Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones

Abstract

RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous ‘polyamide amino acids’ (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy–entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.

Efficient in silico exploration of RNA interhelical conformations using Euler angles and WExplore

“…As protein-bound RNA molecules can also be highly dynamic ( 30 , 71 ), an alternative approach would be to target instead an entire ensemble of RNA conformations that are fast interconverting, such as the entire set of structures in the ‘open’ partition. This would also result in a lower entropic binding cost, and is consistent with recent observations that so-called ‘loose binders’ of TAR are stronger Tat competitors than tight ones ( 29 ).…”

“…To account for conformational heterogeneity of the binding site in TAR, conventional computational docking protocols have been modified to dock to entire ensembles rather than using a single binding site structure ( 28 ). Furthermore, there is also evidence that the structural heterogeneity of RNA can extend into the bound state ( 29 , 30 ), resulting in so-called fuzzy complexes ( 31 ). This motivates an ensemble-based drug targeting strategy that explicitly models the thermodynamic effects of flexibility ( 32 ).…”

Insights into the development of chemical probes for RNA

“…RNA binding assays included: (i) titrations against fluorescently labeled RNA; (ii) biophysical techniques (SPR and NMR); (iii) microarray immobilization; (iv) in-line probing; (v) competition dialysis and (vi) indicator displacement. We note that a lack of correlation in small molecule activity between in vitro RNA binding and RNA:protein displacement assays has sometimes been reported, ( 16 , 51 , 52 ) highlighting the importance of multiple assays and/or choosing the most relevant assay for a particular system. We also note that, as in all screens, a lack of correlation can be observed between in vitro activity and cell culture activity.…”

Ribosome-targeting antibiotics as inhibitors of oncogenic microRNAs biogenesis: Old scaffolds for new perspectives in RNA targeting

“…In particular, the specificity of all antibiotics showing pre-miRNA binding affinity was measured in the presence of a large excess of tRNA or dsDNA, a methodology that has been largely validated in the literature. [37][38][39] In the first case, the target RNA is placed in the presence of a 100-fold excess of Escherichia coli tRNA (stem-loop structured RNA that account for as much as 15% of the total concentration of intracellular RNA) and the binding of the compound is measured. A specific ratio (K d in the presence of tRNA/K d in the absence of tRNA) can then be calculated.…”