Showing posts with label LEAN. Show all posts
Showing posts with label LEAN. Show all posts

08 May 2013

Fragments in Living Cells

GPCRs seem to be popular around here lately.  In this paper, a group of researchers in the UK developed a report-fluorescent assay to be used on living cells to screen fragments.  Recent advances have been made in structural studies of fragments (see Receptos and Heptares) and with this new assay, the entire suite of experiments for prosecuting FBHG exist for GPCRs.  As the authors point out, and I mentioned here, mucking around with GPCRs with things like detergent stabilization removes ancillary proteins which can provide allosteric interactions. As the authors state:
It is now acknowledged that GPCRs can adopt multiple active conformations as a consequence of protein-protein interactions that can lead to the activation or attenuation of different signaling pathways.  Furthermore, different agonists appear able to bias signaling in favor of a particular downstream pathway, including those that do not involve heterotrimeric G proteins . It is also clear that the binding affinity of antagonists can vary depending on the signaling pathway and agonist that is being studied. These data suggest that intracellular signaling proteins can elicit marked allosteric influences on the binding of both agonists and antagonists to a particular GPCR and as a consequence the cellular context in which binding affinities are measured will have a major impact on drug screening strategies. [Emphasis mine].
To build their assay, they used an existing fluorescent existing amine congener that was commercially available and ensured that it had competitive, antagonistic properties against A3AR (Adenosine-A3 receptor).  It exhibited many desirable characteristics for a high-content screening: including high affinity and slow off-rate.  It was able to quantify agonist displacement from A3AR.  

To test for the ability to detect weak binders, they deconstructed the high affinity A3AR antagonist (1) into its component fragments (2-7) [the affinity values are pKi].  They then acquired a 248 subset of the Maybridge fragment library that was Voldemort Rule compliant.
  They found 38 confirmed actives, with the compounds shown below as the top 6:

To me, the interesting part of this paper is NOT the subsequent SAR and the novel compounds that resulted, but instead the fact that there is now an assay for A3-and A1AR that can reliably detect fragments and support SAR studies.  Of course, one major caveat here is something that I learned from a venerable GPCR chemist at Lilly: the natural ligands of GPCRs are fragments, so of course fragment screening works.  I think most people who work in fragments would be very happy with 1 microM actives from a screen; I think most GPCR chemists would not.  I would also imagine that ligand efficiency is even more important when working in this class of compounds. 

18 November 2011

And once more into the breach...

When the market is more than 20 Billion dollars, you will find everyone working there. And so, with this recent publication, we have another entrant into the BACE inhibitor from Fragments competition, discussed previously here. This is the fifth by my count, the first being from Astra Zeneca.

In this paper, Eli Lilly describes their efforts using fragments to generate "the first orally available non-peptidic BACE1 inhibitor that produces profound Abeta-lowering effects in animals." They screened ~8000 compounds at 4.76mM that generated a number of low-affinity, but highly "LEAN" fragments (discussed below). Of most interest were the amino-benzothiazine (1) and amino-thiadiazine (2) compounds.

The authors note that co-crystallilzation was a key advance for their understanding of this system. The co-crystal showed two copies of (1) with high active site occupancy and in the "open-flap" conformation. One copy engaged the catalytic dyad and the other spanned the S1-S3 cavity. This data let them recognize that the planarity of the molecules were not optimal for fragment growth, so they "de-planarized" them, leading to (3). Only one enantiomer of (3) was recognized by BACE. The co-crystal of this compound showed binding identical to the original fragment, one copy engaging the catalytic dyad and one in the S1-S3 region. Addition of the S3 moiety pyrimidine led to (4). Fluorination of the central ring reduced in vivo clearance and and realized a significant increase in potency, while maintaining atom efficiency (5).

The crystal structure of (5) shows that this molecule retains an optimal H-bonding network, efficiently traverses S1, and projects the pyrimidine into S3.

Compound (5) was tested in animal models and pre-clinically in healthy human volunteers given orally. It showed significant reduction in Abeta levels in brain and CSF. Retinal pathology became a concern in longer term animal studies and the compound was not taken any further.

This paper shows the power of Fragments in discovering novel scaffolds for important targets. It is also important to note that the modified fragment hit retained the same binding as the original fragment hit.

The other contribution that the Lilly group brings out in this paper is the concept of LEAN (Ligand Efficiency by Atom Number): -log (IC50)/Number of heavy atoms. This is one of many ways people have developed to gauge the efficiency of their ligand hits, I think this is the simplest to use. As can be seen from the Lilly data, a LEAN of >=0.30 is an efficient molecule. For those of us who don't do logs in our head well, this lends it itself to a simple cheat sheet:

I can send a copy of this spreadsheet to anyone who wants.