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? 

Mass Spec Screening in Solution

Mass spectrometry is a technique that most people are familiar with, as a QC tool.  It also has been demonstrated as a screening/validation tool.  Native mass spectrometry (nMS) has been discussed here, Weak Affinity Chromatography (WAC) here, and Hydrogen-deuterium exchange (HDX) here.  All of these methods have advantages and disadvantages.  A "new" method is the ligand-observed MS screening (LO-MS).  [I put new in quotes because I know of at least one company that has been using this method for screening for years via a CRO.]

The concept of LO-MS is straight forward (Figure 1) and very similar to WAC.  A mixture of fragments, in this case 384, are mixed with target (NS5B), incubated, and the ultrafiltrated (50kDa cutoff).  This step eliminates the need for the immobilization step in WAC, ensuring the native conformation.  The fragments were at 25 uM, while the target was at 50 uM. 

Figure 1.  Fragments MW 165 and 130 are binders.  MW162 and 150 are not. 

Retained fragments are then dissociated with 90% methanol and those showing intensity higher than the protein-minus control are considered binders (S/N  greater than 10).  In their library, 5% of the compounds were not amenable to mass spec detection, but they included them to increase the complexity of the mixture.  In the end, they ended up with 20 binders in 20 minutes!  They repeated the screen with smaller mixtures (50 and 84 fragments) where they found 12 binders (a subset of the original 20).  As a follow up, they ran the binders by SPR, validating 10 of the binders (50%).  5 out of these 10 gave useable crystals (observable electron density for the fragment) (50%).  They also show how the data can be used to generate Kds (like WAC).

This method raises some issues with me, but first let me say, it sure seems to work, and fast to boot.  From people I know who have used this to screen, they have been very happy.  Here is what bothers me: self-competition in the tube a discussed here and here, this is a non-equilibrium method (variable protein concentration during the ultrafiltration), and it is an indirect method.  For me, I prefer methods that directly detect ligand-target interactions, like NMR, SPR, and nMS.