X-ray crystallography plays a starring role in fragment-based lead discovery. But, with a few exceptions, it is rarely the primary screen. One of these exceptions was reported recently in Acta Crystallogr. D by Wim Hol and coworkers at the University of Washington, Seattle.
The researchers were interested in the histidyl-tRNA synthetase (HisRS) from Trypanosoma cruzi, the parasite that causes Chagas disease. They had previously constructed a library of 680 fragments in pools of 10, designed such that the fragments within each pool had different shapes to facilitate crystallographic screening. Crystals of HisRS•His (ie, the enzyme in complex with its histidine substrate) were soaked with the pools, such that the final concentration of each fragment was 1.5 mM. Fifteen of these pools showed new electron density, and these in turn yielded 15 different fragment hits when the pools were deconvoluted. Two additional fragments from the deconvolution process showed weak density, though these were not further pursued.
Strikingly, all 15 fragments bound to the same site, a site not observed in the absence of the fragments. This is a narrow groove described by the researchers as a “document sleeve,” and in fact all the hits are single six-membered aromatics or double aromatics with few or no aliphatic substituents. Most of the fragments are also quite small, with the majority having just 8 or 9 non-hydrogen atoms. Although all the fragments bind in roughly the same plane, there is considerable variation in the positions of substituents, and some of the fragments appear to bind in multiple orientations.
Next, the researchers tested their hits in orthogonal assays. Only one fragment showed thermal stabilization of the enzyme, and only three showed any inhibition in a functional assay (at most 39% at 2 mM of fragment). Thus, these are very weak binders.
The fragment-binding pocket is a few Ångstroms away from the histidine substrate, making linking the two ligands feasible. In preparation for this step, the researchers acetylated the amino group of the most potent fragment. This caused little change in the functional activity, but crystallography revealed that the fragment’s binding orientation had flipped around, such that the acetamide group pointed away from the histidine and towards a cysteine residue. Attempting to turn lemons into lemonade, the researchers added electrophiles to try to interact with the cysteine residue. Some of these molecules had measurable IC50 values (on the order of 1 mM), and crystallography of one of these showed covalent bond formation between the fragment and the targeted cysteine. This cysteine residue is found in the T. cruzi enzyme but not the human version, and indeed these molecules appear to be more active against the parasitic HisRS.
This is a nice example of fragment screening by crystallography that illustrates one of its main challenges: crystallography is capable of detecting extremely weak binders that may prove difficult to advance. Still, the researchers have taken some promising initial steps, and it will be fun to see what they come up with.