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!

Caveat Emptor...or marketing does not always tell you whats really in the package.

In case you missed it, I spoke at the ACS on Sunday.  It was in a computational session looking at designing libraries and I am pretty sure I was the only non compchemist.  It was about all the problem compchemists have caused in library design.  My talk was even live tweeted by Ash (@curiouswavefn) and was well received.  So, looking at the next paper in my queue, its a computational-focused paper.  So, After spending several hours on a Sunday listening to compchemists, have I softened?  

This paper is the subject of today's post.  It is an extension of this paper which describes their virtual screen.  From a 2 million compound virtual screen, they tested 17 compounds in vitro leading to 2 micromolar compounds.  This paper is the story of the most potent of the two micromolar compounds.  The target is CREBBP, which is another in the long line of epigenetic targets.  Compound A was one of the original in silico actives that was tested.  Three analogs were obtained and tested (B-D).

Figure 1.  Original active, A.  Analogs B-D.  Common structural motif is shown in blue.

Compound B was the most potent and become the focus of their optimization efforts. Of course, my eyes are drawn to that potential michael acceptor, but the authors dismiss it based upon their docking results: the only alkylatable residue in the area of its putative binding is well buried.  It is a moot point anyway because they were able to replace it with a isopthalate group and increase potency by 5x, 0.9uM (Compound 6).  Interestingly,the potency of 6 is different depending on the assay used: 0.8 um in a competition binding assay and 8.7uM in a TR-FRET assay.

Figure 2.  Compound 6

This compound was crystallized and showed that the predicted binding mode was correct. 

They then performed some gobbledy-gook MD calculations (finite-difference Poisson, warning PDF) in order to evaluate the electrostatic contribution of the polar contribution to binding of 6 and 7.  Compound 6 had more favorable electrostatic interactions (0.8 kcal/mol) than 7, which had more favorable van der Waals interactions (1.4 kcal/mol).  With this crucial information AND the crystal structure in hand, they then explored additional chemical space.  

Despite the authors' claim, I don't think they actually improved the potency significantly.  Compound 6 is 8 uM in the TR-FRET assay and the best compounds they claim are 1 or 2 uM.  I really have to call monkeyshines here.  They use the different assays interchangeably, yet never explain the one is used for what purpose.  Its cherry picking values.  When talking about selectivity, they switch to using thermal shift values.  And we all know the value of that.  So, I find it hard to believe their "most potent" this or "selectivity" that. The title of the paper includes "nanomolar", but that is only in one assay.  That's like saying I can run a 6 minute mile, since I did it once under optimum conditions.  Honestly, my typical times (WAY back when) were more 8:30 miles.  That honesty in data reporting.  Since they obviously had access to different assays, why weren't all compounds run in one, or optimally both.  I don't see that the MD calculations had any positive impact.  Maybe its the heat, but this paper is a not a sham, but definitely full of deceptive advertising.

Fragments vs IAPs: resisting the affinity Siren

In 2011, Mike Hann decried “addiction to potency”. Indeed, newcomers to fragment-based methods often have to undergo a psychological shift to work with low affinity binders. But, as we asked last year, how weak is too weak? In a paper just published in J. Med. Chem., Gianni Chessari and colleagues at Astex may have set a new bar.

The researchers wanted to develop leads against inhibitor of apoptosis proteins (IAPs). The BIR3 domains of proteins such as cIAP1 and XIAP bind to and block the action of caspases and other proteins, allowing cancer cells to survive. Several groups have developed molecules that block these protein-protein interactions, usually by starting with an endogenous peptide inhibitor. However, most of these bind preferentially to cIAP1, and some evidence suggests that a balanced antagonist may have advantages.

The BIR3 domain is quite small, just 11.8 kDa, making standard ligand-observed NMR screening difficult. Instead, the researchers looked at line broadening and chemical shift changes of protein protons with δ < 0.4 ppm or 9.8 – 10.4 ppm on addition of fragments. Anticipating very weak binders, they used 0.2 mM protein and 10 mM fragments (in mixtures of two). A total of 1151 fragments were screened, of which 100 had been computationally preselected.

