Review of 2015 reviews

In the Northern Hemisphere the winter solstice has passed but the days are still short, and 2015 is hurtling into history. As we did in 2014, 2013, and 2012, Practical Fragments will spend this last post of the year highlighting notable events as well as reviews we didn't previously cover.

Two major conferences this year were CHI’s Tenth Annual FBDD meeting in San Diego (discussed here and here) and Pacifichem 2015. If you missed these don’t worry – we’ll have an updated list of 2016 events soon.

After a three year drought, two new books published in 2015: Fragment-based methods in drug discovery and Fragment-based drug discovery. And the trend looks set to continue, with a new book edited by Wolfgang Jahnke and me set to publish in early 2016.

In addition to complete books, several book chapters may be of interest to readers, the first being “Fragment-based drug discovery” by Jean-Paul Renaud and NovAliX colleagues, published in Small molecule medicinal chemistry (Wiley). This is a general review of the topic, focused heavily on biophysical techniques, especially SPR, NMR, and native MS. It also includes a couple case studies – one on the clinical compound AT9283 and one on the bromodomain BRD2.

The next three chapters all come from Springer’s massive Methods in molecular biology series. Continuing the biophysical theme is “Biophysical methods for identifying fragment-based inhibitors of protein-protein interactions,” by Michelle Arkin and colleagues at UCSF. This provides background and step-by-step instructions for SPR, differential scanning fluorimetry (DSF), NMR (including STD, WaterLOGSY, and HSQC/HMQC), and X-ray crystallography. A more detailed guide to STD NMR is provided by Hai-Young Kim and Daniel Wyss (Merck) in “NMR screening in fragment-based drug design: a practical guide,” while Byeonggu Han and Hee-Chul Ahn (Dongguk University-Seoul) discuss STD NMR applied to kinases in “Recombinant Kinase Production and Fragment Screening by NMR Spectroscopy.”

Moving on to journals, two reviews focus on protein-protein interactions. The first, by Thomas Magee (Pfizer) in Bioorg. Med. Chem. Lett., briefly touches on challenges and solutions before focusing on several case studies, including navitoclax, Mcl-1, RPA, KRas, Rad, bromodomains, XIAP, HCV NS3, and more. The second, by Chunquan Sheng (Second Military Medical University, Shanghai), Wei Wang (University of New Mexico and East China University of Science and Technology) and colleagues is published in Chem. Soc. Rev. This is much broader, covering not just fragment-based approaches but others as well, and includes 229 references and 21 figures. There’s a lot of good stuff in this paper, but unfortunately the authors do not discuss the numerous false positives that can occur, such as aggregation and PAINS, and some of their examples are artifacts. Caveat lector.

The next two papers focus on specific therapeutic areas. Xinyong Liu and colleagues at Shandong University discuss the application of fragment approaches to HIV targets in Expert Opin. Drug Discov. In addition to recent examples, this covers some of the older literature, as well as less conventional topics such as dynamic combinatorial chemistry. And in Front. Neurol., Jeffry Madura and Christopher Surratt (Duquesne University) discuss the role fragment-based approaches can play in developing drugs that target the central nervous system (CNS). This review is particularly focused on computational methods.

The next three papers continue the computational theme. Dima Kozakov, Adrian Whitty, Sandor Vajda (Boston University) and co-workers have two reviews discussing work we highlighted earlier this year. The first, in Trends Pharmacol. Sci., is an excellent summary of how computational hot spot analysis can predict whether a protein will be ligandable, and includes a number of case studies. The second, a Perspective in J. Med. Chem., is a much more wide-ranging analysis of the approach. This paper also considers difficult targets, some of which may be tackled with larger molecules such as macrocycles, and others of which may simply not be druggable. And in Chem. Biol. Drug Des., Matthew Bartolowits and V. Jo Davisson (Purdue University), focus on “subpockets,” which are essentially the regions surrounding individual amino acid residues in proteins. This paper also includes an extensive list of software tools for analyzing binding sites.

Finally, Chris Murray and David Rees (Astex) have a brief but lively essay in Angew. Chem. Int. Ed. After providing essentially a target product profile for an ideal fragment, they challenge chemists to devise new routes to superior fragments. Although fragments may seem simple, the “precision synthesis” required to elaborate them “is often rate-limiting.” Diversity-oriented synthesis (DOS) is one potential solution, although there does not seem to have been as much activity here as might have been hoped. Some of the problems are prosaic but significant: as we’ve noted, highly water soluble fragments can be hard to isolate. The authors call for new synthetic methodology compatible with small fragments containing diverse hydrogen-bonding functional groups.

And with that, Practical Fragments says farewell for the year. Thanks for reading (and especially for commenting) and may 2016 bring brilliant breakthroughs!

Pacifichem 2015

Last week saw the first-ever fragment-based symposium at Pacifichem. These are massive meetings held in Honolulu every 5 years to bring together scientists from countries surrounding the Pacific. Competing with views like this can be challenging.

