Review of 2022 reviews

The winter solstice is behind us in the Northern Hemisphere, which means 2022 is rapidly drawing to a close. As we have done for the past decade, Practical Fragments will spend this last post of the year summarizing conferences and reviews.

 

The remarkable progress in vaccines against SARS-CoV-2 allowed the full return of in-person conferences, and it was nice to see folks at CHI’s Discovery on Target in Boston and Drug Discovery Chemistry in San Diego. Nearly twenty reviews of interest to this readership were published, and these are covered thematically.

 

Targets

Several reviews cover the use of FBLD to target antiviral and antibacterial targets. Sangeeta Tiwari and colleagues at University of Texas El Paso cover both in an open access Pharmaceuticals review, focusing on tuberculosis and HIV, which often afflict the same individuals, leading to worse outcomes. The paper includes several tables with chemical structures, though the fragment origins of some molecules are not apparent.

 

Tuberculosis is caused by Mycobacterium tuberculosis, but there are more than 170 known members of the Mycobacteriaceae family. In an open access Int J. Mol. Sci. paper, the Tiwari group describes fragment-based approaches against these bugs. In addition to multiple examples, the review provides summaries of fragment finding methods and some of the challenges the field faces.

 

Another organism, Pseudomonas aeruginosa, infects the lungs of people with cystic fibrosis. In an open access Front. Mol. Biosci. paper, Tom Blundell and collaborators at University of Cambridge summarize fragment-based campaigns against this organism and its enzymes. The authors focus on structure-guided methods and note that the work is “at an early stage” but encouraging.

 

Switching to mammalian targets, Katrin Rittinger and colleagues at The Francis Crick Institute review (open access) applications of FBLD for targeting the ubiquitin system in Front. Mol. Biosci. The paper includes a nice table summarizing 15 examples that includes target, enzyme class, fragment binding mode, detection methods, and chemical structures of the fragment hit and optimized compound where applicable. Many of these are covalent modifiers; more on that topic below.

 

Finally, Tarun Jha, Shovanlal Gayen, and collaborators at Jadavpur University discuss “recent trends in fragment-based anticancer drug design strategies” in Biochem. Pharm. In addition to case studies (with chemical structures) of FBLD approaches against 18 oncology targets, the review covers fragment libraries, screening methods, optimization, and challenges.

 

Methods

Many of the targets above are challenging, and it’s always nice to be able to assess how challenging a project might be at the outset. In Curr. Opin. Struct. Biol., Sandor Vajda and collaborators at Boston University and Stony Brook University discuss (open access) “mapping the binding sites of challenging drug targets.” This is a brief, readable account of computational methods to identify hot spots, including allosteric ones. The authors examine the various small-molecule binding sites on KRAS and conclude that, due to “limited druggability,” the “other G12 oncogenic mutants will be very challenging.” Perhaps, but not impossible, as researchers at Mirati demonstrated earlier this year with the (open access) publication of a low (or sub) nanomolar KRAS^G12D inhibitor.

 

Among experimental methods used in FBDD, NMR is a mainstay, as demonstrated by Luca Mureddu and Geerten Vuister (University of Leicester) in Front. Mol. Biosci. (open access). The paper covers methods, successes, and challenges, focusing on three compounds that reached the clinic: AZD3839, venetoclax, and S64315.

 

In contrast to NMR, dynamic combinatorial chemistry (DCC) and DNA-encoded libraries (DEL) are used less frequently in FBLD. In RSC Chem. Biol., Xiaoyu Li and collaborators at University of Hong Kong and Jining Medical University discuss “recent advances in DNA-encoded dynamic libraries.” This concise paper covers lots of ground and does not understate the challenges.

 

Libraries

“The importance of high-quality molecule libraries” is emphasized by Justin Bower and colleagues at the Beatson Institute in Mol. Oncol. This highly readable and wide-ranging open access review covers all aspects of library design and use and includes comparisons of some of the major commercial vendors. An important point is that the “hit rate does not define the success of a library as it is more important to identify ligand-efficient and chemically tractable start points.”

