Fragments vs bacterial DNA ligase: triumph of structure-based design

Fragment approaches have been used successfully against several anti-bacterial targets (see for example here, here, and here). In a recent issue of ACS Med. Chem. Lett., a team of researchers from Astex and GlaxoSmithKline report another potential weapon in the ongoing war against bugs.

The researchers were interested in bacterial DNA ligase (LigA), which is essential for DNA replication, is highly conserved among numerous types of bacteria, and is quite different from its human counterpart. They started by screening ~1500 fragments against S. aureus LigA using a combination of X-ray crystallography (soaking), ligand-observed NMR (WaterLOGSY), and thermal shift assays. Hits that made it through this gauntlet were then evaluated by isothermal titration calorimetry (ITC) and prioritized in part by ligand efficiency. One of the best molecules was compound 3.

The chlorine atom of compound 3 bound in a hydrophobic pocket of the enzyme. Wary of the potential reactivity of this motif, the researchers replaced it with a trifluoromethyl group; they also removed a nitrogen from the pyrazine ring to provide a vector for fragment growing. The resulting compound 10 had slightly improved potency.

Examining the structure of the initial fragment also revealed a water-mediated hydrogen bond, and by enlarging the triazole to a 6-azaindole 6-azaindazole (compound 12) the researchers were able to make this hydrogen bond directly while also more effectively filling the pocket, providing a satisfying 70-fold boost in affinity. However, close inspection of the crystal structure and computational modeling suggested that this molecule was binding in an energetically unfavorable conformation. Simply adding a nitrogen to the pyridine ring alleviated this problem, providing another 15-fold boost in potency (compound 13). This molecule also showed antibacterial activity against a number of Gram-positive pathogens.

This is a brief but elegant paper that demonstrates the power of crystallography and modeling to drive a fragment-derived medicinal chemistry effort. It will be fun to watch this story progress.

Poll results: how do you store your fragment libraries?

The results are in, and it looks like there is wide diversity in how folks store their working fragment libraries:

Of the 79 votes, the largest number, roughly 42%, keep their compounds at -20 °C. The next largest category was room temperature (29%), with -80 °C (16%) and +4 °C (13%) rounding out the list.

There were also some good comments to the original post giving more details as to solvent, use of inert gas, etc. These parameters are more difficult to capture in a multiple-choice poll, so please keep those examples coming.

Druggable is as Druggable Does; Or a Million Ways to use NMR

As we all know, the closure of sites is a bad thing for those of us in Pharma.  One very small silver lining is that this frees up a lot of very nice work to be published.  The former BI site in Laval has been closed for a year and we are still seeing great papers coming out.  In this one in JMed Chem,  LaPlante and co-workers tell us about their fragment efforts against HCV helicase. 

HCV has recently had drugs approved for its treatment, but as with any virus, different modes of treatment are important.  The ATP-dependent helicase activity is found in the C-terminal 2/3 of the NS3 protein. Helicase activity is straight forward to measure and there has been some success in terms of non-viral specific inhibitors.  The inhibitors found to date have been found to act through undesireable mechanisms, but with a wealth of structural information there is no reason why helicase is inherently undruggable.  With this information in hand, they decided to target site 3+4 (green sticks are DNA from the structure), near the most conserved residue W501.  The ATP-binding site is 1+2 for reference. 

 Their first approach was to screen the 1,000,000+ corporate compound collection.  As you would expect for a paper blogged about here, they failed to find anything interesting (all the inhibitors worked by undesireable modes).  So, on to the FBDD campaign, to save the day once more.  The used a "shotgun" approach with their fragment screen:

One source of compounds came from an earlier HTS where they rejected fragment-like molecules for lack of potency, additional HCS screening of in house fragment collection, commercial fragments were screened in an SPR assay, virtual screening, and NMR.  They had a stringent workflow aimed at producing quality compounds for X-ray.  [The in-house fragment collection was 1000 compounds.]  This, along with NMR, validated ligands that bound to site 3+4.  They note one particularly noteworthy problem: high false positive rates due to the high ligand concentrations needed for the assays.  This lead to aggregation, solubility, and promiscuity.  This lead them to implement specific assays designed to eliminate these compounds (two NMR papers published in 2013, ref 18). 

