Help develop new antibiotics from fragments!

The state of antibiotic drug discovery is – to put it mildly – dangerously poor. Not only do you have all the challenges inherent to drug discovery, you’re dealing with organisms that can mutate more rapidly than even the craftiest cancer cells. And then there’s the commercial challenge: earlier this month the biotech company Achaogen filed for Chapter 11 bankruptcy, less than a year after winning approval for a new antibiotic.

As Douglas Adams’s Golgafrincham learned, complacency about microbial threats is suicidal. But what can any one of us do? Chris Swain, whom we’ve previously highlighted on Practical Fragments, is involved with a consortium of researchers called Open Source Antibiotics. Their mission: “to discover and develop new, inexpensive medicines for bacterial infections.” And they are asking for our help. More on that below.

The researchers initially chose to focus on two essential enzymes necessary for cell wall biosynthesis, MurD and MurE, both of which are highly conserved across bacteria and absent in humans. They conducted a crystallographic fragment screen of both enzymes at XChem, soaking 768 fragments individually at 500 mM concentration. As we’ve written previously, you’ll almost always get hits if you screen crystallographically at a high enough concentration.

For MurD, four hits were found, all of which bind in the same pocket (in separate structures). Interestingly, this pocket is not the active site, but adjacent to it. The binding modes of the fragments are described in detail here, and the researchers suggest that growing the fragments could lead to competitive inhibitors. The fragments also bind near a loop that has been proposed as a target for allosteric inhibitors, so growing towards this region of the protein would also be an interesting strategy.

MurE was even more productive, with fragments bound at 12 separate sites. (Though impressive, that falls short of the record.) Some of these sites are likely artifacts of crystal packing, or so remote from the active site of the enzyme that they are unlikely to have any functional effects. However, some fragments bind more closely to the active site, and would be good candidates for fragment growing.

If this were a typical publication one might say "cool," and hope that someone picks up on the work sometime in the future. But this, dear reader, is different.

The researchers are actively seeking suggestions for how to advance the hits. Perhaps you want to try running some of these fragments through the Fragment Network? Or do you have a platform, such as “growing via merging,” AutoCouple, or this one, that suggests (and perhaps even synthesizes) new molecules? Perhaps you want to use some of the fragments to work out new chemistry? The consortium has a budget to purchase commercial compounds, and will also accept custom-made molecules. In addition to crystallography, they have enzymatic assays, and are building additional downstream capabilities.

The Centers for Disease Control identifies antibiotic resistance as one of the most serious worldwide health threats. Some have called for a global consortium—modeled after the International Panel for Climate Change—to tackle the problem. But in the meantime, you can play a role yourself. If you would like to participate, you can do so here. The bugs are not waiting for us – and they are already ahead.

A bestiary of fragment hits

What do fragment hits look like, and how do they bind? Fabrizio Giordanetto, David Shaw, and colleagues at D. E. Shaw Research were interested in these questions, and their answers are provided in a recent J. Med. Chem. paper (open access, and also covered well by Derek Lowe).

The researchers started by searching the protein data bank (PDB) for the word “fragment” and selecting higher resolution structures (at least 2.5 Å) with a ligand containing 20 or fewer non-hydrogen atoms. Those of you who have done bibliometric searchers will appreciate that a lot of manual curation is required, and the initial list of 5115 complexes was ultimately winnowed down to 489, with 462 unique fragments and 126 unique proteins, about two-thirds solved to ≤2.0 Å resolution.

In contrast to a previous study, only a minority (18%) of proteins contained more than one binding site, suggesting that secondary (possibly allosteric) sites may be less common than hoped.

As to the fragments themselves, 21 bound in more than one pocket (not necessarily on the same protein), including the universal fragment 4-bromopyrazole. The fragments ranged in size between 6 and 20 non-hydrogen atoms, with 81% having 10 to 16, consistent with our poll last year. Given these sizes, it is perhaps not surprising that the vast majority of fragments conformed to the rule of three.

Roughly two thirds of the fragments were uncharged, while 22% contained a negative formal charge (usually a carboxylic acid) and 11% contained a positive formal charge such as an aliphatic amine. Interestingly, more than 90% of the fragment hits were achiral. Since our 2017 poll found that most fragment libraries contain chiral compounds, these results might suggest lower hit rates for these compounds. Fragment hits also tended to have lower Fsp³ scores than those in a popular commercial library, which is consistent with the observation that “less shapely” fragments give higher hit rates.