 

Thus, even though shapely fragments may have lower hit rates than more planar aromatic fragments, they may still be worth including – if you can make them. In Drug Discov. Today (open access), Peter O’Brien and collaborators at University of York and Vrije Universiteit Amsterdam review synthetic strategies behind 25 “3D” fragment libraries. The tabular summary showing all the scaffolds emphasizes that most of these libraries are modest in size, with the largest being 102 members. Chemists will particularly enjoy the multiple synthetic schemes. The authors note the importance of “fragment sociability” to facilitate SAR and elaboration.

 

Covalent fragments

Special libraries are required for covalent fragment-based drug discovery, the most notable feature being the “warhead” that reacts with the protein target. These are the focus of a chapter in Adv. Chem. Prot. by Péter Ábrányi-Balogh and György Keserű of the Hungarian Research Centre for Natural Sciences. The review includes a table containing more than 100 warheads with associated mechanisms and amino acid selectivity.

 

The “reactivity of covalent fragments and their role in fragment-based drug design” is the focus in an (open access) Pharmaceuticals review by Kirsten McAulay and colleagues at the Beatson Institute. This is a nice overview of the field and contains several case studies. The authors conclude that “striking a balance between reactivity, potency and selectivity is key to identifying potential candidates.”

 

“Advances in covalent drug discovery” are reviewed (open access) by Dan Nomura and colleagues at University of California Berkeley in Nat. Rev. Drug Disc. This is a highly readable and comprehensive overview of the field. The authors differentiate between “ligand-first” approaches, in which a covalent warhead is appended to a known binder (such as here) and “electrophile-first,” in which “the initial discovery process is rooted in finding a covalent ligand from the outset,” such as for KRAS^G12C inhibitors.

 

Another broad overview of covalent inhibitors is provided by Juswinder Singh (Ankaa Therapeutics) in J. Med. Chem. Jus is a pioneer in the field, having published the first targeted covalent inhibitor in 1997. Of 1673 small molecules approved as drugs by the US FDA, only about 7% are covalent, and it wasn’t until recently that these have been intensively pursued. Part of the reluctance has been concerns over toxicity, but the paper suggests that – at least among kinase inhibitors – covalent drugs may actually be safer, perhaps due to conjugation of glutathione to the warhead and rapid clearance rather than formation of reactive metabolites.

 

Other

Whether covalent or not, thermodynamics plays a fundamental role in protein-ligand interactions, and this is the topic of an (open access) review in Life by Conceição Minetti and David Remeta of the State University of New Jersey. The paper covers a lot of ground, including drug discovery approaches, metrics (such as LE, LLE, etc.), isothermal titration calorimetry, case studies, and more. Importantly, the authors acknowledge the many challenges of applying thermodynamics to drug discovery, some of which we highlighted here.

 

Thermodynamics explains the potency increases longed for when doing fragment-linking, the subject of two reviews. In Chem. Biol. Drug Des. Anthony Coyne and colleagues at University of Cambridge provide a broad overview, starting with the historical theoretical background and newer developments. The bulk of the paper surveys published examples of fragment linking, with structure-based methods (whether X-ray, NMR, or computational) separated from target-guided methods such as DCC.

 

The second review, published in Bioorg. Chem. by Junmei Peng and colleagues at University of South China, is broader in scope, encompassing not just FBLD but also linkers used in PROTACs and even antibody-drug conjugates. The paper is organized by chemical structure of the linker.

 

Finally, in J. Med. Chem., Peter Dragovich, Wolfgang Happ, and colleagues at Genentech and Roche examine “small-molecule lead-finding trends” at their organizations between 2009 and 2020. (Although Genentech is fully owned by Roche, its research organization operates independently.) Fragment-based approaches led to only a small fraction of chemical series at Genentech and none at Roche. The authors note that leads derived from public sources such as patent applications were often found and pursued earlier, and that “purposeful dedication” of resources to fragment approaches may be necessary. Another major source of leads at Genentech is in-licensing, and some of these are fragment-derived.

 

And that’s it for 2022, year three of COVID-19. Thanks for reading and special thanks for commenting. May the coming year bring health, peace, and significant scientific progress.

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.

Review of 2018 reviews

As 2018 recedes into history, we are using this last post of the year to do what we have done since 2012 – review notable events along with reviews we didn’t previously cover.

