Fragment-Based Drug Discovery

This is the straight-to-the-point title of a new book published by the Royal Society of Chemistry, edited by Steven Howard (Astex) and Chris Abell (University of Cambridge). It is the second book on the topic published so far this year, and it is a testimony to the fecundity of the field that the two volumes have very little overlap.

After a brief forward by Harren Jhoti (Astex) and a preface by the editors, the book opens with two personal essays. The first, by me, is something of an apologia for Practical Fragments and the growing role of social media in science (and vice versa). If you’ve ever wondered how this blog got started or why it keeps going, this is where to find out. The second essay is by Martin Drysdale (Beatson Institute). Martin is a long-time practitioner of FBDD, dating back to his early days at Vernalis (when it was RiboTargets) and he tells a fun tale of “adventures and experiences.”

Chapter 1, by Chris Abell and Claudio Dagostin, is entitled “Different Flavours of Fragments.” With a broad overview of the field it makes a good introduction to the book. There are sections on fragment identification, including the idea of a screening cascade, as well as several case studies, some of which we’ve covered on Practical Fragments, including pantothenate synthetase, CYPs, RAD51, and riboswitches.

The next two chapters deal with two of the key fragment-finding methods. Chapter 2, by Tony Giannetti and collaborators at Genentech, GlaxoSmithKline, and SensiQ, covers surface plasmon resonance (SPR). This includes an extensive discussion of data processing and analysis, which is critical for improving the efficiency of the technique. Competition studies are also described, as are advances in hardware, notably those from SensiQ. This is a good complement to Tony's 2011 chapter.

Chapter 3, by Isabelle Krimm (Université de Lyon), provides a thorough description of NMR methods, both ligand-based (STD, WaterLOGSY, ILOE, etc) and protein-based (mostly HSQC). The chapter does a nice job of describing techniques in terms a non-specialist can understand while also providing practical tips on matters such as optimal protein size and concentration.

Chapter 4, by Ian Wall and colleagues at GlaxoSmithKline, provides an overview of FBLD from the viewpoint of computational chemists. The chapter includes some interesting tidbits, such as the observation that fragment hits that yield crystal structures tend to be less lipophilic but also contain a smaller fraction of sp³ atoms and more aromatic rings. The researchers note that the current fashion for “3D” fragments is yet to be experimentally validated. They also include accessible sections on modeling, druggability, and integrating fragment information into a broader medicinal chemistry program.

The remaining chapters focus on specific types of targets. Chapter 5, by Miles Congreve and Robert Cooke (both at Heptares) is devoted to G protein-coupled receptors (GPCRs). This includes descriptions of how to screen fragments against these membrane proteins using SPR, TINS, CE, thermal melts, and competition binding. It also includes a detailed case study of their β1 adrenergic receptor work (summarized here). Congreve and Cooke assert that, although many of the GPCR targets screened to date have been highly ligandable, technical challenges only now being addressed have caused this area of research to lag about a decade behind other targets. They predict a bright future.

Rod Hubbard (Vernalis and University of York) turns to protein-protein interactions in Chapter 6. After describing why these tend to be more challenging than most enzymes and covering some of the methods for finding and advancing fragments, he then presents several case studies, including FKBP (one of the first targets screened using SAR by NMR), Bcl-2 family members (including Bcl-x_L and Mcl-1), Ras, and BRCA2/RAD51. He concludes with a nice section on “general lessons,” which boils down to “patience, pragmatism, and integration.” As Teddy recently noted, this can lead to substantial rewards.

Allosteric ligands have potential advantages in terms of selectivity and addressing otherwise challenging targets, and in Chapter 7 Steven Howard (Astex) describes how fragments can play a role here. This includes how to establish functionality of putative allosteric binders, as well as case studies such as HIV-1 RT, FPPS, and HCV NS3. Astex researchers have recently stated that they find on average more than two ligand binding sites per protein, and this chapter includes a table listing these (including 5 binding sites each on bPKA-PKB and PKM2).

The longest chapter, by Christina Spry (Australian National University) and Anthony Coyne (University of Cambridge) describes fragment-based discovery of antibacterial compounds. After discussing some of the challenges, the authors report several in depth case studies including DNA gyrase, DNA ligase, CTX-M, AmpC, CYP121, and pantothenate synthetase, among others. At least one fragment-derived antibacterial agent entered the clinic; hopefully more will follow.

Chapter 9, by Iwan de Esch and colleagues at VU University Amsterdam, focuses on acetylcholine-binding proteins (AChBPs), both as surrogates for membrane-bound acetylcholine receptors and as well-behaved model proteins on which to hone techniques (see for example here, here, and here). Since AChBPs have evolved to bind fragment-sized acetylcholine, these proteins can bind tightly to small ligands; 14-atom epibatidine binds with picomolar affinity, for example, with a ligand efficiency approaching 1 kcal mol^-1 atom^-1.

