Fragment Ontology

We here at Practical Fragments look for papers in the literature about fragments.  Typically, it is a Web of Science search, or I see something come in an alert that has "fragment" in the title.  Well, not everything with fragment in the title is not really about fragments as we typically think of them. So, I recently came across this paper titled : "Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins".  I decided to give the paper a good perusal, largely because one of the authors is from where I did my post-doc.

The authors are interested in making competitive inhibitors of carbohydrate recognition domains for the treatment of a variety of diseases.  The challenge with lectin inhibitors is that the native carbohydrate has relatively low affinity and are synthetically complex.  As you would think, you can use the carbohydrate for binding specificity and then add something more "drug-like" to increase affinity through other interactions.  This approach has been successful but require complex multistep syntheses.  In this paper, they decided to search for peptides which can synergize with carbohydrates rather than serving solely as a linker or standalone recognition element.  To do this, and increase throughput they used a genetically encoded library, phage display.  In short, they created a glycopeptide library of 10^8  molecules through derivatization of a peptide library with carbohydrate.  This approach allows the addition of different carbohydrates (targeting different lectins) with the same peptide library.  

Figure 1.  Library Screening Approach for Genetically Encoded Glycopeptide Libraries.

In this approach, the first library (Man-X7) is screened against the target and anti-target.  The second library (methyl-X7) and the third library (Ser-X7) are screened only against the target.  After the first round of panning, they identified a weak consensus of Man-[WYF]Y[SDEA].  These peptides were made and able to compete with ConA for ligand in SPR, the mannose was shown to be essential to activity, the specific peptide sequence was required for synergistic binding.  Further work showed that the final four residues of the peptide contributed minimally to binding, so they lopped them off.  

They then performed two more rounds of panning with Man-WY[D/E]-X7.  All of the hits from these rounds had single digit micromolar affinity and the glycan-proximal ligands are responsible for most of the affinity.  How did they know if this is actuallly binding to where they want it to?

Figure 2.  Man-WYD co-crystalllized with ConA. 

Figure 2. shows the crystal structure of Man-WYD.  The mannose moiety binds where it is expected.  However, the peptide is not binding in the remainder of the trisachharide binding site, but instead in a somewhat deeper cavity near Y12.  Additionally, a latent hydrophobic site is opened up through induced fit (asterisk), filled by the Y residue of the glycopeptide.  

This approach led to the discovery of a novel class of compounds which would not have been discoverable by "standard" approaches.  But, is this fragments?  In my eyes, fragments takes simple compounds and screen them against the target.  It then optimizes the actives as quickly as possible and does iterations.  A key component to FBDD is SBDD and identification of how the actives/hits bind.  To me, this approach adheres to all the tenets of FBDD.  We have seen super huge screening molecules before, so that should not be an issue. As I have said, FBDD is about small little things being screened effectively.  I think this paper shows it is more about how you think about your system.