Fragments in cells

A couple months ago I considered writing an April Fools’ post on screening fragments in vivo. A recent paper in J. Med. Chem. reports something similar, only it’s no joke.

Alexander Shekhtman and colleagues at SUNY Albany have developed a method they call “screening of small molecule interactor library by using in-cell NMR”, or SMILI-NMR. The process starts by overexpressing two proteins within cells (E. coli, in this case). If the proteins are sequentially expressed, one of them can be selectively labeled with NMR-active isotopes. To test their system, the researchers overexpressed the model proteins FKBP and FRB. These proteins interact only weakly by themselves, but in the presence of the small molecule rapamycin they form a high affinity complex. By performing NMR on the cells, the researchers could observe changes in NMR peaks corresponding to formation of the ternary complex inside the cells when rapamycin was added. They could also do competition studies: adding the small molecule ascomycin to this complex causes a change in the NMR peaks corresponding to the rapamycin being competed away by the ascomycin.

The next step was to look for new molecules that would modulate the interaction between FKBP and FRB, and the researchers chose a library of 289 dipeptides, which are actively transported into cells. The dipeptides were mostly fragment-sized, ranging from a low molecular weight of 132 (Gly-Gly) to a high of 390 (Trp-Trp). The dipeptides were screened in pools (organized in a matrix) and then deconvoluted to identify the most active molecules. Interestingly, none of the molecules caused discrete changes to the NMR spectra as observed with rapamycin or ascomycin, but several caused some of the NMR peaks to disappear and the remaining peaks to broaden dramatically. The most potent compound was Ala-Glu (MW 218), which caused this phenomenon at 5 mM concentration. The authors interpret this effect as being caused by the formation of a large complex consisting of many molecules of FKBP, FRB, and Ala-Glu. Interestingly, although ascomycin could reverse the effect of Ala-Glu, rapamycin could not.

The dipeptide Ala-Glu also behaved similarly to rapamycin in yeast cells: both molecules prevented growth by yeast expressing FKBP, while having no effect on yeast lacking FKBP. This was attributed to both molecules facilitating complex formation between FKBP and FRB within yeast.

The Ala-Glu “fragment” has some issues (ClogP = -4, for example); it would be interesting to see how some of the original FKBP fragments discovered at Abbott behave in this assay. And although not everyone has access to a 700 MHz NMR with a cryoprobe, this is an intriguing approach for studying protein-protein interactions in a very biologically relevant milieu.