They then clustered the best hits into 9 chemotypes:

 They used an "Analog by Catalog" approach and soaked or co-crytallized the best compounds into crystals.  S6, S7, and S9 were not found to bind to helicase in the crystallization trials and were deprioritized.  S5 was found at Site 3+4, but also others.  S1-4, and S8 were found to bind solely to site 3+4 (12 examples shown overlain). The key feature of this is the compounds are centralized in a wide groove over W501.  The topology of the binding site (wide groove and small lipophilic pocket) meant that optimizing for potency could be challenging.

From this, they decided S2-S4 were the most promising.  In the end, the focused on the S2 indole series as the most promising.  The S2 stereotype 1

was found from an STD-NMR screen of 3 fragment per sample (300 uM fragment and 3.5 uM helicase).  They then, much to my heart's delight, they reached into the NMR cabinet for line broadening and competition experiments confirming it binds in site 3+4.  X-ray confirmed the binding mode, but potency was not improved with chemistry.  So back into the NMR cabinet they went: a methyl resonance assay, 

 15N TROSY showing peaks shifting upon addition of a derivative of 1, and 19F NMR!  OMG, how awesome is this?  

In terms of the chemistry, removing the Br does not change the potency, but did change the orientation of the compound in the binding site.  Further elaboration led to this compound 19 (3 uM and 0.23 LE):

It contains a nitro group, think what you may.  In order to confirm the binding affinity of the compound without immobilizing protein, they used the methyl resonances to do the titrations.  The two separate peaks they followed gave values of 32 and 28 uM (+/- 8).  Given the broadness of these peaks, I think this is a pretty decent assay, although it is an order of magnitude different than the biochemical Kd.  However, subsequent structural studies revealed that there is significant structural dynamic differences between pH 6.5 and 7.5.  ITC gave the same number (33 uM and enthalpy driven); however, the ITC had to be run at high compound concentration and a different pH.  They then went off the deep end and decided to use CD (I can't link to a previous post of using CD because we have never had a post where someone used it).  With a horrible assay (don't even get me started on near-UV CD as a readout of tertiary structure), they got reasonably close to the Kds determined by ITC and methyl-NMR.  

This is a very nice example of not being afraid of a target and using all available tools to advance hits against it.  It also shows the WIDE range of NMR experiments that can be used and that are easy and practical.  In terms of full disclosure, Steven LaPlante is a FOT (Friend of Teddy) and I have been working with him. 

Fragments vs NAMPT, maximum ligand efficiencies, and off-target activities

The enzyme nicotinamide phosphoribosyltransferase (NAMPT) is essential for the synthesis of the important cofactor NAD and thus an intriguing target for blocking cancer cell metabolism. In two recent papers, researchers from Genentech, Forma Therapeutics, Pharmaron Beijing, and Crown Biosciences describe how they used fragment-based approaches to discover new inhibitors of this enzyme.

In the first (J. Med. Chem.) paper, Peter Dragovich (Genentech) and collaborators start with a screen of 5000 fragments using surface plasmon resonance (SPR) at the relatively low concentration of 100 µM. This yielded 283 hits which were retested at 150 µM and also competed with a known high-affinity inhibitor; noncompetitive fragments, which presumably bind outside the active site, were discarded. This winnowed interesting hits to 118 fragments, each of which was characterized in full dose-response curves. Only 6 were extremely weak (K_D> 2 mM) or nonspecific, while 35 were quite potent (K_D< 100 µM).

As an interesting aside, the substrate for NAMPT is nicotinamide, and this was characterized by SPR as having a remarkably high ligand efficiency (LE) approaching the “soft limit” Teddy recently discussed. The researchers suggest:

The LE exhibited by nicotinamide for NAMPT is the highest we have observed for a fragment lead and, given that NAMPT is highly optimized to efficiently bind this substrate, may approach an upper limit of this parameter for such molecules.

Keep in mind that Genentech has done lots of screens, so this is a significant statement. Indeed, I can think of only a few fragments (here and here) with comparable LE values.

