respondents to this question, 116 identified themselves as practicing FBLD, and we assumed they also answered the second question. The overall number of responses is slightly higher than in 2013 but a bit lower than in 2016.

Readership demographics have shifted from previous years, with about two thirds of respondents hailing from industry, up from just over half historically. The fraction of respondents who actively practice FBLD is also up modestly, to 85%.

But the question probably of most interest is on screening methods, summarized here.

As we also saw in 2013 and 2016, nearly all fragment-finding techniques are being used more, with the average respondent employing 6 methods today compared with 4.1 in 2016, 3.6 in 2013, and 2.4 in 2011.

X-ray crystallography has leapt to first place, likely driven in part by increasing speed and automation as well as by studies suggesting that crystallography can give impressively high hit rates.

As in 2016, ligand-detected NMR, SPR, and thermal shift assays are all very popular. Use of computational approaches has increased, though perhaps not as much as might be expected given recent advances. Functional screening is the only technique for which use has remained constant, or perhaps even declined very slightly from 2013.

For the first time we asked about use of literature to identify fragments, and nearly a third of respondents said they incorporate previously published fragments into their work. As the amount of publicly available information continues to increase it will be interesting to see whether this number grows.

More niche methods such as mass spectrometry, MST, affinity selection, and biolayer interferometry are gaining adherents; 30 respondents reported using mass spectrometry, for example. While fewer than 20% of respondents are using affinity chromatography (including WAC), CE, or ultrafiltration, that proportion has nearly quadrupled from our previous three polls, though we can’t say which of these related methods accounts for the increase.

Finally, only four respondents reported using “other” methods, such as SHG. Perhaps we’ll ask about this and other emerging methods explicitly next time.

Do the results surprise you, or are they consistent with what you are using at your organization?