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First-generation RET inhibitors were multi-tyrosine kinase inhibitors (TKIs) derived from secondary pharmacology targeting the adenine-binding pocket that resulted in poor clinical outputs. Recently developed second-generation RET inhibitors (primary pharmacology derived) exploit in addition further vulnerabilities within the active site e.g. the post-lysine pocket (PKP). We define the structural and dynamical determinants conferring high selectivity to these inhibitors agaisnt RET by targeting the PKP, making them clinically successful. [Display omitted] • The druggability landscape of the RET active site is determined by the structural dynamics of the P-loop and the αC helix. • RET selectivity is achieved by the occupancy of a small cryptic pocket adjacent to catalytic lysine defined by K758, L760, E768 and L772: the post-lysine pocket. • Efficient occupancy of the post-lysine pocket is restricted to a synchronous P-loop open and αC-in configuration, a distinctive feature of RET crystal structures. • LOXO-292 and BLU-667 target the post-lysine pocket and exploit extensive ATP mimicry. • Engineered mutants targeting the post-lysine pocket impact on inhibitor binding and sensitivity, as well as RET tyrosine kinase activity. The structural and dynamic determinants that confer highly selective RET kinase inhibition are poorly understood. To explore the druggability landscape of the RET active site in order to uncover structural and dynamic vulnerabilities that can be therapeutically exploited. We apply an integrated structural, computational and biochemical approach in order to explore the druggability landscape of the RET active site. We demonstrate that the that the druggability landscape of the RET active site is determined by the conformational setting of the ATP-binding (P-) loop and its coordination with the αC helix. Open and intermediate P-loop structures display additional druggable vulnerabilities within the active site that were not exploited by first generation RET inhibitors. We identify a cryptic pocket adjacent to the catalytic lysine formed by K758, L760, E768 and L772, that we name the post-lysine pocket, with higher druggability potential than the adenine-binding site and with important implications in the regulation of the phospho-tyrosine kinase activity. Crystal structure and simulation data show that the binding mode of highly-selective RET kinase inhibitors LOXO-292 and BLU-667 is controlled by a synchronous open P-loop and αC-in configuration that allows accessibility to the post-lysine pocket. Molecular dynamics simulations show that these inhibitors efficiently occupy the post-lysine pocket with high stability through the simulation time-scale (300 ns), with both inhibitors forming hydrophobic contacts further stabilized by pi-cation interactions with the catalytic K758. Engineered mutants targeting the post-lysine pocket impact on inhibitor binding and sensitivity, as well as RET tyrosine kinase activity. The identification of the post-lysine pocket as a new druggable vulnerability in the RET kinase and its exploitation by second generation RET inhibitors have important implications for future drug design and the development of personalized therapies for patients with RET-driven cancers. [ABSTRACT FROM AUTHOR]

Ribosome synthesis is a complex process that involves a large set of protein trans-acting factors, among them DEx(D/H)-box helicases. These are enzymes that carry out remodelling activities onto RNAs by hydrolysing ATP. The nucleolar DEGD-box protein Dbp7 is required for the biogenesis of large 60S ribosomal subunits. Recently, we have shown that Dbp7 is an RNA helicase that regulates the dynamic base-pairing between the snR190 small nucleolar RNA and the precursors of the ribosomal RNA within early pre-60S ribosomal particles. As the rest of DEx(D/H)-box proteins, Dbp7 has a modular organization formed by a helicase core region, which contains conserved motifs, and variable, non-conserved N- and C-terminal extensions. The role of these extensions remains unknown. Herein, we show that the N-terminal domain of Dbp7 is necessary for efficient nuclear import of the protein. Indeed, a basic bipartite nuclear localization signal (NLS) could be identified in its N-terminal domain. Removal of this putative NLS impairs, but does not abolish, Dbp7 nuclear import. Both N- and C-terminal domains are required for normal growth and 60S ribosomal subunit synthesis. Furthermore, we have studied the role of these domains in the association of Dbp7 with pre-ribosomal particles. Altogether, our results show that the N- and C-terminal domains of Dbp7 are important for the optimal function of this protein during ribosome biogenesis. [ABSTRACT FROM AUTHOR]

The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia , we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia . Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size. [ABSTRACT FROM AUTHOR]

Nonsense-mediated decay recognises mRNAs containing premature termination codons. One of its components, UPF3, is a molecular link bridging through its binding to the exon junction complex nonsense-mediated decay and splicing. In protists UPF3 has not been identified yet. We report that Paramecium tetraurelia bears an UPF3 gene and that it has a role in nonsense-mediated decay. Interestingly, the identified UPF3 has not conserved the essential amino acids required to bind the exon junction complex. Though, our data indicates that this ciliate bears genes coding for core proteins of the exon junction complex. [ABSTRACT FROM AUTHOR]