But back to NAMPT. More than 30 co-crystal structures of fragments bound to the enzyme were solved, and several of these fragments were advanced. In doing so a variety of information was used, including data from molecules previously discovered in-house and elsewhere. Lots of nice SAR are presented, and if you’re into structure-based drug design I’d strongly encourage a close reading of the paper. Just to give you a flavor, compounds 12 and 13 (blue), despite their structural similarity, bound in very different orientations. A bit of engineering led to compound 15, and crystallography revealed that only a single enantiomer of a racemic mixture binds to the enzyme. Borrowing information from other NAMPT inhibitors led to the potent single enantiomer compound 17; the other enantiomer is 250-fold less active. Further modification yielded an orally active molecule with activity in a mouse xenograft model.

In the Bioorg. Med. Chem. Lett. paper, members of the same team describe two other series of molecules derived from fragments – and provide some important warnings about interpreting data.

One series (not shown here) was optimized to nanomolar potency in biochemical assays and antiproliferative cell assays. However, the team did a series of careful follow-up studies to show that these molecules are probably acting through off-target mechanisms. For example, the molecules do not reduce NAD levels as they should, and addition of the product of NAMPT did not rescue the cells, as it would were NAMPT the primary target.

For the other series, compound 7 (red above) was characterized crystallographically bound to NAMPT. Initial attempts to improve affinity were unsuccessful, but the co-crystal structure of another fragment suggested that replacing the pyrazole moiety with a simple phenyl group would be tolerated, leading to compound 25. Subsequent fragment growing ultimately led to Compound 51, with low nanomolar potency in both biochemical and cell-based assays. Importantly, this molecule did reduce NAD levels in cells, and the antiproliferative effects could be rescued by adding the product of NAMPT. Taken together, these data show that compound 51 is a nanomolar inhibitor of NAMPT both biochemically and in cells.

The importance of such rigorous characterization is driven home by a footnote, in which the researchers reveal that compound 51 was previously alleged to be an inhibitor of glucose transporter 1 (GLUT1). This was published in a high-profile journal, and several chemical suppliers now sell this compound (called STF-31). Although the current paper does not explicitly say so, it is possible the results in the earlier paper could be attributed to NAMPT inhibition rather than GLUT1 inhibition.

In the hope that views on STF-31 will evolve, I’ll close this Darwin Day post with a quote from The Descent of Man:

False facts are highly injurious to the progress of science, for they often long endure; but false views, if supported by some evidence, do little harm, as every one takes a salutary pleasure in proving their falseness; and when this is done, one path towards error is closed and the road to truth is often at the same time opened.

Pushing the Limit

Dan's recent post discusses the limits of fragments binding to PPIs.  This paper from a while back came to my attention recently and I think it is important to bring up for discussion.  In it, they discuss the physical limits of binding.  They start with this:

Protein−ligand binding is a delicate balance between the loss of entropy resulting from complexation and the enthalpy gained by forming favorable contacts with the protein.

Entropy changes comes from linking two fragments, loss of internal flexibility, and reorganizing water in the binding site.  Current thinking (2 years ago) provides that in terms of favorable energy, van der Waals forces are the primary driver of affinity, while H-bonding and electrostatic interactions drive specificity.  Then they revisit this seminal paper from Kuntz et al but with the intent of exploring ALL biophysical properties rather than drug like ones.  For this study, Ligand Efficiency is DeltaG divided by the HAC. 

If van der Waals forces are the primary driver of affinity, there should be a correlation between affinity and size/contact area. 

There is not.  In terms of efficiencies, the median efficiency is -0.34 kcal/mol*atom.  Putting that in terms of buried surface area (BSA), they determined that the median efficiency is -23 cal/mol*A^2 (Angstrom squared).  To compare, if you look at only solvent accessible area, this value goes down to -7 cal/mol*A^2.  However, despite their inherently larger binding areas, macromolecules do not bind with greater inherent affinity than small molecules.  They argue that this is due to better "burying" of the small molecules.  As expected the most ligand efficient compounds are small, highly charged compunds buried in highly charged sites.  The limit is -1.75kcal/mol*atom, but a soft limit of -0.83 kcal/mol*atom is proposed.  

For maximal binding efficiency, they found that 90% of these interactions involve a charge-charge interaction or a metal ion.  In fact, the most efficient have several charge-charge interactions.  It is known the most efficient ligands are small, but not all small ligands are highly efficient.  So, what makes this difference?  As would be expected (at least I expected it), the longer the distance between charged groups the less efficient the interaction. 

