Screening MiniFrags by NMR

Small is becoming big. Five years ago we highlighted MiniFrags, consisting of just 5-7 non-hydrogen atoms; FragLites and MicroFrags soon followed. Screening these tiniest of fragments at high concentrations can thoroughly explore hot spots on a protein and identify favorable molecular interactions. But because they are so extraordinarily small, experimental methods for screening them have been mostly limited to crystallography. In a new J. Med. Chem. paper, Annagiulia Favaro and Mattia Sturlese (University of Padova) turn to the most venerable of fragment-finding methods, NMR.

 

The researchers started with the 81 reported MiniFrags and removed those with aqueous solubility less than 250 mM or without protons observable by NMR (such as phosphate). The remaining 69 fragments were dissolved directly in phosphate buffer, mostly at 1 M concentration, though lower solubility fragments were dissolved at 250 mM. Importantly, the pH of each sample was carefully adjusted to 7.1 to ensure that any signals correspond to MiniFrag binding and not to changes in experimental conditions.

 

As a test case, the researchers chose the antiapoptotic target BFL1. This protein is related to BCL2, the target of venetoclax, which was discovered using SAR by NMR. BFL1 has a hydrophobic cleft with five subpockets and has been studied by NMR. Like other BCL2 family members it is a difficult target, as we noted earlier this year.

 

The actual screen was done using chemical shift perturbation (CSP) detected by two-dimensional ¹H-¹⁵N HMQC. Fragments were screened at 100 mM, a 5000-fold excess above the protein concentration. Hits were confirmed at 20 mM (more on that below). As with the library preparation, pH was carefully controlled.

 

At such high ligand concentrations, any impurities could become a problem: a 2% contaminant would be present at 2 mM. To weed these out, the researchers performed WaterLOGSY experiments. These only produce a signal at ligand to protein ratios much lower than 1000 to 1, so any hits could only come from impurities.

 

Even at high concentrations, CSPs caused by weak fragments are small, so the researchers developed an analysis method to identify those that shift more than at least one standard deviation from the average. CSPs can shift in any direction on a two-dimensional map, but any one protein-ligand interaction should shift signals in the same direction. Here is where the 20 mM confirmation experiment comes into play: a “cosine similarity” assesses whether two CSPs are in the same direction and thus likely to be real.

 

Screening BFL1 led to 53 hits, a hit rate of 78%, similar to crystallographic screens of MiniFrags against other targets. Forty percent of MiniFrags bound to multiple sites on the protein; only 11 (16%) bound to a single site. The five subpockets were each liganded by 6-17 MiniFrags. In subsequent experiments, the researchers were also able to measure binding of two different fragments to different pockets simultaneously, akin to SAR by NMR.

 

This is an interesting approach, but while fragments with >5 mM dissociation constants have been advanced to drugs, the utility of a 100 mM binder remains to be seen. That said, the technique could be a boon for understanding protein-ligand interactions, and I look forward to seeing it applied more broadly. In particular, screening the same set of MiniFrags on the same protein by NMR, crystallography, and computational methods could be quite informative.