A sharp NMR trick for rapidly measuring affinities

As noted in our poll last year, ligand-detected NMR ranks among the most popular fragment-finding approaches. The various methods are able to detect even weak binders, so determining affinities is important to effectively prioritize hits. This, however, can be time-consuming. In a recent J. Am. Chem. Soc. paper, Ridvan Nepravishta, Dušan Uhrín, and collaborators at CRUK Scotland Institute, University of Edinburgh, and Universidad de Sevilla present a clever way to speed up the process.

 

Normally, NMR spectra of small molecules show multiple spectral lines, with each line corresponding to a different atom or atoms (typically protons). Indeed, depending on the details, the signal from a single proton might be split into multiple peaks. All these signals are great for understanding the details of individual atoms, but the more lines there are, the lower the signal to noise ratio. For maximum sensitivity it would be nice to combine all the lines from all the atoms in a given molecule into a single, intense singlet. This is exactly what the researchers have done.

 

The approach is called Sensitive, Homogeneous And Resolved PEaks in Real time, or SHARPER. For the NMR aficionados out there, “when placed before the acquisition of the NMR signal, a train of spin-echoes in the form of the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence suppresses evolution due to chemical shifts and J couplings…. All these attributes of the CPMG pulse sequence are maintained when the spin-echo train is employed during the acquisition of the NMR signal. However, this time, the outcome is not a regular spectrum, but under certain conditions, a single spectral line formed as a sum of Lorentzian lines of contributing spins.”

 

The researchers initially applied SHARPER to two commonly used ligand-detected methods: ¹H STD, which we wrote about here, and ¹H CPMG, which we wrote about here. The first test system was human serum albumin (HSA) binding to naproxen. Keeping protein concentration constant at 9 µM and varying ligand concentration gave similar K_D values (210-280 µM) for standard STD, STD SHARPER, and CPMG SHARPER (conventional CPMG failed due to insensitivity at lower ligand concentrations). These values are an order of magnitude higher than those reported using SPR and ITC (25 and 10 µM, respectively) because of the high protein and ligand concentrations needed for conventional NMR approaches; when the SHARPER experiments were rerun at 1 µM HSA, the K_D values were 39 µM. Several other HSA ligands also gave good agreement with the literature.

 

Next, the researchers applied STD SHARPER to the anti-cancer target fascin, which we wrote about in 2019. An examination of 11 ligands from that study gave good agreement with the published dissociation constants. Importantly, SHARPER was faster than conventional approaches, with 15 K_D determinations per day instead of four.

 

Not content with this four-fold improvement in throughput, the researchers developed a new experiment based on line broadening called ¹H LB SHARPER. This allows the determination of 48 dissociation constants per day, and the results for HSA and fascin agreed with the other methods.

 

One of the most time-consuming aspects of most NMR-based affinity measurements is preparing and analyzing samples at multiple ligand concentrations, so the researchers turned to machine learning to choose which ligand concentrations would be most informative and choose just two of them rather than the six or more commonly used. This worked too, thereby potentially increasing throughput to 144 dissociation constants per day.

 

The researchers suggest that SHARPER could also be applied to some of the other recent NMR techniques we’ve discussed, such as PEARLScreeen and photo-CIDNP. Although I always emphasize that I’m no NMR spectroscopist, this strikes me as a neat, practical approach. What do you think?

LO-MS...Coming of Age.

There are many ways to screen for fragments.  One of the really emerging areas uses mass spec detection: WAC, native mass spec, HDX, and ligand-observed MS.  The group that we highlighted back in March has a new paper where they look at the method in terms of accuracy of Kd determination and compare the results to other biophysical methods.  

Their previous work they looked at relative affinity ranking of bound fragments.  In this study, they compared the accuracy of Kd determination in this method to ITC.  First they used a pool of 3 or 4 known CAI inhibitors and a pool of 50 fragments (from their collection).  The conditions were defined:

The hCAI protein was incubated with each inhibitor mixture in the binding buffer with a total volume of 50 mLat room temperature for 40 min. The protein concentration was maintained at 25 mM and the inhibitor concentration increased from 1 mM to 50 mM. The control was prepared by using the binding buffer substitute for hCAI during incubation. The incubation solution was then filtered through a 10 kDa MW cutoff ultrafiltration membrane by centrifugation at 13,000 g for 10 min at 4 C followed by a quickwash with 10mM ammonium acetate (pH 8.0) to remove the unbound compounds.

 Two different methods were used to calculate Kd: 1. saturation curves and 2. measuring the unbound fraction of ligand .  Method 1 was deemed unsuitable for determining Kds for ligands with largely different Kds.  Method 2 did not depend on saturation curve fitting, instead using a calibration curve and did not observe any fragment competition at higher P:L ratios (6:1 or 8:1).  This approach also improved the sensitivity of the assay allowing better detection of lower affinity ligands.  Kds determined using this method matched those determined by ITC.  

To further test the method, they ran a pool of 50 fragments against HCV RNA polymerase NS5B.  This gave, as expected, a complicated chromatographic baseline.  To exclude promiscuous binders, they ran against BSA in parallel.  Eight fragments in the mixed pool showed selective binding to NS5B using unbound fraction analysis vs. 2 from the bound fraction analysis.  7 of 8 fragments were confirmed by SPR (ITC could not be used).  The 1 BFA fragment to be analyzed by SPR showed that it was a very weak binder.  

This sort of work makes me happy.  Methods always need to be pushed and evaluated.  When evaluating methods, if this sort of cross validation hasn't been done, question as to why not?