Nonetheless, Practical Fragments is happy to report that the symposium was popular, with some talks at close to standing-room-only capacity. There were over 40 presentations and posters from eight countries, and Derek Cole (Takeda) and Chris Smith (Coi) also chaired a lively round-table discussion. I’ll just try to convey a few broad themes.

The utility of “three-dimensional” fragments (as opposed to “flatter” aromatic fragments) came under fire. Jane Withka of Pfizer reported that a small library of 400 fragments, 80% of which had chiral centers, produced lower hit rates and lower confirmation rates in SPR screens than her company’s original fragment library, consistent with what Astex reported.

Another theme was decreasing the concentration at which fragments are screened. Tom Peat (CSIRO) said that even weak (1-10 mM) hits can be found by screening fragments at 100-200 µM using SPR. This seems to be something of a “sweet spot;” aggregation artifacts become significantly more problematic at higher concentrations. For native mass spectrometry, even 10 µM fragment seems to work well, though Tom has a rather impressive MS instrument. Similarly, commentator sgcox noted that DSF is best conducted below 100 µM fragment concentration.

As we noted six years ago, fluorine NMR is also ultrasensitive. Brad Jordan (Amgen) stated that he routinely detects 4-5 mM binders even when screening fragments at 20 µM. Brad also discussed an update of work we covered previously, in which a fragment-linking approach ultimately led to picomolar inhibitors of BACE1. Continuing the fluorine theme, Ray Norton (Monash Institute of Pharmaceutical Sciences, MIPS) described his group’s work with protein-observed ¹⁹F NMR. Clearly more people are catching the fluorine bug, as attested by its popularity in our recent poll.

NMR in general was well-represented. In addition to standard approaches, Bill Marathias (Beryllium) used NMR to find hits against microRNA 21, Ivanhoe Leung (University of Auckland) used boron NMR as part of a dynamic combinatorial chemistry program, Biswaranjan Mohanty (MIPS) described methyl-specific labeling, and Shigeru Matsuoka (Osaka University) discussed solid-state NMR.

Crystallography remains king when it works, though several speakers noted that they had obtained dozens or even hundreds of structures of their protein without capturing a bound fragment. And even successful protein-ligand structures can mislead; Carsten Detering (BioSolveIT) reported that his computational approach detected problems in about half of 107 published structures. Still, structures can be extraordinarily useful: we recently highlighted an AstraZeneca paper that released dozens of structures, and Greg Warren (OpenEye) used these to address questions about solvation. What’s more, crystallographers are looking to improve things: Janet Newman (CSIRO) highlighted an app called Cinder (“Crystallographic Tinder”) to speed up the identification of protein crystals. It’s available for Android, with an iOS version coming soon.

Of course, although the science is fun, the ultimate goals of fragment-based drug discovery are better drugs, and here too we are making progress. Jane Withka noted that several Pfizer kinase candidates had come from fragments. Tatsuya Niimi provided an overview of fragment projects at Astellas between 2009 and 2014: of 88 programs, 15 have produced compounds with IC₅₀ values < 200 nM. Not counting projects that were dropped for strategic reasons or are still in progress, this is an overall success rate of 43%. As expected, the successful targets were computationally predicted to be more tractable than those that failed, though unexpected conformational changes or covalent approaches proved that at least one “undruggable” target may need to be reclassified.

Gianni Chessari (Astex) provided an update of their cIAP/XIAP program and revealed that ASTX660 has recently entered a phase 1-2 clinical trial for cancer. I learned of another drug that has just entered clinical trials, though as its fragment origins have not yet been disclosed I’ll defer naming it. In any case, I’m looking forward to adding several new molecules the next time I update the list of fragment-derived clinical programs.

At the other end of the clinical spectrum, Chaohong Sun (AbbVie) briefly touched on their late-stage ABT-199, which is expected to be approved in the near future. And Daniel Wyss discussed Merck’s BACE1 inhibitor MK-8931, or verubecestat (see here for a nice summary in C&EN), which is in phase 3 clinical trials for Alzheimer’s disease (AD). The results, expected in early 2017, will be either a new hope for the millions of patients with AD – and the billions of people who hope to live long enough to one day be at risk – or a colossally expensive disappointment. Either way, they will provide the best test yet of the amyloid hypothesis.

I could go on but will instead end here – just as higher fragment concentrations lead to more artifacts, more words likely lead to fewer readers. Thanks to all who presented, organized, and sponsored the symposium. If you attended, please share your thoughts!

Fragments vs MKK3: modeling all the way to low nanomolar

The mitogen-activated protein kinase (MAPK) signaling pathway is a rich source of targets, particularly for inflammation. Within this cascade the p38 kinases have been heavily studied, but many of the inhibitors that entered the clinic derailed for various reasons, including efficacy. Thus, some groups have sought to block the pathway upstream of p38. A paper just published online in Bioorg. Med. Chem. Lett. by Steve Swann and colleagues at Takeda describes some of their efforts to accomplish this.

