Fragments vs RNA revisited: the power of two

RNA can assume complex three-dimensional structures just like proteins, and given the many roles it plays it is perhaps surprising that there are so few drugs that target this class of biomolecules. One problem is that ribonucleic acids are less diverse than amino acids, so there is less scope for developing small molecules that bind to specific regions of RNA. Nonetheless, a few brave souls have tried, some using fragment-based approaches. The latest such effort appears in J. Mol. Biol.

The researchers, led by Gabriele Varani of the University of Washington, Seattle, took a two-step approach to find two fragments that could simultaneously bind to the TAR element of HIV-1, a short stem-loop element essential for viral replication. The protein that normally binds to TAR contains a critical arginine residue, so the researchers started by purchasing a set of 16 arginine mimetics and using NMR to determine if any of them bound to TAR RNA. Several did, and one guanidine-containing molecule (MV2003) gave a strong NMR signal and also contained a hydrophobic element. The researchers decided to use this to hunt for a second fragment.

To find the second binder, the team screened 250 generic (ie, not targeted to RNA) fragments from Maybridge in pools of 5-8 in the presence of the first fragment. Remarkably, saturation transfer difference (STD) experiments, which detect changes in ligand NMR signals upon binding to macromolecules, suggested that more than 100 of these generic fragments appeared to bind to TAR RNA. However, more careful study of 20 representative fragments from 13 different scaffolds rapidly winnowed the set: 5 didn’t repeat when tested outside the pool, 6 gave signals in the absence of RNA, and 3 were not dependent on the presence of MV2003, suggesting that they bind nonspecifically. However, the remaining 6 only produced signals in the presence of both TAR RNA and MV2003, indicating a specific ternary complex. Although two of these fragments contain a (positively charged) primary amine, the rest are likely either neutral or only partially protonated at physiological pH. Interestingly, one of these is closely related to an RNA-binding fragment identified in previous work by a different group.

Next, the researchers constructed a model of how MV2003 bound to RNA. They used NMR data (nuclear Overhauser effects, or NOEs) to determine which atoms of MV2003 were close to which atoms of TAR RNA. Unfortunately no intermolecular NOEs were observed between any of the six fragments and the RNA, but it was possible to observe interligand NOEs (ILOEs) between the fragments and MV2003, and this enabled additional modeling suggesting that the fragments bind in a small pocket that only forms when MV2003 binds to RNA.

The paper ends with a cliff-hanger:

The formation of a new binding pocket allows binding of other fragments and suggests that more powerful ligands can be generated by linking the fragments together.

Although fragment linking is easier said than done, the hydrophobic moiety in MV2003 may improve the odds here, as described in the previous post. Practical Fragments hopes they will give it a shot!