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.

Dynamic combinatorial chemistry revisited: why it’s so difficult

Last year we discussed the application of dynamic combinatorial chemistry (DCC) to fragment linking. The idea is that a protein will shift the equilibrium of a reversible reaction, selecting the tightest binder. Over the past twenty years practitioners of DCC have generated plenty of papers, some quite nice, but I do not recall seeing examples of the technique generating novel and attractive chemical leads. A new paper in Chem. Eur. J. by Beat Ernst and colleagues at the University of Basel explains why it is so difficult.

The researchers were interested in the bacterial protein FimH, which helps microbes colonize the urinary tract by adhering to human proteins that are decorated with mannose. The chemistry the researchers decided to explore for DCC was the reaction of aldehydes with hydrazides to form acylhydrazones. This reaction is slowly reversible at pH 7, allowing exchange between library members to occur, but it can be essentially frozen by raising the pH.

To try to understand every aspect of their system, the researchers focused on a tiny library. Two aldehydes were chosen, one based on mannose, the other based on glucose. Four (quite similar) commercially available hydrazides were purchased.

The researchers made and tested the affinity of each of the eight possible library members using surface plasmon resonance (SPR). The four acylhydrazones based on mannose had dissociation constants (K_D) ranging from 0.33 to 0.76 µM, while the mannose aldehyde came in at 3.2 µM. In contrast, the four acylhydrazones based on glucose had K_D values between 152 and 735 µM, comparable to the glucose aldehyde itself (194 µM). Since mannose is the natural ligand for FimH while glucose is not, this was expected.

One challenge of DCC is separating library members from the protein for analysis; releasing bound ligands can be particularly challenging if they bind tightly to the protein. A variety of methods were tested, including microfiltration, but this gave “massive alterations in composition.” Various attempts at protein denaturation and precipitation using organic solvents or heat also failed. The fact that this step was so difficult, even for closely related ligands (the difference between mannose and glucose is the stereochemistry around a single hydroxyl group) underscores the challenge of analyzing DCC mixtures.

The problem was finally solved by using a biotinylated version of FimH which could be captured using commercial streptavidin agarose beads.

The most general approach works as follows.

1. Incubate 100 µM FimH protein with library (with each aldehyde and hydrazide at 50-200 µM) at pH 7 for 3 days in the presence of 10 mM aniline, which catalyzes the acylhydrazone exchange.

2. Raise the pH to 8.5 to stop the reaction, add streptavidin agarose, centrifuge, and discard the supernatant containing the unbound molecules.

3. Resuspend the agarose beads containing the protein, add a competitor to release bound ligands, increase the pH to 12 to ensure release, and analyze the product ratios using HPLC.

Although cumbersome, this protocol does work: mannose-derived compounds were enriched relative to glucose-derived compounds, as expected due to their higher affinities, and the most potent compound was enriched over the less potent ones. That said, the robustness of the results were dependent on the ratios of library components.

So will DCC ever be practical? I’m not so sure. But, as the researchers end hopefully but not hypefully, their work “is a contribution to this challenge.”

Molecules special issue: Developments in Fragment-Based Lead Discovery

Last December the first-ever Pacifichem symposium on FBLD was held in Honolulu. Two of the organizers, Martin Scanlon and Ray Norton, invited participants to submit manuscripts to a special issue of Molecules, which has now published.

The collection starts with a very brief Foreword by me describing the Symposium itself. The first actual paper, from Qingwen Zhang and collaborators at the Shanghai Institute of Pharmaceutical Industry, WuXi AppTec, and China Pharmaceutical University, focuses on kinase inhibitors. The researchers examine fragment-sized substructures of 15 approved drugs that inhibit kinases and use these to design a high-nanomolar inhibitor of the V600E mutant form of BRAF, which modeling suggests should bind to the protein in the “DFG-out” conformation.

Next comes a fragment-finding paper from Thomas Leeper and collaborators at the University of Akron and the University of North Carolina, Chapel Hill. The researchers were interested in finding inhibitors of the glutaredoxin protein (GRX) from the pathogen Brucella melitensis, which causes brucellosis. An STD NMR screen of 463 fragments (each at 0.5 mM in pools of 5-7) resulted in 84 hits, though 75 also hit human GRX. Subsequent experiments including chemical shift perturbation and modeling identified a mM binder with modest selectivity over the human enzyme. Next, the researchers introduced several covalent warheads (including a rather exotic ruthenium analog), one of which led to improved affinity, though the stoichiometry was not determined.

The remaining papers are all reviews, starting with one on native mass spectrometry (MS) by Liliana Pedro and Ronald Quinn at Griffith University. This provides a good historical, theoretical, and practical overview of the technique generally, as well as various applications for fragment-screening. It also covers most of the published examples and discusses both the strengths (such as speed and low protein consumption) as well as the weaknesses (false positives and false negatives) of native MS.

NMR is up next, with a paper by Pacifichem organizer Ke Ruan and colleagues at the University of Science and Technology of China, Hefei. This provides a concise but detailed description of library design, ligand- and protein-detected fragment screening, structural model generation, and hit to lead optimization.