And Chapter 10, by Chun-wa Chung and Paul Bamborough at GlaxoSmithKline, concisely covers epigenetics. Bromodomains are well-represented, including a table of ten examples (see for example here, here, here, here, here, and here). Happily, although some of these projects started from similar or identical fragments, the final molecules are quite divergent. However, the authors note that much less has been published on histone-modifying enzymes, such as demethylases and deacetylases, perhaps reflecting the challenges of achieving specificity with what are often metalloenzymes.

Finally, this is the 500^th post since Teddy founded Practical Fragments way back in the summer of 2008. Thanks for reading, and special thanks for commenting!

Growing into closed pockets

Fragment-growing is a popular way to increase the activity of fragments, all the more so when there is an obvious place towards which to grow. In a paper published online in the J. Am. Chem. Soc., Iwan de Esch and colleagues at VU University Amsterdam describe the structural and thermodynamic consequences of one such effort, and conclude that binding in a certain normally closed pocket is enthalpically rather than entropically driven.

The researchers were interested in acetylcholine-binding protein (AChBP), a soluble and crystallizable homolog of an important class of ligand-gated ion channels. This is the same protein (and the same group) highlighted last year in the context of ligand efficiency hot spots. In the current work, a fairly potent fragment, compound 1, was co-crystallized with AChBP and the structure solved. This fragment binds in roughly the same position as the more potent natural product lobeline (compound 2), but lobeline contains a hydroxyphenethyl group that the fragment lacks (see figure). This moiety binds in a hydrophobic pocket that does not appear in the fragment complex due to the movement of a tyrosine residue. Recognizing the potential for the pocket to form, the researchers introduced this moiety into their own molecule, producing compound 3.

Gratifyingly, compound 3 binds about 50-fold more tightly than the initial fragment. This molecule was also co-crystallized with AChBP, and, as designed, the phenyl group binds in the “lobeline pocket". Moreover, compound 3 does not show a corresponding increase in affinity towards a version of AChBP from a different organism that does not have this pocket.

To correlate binding mode with thermodynamics, the researchers also characterized the binding of their compounds using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). ITC measures enthalpy directly, and performing SPR analyses at different temperatures can also be used to dissect enthalpic and entropic terms. The two methods generally concurred, though the numbers did jump around a bit, and in a few cases SPR predicted negative (ie, unfavorable) entropy where ITC suggested positive (favorable) entropy for the same compound.

Both SPR and ITC indicated that the increase in potency for compound 3 over compound 1 is driven by enthalpy, not entropy. This is somewhat unexpected, as the contacts made by compound 3 are largely hydrophobic, and the simplistic view is that such contacts are usually entropy-dominated. (The added hydroxyl in compound 3 doesn’t appear to be doing anything useful, and in fact removing it increases potency roughly three-fold). The researchers suggest that, for poorly solvated hidden pockets such as this, enthalpy may dominate. Perhaps also the protein rearrangement necessary to open the pocket is entropically costly.

There is much more data in the paper than can be summarized here, and the notion that ligands that induce conformational changes in proteins could be enthalpic rather than entropic binders is an intriguing hypothesis. However, as a vigorous debate last year demonstrates, it is still unclear whether knowing the answer – as scientifically interesting as it may be – will have practical implications for drug discovery.

Ligand efficiency hot spots

Hot spots are regions of a protein with a particular predilection for binding to small molecules – thermodynamic sinkholes, so to speak. Discovering one of these can get you to potent molecules very quickly. In an effort to better understand hot spots, Iwan de Esch and colleagues at VU University in Amsterdam and collaborators at Beactica have deconstructed a potent ligand for nicotinic acetylcholine binding protein (AChBP), a model protein for ligand-gated ion channels, which are implicated in a variety of neurological diseases. They report their results in a recent issue of J. Med. Chem.

The researchers started with the previously reported quinuclidine compound 6 (see figure) and fragmented this into 20 analogs. They tested these in a surface plasmon resonance (SPR) assay as well as in a more conventional radioligand binding assay; the agreement between these very different assay formats was excellent, further validating the utility of SPR as a useful tool for discovering fragments.

Not surprisingly, some of the fragments have higher ligand efficiencies than the larger, more potent molecule, suggesting that there is a hot-spot that recognizes the core fragment 25 (which is structurally related to nicotine). This concept of “group efficiency” has been described previously, and can be useful for optimizing fragments. For example, compound 22, without a basic nitrogen atom, has the lowest ligand efficiency in the bunch; presumably, simply adding the nitrogen would give a sizable boost in potency.

However, one needs to be cautious. The researchers use computer docking to develop models of how each of these fragments bind, but as we have seen before, isolated fragments do not always recapitulate the binding modes of fully elaborated molecules. Still, particularly in the absence of structure (as is the case with many ion channels), exercises such as this could provide useful ideas for what to do with fragment hits.