Fragments in Brazil

Most of the fragment events we’ve highlighted are in the US, Europe, and Australia, but that does not fully reflect where all the good science is happening. In a recent ACS Med. Chem. Lett. paper, Carolina Horta Andrade, Maria Cristina Nonato, and Flavio da Silva Emery introduce CRAFT: the Center for Research and Advancement in Fragments and molecular Targets.

 

Established in 2021, CRAFT is a collaboration between the University of Saõ Paulo and the Federal University of Goiás. The center is focused on endemic diseases of Brazil. As the researchers note, only one of the 60 or so fragment-derived drugs that have entered the clinic is an anti-infective, so there is clearly significant need. CRAFT also has an educational and training component reminiscent of the European FragNet and the Australian Centre for Fragment-Based Design.

 

One focus of CRAFT is fragment library design, including underexplored heterocyclic systems. Importantly, the researchers are investigating new synthetic methodologies to be able to functionalize different regions of the fragments. They are also exploring fragments similar to or derived from natural products.

 

Targets are of course essential, and CRAFT is investing in protein production and characterization, such as the enzyme DHODH from Leishmania; we’ve written recently about a fragment approach to the mammalian counterpart.

 

Finally, CRAFT is investing in structure-based design, ligand-based design, and phenotypic screening. And in 2024 no venture would be complete without use of machine learning.

 

Academic laboratories often struggle with downstream drug discovery efforts such as drug metabolism and pharmacokinetics. CRAFT recognizes this and has partnered with the Welcome Centre for Anti-Infectives Research to train participants in DMPK.

 

The researchers “invite the global scientific community to collaborate with us in addressing neglected diseases.” I hope they succeed. Five years ago we highlighted the consortium Open Source Antibiotics, but that site seems to be updated infrequently. The COVID Moonshot has been more successful but is arguably less urgent given the billions of dollars of industry money that poured into research on SARS-CoV-2. From an ethical perspective society should invest more on combating tropical diseases. And as the planet warms, these diseases will increasingly move out of the tropics.

Fragments vs metallo-beta-lactamases

With all the attention on COVID-19 over the past few years, it’s easy to forget about bacteria. But they haven’t forgotten about us. Indeed, antibiotic resistance genes are continuing to spread throughout the microbial world. One class of genes encode the metallo-beta-lactamases (MBLs), which hydrolyze beta-lactam antibiotics. In a recent J. Med. Chem. paper, Mihirbaran Mandal, Li Xiao, and colleagues at Merck describe their efforts against these enzymes. (Mihirbaran spoke about this work at the CHI DDC meeting a few weeks ago.)

 

The researchers started by turning to the literature and previous internal efforts. Tetrazole-containing molecules had been identified as MBL inhibitors, and a virtual screen led to the selection of 76 compounds for testing, of which 29 inhibited the enzyme NDM-1, one of several known MBLs. These included fragment-sized compound 13, and a subsequent screen also identified compound 14.

 

Crystal structures of both these molecules revealed that they bind in the active site. The tetrazole makes interactions with one of the catalytic zinc ions, while the proximal sulfonamide moiety in compound 14 makes interactions with both zinc ions by displacing a bridging hydroxide ion. Merging compounds 13 and 14 led to compound 18, with nanomolar activity against NDM-1 as well as two other MBLs.

 

Despite its metal-chelating sulfonamide moiety, compound 18 was inactive against 34 mammalian metalloenzymes tested. It also didn’t inhibit any of 114 potential off-targets in a Eurofins screen. As expected, the molecule alone had no bactericidal activity, but it did enhance the activity of the beta-lactam antibiotic imipenem in MBL-expressing bacteria. Increasing the polarity of the molecule by adding a hydroxyl moiety to a solvent-exposed region and converting a phenyl to a pyridyl ring resulted in compound 23. While less active against isolated MBL enzymes, this molecule was more effective at inhibiting bacterial growth in the presence of imipenem, possibly due to accumulation in the periplasmic space.

 

Compound 23 is clean against off-targets such as hERG, ion channels, and various CYP enzymes. It has reasonable pharmacokinetic properties in mice when dosed intravenously. In a preliminary mouse efficacy study, the compound reduced levels of bacteria in spleen and kidney when dosed with imipenem.

 

This is a nice example of structure-based design starting from fragment-sized molecules. With an abundance of nitrogen atoms and a ClogP < 0, compound 23 looks unusual. Nonetheless, the researchers write that “further evolution of this class of molecules… eventually led to the discovery of several clinical candidates.” I look forward to seeing these described.