Digging more deeply into the chemical structures themselves, nearly a third of the fragments contained a phenyl ring, while 6% contained a pyridine and 5% contained a pyrazole. Thiophenes, indoles, indazoles, piperidines, furans, and pyrrolidines were present in 2-3% of fragment hits. But there was also plenty of diversity: more than half of fragments contained a unique ring system.

So that’s what fragment hits look like. How do they bind? Deeply, for starters: about three quarters of the fragment hits buried more than 80% of their solvent-accessible surface area, and 21 fragments were completely engulfed within a protein.

Not surprisingly, more than 90% of complexes showed at least one polar interaction, such as a hydrogen bond or a coordination bond to a metal ion. Many complexes contained more than one, and one had seven! Interestingly, these polar interactions also tended to be buried. Nitrogen and oxygen atoms from the fragments were equally likely to form hydrogen bonds. Interactions with bound water molecules were considered for high-resolution (≤1.5 Å) structures, and nearly half of these contained a water molecule with at least two hydrogen bonds to the protein and one to a fragment.

Beyond these conventional sorts of polar interactions, there were also less traditional interactions such as arene-mediated contacts, which occurred in nearly half of cases. As we’ve noted, these are often under-appreciated but can be useful for improving affinity. The subject of halogen bonds came up recently, but these turned out to be quite rare, appearing in just 3% of cases. Sulfur-mediated contacts and carbon hydrogen bonds were more common, appearing in 11-12% of complexes, respectively.

All of this has important implications for fragment library design. As the researchers note, this set of 462 fragments could be used as the basis for a library, and laudably all the structures are provided in the supporting information. Generalizing beyond these specific molecules, roughly a quarter of the atoms in the fragment hits are polar (nitrogen or oxygen) and thus more likely to form classic hydrogen bonds. The researchers “strongly suggest” maintaining this ratio in designing new fragments.

The researchers also suggest presenting “a minimum set of individual polar pharmacophoric elements, as opposed to distributing several pharmacophores on a given fragment,” which is essentially the minimal pharmacophore strategy described here.

The one category of data I would have liked to see was affinity. Many binding measurements were probably not reported, and experimental error can be particularly confounding for weaker interactions, but even a subset of the data should allow some conclusions about the strength of various molecular interactions. Hopefully this will be the basis of a follow-up publication.

Third fragment-based drug approved!

Last Friday the US FDA approved erdafitinib (Balversa) for certain bladder cancers with FGFR2 or FGFR3 mutations. Although the fragment-to-lead story has yet to be published, those of you who were fortunate enough to attend Fragments 2019 last month heard some of it from Harren Jhoti.

Congratulations to the folks at Astex and J&J for a new tool in the campaign against cancer!

And earlier in the pipeline, several more drugs have entered the clinic starting from fragments, taking the number above 45.

Fourteenth Annual Fragment-based Drug Discovery Meeting

CHI’s Drug Discovery Chemistry (DDC) meeting took place last week in San Diego. I think this was the largest yet, with >825 attendees, a third from outside the US, and nearly 70% from industry. The initial DDC meeting in 2006 had just four tracks, of which FBDD is the only one that remains. This one had nine tracks and four one-day symposia, so it was obviously impossible to see everything. Like last year, I’ll just stick to broad themes.

Success Stories

As always, clinical compounds received deserved attention. Among two I’ve covered recently, Paul Sprengeler described eFFECTOR’s MNK1/2 inhibitor eFT508, while Wolfgang Jahnke discussed Novartis’s allosteric BCR-ABL1 inhibitor ABL001. As previously mentioned, ABL001 is a case study in persistence: the project started in stealth mode and was put on hold a couple times until seemingly intractable problems could be overcome.

Another story of persistence, albeit with a less happy outcome, was presented by Erik Hembre, who discussed Lilly’s BACE1 program. Teddy wrote about their first fragment-derived molecule to enter the clinic, LY2811376, back in 2011. Unfortunately this molecule showed retinal toxicity in three-month animal studies, so the researchers further optimized their molecule to LY2886721, which made it to phase 2 studies before dropping out due to elevated liver enzymes. Reasoning that a more potent molecule would require a lower dose and thus lower the risk of toxicity, the researchers used structure-based drug design to get to picomolar LY3202626, which also made it to phase 2 before being scuttled due to the apparent invalidation of BACE1 as an Alzheimer’s disease target.