For every 1A drop in average contact distance, the maximal efficiency goes down -0.41 kcal/mol*atom.  Wait!  What about desolvation you ask?  Isn't the entropic cost of desolvation in charged molecules very high?  In many of the highest efficiency complexes, there is water in the binding sites, so not all of the water is displaced, the authors state.  They also state that the charges in the binding pocket may not be fully solvated because the pockets around the charge are so small.  Yeah, I am not happy with that explanation either. 

So what makes maximally affinity?  Kuntz et al. said after 15 heavy atoms your affinity plateaus.  In fact, Kuntz showed that it is exceedingly rare to find an affinity >-15 kcal/mol, arguing that this is due to biological effects, namely clearance.  This paper argues that that cutoff is "seredipitously random or manmade".  Other people have argued that as ligands increase in size the maximal efficiency would drop because the number of interactions that need to be optimized increases and the only way to do this is through structural compromises and thus reduced affinity.  The authors of this paper begrudgingly admit this hyopthesis fits their data.

So, what implications does this mean for fragments? Does Kuntz's data mean that fragment libraries should be no bigger than 15 heavy atoms? Should we consider adding charged moieties or even metals?  They argue that this is a source of vast potential improvement for drug design. 

How weak is too weak for PPIs?

Ben Perry brought up an interesting question in a comment to a recent post about fragments that bind at a protein-protein interface: “At what level of binding potency does one accept that there may not be any functional consequence?” I suspect the answer will vary in part based on the difficulty and importance of the target, and many protein-protein interactions (PPIs) rank high on both counts. In a recent (and open-access!) paper in ACS Med. Chem. Lett., Alessio Ciulli and collaborators at the University of Dundee, the University of Cambridge, and the University of Coimbra (Portugal) ask how far NMR can be pushed to find weak fragments.

The researchers started with a low micromolar inhibitor of the interaction between the von Hippel-Lindau protein and the alpha subunit of hypoxia-inducible factor 1 (pVHL:HIF-1α), an interaction important in cellular oxygen sensing. The team had previously deconstructed this molecule into component fragments, but they were unable to detect binding of the smallest fragments.

In the new study, the researchers again deconstructed the inhibitor into differently sized fragments and used three ligand-detected NMR techniques (STD, CPMG, and WaterLOGSY) to try to identify binders. As before, under standard conditions of 1 mM ligand and 10 µM protein, none of the smallest fragments were detected. However, by maintaining ligand concentration and increasing the protein concentration to 40 µM (to increase the fraction of bound ligand) or increasing concentrations of both protein (to 30 µM) and ligand (to 3 mM), the researchers were able to detect binding of fragments that adhere to the rule of three.

Of course, at these high concentrations, the potential for artifacts also increases, but the researchers were able to verify binding by isothermal titration calorimetry (ITC) and competition with a high-affinity peptide. They were also able to use STD data to show which regions of fragments bind to the protein, suggesting that the fragments bind similarly on their own as they do in the parent molecule. (Note that this is in contrast to a deconstruction study on a different PPI.) Even more impressively for a large (42 kDa) protein, the researchers were able to use 2-dimensional NMR (¹H-¹⁵N HSQC) to confirm the binding sites.

Last year we highlighted a study that deconstructed an inhibitor of the p53/MDM2 interaction. In that case, the researchers were only able to find super-sized fragments, and they argued that for PPIs the rule of three should be relaxed. The current paper is a nice illustration that very small, weak fragments can in fact be detected for PPIs, though you may need to push your biophysical techniques to the limit.

But back to the original question of how weak is too weak. With K_d values from 2.7-4.9 mM, these are truly feeble fragments. Nonetheless, they could in theory have been viable starting points had they been found prospectively. That assumes, though, that these fragments would have been recognized as useful and properly prioritized. The ligand efficiencies (LE) of all the fragments, while not great, are not beyond the pale for PPIs. Previous research had suggested that much of the overall binding affinity in compound 1 comes from the hydroxyproline fragment (compound 6, which was originally derived from the natural substrate). Not discussed in the paper, but perhaps more significantly, the LLE (LipE) and LLE_AT values are best for compound 6, which despite having the lowest affinity is the only compound that could be crystallographically characterized bound to the protein. In the Great Debate over metrics, this suggests that LLE and LLE_AT may be more useful than simple LE for comparing very weak fragments.