Twenty seven hits against four tuberculosis targets

1.2 million deaths. If you did not read the title of this post carefully you may assume this statistic refers to COVID-19. In fact, it is the number of people who died of tuberculosis in 2019. Worse, drug resistant forms of Mycobacterium tuberculosis, the organism that causes TB, are spreading far faster than new treatments are being developed. Initial efforts at addressing this problem are reported (open access) in Comp. Struct. Biotech. J. by Sangeeta Tiwari (University of Texas El Paso), Vitor Mendes (University of Cambridge) and a multinational team of collaborators.

 

M. tuberculosis is capable of making all 20 amino acids. The bug can also scavenge arginine from its host, but only inefficiently: knocking out the biosynthetic pathway abolishes virulence. Thus, targeting this pathway might lead to new drugs.

 

In total eight enzymes are needed to synthesize L-arginine from L-glutamate, and the researchers targeted four of them. The proteins were screened against a library of 960 fragments (each at 5 mM) using differential scanning fluorimetry (DSF). Depending on the specific target some of the hits were validated by SPR or ligand-based NMR before being taken into crystallography, which yielded structures of all the enzymes. In total 13 fragments were found to bind to ArgB, 4 bound to ArgC, 2 bound to ArgD, and 8 bound to ArgF. All the coordinates have been deposited in the protein data bank, though they don’t seem to have been released as of June 28.

 

The paper details the binding interactions for all the hits. Most of them are quite weak, though two hits against ArgB have low micromolar dissociation constants as assessed by ITC. Tantalizingly, these inhibit the growth of M. tuberculosis, and one of them seems to be on-target (adding arginine to the media rescues the inhibition). All the ArgB fragments bind not at the active site but rather at an interface between protein subunits. Unfortunately this site is quite hydrophobic, as are the fragments, suggesting an uphill battle in optimization.

 

A good antibiotic should not hit human proteins, and neither ArgB nor ArgC have human orthologs. ArgF does, but the region where the fragments bind is quite different. ArgD, with only two crystallographically-confirmed hits and 36% identity to the human enzyme, is probably the least attractive.

 

A year before the COVID-19 Moonshot launched we highlighted the Open Source Antibiotics initiative. I don’t think that team was involved with this research, but they would seem to be a natural fit. If you have spare bandwidth and are looking to do some fragment to lead optimization, this paper provides more than two dozen starting points.

Fragments vs LpxC, two ways

Gram negative bacteria such as Pseudomonas aeruginosa are a continuing threat, and antibacterial drug discovery is not keeping pace. The enzyme UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is critical for the synthesis of the bacterial cell wall lipopolysaccharide. In a new J. Med. Chem. paper, Yousuke Yamada, Rod Hubbard, and collaborators at Taisho and Vernalis describe progress against this target.

 

LpxC is a zinc hydrolase, and although previous potent inhibitors have been reported against the metalloenzyme, these contained hydroxamate moieties. Unfortunately, hydroxamic acids are rather nonspecific zinc binders, and many of them hit human enzymes such as HDACs and MMPs. Thus, the researchers turned to fragments to find new metallophilic starting points.

 

The 1152 members of the Vernalis fragment library were screened against LpxC using three NMR experiments: STD, WaterLOGSY, and CPMG in pools of six. This yielded a remarkable 252 hits in at least one assay. These were retested individually and for competition with a substrate pocket-binding small molecule, resulting in 28 hits, two of which were advanced.

 

A crystal structure of compound 6 bound to LpxC suggested that adding a hydroxyl group could make additional interactions with the protein, and this was confirmed in the form of compound 10. Further fiddling in this region of the molecule was not successful, and the phenyl ring did not provide good vectors to a hydrophobic tunnel. However, replacing the phenyl with a more shapely piperidine yielded compound 17. Although this molecule had slightly lower affinity, it did provide a better starting point for further optimization, ultimately leading to compound 21, with low nanomolar potency against LpxC. Unfortunately, this and other members of the series showed only weak antibacterial activity.

 

Compound 9 was weaker than the other fragment starting point, but making and testing related compounds led to improved binders such as compound 27. This was the first molecule in this series to be structurally characterized, and crystallography revealed that the imidazole was making a single interaction with the zinc at the heart of the LpxC active site. Adding a hydroxyl led to bidentate chelator 29 (i.e. two interactions with the zinc) that had better activity, and further structure-based design ultimately led to low nanomolar inhibitors such as compound 43. In contrast to the other series, this one did show antibacterial activity, and the researchers eventually discovered molecules with in vivo efficacy. Both series were also selective against a small panel of human metalloproteases.

