Fragments vs CYP125 and CYP142 for M. tuberculosis

Although 2020 and 2021 were baleful exceptions, tuberculosis is normally the world’s deadliest infectious disease. The pathogen Mycobacterium tuberculosis (Mtb) makes its home inside macrophages, the very cells that normally destroy microorganisms. Worse, some strains have become resistant to approved drugs. In a recent open-access J. Med. Chem. paper, Madeline Kavanagh, Kirsty McLean, and collaborators at University of Manchester, University of Cambridge, and elsewhere explore a new mechanism to fight this ancient disease.

 

An important nutrient source Mtb exploits inside human cells is cholesterol, which bacteria oxidize with the cytochrome P450 enzyme CYP125. A second enzyme, CYP142, is also present in some strains and is functionally redundant. Thus, the researchers set out to make a dual inhibitor.

 

Mtb has some 20 CYPs, and the Cambridge researchers have been studying them for a long time: we wrote about their work on CYP121 in 2016 and their work on CYP126 in 2014. All these enzymes contain a heme cofactor, and much is known about targeting the bound iron. However, some ligands are promiscuous, hitting human P450 enzymes, or they are rapidly effluxed out of cells. Thus, the researchers built a fragment library of just 80 likely heme binders but excluded particularly promiscuous moieties, such as imidazoles. The library was screened using UV-vis spectroscopy; ligands that bind to the heme group cause a red-shift in the λ_max. Only four hits were found for CYP125, while a dozen were found for CYP142, including three of the four CYP125 hits. Compound 1a had modest affinity for CYP125 and low micromolar affinity for CYP142.

 

Compound 1a was soaked into crystals of CYP142, and interestingly two molecules bound at the active site: one coordinating to the iron atom as expected, the other binding near the entrance of the active site. This suggested a linking or merging strategy, so the researchers made small libraries based on compound 1a and tested these against the two enzymes. Compound 5m was the most potent against both. Crystal structures of this molecule bound to both CYP125 and CYP142 confirmed that the pyridine nitrogen maintained its interaction with the heme iron, while the added bit nicely filled the space previously occupied by the second copy of compound 1a.

 

Functional assays revealed that compound 5m inhibited both enzymes with nanomolar activity, comparable to their affinities. It also inhibited the growth of Mtb grown on media containing cholesterol as the sole source of carbon. More impressively, it even inhibited the growth of Mtb in standard media spiked with just low concentrations of cholesterol. Oddly though, it also inhibited the growth of Mtb grown on media not containing cholesterol, albeit at a higher concentration, suggesting perhaps other targets. But one reason tuberculosis is so hard to treat is that the bacteria persist inside human cells. Encouragingly, compound 5m inhibited the growth of Mtb in human macrophages at low micromolar concentrations, and it  did not show cytotoxicity up to 50 micromolar concentration.

 

Unfortunately, compound 5m did show cytotoxicity to human HepG2 cells, and it also inhibited several human P450 enzymes at high nanomolar concentrations, which could cause drug-drug interactions. Also, selectivity against other MTb P450 enzymes is unclear. Finally, no in vitro ADME data are reported. Nonetheless, this is a nice fragment to lead story, and compound 5m could be used – cautiously – as a chemical probe to study Mtb biology.

Fragments vs LpxC revisited

Back in 2020 we described fragment-derived inhibitors of the highly conserved bacterial enzyme LpxC, which is essential for biosynthesis of the outer membrane in Gram-negative bacteria. In a recent (open access) paper in J. Med. Chem., a different group consisting of Ralph Holl and collaborators at Universität Hamburg and several other academic centers describe a new series.

 

The researchers started with compound 9, a molecule they had previously discovered. The substrate for LpxC is a rather large small molecule called (UDP)-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine. Compound 9 does not occupy the UDP-binding site, so the researchers initially tried building towards it with a series of simple linkers connected to a phenyl group. The (S) enantiomers tended to be more active than the (R)-enantiomers, and the most potent was compound (S)-13a, which showed sub-micromolar activity against LpxC from E. coli as well as P. aeruginosa in an enzymatic assay. (For simplicity only the E. coli data are shown here.)