The researchers focused on MKK3 and to a lesser degree the related MKK6, both of which phosphorylate and activate p38. They began by screening their 11,012 fragments in a biochemical assay at 100 µM each. Hits were prioritized by estimating the IC₅₀ values and thus approximate ligand efficiency (LE) and lipophilic ligand efficiency values (LLE) for each compound that inhibited >30%. Of these, 93 gave LE ≥ 0.35 kcal/mol per heavy atom and LLE ≥ 4. (Incidentally, this seems like a perfectly reasonable use of metrics to triage a large number of compounds, and the speed and simplicity is a good counterargument to more complicated proposals.) Some hits were tested using full dose-response curves to determine actual IC₅₀ values and surface plasmon resonance assays to determine K_d values; compound 1 was particularly compelling.

Readers may recall that Takeda found this very same fragment as an inhibitor of BTK (a kinase in an unrelated pathway), and they used the compound/BTK crystal structure along with the published crystal structure of MKK6 to develop a binding model. In their pursuit of MKK3/6 inhibitors, the Takeda team performed biochemical screens of available related compounds. This led to compound 2, which modeling predicted would bind in a similar fashion. The binding model also suggested the possibility of picking up a hydrogen bond to a lysine residue, leading to the more potent compound 3. Further optimization led to compounds 4 and 6, both with low nanomolar potency against MKK3 and low micromolar or high nanomolar cell-based activity. Profiling these against a dozen other kinases within the p38 signaling pathway revealed good selectivity against all except MKK6.

This is a nice, concise paper that illustrates how modeling, even without direct structural information, can be used to advance a fragment to low nanomolar inhibitors, albeit in a well-studied class of targets. It is also another illustration that the same fragment can be used to develop completely different series. And finally, these molecules look promising as chemical probes and possibly drug leads; it will be fun to watch as more data are disclosed.

Fragments vs PDE10A revisited

Independent teams have reported using fragments to identify structurally distinct inhibitors against a popular psychiatric target.

Last month Practical Fragments highlighted a paper from Merck describing researchers’ success in advancing a fragment to a potent selective inhibitor of PDE10A, a potential target for schizophrenia. The final molecule had picomolar activity but suffered from various shortcomings, and the post ended by stating that “there is still plenty of work to do, and it will be fun to watch this story unfold.” Well, we didn’t have to wait long: a new paper in Bioorg. Med. Chem. Lett by Izzat Raheem and Merck colleagues describes further optimization of this series – again using fragments.

The team started by making various changes to compound 15h (shown in the previous post), ultimately leading to compound 4. Although this had lower affinity, it had significantly improved solubility and pharmacokinetic properties. Unfortunately, although selective against other PDEs, it was less selective against a broader panel of off-targets and inhibited both CYP2C9 and CYP3A4. In fact, 1000 analogs (!) containing the central fragment also hit these two enzymes, suggesting the problem was inherent to this core.

At this point the researchers returned to their original fragment screen and recognized that compound 5 had a similar structure to the original fragment. Appending the two “arms” of compound 4 onto this core led to the compound called Pyp-1, with good potency, solubility, and >5800-fold selectivity against other PDEs. Importantly, this molecule did not show the CYP activity of the previous series, and also displayed good pharmacokinetic properties in rats, dogs, and rhesus monkeys. A rat toxicity study didn’t reveal any red flags, and the molecule showed good pharmacodynamic effects in several animal models. The researchers acknowledge that this is a crowded field, with at least 7 compounds having entered the clinic, but Pyp-1 looks promising; at the very least it is a worthy chemical probe.

Continuing the theme of PDE10A, a second paper in Bioorg. Med. Chem. Lett. by Jeffrey Varnes and Jeffrey Albert reports an earlier-stage program from AstraZeneca. In this case, the researchers used a fragment-assisted drug discovery approach, integrating fragment information with data from high-throughput screening.

A functional screen of 3000 fragments led to a fairly high hit rate, with 414 compounds having ligand efficiencies ≥ 0.3 kcal/mol per atom. Many of these were similar to previously described PDE10A inhibitors and were thus deprioritized. On the other hand, compounds 6 and 7 were rather unusual structurally.

A high-throughput screen was conducted at the same time, and this also generated a high hit rate: ~5%, or 11,000 compounds. Unlike the Merck group, the AstraZeneca researchers were unable to obtain crystal structures of their fragments bound to PDE10A, so instead they looked for HTS hits similar to fragments 6 and 7, resulting in 14 compounds. Most of these were false positives or contained unattractive functionalities, but compound 8 turned out to inhibit significantly better than either fragment. Further medicinal chemistry led to compound 12, which is both potent and structurally distinct from other PDE10A inhibitors.

Together these papers reveal how fragments can be exploited to develop quite different molecules against the same target. Although the Merck series is clearly more advanced, it is impressive that the AstraZeneca work was done in the absence of crystallographic support. And in both cases, medicinal chemistry played an essential role: Valinor may beckon, but it will have to wait.