Talks on BCL2 and MCL1 inhibitors from Vernalis, AstraZeneca, and Servier all involved fragments in some capacity, but unfortunately they were in the protein-protein interaction track which was held concurrently with the FBDD session I was chairing. Suffice it to say you can expect to hear more about the phase 1 compounds AZD5991 and S654315.

A few earlier-stage success stories included Till Maurer’s discussion of the Genentech USP7 program (see here), Santosh Neelamkavil on Merck’s Factor XIa inhibitors, and Rod Hubbard on Vernalis DYRK1A, PAK1, and LRRK2 inhibitors. We have previously written about how displacing “high-energy” water molecules can be useful, and this tactic was used by Sven Hoelder at the Institute of Cancer Research for their BCL6 inhibitors. Last week we highlighted halogen bonds, which proved important for transforming molecules that simply bind to MEK1 to molecules that bind and inhibit the protein, as described by AstraZeneca’s Paolo Di Fruscia.

Methods

The MEK1 story Paolo told began with a very weak (0.45 mM) fragment that the team was able to advance to 300 nM in the absence of structure, though they did eventually obtain a crystal structure that supported further optimization. On the topic of crystallography, Marc O’Reilly discussed the Astex MiniFrag approach, which we recently wrote about here. Only a couple of these fragments contain a bromine atom, but Marc did mention that, of the 10,051 X-ray complexes solved at Astex, a number show halogen bonds, including some to the hinge region in kinases.

At FBLD 2018 Astex’s Chris Murray showed the first cryo-EM structure of a fragment bound to a protein, and Marc confirmed that they have now obtained structures of fragments bound to two targets, with fragments as small as 120 Da and resolution as good as 2.3 Å. They are increasing automation, with turnaround times of less than 24 hours in some cases. Santosh also mentioned that Merck is applying cryo-EM to fragments.

Frank McCormick (UCSF) highlighted multiple fragment-finding methods used to discover inhibitors against RAS family proteins, which are responsible for more than a million cancer deaths each year. In addition to stalwarts such as crystallography and NMR, these include less common methods such as Tethering and the second harmonic generation (SHG) approach for detecting conformational changes used by Biodesy. RAS was reported as a cancer driver almost forty years ago, but only now are the first direct inhibitors entering the clinic – a testimony to both the challenging nature of the target and how far we’ve come.

SHG and Tethering were also highlighted elsewhere: Charles Wartchow described how SHG identified 392 hits from a collection of 2563 fragments against an E3 ligase bound to a target protein at Novartis, while Michelle Arkin described her use of Tethering at UCSF to find molecules that could stabilize a complex of 14-3-3 bound to a specific client protein (see here).

An effective sponsored talk was presented by Björn Walse of SARomics Biostructures and Red Glead Discovery, who described weak affinity chromatography (WAC). Once they saw the schedule for DDC, they looked for a target that would be presented shortly before their presentation, and chose the protein USP7 as a test case. Beginning in January, they screened a library of 1200 fragments to obtain 34 hits, of which 7 confirmed in a thermal shift assay. This led to an SAR-by-catalog experiment, and 11 of the 31 fragments tested showed activity, as did a Genentech positive control compound.

All methods can generate false positives and false negatives (see for example here and here), some of which were described in an excellent talk by Engi Hassaan of Philipps University. Engi discussed how improving the sensitivity of an STD assay by decreasing salt concentration identified more fragments that had previously been found by crystallographic screening. She also presented a case study of how introducing a tryptophan residue into a small protein to facilitate purification led to problems down the road when the tryptophan side chain blocked a key pocket in the crystal lattice. Gregg Siegal (ZoBio) also highlighted a case where a fragment bound to the dimer interface in a crystal structure, whereas in solution the fragment bound to the active site, as observed by NMR.

Finally, among computational methods, Pawel Sledz (University of Zurich) gave a nice overview of the SEED and AutoCouple methods, while Paul Hawkins (OpenEye) described rapid searching of more than 10 billion chemical structures using ROCS (rapid overlay of chemical features). SkyFragNet is looking closer with each passing year.

There is much more to say, so please feel free to comment. Several good events are still coming up this year, and mark your calendar for 2020, when DDC returns to San Diego April 13-17!

Helpful halogens in fragment libraries

A couple weeks ago we highlighted a small fragment collection (MiniFrags) designed for crystallographic screening. We continue the theme this week with two more papers on the topic, with an emphasis on halogens.