Among the best hits were those containing an alanine residue; one of these had mid-micromolar affinity and reasonable ligand efficiency. However, these bound preferentially to the BIR3 domain of cIAP1, in common with other reported inhibitors that also contain an alanine. In contrast, compound 1 appeared to have more balanced activity. Its affinity was risibly weak, but crystal soaking led to interpretable electron density, and also suggested that adding a suitably positioned methyl group could fill the small hydrophobic pocket normally occupied by the alanine side chain. The resulting compound 5 showed measurable – and balanced – activity against both proteins.

Next, a small virtual library was constructed in which the pyrrolidine was replaced with substituents to both improve affinity and create a scaffold for reaching into the P4 pocket. Thirty compounds were made, but disappointingly none of these had significantly improved affinity. Undeterred, the researchers obtained crystal structures of some of them bound to XIAP-BIR3 and performed careful modeling. The phenyl ring of compound 7 binds in a region of the protein with an electronegative potential, and by simply adding electron withdrawing substituents the affinity could be improved by >50-fold. Further growth ultimately led to compound 21, with nanomolar potency against both XIAP and cIAP1, though with a preference for the latter. This compound also showed on-target activity in cell-based assays as well as activity in mouse xenograft models.

It would have been easy to overlook compound 1; indeed, it took rather strenuous efforts to find it. Yet, comparing the structures of compound 1 and 21 bound to XIAP-BIR3 reveals that the initial fragment maintains its position and binding interactions in the elaborated molecule. This is a clear example that, with persistence and creativity, it is possible to advance even the weakest of fragments. The researchers note in the conclusion (and have reported at conferences) that they were able to optimize this series to low nanomolar inhibitors against both targets. Whether or not this leads to a drug, it does look like another candidate for a useful chemical probe.

Silver Ain't Bad

For those of you who have been reading this blog for a while, you are familiar with the "Fragments in the Clinic" posts, Jan2015, Jan2013, and Jan2010.  Two of  those slowly making its way through the pipeline is navitoclax, ABT-263, and venetoclax, ABT-199.  Today Abbvie and Genentech announced that it had met its end point in phase II trials.  The companies plan to file for approval by the end of this year.  At that point we will then have TWO compounds approved from fragments.  Congratulations to all of those folks who have worked on this over the years!!!

Fragments vs PAK1 – allosterically

Some of our recent posts have discussed the use of fragment-based approaches to discover selective new chemical probes. Continuing this theme, a paper from Alexei Karpov and colleagues at Novartis in ACS Med. Chem. Lett. describes a success story against the kinase PAK1.

The six p21-activated kinases (PAKs) have been implicated in a variety of indications, from cancer to neurodegenerative diseases. Unfortunately, most reported inhibitors are not sufficiently selective to elucidate the biology. The researchers were particularly interested in PAK1, against which they performed a fragment screen (no details of methods or libraries are provided). One of the more interesting hits was fragment 1. Although a bit chunky (24 heavy atoms, MW = 328 Da) with only modest ligand efficiency, it is structurally unusual for a kinase binder.

In fact, crystallography revealed that the fragment binds not in the active site at all, but rather in an allosteric site adjacent to the ATP-binding site, with the so-called DFG-loop in the “out” conformation. Not surprisingly, this molecule was more active against the inactive form of the enzyme. Despite its binding mode, it did appear to be ATP-competitive, perhaps because the DFG-out conformation of the protein is not able to bind ATP.

Aficionados of GPCRs will not be surprised to learn that fragment 1 – a dibenzodiazepine – bound several with high affinity, but the researchers used structural information both to ablate this off-target activity as well as improve affinity for PAK1, resulting ultimately in compound 3. This molecule was completely selective for PAK1 when tested at 10 µM concentration against a panel of 442 kinases, as well as against a panel of 22 other potential off-targets. Unexpectedly, it was even >50-fold selective against the closely related PAK2. It also displayed good permeability and acceptable solubility, though the stability could be improved.

Compound 3 blocked PAK1 autophosphorylation in cells, but was only modestly effective at blocking proliferation of a pancreatic cancer cell line with high levels of PAK1. As it turns out, all known cancer cell lines that are dependent on PAK1 also seem to use PAK2, so perhaps this chemical probe is too selective. Nevertheless, it will be useful to help disentangle the overlapping roles of these two kinases.

