Getting from a fragment to something more useful can be time consuming, and frustrating. Often it requires making lots of inactive analogs. It is easy to set up multiple chemical reactions in parallel, but purifying products is tedious. Thus, there has been a trend towards screening crude reaction mixtures. Off-rate screening (ORS) using SPR was published back in 2014, and other techniques have been used as well. Now, two recent papers describe using crystallography as the assay.
In a J. Med. Chem. paper, Bradley Doak, Martin Scanlon, and collaborators at Monash University, La Trobe University, University of Wollongong, and Queensland University of Technology describe Rapid Elaboration of Fragments into Leads using X-ray crystallography (REFiLx; see also here). The researchers note that ORS is best for identifying reasonably potent compounds, with low (<10) micromolar potency or better. For some targets, this is a bar too high. Specifically, they were interested in E. coli disulfide bond forming protein A (EcDsbA), an anti-virulence target for which they had found – after considerable effort – a 490 µM fragment.
The fragment contained a carboxylic acid, which could be conveniently used for amide bond formation, and the researchers decided to make a bespoke library around it. A collection of 93 small (5-12 heavy atoms) amines was assembled and each member was reacted with 2 micromoles (about 0.5 mg) of the fragment in a 96 well plate. The reactions were then evaporated and dissolved in DMSO to 100 mM concentration (assuming the reactions went to completion). HPLC-MS analyses revealed likely product for 83 reactions with yields up to 90%, though the average was closer to 25%. These crude reaction mixtures were then soaked into previously grown crystals of EcDsbA and analyzed crystallographically using both manual and automatic processing (including PanDDA).
The result was four hits, all of which were resynthesized and characterized in detail. Crystallography of the pure compounds confirmed the binding modes found in the crude reaction mixtures. Gratifyingly, one of the compounds bound 8-fold more tightly than the initial fragment, as assessed by two dimensional NMR.
The four hits all had purities >50% in the crude reaction mixtures, so the researchers resynthesized a few other non-hits that had lower yields. One of these was about 2-fold better than the original fragment, suggesting a false negative in the first screen. Experiments in which the most active hit was spiked into faux reaction mixtures at increasingly lower concentrations revealed – as expected – that detection was more difficult at lower concentrations.
A similar approach is described in Communications Chemistry by Rod Hubbard and collaborators at Vernalis, University of York, Diamond Light Source, University of Oxford, and University of Johannesburg. However, whereas the first paper focuses on trying to find improved hits against a difficult target, this paper focuses more on methodology. The researchers used two of the same protein targets for which they had previously performed off-rate screening, HSP90 and PDHK2. Conveniently, both these ATPases share some inhibitors, so the same set of 83 crude reaction mixtures could be used for both enzymes. Also in contrast to the EcDsbA example, the starting fragments (found in prior screens) were considerably more potent, as were the final molecules.
The researchers note that “as with any high-throughput experimental approach, there are false positives and false negatives.” Because off-rates were measured for all the crude reaction mixtures, the researchers could assess false positives and false negatives in the crystallographic screens. In false positives, products were seen in the crystal structures even though their affinities were no better than the reactant. In false negatives, starting material was seen in the crystal structures even though the products had better affinities. Together, these accounted for about half the results. Digging into the details, poor reaction yields and low solubility for the products accounted for many of the false negatives. False positives are harder to explain, though the researchers note that the buffer used for crystal soaking is different from that used for SPR.
There were also some notable successes: for one racemic compound, crystals of PDHK2 “correctly” selected the more potent enantiomer (Kd = 0.14 µM) over the less potent one (Kd = 17 µM). As in the first paper, there is a wealth of experimental details, including improvements to PanDDA protocols. The researchers performed the soaks in triplicate, and although this did lead to an increased number of hits, they note that singleton screening would probably be sufficient for most applications.
At the CHI FBDD meeting last month Frank von Delft, who is an author of the second paper, noted that he is increasingly using crude reaction screening to progress fragments, including against SARS-CoV-2. I look forward to seeing this approach, and the leads that come from it, advance.