This was a busy year for meetings, starting in January with a FragNet event in Barcelona, then moving to San Diego in April for the annual CHI FBDD meeting. Boston saw an embarrassment of riches, from the first US-based NovAliX meeting, to a symposium on FBDD at the Fall ACS meeting, followed closely by a number of relevant talks at CHI’s Discovery on Target. Finally, the tenth anniversary of the renowned FBLD meeting returned to San Diego. Look for a schedule of 2019 events later this month.

If meetings were abundant, the same can be said for reviews.

Lead optimization

Writing in J. Med. Chem., Dean Brown and Jonas Boström (AstraZeneca) asked “where do recent small molecule clinical development candidates come from?” For three quarters of the 66 molecules published in J. Med. Chem. in 2016 and 2017 the answer is from known compounds or HTS, though fragments accounted for four examples. Although average molecular weight increased during lead optimization, lipophilicity did not, suggesting the importance of this parameter.

The importance of keeping lipophilicity in check is also emphasized by Robert Young (GlaxoSmithKline) and Paul Leeson (Paul Leeson Consulting) in a massive J. Med. Chem. treatise on lead optimization. Buttressed with dozens of examples, including several from FBLD, they show that the final molecule is usually among the most efficient (in terms of LE and LLE) in a given series, even when metrics were not explicitly used by the project team. Perhaps with pedants like Dr. Saysno in mind, they also emphasize the complexity of drug discovery, and note that “seeking optimum efficiencies and physicochemical properties are guiding principles and not rules.”

Lipophilic ligand efficiency (LLE) is also the focus of a paper in Bioorg. Med. Chem. by James Scott (AstraZeneca) and Michael Waring (Newcastle University). This is based largely on personal experiences and provides lots of helpful tips. Importantly, the researchers note that calculated lipophilicity values can differ dramatically from measured values, and go so far as to say that “this variation is sufficient to render LLEs derived from calculated values meaningless.”

Turning wholly to fragments, Chris Johnson and collaborators (including yours truly) from Astex, Carmot, Vrije Universiteit Amsterdam, and Novartis have published an analysis in J. Med. Chem. of fragment-to-lead success stories from last year. This review, the third in a series, also summarizes all 85 examples published between 2015 and 2017, confirming and expanding some of the trends we mentioned last year.

Targets

Two reviews focus on specific target classes. Bas Lamoree and Rod Hubbard (University of York) cover antibiotics in SLAS Discovery. After a nice, concise review of fragment-finding methods, the researchers discuss a number of case studies, many of which will be familiar to regular readers of this blog, including an early example of whole-cell screening.

David Bailey and collaborators from IOTA and University of Cambridge discuss cyclic nucleotide phosphodiesterases (PDEs) in J. Med. Chem. The researchers provide a good overview of the field, including mining the open database ChEMBL for fragment-sized inhibitors. As they point out, the first inhibitors discovered for these cell-signaling enzymes were fragment-sized, so it is no surprise that FBLD has been fruitful – see here for an example from earlier this year. Interestingly though, although at least six fragment-sized PDE inhibitor drugs have been approved, none of these were actually discovered using FBLD.

PDEs are an example of “ligandable” targets, for which small molecule modulators are readily discovered. In Drug Discovery Today, Sinisa Vukovic and David Huggins (University of Cambridge) discuss ligandability “in terms of the balance between effort and reward.” They use a published database of protein-ligand affinities to develop a metric, LIG_exp, for experimental ligandability, and also describe their computational metric, Solvaware, which is based on identifying clusters of water molecules binding weakly to a protein. Comparisons with experimental data and with other predictive metrics, such as FTMap, reveal that while the computational methods are useful, there is still room for improvement.

We have previously written about how target-guided synthesis methods such as dynamic combinatorial chemistry have – despite decades of research – yielded few truly novel, drug-like ligands. Is this because the targets chosen were simply not ligandable? In J. Med. Chem., Anna Hirsch and collaborators at the University of Groningen, the Helmholtz Institute for Pharmaceutical Research, and Saarland University review some (though by no means all) published examples and examine their computationally determined ligandability scores. There seems to be no difference between these targets and a set of traditional drug targets.