More broadly, this is a nice illustration of the selectivity achievable with allosteric kinase inhibitors. Indeed, as we recently noted in a comment to a post earlier this year, Novartis has put at least one allosteric kinase inhibitor into the clinic.

The Value of DSF

Science is based upon incremental advances of previous work.  A year ago, Dan blogged about worked on BioA.  The key take home from that work was that a hydrazine fragment ended up destabilizing the target by 18C.  It ended up being, as expected, a reversible, SAM-competitive inhibitor with modest potency.  As Dan concluded:

This is a very nice paper, and it will be fascinating to try to understand how the fragments so effectively destabilize the protein despite binding tightly, and how this translates into inhibition. The researchers suggest that finding ligands that destabilize proteins could be generally useful for turning off proteins.

In this paper, the same group is back (This work was also presented at DDC in San Diego in April). Interestingly, they seemed to have abandoned the hydrazine.  Taking the same approach (DSF-Xray-ITC) they identify different fragments (2% hit rate from a 1000 screened).  9 were stabilizers (average of +3.8C) and 12 were destabilizers (average of -13.8C(!)).  5 fragments were able to be crystallized by soaking, co-crystallization was able to add one more structure (Figure 1).  Interestingly, the calorimetry showed that only F5's binding is strongly, enthalpically driven.

Figure 1.  Crystallographically Confirmed Fragment Hits

The authors make several interesting observations:

• Little correlation between magnitude of Tm shift and confirmation by crystallization
• Stabilizing and destabilizing compounds were confirmed by Xray
• No correlation between magnitude of the Tm shift and calorimetry determined Kd.
• Conformational flexibility in the target active site need to be taken into account.

This is not surprising to me; I have seen/heard this many times.  What does this mean for DSF in general? 

Fragments and HTS vs BCATm

One of the themes throughout this blog is that fragments are useful not just in and of themselves, but as part of a broader tool kit, what Mark Whittaker referred to as fragment-assisted drug discovery, or FADD. A nice example of this has just been published in J. Med. Chem. by Sophie Bertrand and colleagues at GlaxoSmithKline and the University of Strathclyde.

The researchers were interested in mitochondrial branched-chain aminotransferase (BCATm), an enzyme that transforms leucine, isoleucine, and valine into their corresponding α-keto acids. Knockout mouse studies had suggested that this might be an attractive target for obesity and dyslipidemia, but there’s nothing like a chemical probe to really (in)validate a target. To find one, the researchers performed both fragment and high-throughput screens (HTS).

The full results from the fragment screen have not yet been published, but the current paper notes that the researchers screened 1056 fragments using biochemical, STD-NMR, and thermal shift assays. Compound 1 came up as a hit in all three assays, and despite modest potency and ligand efficiency, it did have impressive LLE_AT. The researchers were unable to determine a crystal structure of this fragment bound to the protein, but STD-NMR screens of related fragments yielded very similar hits that could be successfully soaked into crystals of BCATm.

The HTS also produced hits, notably compound 4, which is clearly similar to compound 1. In addition to its increased biochemical potency, it also displayed good cell activity. Moreover, a crystal structure revealed that the bromobenzyl substituent bound in an induced pocket that did not appear in the structure with the fragment, or indeed in any other structures of BCATm.

The researchers merged the fragment hits with the HTS hits to get molecules such as compound 7, with a satisfying boost in potency. Interestingly, the fragment-derived core consistently gave a roughly 10-fold boost in potency compared to the triazolo compounds from HTS. Comparison of crystal structures suggested that this was due to the displacement of a high-energy water molecule by the nitrile.

Extensive SAR studies revealed that the propyl group could be extended slightly but most other changes at that position were deleterious. The bromobenzyl substituent was more tolerant of substitutions, including an aliphatic replacement, though this abolished cell activity. Compound 61 turned out to be among the best molecules in terms of potency and pharmaceutical properties, including an impressive 100% oral bioavailability and a 9.2 hour half-life in mice. Moreover, this compound led to higher levels of leucine, isoleucine, and valine when mice were fed these amino acids.

This is a lovely case study of using information from a variety of sources to enable medicinal chemistry. Like other examples of FADD, one could argue as to whether the final molecule would have been discovered without the fragment information, but it probably at least accelerated the process. More importantly, molecules such as compound 61 will help to answer the question of whether BCATm will be a viable drug target.