 

Seeking to improve affinity, the researchers screened 650 fragments in pools of five against LpxC in the presence of compound 9 using STD NMR and WaterLOGSY. After deconvolution, this led to 97 hits. STD-based epitope mapping, which we wrote about here, was used to prioritize fragments likely to have a single, well-defined binding mode, culling the number to 19. Finally, NMR-ILOE experiments (see here) suggested that nine of this set bound in close proximity to compound 9, while the other ten did not. Four of these fragments, including the simple indole F3, were then linked to compound 9 at various positions. This is akin to SAR by NMR, but with less information about the relative binding modes so more trial and error is necessary.

 

Among the roughly two dozen molecules made, compound (S)-13j was the most potent against LpxC, with low nanomolar activity. This compound (and several others) also showed antibacterial activity against E. coli and several other strains of Gram-negative bacteria. In vitro stability studies of compound (S)-13j were promising, though the researchers noted the need for improvement. And, since the molecule contains a hydroxamic acid moiety potentially capable of binding to multiple metalloproteins, it was tested against a handful of mammalian zinc-dependent enzymes and shown to be nearly inactive.

 

Compound (S)-13j is 15-fold more potent than the simple phenyl analog (S)-13a, and molecular modeling suggested this may be due to a hydrogen bond from the protein to the indole NH. Although one could argue that it would have been possible to arrive at compound (S)-13j using standard medicinal chemistry starting from (S)-13a, this may have taken longer without knowledge of the indole fragment. Whether or not the molecules advance further, this is a nice example of using fragment screening to find a second-site binder to improve affinity of an existing lead.

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.

Fragment merging vs bacterial SAICAR synthetase

People living with cystic fibrosis are susceptible to lung infections from a rogues’ gallery of bacterial species, one of which is Mycobacterium abscessus. It is often antibiotic resistant, and even when it responds, a course of antibiotics can take two years to resolve the infection. In a recent ACS Infect. Dis. paper Tom Blundell, Anthony Coyne, and collaborators at University of Cambridge and elsewhere describe progress against this organism.

 

The researchers chose to target a protein called SAICAR synthetase, or PurC, which is essential for purine biosynthesis and thus bacterial growth, as shown by genetic knockout studies. The enzyme is significantly different from the human ortholog, but similar to the Mycobacterium tuberculosis ortholog, giving the potential for a twofer.

 

Fragment screening was conducted both in-house using thermal shift assays as well as at XChem using crystallography; we discussed the differing outputs of these screens in this 2019 post. Compound 1, from the in-house screen, was found crystallographically to bind in the ATP-binding site, and ITC studies revealed it to have high micromolar affinity for the protein. Meanwhile, compound 2 was identified from the crystallographic screen, and while the affinity wasn’t measured, the pyridyl ring is located a short distance from where compound 1 binds.

 

Initial SAR around fragment 1 revealed that growing toward the binding site of compound 2 would be possible, as illustrated by compound 9. Appending a pyridyl ring onto this molecule led to compound 16, with low micromolar affinity. The pyridyl moiety stacks onto an arginine side chain, and improving this interaction by replacing the pyridyl with a phenyl appended with electron-withdrawing fluorine atoms led to compound 27, with submicromolar activity. Overlaying the crystal structures of compounds 1 (cyan), 2 (magenta), and 27 (gray) reveals that the merged molecule does indeed bind in a similar manner to the component fragments.

Unfortunately, despite good biochemical activity against PurC, none of the compounds were particularly effective at inhibiting growth of either M. abscessus or M. tuberculosis. Such disconnects between biochemical and cell potency are unfortunately all too common, particularly for antimicrobial targets, as we wrote about here. The researchers suggest possible reasons including efflux and physicochemical properties. The paper ends by noting that work is continuing, and we look forward to hearing more.

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.

Fragments vs Gram-negative bacterial PPAT

Of the 30+ fragment-derived drugs that have entered the clinic, only one is an antibiotic. In part this reflects a shift away from this therapeutic area by many companies. Novartis, though, has continued to invest, as demonstrated by two consecutive papers in J. Med. Chem.

The researchers were interested in the enzyme phosphopantetheine adenylyltransferase (PPAT, or CoaD), which catalyzes the penultimate step of coenzyme A biosynthesis from ATP and 4'-phosphopantetheine. Although the enzyme is present in all organisms, the bacterial form is highly conserved across prokaryotes and significantly different than the human form. It is also essential for bacterial growth, thus making it an attractive target.