The first, published in J. Med. Chem. by Martin Noble, Michael Waring, and collaborators at Newcastle University, describes a library of “FragLites.” These small (< 14 non-hydrogen atom) fragments are designed to explore “pharmacophore doublets,” such as a hydrogen bond acceptor (HBA) next to a hydrogen bond donor (HBD). For example, the universal fragment 5-bromopyrazole contains an HBA separated by one bond from an HBD. The researchers constructed a set of compounds with either two HBAs or an HBA and an HBD separated by 1 to 5 bonds. Importantly, all compounds also contained either a bromine or iodine atom, the idea being that anomalous dispersion could be used to help identify the fragments using crystallography. A total of 31 FragLites are described, with between 1 to 9 examples for each type of connectivity.

As a test case, these were screened against the kinase CDK2, which has previously been screened crystallographically. FragLites were soaked into crystals at 50 mM, and 9 of the FragLites were found to bind in a total of 6 sites, 4 of which had not been previously observed. The anomalous signal provided by the halogens was important: when the researchers used only normal scattering they identified just 10 of the 16 binding events even when using the powerful PanDDA background correction method. The anomalous signal also helped clarify the binding modes.

The ATP-binding site is where 7 of the 9 FragLites bound, with all but one of them making hydrogen bonding interactions to the hinge region. While not surprising, this does demonstrate that the FragLites can be used experimentally to identify the best binding site. Interestingly, (2-methoxy-4-bromophenyl)acetic acid bound in the active site as well as three other secondary sites; one of these sites hosted three copies of the ligand! It will be interesting to see whether this fragment is generally promiscuous in other proteins too.

As the researchers note, the composition of the FragLite library can be optimized. For example, both of the HBA-HBD fragments with 1-bond separation were identified as hits, while only 3 of the 9 HBA-HBD fragments with 2-bond separation were. Is this due to the choice of fragments, the target tested, or both? The approach is conceptually similar to the Astex minimal pharmacophore concept, so it will be useful to include other types of pharmacophores too (a single HBA or HBD, for example).

A related paper was published in Front. Chem. by Frank Boeckler and colleagues at Eberhard Karls Universität Tübingen. Long-time readers will recall his earlier halogen-containing library designed for identifying halogen bonds: favorable interactions between halogens and Lewis bases such as carbonyl oxygen atoms. Perhaps because they have relatively stringent geometric requirements (2.75 – 3.5 Å, and a bond angle of 155-180°), halogen bonds are often ignored; the FragLite paper doesn’t even mention them.

The new Boeckler paper describes the construction of a library of 198 halogen-containing fragments, all of which are commercially available and relatively inexpensive. Most of these are rule-of-three compliant, though quite a few also contain more than three hydrogen bond acceptors. Also, given that each fragment contains a halogen, the molecular weights are skewed upward. Solubility was experimentally determined for about half of the fragments, but the highest concentration tested was only 5 mM, and even here several were not fully soluble.

Although no screening data are provided, the researchers note that their “library is available for other working groups.” In the spirit of international cooperation, I suggest a collaboration with the FragLite group!

Machines, fixing human disease

Last year we highlighted the secretive juggernaut DREADCO's move into drug discovery. Today they announced the launch of their new division SkyFragNet (not to be confused with the European graduate training program FragNet). Its audacious mission: “to eradicate human disease."

SkyFragNet will automate every aspect of drug discovery. The approach starts with a powerful docking method, in which all 166 billion members of GDB-17 will be docked against a target of interest. Synthetic schemes for the virtual hits will be computationally generated, and the compounds will be synthesized using automated flow synthesis and mass-directed purification.

Fragment hits that confirm in a panel of biophysical techniques will then undergo computational-based growing; SkyFragNet incorporates the latest AI algorithms to maximize the likelihood of success. As with the fragments, designed molecules will be synthesized and tested, first in biochemical and then in cell-based assays.

Although the folks at Mordor State College are trying to make animal testing obsolete, SkyFragNet will still rely on pharmaokinetic and pharmacodynamic studies. However, they have built a fully mechanized vivarium run entirely by robots - think of The Matrix but with mice in place of humans.

Finally, compounds that make it through this gauntlet will be scaled up under GMP conditions (automated, of course) for clinical trials. It remains to be seen how many compounds SkyFragNet will take into the clinic, or whether the success rates will be higher than those of their human counterparts.

Of course, with all this power comes enormous responsibility. If things go wrong, hopefully DREADCO will have the wisdom to Terminate the program. Eradicating human disease could be done in two very different ways.