Protein-directed dynamic combinatorial chemistry (DCC) is tackled by Renjie Huang and Ivanhoe Leung, both at the University of Auckland. In addition to summarizing the theory and various literature examples, the authors do an excellent job covering the pros and cons of different types of chemistries and analytical techniques.

Next comes a review by Begoña Heras and collaborators at La Trobe University and Monash University on the subject of bacterial Dsb proteins, which are essential for disulfide bond formation in virulence factors. The review covers the biology as well as several approaches to finding inhibitors, some of which we’ve previously covered (here and here). There is much more to do: as the researchers conclude, “the development of Dsb inhibitors is still in its infancy.”

Finally, Ray Norton and colleagues at Monash University discuss applications of ¹⁹F NMR for fragment-based lead discovery. In addition to covering fluorine-containing fragments, the researchers also discuss using fluorine-containing probe molecules and – even more unusual – fluorine-labeled proteins, in this case using 5-fluorotryptophan. The paper includes previously unpublished results on how these latter two approaches can be used to understand protein-ligand interactions.

One nice feature of this journal is that it is open-access, so if you are lucky enough to be back in Hawaii this December you can pull up the papers on your smartphone while lying on the beach.

Dynamic combinatorial chemistry and fragment linking

Dynamic combinatorial chemistry (DCC) sounds incredibly cool. The idea is that libraries spontaneously form and reform. Add a protein and Le Châtelier's principle favors the formation of the best binders. In other words, not only does cream rise to the top, more cream is actually created.

The applications of DCC for fragment linking are obvious, and indeed early reports date back nearly twenty years to the dawn of practical FBDD. The latest results are described in a new paper in Angew. Chem. Int. Ed. by Anna Hirsch and collaborators mostly at the University of Groningen.

The researchers were interested in the aspartic protease endothiapepsin, which is a model protein for more disease-relevant targets. This is a dream protein: it is easy to make in large amounts, crystallizes readily, and is stable for weeks at room temperature. Readers will recall that this protein has also been the subject of multiple screening methods. Previous efforts using DCC had generated low micromolar inhibitors such as 1 and 2. These acylhydrazones form reversibly from hydrazides and aldehydes. Crystallography had also previously revealed that compound 1 binds in the so-called S1 and S2 subsites of endothiapesin while compound 2 binds in the S1 and S2’ subsites. In the current paper, the researchers enlisted DCC to try to combine the best of the binding elements.

To do this, the researchers chose isophthalaldehyde, which contains two aldehyde moieties, and nine hydrazides, which could give a total of 78 different bis-acylhydrazones. They incubated 50 µM of isophthalaldehyde with either four or five of the hydrazides (each at 100 µM), with or without 50 µM protein, and in the presence of 10 mM aniline to accelerate the exchange. Reactions were allowed to incubate at room temperature at pH 4.6 for 20 hours, after which the protein was denatured and the samples were analyzed by HPLC to see whether some products were enriched in the presence of protein.

Biologists may want to consider whether their favorite proteins would remain folded and functional under these conditions, and chemists may also balk at molecules containing an acylhydrazone moiety – let alone two. Leaving aside these concerns, though, what were the results?

As one would hope, some molecules were enriched over others when protein was present, though only by a modest two or three-fold. Two of the enriched molecules – both homodimers – were resynthesized and tested. Compound 13 was quite potent, and crystallography revealed that it binds in a similar fashion to compound 1, though electron density is missing for part of the molecule. Compound 16, on the other hand, is only marginally more potent than the starting molecules. Unfortunately the researchers do not discuss the activities of molecules that had not been enriched at all.

The paper ends by stating rather hopefully that DCC “holds great promise for accelerating drug development for this challenging class of proteases, and it could afford useful new lead compounds. This approach could be also extended to a large number of other protein targets.”

I’m not so sure.

This is an interesting study; the work was carefully done and thoroughly documented—but I’m less sanguine about whether DCC will actually ever be practical for lead generation. Indeed, the very fact that the experiments were done well yet are incapable of distinguishing a strong binder from a weaker one argues that the technique is inherently limited. I would love to see DCC work, but it seems to me that, even after two decades of effort, DCC has not been able to move beyond proof of concept studies. Does anyone have a good counterexample?

Fragment linking in crystallo

Of the many ways to link fragments, one of the most intriguing is when the protein itself catalyzes or templates the assembly of two fragments (see for example here and here). The latest example of such target-directed fragment linking was published in last month’s issue of J. Appl. Cryst.

The researchers, led by Isao Tanaka at Hokkaido University, were interested in ligating fragments together in protein crystals. They first took crystals of the model protein trypsin and soaked these with an “anchor molecule,” in this case one of two benzamidine-containing aldehydes (benzamidines are classic trypsin binders). The crystals were then transferred to a second solution containing a “tuning molecule,” each of which contained either an aminooxy or hydrazine moiety that could react covalently with the aldehyde of the anchor molecule. Finally, the crystals were analyzed by X-ray and structures of any bound ligands solved.

