Universal fragments for discovering hot spots and aiding crystallography

A couple years ago we highlighted a paper from Eddy Arnold’s group at Rutgers University in which crystallographic fragment screening revealed over a dozen secondary ligand binding sites on HIV-1 reverse transcriptase (RT). Shockingly, the fragment 4-bromopyrazole bound to every single site, which led us to ask “is this a privileged fragment or a promiscuous binder? And as for the sites with no known functional activity, are these useful?” The Arnold group asked themselves these same questions, and provide answers in a new paper in the open-access journal IUCrJ.

The researchers first considered whether 4-bromopyrazole is special. They collected about 20 halogenated aromatic fragments and soaked these into crystals of HIV-1 RT at concentrations ranging from 20 to 500 mM. Of these, 4-iodopyrazole also bound at multiple sites, but most of the others – even closely related molecules such as 3-bromopyrrole or 4-bromothiazole – did not bind to any.

Next, the authors extended these observations to other proteins. When they soaked their molecules into crystals of the endonuclease from the 2009 pandemic influenza strain, they found that 4-bromopyrazole bound to four sites, including two of three identified in a previous crystallographic fragment screen. In one case, a phenylalanine side chain shifted to open up a new hydrophobic binding site. A similar and previously unobserved shift occurred with a tyrosine side chain when 4-bromopyrazole was soaked into the protein proteinase K. Thus, this fragment is able to identify otherwise cryptic binding sites.

Interestingly, the 4-bromopyrazole binding sites could be strikingly dissimilar, ranging from hydrophobic to mildly electropositive to strongly electronegative. The researchers note that the halogen can form either hydrophobic or polar interactions. Also, one pyrazole nitrogen can act as a hydrogen-bond acceptor while the other can independently act as a donor, and these interactions can be with the protein directly or through bridging water molecules.

Last week we highlighted work from Astex suggesting that secondary binding sites in proteins are common, but in most of those cases the proteins had only one additional site, and only a couple had five or six. In contrast, 4-iodopyrazole bound to 21 sites in HIV-1 RT, although only five of these had sufficiently good electron density to allow the entire fragment to be built. (That is, crystallography only clearly revealed the location of the iodine atom in the others.) How many of these sites are bona fide hot spots, and how many could be predicted using computational techniques such as FTMap?

This is all quite interesting, but, as we asked previously, is it useful? The researchers provide two applications.

First, 4-bromopyrazole may be a general probe to assess whether a protein is ligandable. Soaking crystals of the catalytic core domain of HIV-1 integrase in 500 mM of 4-bromopyrazole revealed no binding sites, in sharp contrast to HIV-1 RT, endonuclease, and proteinase K. Integrase also showed a very low hit rate in a general fragment screen, and a plot of binding sites vs fragment-screening hit rate for three proteins showed a linear correlation. Obviously this is a tiny data set, but if it holds up it could be an easy experimental way to assess the difficulty of targets.

Second, the bromine or iodine atoms in the pyrazole fragments could be used in single-wavelength anomalous dispersion phasing, a useful approach for solving crystal structures. The researchers demonstrated this experimentally for HIV-1 RT, endonuclease, and proteinase K, and suggest that 4-bromopyrazole and 4-iodopyrazole could be inexpensive and helpful additions to a “crystallographer’s toolkit.”

Thus, unlike other frequent-hitters such as PrATs, 4-halopyrazoles might be promiscuous yet specific – and useful. I look forward to seeing whether these "universal fragments" catch on.