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. 

Fragment Design Done Right

As many of you probably know, I am not a fan of virtual screening, computational design, in silico much of anything.  I think it tends to be poorly applied, or academic.  Now, don't mark me as a Luddite, I think that computational tools can be quite useful, when appropriately applied.  What is appropriate?  Read on and let Hoffmann-LaRoche-Nutley show you in this beautiful paper.  

This is one of a line of great papers coming out of the closing Nutley site, so that is the one upside.  In this paper, the authors present how they leveraged the expertise of their chemists to design fragments against HCV NS5B, a well known drug target.  The story starts (I hesitate to say "their efforts start...") with a screen of 2700 fragments by SPR.  They identified 163 hits of which 29 were selected (criteria unstated) for co-crystallization.  Only one fragment delivered, 1.   

Fragment 1 had a 78uM KD, 130 uM IC50, but it could not be optimized for affinity, physicochemical or ADME properties at all.  They one important discovery from the co-crystal of 1 was an unexpected, and they believe, first ever interaction of its type: the NH hydrogen interacting with Q446 (Figure 1). 

Figure 1. 

Using this and the published structures internally (2-3) and externally (4-6) the built a model. The following guidelines were proposed for the new fragments: 1. Satisfy carbonyl of Q446 and NH of Y448, optionally displacing or engaging the conserved water molecule, 2. Occupy large hydrophobic pocket, exploring its size, 3. Position aromatic chain to make edge to face interaction with Y448, 4. And at least one hydrophobic interaction with G410 and/or M414.

Figure 2.

In a triumph of democracy and teamwork, the chemist woud discuss his ideas with the compchemist and have them modeled.  The ideas were presented to the team and the best ideas selected for synthesis.  They also chose to avoid acidic functionality.  Since 1 was the only known binder in the region without acidic functionality, they focused on incorporating the unique Q446 interaction.  What they found was that compounds capable of 1,2 and 1,3 interactions were best.  Table 1. shows their SAR. 

Their first two compounds were dead, dead as a this parrot.  Compound 9 satisfied 3 of the 4 criteria they established, yet showed very poor activity and bad ligand efficiency. Using LE was crucial, the authors state, because it allows the to distinguish affinity through bulk, vs. affinity through efficiency.  Finally, adding substituents to the hydantoin to explore the hydrophobic pocket showed significant increases in activity, e.g. 9->12.  Fragment 12 was co-crystallized and confirmed the expected binding mode.  A 2-pyridone fragment (13) gave similar activity to 9.  So, starting with a Pfizer-inspired compound gave 14 which demonstrated an increase in potency, but NOT ligand efficiency.   They next tried 15 and voila! a 100x increase in affinity with four less heavy atoms, the ligand efficiency went way up!  Co-crystallization showed that 15 bound as expected.  Two more heavy atoms added to 15 led to 16 and showed increased potency and ligand efficiency while retaining the desireable phyiscochemical properties of 15.  Compound 16 was further optimized and entered clinical trials with all of the atoms presented in it. 

So, what makes this "Fragment Design Done Right" in my eyes?  In this case, they utilized fragment docking as an aid to chemist's designing ligands.  They used in silico tools, like modeling, to test the potentially validity of the chemist's hypotheses.  In the end, their computation was as good as their experimental follow up, in this case X-ray.  What differentiates humans from the brute beasts (except for all the exceptions out there where tool usage has been shown) is that we use tools.  Using tools correctly, is what differentiates the smart humans from the herd.