A total of 33 different tuning molecules were examined, and two of these produced clear electron density in the active site showing that ligation with the anchor molecule had occurred (for example ALD2 and OXA9). Three others produced structures that suggested some disorder in the binding mode of the tuning molecule, and a fourth showed an assembled product that extended from the active site to a second trypsin molecule in the crystal lattice.

A study similar to this was published a number of years ago, but in that case it was not clear whether the ligation occurred in the crystal or in solution. In the present case, soaking pre-assembled molecules into the crystals produced inferior electron density to the two-step process. More excitingly, time-resolved experiments actually showed structural snapshots of the complex forming, both in the active site (which occurred in under a minute) as well as at the dimer interface (which took over an hour).

Unfortunately, the assembled products are not notably better binders than the initial fragment. The authors attribute this to the fact that their library of tuning molecules was very small. However, it is also possible that the approach selects not for the best binders but for those that can best form complexes within a fairly rigid crystal lattice. As we’ve seen before, protein crystals are far from physiological. It will be interesting to see whether in-crystal chemical ligation can generate superior binders.

DCC and FBDD

Dynamic combinatorial chemistry (DCC) has grown alongside of and often intersected with FBDD. In a recent issue of Angewandte Chemie, Jörg Rademann and colleagues at the Leibniz Institute of Molecular Pharmacology describe the latest example.

Put simply, DCC generates new molecules with some desired property by allowing smaller molecules to assemble reversibly under selection pressure. If the selection pressure is binding to a protein target and the molecules undergoing reactions are fragments, DCC can be used for FBDD. As we previously noted, Huc and Lehn published one of the earliest demonstrations of this. DCC has also been used at a few companies, including Astex and Sunesis, and even formed the basis of the (sadly) short-lived Therascope.

Rademann’s approach, "dynamic ligation screening", is based on labeling one fragment with a fluorescent probe and then screening it in a fluorescence polarization assay with other test fragments. If the labeled fragment binds competitively with a test fragment, this implies that the two fragments bind to the same site. However, if the fluorescence polarization signal increases in the presence of the test fragment (indicating increased binding of the labeled fragment), this suggests that the test fragment and the labeled fragment are binding cooperatively.

The researchers applied dynamic ligation screening to the protease caspase-3, a key mediator of apoptosis relevant for many diseases. As their labeled “fragment,” they chose a high-affinity tetrapeptide containing an alpha-ketoaldehyde: the ketone interacts covalently with the catalytic cysteine of the enzyme, while the aldehyde can form imines with amine-containing fragments. Interestingly, this strategy selects for fragments that bind in the S1’ subsite of the enzyme, which has not received as much attention as the tetrapeptide binding sites S1-S4.

A fluorescently labeled version of the tetrapeptide was screened against a library of 7,397 fragments, of which 4,019 contained primary amines. Of these, 78 fragments caused a decrease in the fluorescence polarization signal, suggesting that they compete with the tetrapeptide for binding. These were tested in an enzymatic assay: 21 of them were active at 10 micromolar concentrations, and four had Ki values from 3.1 to 5.5 micromolar; these four molecules have electrophilic carbons, making it likely that they bind to the catalytic cysteine residue.

Of greater interest, 176 fragments were cooperative, increasing the fluorescence polarization (FP) of the labeled tetrapeptide fragment by at least 20%. 50 of these were tested in an enzymatic assay, with the amine shown below emerging as the most potent FP enhancer and a Ki of 120 micromolar alone. A series of experiments guided by mathematical modeling suggested that the protein was templating the formation of an imine bond between the aldehyde of the tetrapeptide and the amine. Moreover, the reduced (amine) version of this conjugate exhibits a very high affinity for caspase-3, with a Ki of 80 picomolar.



Of course, affinity is not everything: with a molecular weight of 767 Da and a clearly peptidic nature, the pharmaceutical properties of this molecule, and even its cell activity, are questionable.

This study is reminiscent of some work we did at Sunesis, using caspase-3 to template the assembly of a non-peptidic inhibitor using Tethering. In that case we built molecules in the S1-S4 pockets, but did not do much work to extend into the S1’ pocket. It would be interesting to see if the fragment Rademann and colleagues discovered also boosts the potency of the molecules we identified.

For dynamic ligation screening to be general it needs to surmount at least two major potential limitations. First, it remains to be seen whether the technique will work with actual fragments, which are likely to have far lower affinities than the 25 nM tetrapeptide used in this study. Second, cooperative binding of the fragments does not translate to synergy in the final molecule: the conjugate has a lower ligand efficiency than either of the fragments, despite the apparent cooperativity of the two fragments binding to the target. This could be because the conjugate contains an amine, whereas the two fragments in solution presumably were linked by an imine; the differences in geometry and chemical nature between these two moieties are profound, and one could imagine that many amine-linked compounds would not be selected as imines, and vice versa.

Still, this is an interesting approach to tackle the long-standing challenge of linking fragments, and it will be fun to watch for new developments.