Finding fragments

Crystallography continues to be a key tool for FBLD: as we noted in the review of the 2017 literature, 21 of the 30 examples made use of a crystal structure of either the starting fragment or an analog, and only 3 projects didn’t use crystallography at all. That said, FBLD is possible without crystallography, as illustrated through multiple examples in a Cell Chem. Biol. review by Wolfgang Jahnke (Novartis), Ben Davis (Vernalis), and me (Carmot).

In the absence of a crystal structure, NMR is best suited for providing structural information, and this is the subject of a review in Molecules by Barak Akabayov and colleagues at Ben-Gurion University of the Negev. The researchers provide a nice summary of NMR screening methods and success stories within a broader history of FBLD. They also include an extensive list of fragment library providers as well as a discussion of virtual screening.

Speaking of virtual screening, three reviews cover this topic. In Methods Mol. Biol., Durai Sundar and colleagues at Indian Institute of Technology Delhi touch on a number of computational approaches for de novo ligand design, though the lack of structures sometimes makes it challenging to read. A broader, more visually appealing review is published in AAPS Journal by Yuemin Bian and Xiang-Qun Xie at University of Pittsburgh. In addition to an overview and case studies, the researchers also provide a nice table summarizing 15 different computational programs. One of these, SEED, is a main focus of a review in Eur. J. Med. Chem. by Jean-Rémy Marchand and Amedeo Caflisch (University of Zürich). The researchers describe how this docking program can be combined with X-ray crystallography (SEED2XR) to rapidly identify fragments; we highlighted an example with a bromodomain. Their ALTA protocol uses SEED to generate larger, more potent molecules, as we described for the kinase EphB4. The researchers note that together these protocols have led to about 200 protein-ligand crystal structures deposited in the PDB over the past five years.

Rounding out methods, Sten Ohlson and Minh-Dao Duong-Thi (Nanyang Technological University) provide a detailed how-to guide in Methods for performing weak affinity chromatography, and how this can be combined with mass spectrometry (WAC-MS), as we noted last year.

Chemistry

One drawback of some computational approaches for fragment optimization is that they do not consider synthetic accessibility. In Mol. Inform., Philippe Roche, Xavier Morelli, and collaborators at Aix-Marseille University and Institut Paoli-Calmettes focus on hit to lead approaches that do, and provide a handy table summarizing nearly a dozen computational methods. We highlighted one from the authors, DOTS, earlier this year.

DOTS is an example of using DOS, or diversity-oriented synthesis. In Front. Chem., David Spring and colleagues at University of Cambridge review recent applications of DOS for generating new fragments, some of which we recently highlighted. Only a couple examples of successfully screening these new fragments are described, but the authors note that this is likely to increase as virtual library screening continues to advance.

Perhaps the most productive fragment of all time is 7-azaindole, the origin of three fragment-derived clinical compounds. (The moiety appears in both approved FBLD-derived drugs, vemurafenib and venetoclax.) Takayuki Irie and Masaaki Sawa of Carna Biosciences devote their attention to this little bicycle in Chem. Pharm. Bull. The researchers count six clinical kinase inhibitors that contain 7-azaindole (not all from FBLD) as well as more than 100,000 disclosed compounds containing the fragment. More than 90 kinases have been targeted by molecules containing 7-azaindole, and the paper provides a list of 70 PDB structures of 37 different kinases bound to molecules containing the moiety.

Finally, in J. Med. Chem., Brian Raymer and Samit Bhattacharya (Pfizer) survey the universe of “lead-like” drugs. Among the most highly prescribed small molecule drugs, 36% have molecular weights below 300 Da. Only 28 of 174 drugs approved between 2011 and 2017 fall into this category, consistent with the increasing size of newer drugs. The researchers discuss 16 recently approved drugs, and find that 13 have very high ligand efficiencies (at least 0.4 kcal mol^-1 per heavy atom). As noted above, optimization often entails adding molecular weight by growing or linking, and the researchers suggest that alternative strategies such as conformational restriction and truncation also be investigated.

And with that, Practical Fragments wishes you a happy new year. Thanks for reading some of our 686 posts over the past decade plus, and please keep the comments coming!

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.