In the first paper, Robert Moreau and colleagues start big: a high-concentration screen (at 500 µM) of 25,000 fragments as well as NMR-based screens of their core 1408 fragment library. Triaging both hit sets led to a cornucopia of 39 crystal structures of bound fragments; the chemical structures of a dozen are provided in the paper, with IC₅₀ values from 31 to >2500 µM. Perhaps surprisingly, all of these bound at the pantetheine binding site of the enzyme, suggesting that this is a “hotter” hot spot than the ATP-binding site.

Three of the fragments are described in more detail. The first was optimized from 273 µM to 4.3 µM, but subsequent advancement was unsuccessful. The second fragment, with an IC₅₀ of 230 µM against E. coli PPAT, could be optimized to mid-nanomolar inhibitors; unfortunately these were much less active against PPAT from P. aeruginosa, so this series was also abandoned. But the third fragment discussed, compound 6, proved to be more tractable.

Initial optimization based on other hits led to compound 32, and addition of a methyl to the benzylic linker provided a satisfying 30-fold improvement in potency for compound 33. This “magic” methyl appeared to help desolvate the adjacent NH as well as pre-orient the molecule in the bound conformation. Further growing from this position led to compound 53, which provided a further 7-fold improvement in potency. Crystallography revealed a hydrogen bond between the nitrile nitrogen and a protein backbone amide. Unlike the previous series, this compound was active against PPAT from both E. coli and P. aeruginosa.

The second paper, by Colin Skepper and colleagues, describes further optimization of the molecules to picomolar binders. There’s a lot of lovely medicinal chemistry in both papers, but unfortunately all the molecules displayed at best only modest antibacterial activity. One problem is that Gram-negative bacteria have two membranes: an outer one which blocks lipophilic molecules and an inner one which blocks hydrophilic molecules. Compounds that can make it past these barriers also face an array of diverse efflux pumps, and these seemed to be the downfall of this project. The core of the molecule makes multiple hydrogen bonds to PPAT; about twenty different heterocycles were tested, but most of these had significantly lower potency, and the active ones were efflux pump substrates.

These difficulties in part explain why companies have been moving away from antibiotics. This was not a minor effort: each paper listed more than twenty authors. The second ends somewhat wistfully. “Although none of the series disclosed… yielded a clinical candidate, it is our hope that these studies will help pave the way toward the discovery of new Gram-negative antibacterial agents with novel modes of action.” It is a worthy – if arduous – quest.

Stopping Virulence...One Fragment at a Time.

The best way to not get an infectious disease is vaccinate.   Streptococcus pneumoniae is repsonsible for a million deaths world-wide every year.  For Streptococcus pneumoniae, there are a numbers of vaccines on the market.  These vaccines are bacterial polysaccharides either naked or conjugated to a protein.  They are highly effective, but don't cover all serotypes (there are ~100).  And sometimes a novel serotype arises.  So, if you do get infected treatment is key.  Beta-lactams are the first line of defense, but multi-drug resistance is on the rise, so alternate forms of treatment are needed. Targeting virulence factors has become a recent line of research.  Pneumococcal surface antigen A (PsaA) is strictly conserved surface-exposed lipoprotein expressed by all known pneumococcal serotypes and is essential for colonization and pathogenesis.  PsaA is an integral part of an ATP-binding cassette(ABC) transporter protein complex known as the PsaBCA permease, which is involved in manganese (Mn2+) transport across the bacterial cell membrane. (See there's always a metal involved in cool biology.)  This makes PsaA a good target for pneumococcal infections.  In this paper, a group from down under presents their results using fragments to target PsaA.

They custom built a fragment library (via outsourcing) ~1500 fragments.  This struck me as unusual, if not unique.  Typically, academics make their own or just buy one off the shelf.  I would love to hear why this path was chosen.  In the SI, they do say they used "relaxed" Ro3, but the only relaxation seems to be on the MW.  Have other academics gone this route?  I would love to know more (you can be anonymous in the comments, hint hint).  These were docked into the PsaA metal binding site (Figure 1) based on 3D shape and electrostatic similarity. These were then scored using FlexX. 

Figure 1.  Structure of PsaA. 