 

 

This is a nice fragment to lead story (expect it to be included in the next compilation). As the researchers note, it provides two important lessons. First, fragments can provide multiple different starting points for a target. Second, because fragment libraries tend to be small, it can be valuable to take some time to refine a fragment before launching into fragment growing or merging. Indeed, compound 38 (itself fragment-sized) contains only four more atoms than the initial fragment hit, yet has more than a thousand-fold higher affinity. During lead optimization you often need to add molecular weight, lipophilicity, and possibly polar atoms, so it is crucial to get the core binding elements as good as possible.

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.

Kill Them Bugs!

Bugs are bad.  I hate bugs.  Bugs of all kinds.  In our part of the world we have a particularly noxious, invasive bug called the stink bug.  Ewwww.  And they are everywhere.  And in the winter they are particularly prevalent because they get in your attic, soffets, etc. and then creep into your house.  I would love to be part of a global effort to eradicate these horrible creatures.  I may lose my green bona fides advocating the genocide of an entire species, but so be it.  It is also not so practical, so really not germane to this blog.

However, targeting bacteria is practical, and important.  Antibiotic resistance is on the rise globally and only two antibiotics with novel modes of action have been approved in this century.  Dire consequences meet pressing need.  Many antibiotics with improved efficacy are due to higher to higher potency or resistance to degradation.  But, this avenue has a limited lifespan and novel targets are needed.  Into this breach steps Astra Zeneca, with this paper.  The topoisomerases DNA gyrase and Topisomerase IV Top IV) have already been shown clinically to be validated targets.  The A subunits contains the DNA cleavage domain while the B subunits contain the ATP binding and hydrolysis domain.  DNA gyrase inhibitors also typically inhibit TopIV.  Fluoroquinolones (the DNA complex) and aminocoumarins (the ATP site) target these enzymes. Aminocoumarins have not received much attention to due PK and safety issues. There are a wide variety of ATP-targeting compounds.

Cpds 1 and 2 have been shown by X-Ray to bind in the ATP site and extend outside that site to generate additional interactions with R144.  The team's design goal was a new scaffold that would merge these two compounds attributes.  They chose 2-pyridylureas which had not previously been explored.  Modeling showed that 5-substitution reaches towards R144 with a carboxylate and 4-substitution allows for exploration into more open space.  6-substitution abuts a hydrophobic region and should not be messed with.

Cpds 3-13 were synthesized (or were commercially available) to test these hypotheses with Cpd 6 clearly the best.  Then they explored the 4 and 5 substitutions 9see the actual paper for Tables 1-3).  The chemistry and isozyme exploration they performed was very detailed.  The two best compounds ended up being 31 and 35. 

Then the paper gets into the details (it's 24 pages long and the results/discussionare pp 5-13).  I highly recommend reading that part on your own.  I am really impressed by the work.  As they discuss, the Xtal structures support many of the design hypotheses.  This cannot be understated.  Fragment-based drug design (and in this case it really is DESIGN) was effective and robust.  In the end, their compounds were able to realize potent inhibition of 4 topoisomerases across three bacterial species. Importantly, bacterial growth was realized through inhibition of both the gyrase and Top IV which is the key criterion for continued optimization.  Efficacy in a mouse model was demonstrated with 35.   

Natural linking – though not of fragments

We’ve previously discussed the appeal and challenges of fragment linking. A new paper in Science describes how a naturally occurring antibiotic makes use of a linking strategy, albeit using rather Brobdingnagian fragments.

Simocyclinone D8 (SD8) is a dumbbell shaped molecule isolated several years ago from that ultimate micro-pharma, Streptomyces. Although SD8 blocks the action of bacterial DNA gyrase, which is also the target of fluoroquinolones such as ciprofloxacin and aminocoumarins such as novobiocin, it is mechanistically distinct from these older antibiotics. To understand why, Anthony Maxwell and colleagues at the John Innes Centre in Norwich, UK, solved the co-crystal structure of SD8 bound to GyrA. The structure reveals that the protein forms a dimer of dimers, with four molecules of SD8 bound to the four subunits of GyrA. Weirdly, each molecule of SD8 cross-links two separate subunits of GyrA, although mass-spectrometry, analytical ultracentrifugation, and modeling suggest that a single molecule of SD8 could also bind to a single GyrA subunit. The crystal structure shows that SD8 binds near – but not at – the fluoroquinolone binding site, blocking the DNA-binding portion of GyrA.


What caught my eye is the fact that both halves of the molecule are active by themselves, albeit with a loss in potency (see figure). The linker is over one nanometer long and doesn’t appear to make significant interactions with the protein; it would be fun to know how something like this evolved.

Antibiotics gleefully seem to ignore the Rule of 5, but it wouldn’t hurt to get a smaller, less complicated analog. Replacing either of the two ends with smaller fragments may be a productive approach, as would optimizing the individual “fragments” themselves.