The top 300 fragments were manually inspected and then subjected to a cluster analysis.  The 60 most diverse fragments were then tested in a competitive Zn-binding assay.  Zn is a irreversible inhibitor of PsaA and the assay uses this to test for compound binding.  10 of the 60 fragments exhibited greater than 15% inhibition at 100 microM.  Two of these compounds showed greater than 50% inhibition at 1mM (Cpd 15 and 58, Figure 2.)

Figure 2.  Fragments with greater than 50% activity at 1 mM.  Hydrogen bond acceptors are shown in red, H-bond donors in Blue.

So, with crystal structures available, the authors decided to inspect the docked poses rather than actually trying to obtain a structure of the fragments bound to the protein. So even though docked fragments can, and do tend to, keep their original locations, experimental data is key to confirming in silico predictions.  The made 31 compounds around 15, and one that replaced the p-nitro, o-methoxy phenyl with o-hydroxypphenyl was the best 15h (28 microM, pIC50/HAC=0.37).  To that end, they tried to soak apo-crystals with cpd 15h and were unsuccessful due to limited compound solubility and affinity for the target.  They did not attempt soaking compound 58, which they was unable to be further "optimized" with simple SAR.  Cpd 15h did have antimicrobial activity: significant growth inhibition at 180 ug/ml and total growth inhibition at ~800 ug/ml.  They did a further round of optimization.

This is an example of real FBDD approach, in contrast to just using the words.  However, I think this is really a MPU (minimal publishable unit).  If we are lucky, we can expect to see future papers coming out describing their success (or failure) against this target. 

It's A Start

As the readers of this blog know, I tend to be harsh on academic "Drug discovery" papers.  Sometimes, there is a really worthwhile academic paper, but by and large I find that they tend to publish things that are barely "drug discovery" and more the For Dummies...of what they think drug discovery is.  Which way will I swing on this paper from researchers Down Under?  

The bacterial Sliding Clamp, aka polymerase 3beta, is a key player in bacterial replication and is an "emerging" target. It interacts with other proteins via LM (Linear Motifs): 4-10 amino acid disordered regions.  This is typically a weak interaction (1-100 uM). These LM exist at termini, but sometimes in loops.  A consensus sequence for the LM that interacts with the Sliding Clamp has been identified: QLx1Lx2F/L (S/D preferred at x1; x2 may be absent).  Two classes of compounds have been identified previously but with >10 uM affinity and no -cidal activity. 

So, these authors went after this target using X-ray as the primary screen.  The Zenobia Fragment Library was used (352 molecules) to soak into crystal in pools of 4 fragments.  Four fragments (below) were found to bind to Subsite I on Chain A.  However, no changes in the main chain density were observed.

They also found several other fragments with weak density, and several that were deemed crystallographic artifacts.  None of these compounds showed significant activity below 1 mM in their competition assay.  So, the story then continues that they "sought to improve binding affinity by identifying fragments that could more completely occupy" the binding site.  

[An aside:  To me, this brings up an important point about the choice of fragment collection.  Fragments that are designed for X-ray soaking tend to be small (10-12 HAC).  Just from a theoretical standpoint, those fragment would have to have an affinity in the 250 uM range (LEAN 0.3).   This was covered in a poll and most most people are happy going < 10 HAC.  My question is how often is a very small fragment found as an active?]

To do this, they noted that the fluoro-phenyl group in 1 was previously reported, leading to investigations with compound 5. It was found to fully occupy the binding site.

They searched ZINC for compounds similar to 1-5.  Their initial purchases failed to find any compounds with activity < 1mM.  Eventually, they landed on the hypothesis that chlorocarbazoles were "promising", leading to compound 6.  At this point, I think Dan's head exploded, Scanners-style.  Yes, that is an epoxide.  The co-crystal structure showed that it was binding in the active, albeit with weak electron density.  Their SAR, wisely, did not include the N-alkyl epoxide. 

Both 7 and 8 show good LE and LLEAT.  Only the R enantiomer of 8 caused movement in the main chain.  It also was the most potent in the replication inhibition assay (64 uM).  It was also the most potent in terms of -cidal activity against both Gram positive and negative microbes. 

So, is this a good or bad paper?  I would say it is a start, but if I had been a reviewer I would have made them change the title "Discovery of Lead Compounds Targeting the Bacterial Sliding Clamp
Using a Fragment-Based Approach" to "Discovery of ACTIVE Compounds Targeting the Bacterial Sliding Clamp Using a Fragment-Based Approach".