Selpercatinib (LOXO-292) and pralsetinib (BLU-667) are highly potent RET-selective protein tyrosine kinase inhibitors (TKIs) for treating advanced RET-altered thyroid cancers and non-small-cell lung cancer (NSCLC). It is critical to analyze RET mutants resistant to these drugs and unravel the molecular basis to improve patient outcomes. Cell-free DNAs (cfDNAs) were analyzed in a RET-mutant medullary thyroid cancer (MTC) patient and a CCDC6-RET fusion NSCLC patient who had dramatic response to selpercatinib and later developed resistance. Selpercatinib-resistant RET mutants were identified and cross-profiled with pralsetinib in cell cultures. Crystal structures of RET-selpercatinib and RET-pralsetinib complexes were determined based on high-resolution diffraction data collected with synchrotron radiation. RET^G810C/S mutations at the solvent front and RET^Y806C/N mutation at the hinge region were found in cfDNAs of an MTC patient with RET^{M918T/V804M/L}, who initially responded to selpercatinib and developed resistance. RET^G810C mutant was detected in cfDNAs of a CCDC6-RET-fusion NSCLC patient who developed acquired resistance to selpercatinib. Five RET kinase domain mutations at three non-gatekeeper residues were identified from 39 selpercatinib-resistant cell lines. All five selpercatinib-resistant RET mutants were cross-resistant to pralsetinib. X-ray crystal structures of the RET-selpercatinib and RET-pralsetinib complexes reveal that, unlike other TKIs, these two RET TKIs anchor one end in the front cleft and wrap around the gate wall to access the back cleft. RET mutations at the solvent front and the hinge are resistant to both drugs. Selpercatinib and pralsetinib use an unconventional mode to bind RET that avoids the interference from gatekeeper mutations but is vulnerable to non-gatekeeper mutations.

Holliday junctions are important structural intermediates in recombination, viral integration, and DNA repair. We present here the single-crystal structure of the inverted repeat sequence d(CCGGTACCGG) as a Holliday junction at the nominal resolution of 2.1 Å. Unlike the previous crystal structures, this DNA junction has B-DNA arms with all standard Watson–Crick base pairs; it therefore represents the intermediate proposed by Holliday as being involved in homologous recombination. The junction is in the stacked-X conformation, with two interconnected duplexes formed by coaxially stacked arms, and is crossed at an angle of 41.4° as a right-handed X. A sequence comparison with previous B-DNA and junction crystal structures shows that an ACC trinucleotide forms the core of a stable junction in this system. The 3′-C⋅G base pair of this ACC core forms direct and water-mediated hydrogen bonds to the phosphates at the crossover strands. Interactions within this core define the conformation of the Holliday junction, including the angle relating the stacked duplexes and how the base pairs are stacked in the stable form of the junction.

PyMOL is often used to generate images of biomolecular structures. Hundreds of parameters in PyMOL provide precise control over the appearance of structures. We developed 241 Python functions—called “shortcuts”—that extend and ease the use of PyMOL. A user runs a shortcut by entering its name at the PyMOL prompt. We clustered the shortcuts by functionality into 25 groups for faster look‐up. One set of shortcuts generates new styles of molecular representation. Another group saves files with time stamps in the file names; the unique filenames avoid overwriting files that have already been developed. A third group submits search terms in the user's web browser. The help function prints the function's documentation to the command history window. This documentation includes the PyMOL commands that the user can reuse by copying and pasting onto the command line or into a script file. The shortcuts should save the average PyMOL user many hours per year searching for code fragments in their computer or on‐line.Statement for Lay Public: Computer‐generated images of protein structures are vital to the interpretation of and communication about the molecular structure of proteins. PyMOL is a popular computer program for generating such images. We made a large collection of macros or shortcuts that save time by executing complex operations with a few keystrokes.

Myotonic dystrophy (DM) type 1 is associated with an expansion of (>50) CTG repeats within the 3′ untranslated region (UTR) of the dystrophin myotonin protein kinase gene ( dmpk ). In the corresponding mRNA transcript, the CUG repeats form an extended stem-loop structure. The double-stranded RNA of the stem sequesters RNA binding proteins away from their normal cellular targets resulting in aberrant transcription, alternative splicing patterns, or both, thereby leading to DM. To better understand the structural basis of DM type 1, we determined to 1.58-Å resolution the x-ray crystal structure of an 18-bp RNA containing six CUG repeats. The CUG repeats form antiparallel double-stranded helices that stack end-on-end in the crystal to form infinite, pseudocontinuous helices similar to the long CUG stem loops formed by the expanded CUG repeats in DM type 1. The CUG helix is very similar in structure to A-form RNA with the exception of the unique U-U mismatches. This structure provides a high-resolution view of a toxic, trinucleotide repeat RNA.

Rearranged during transfection (RET) is involved in the physiological development of some organ systems. Activating RET alterations via either gene fusions or point mutations are potent oncogenic drivers in non-small cell lung cancer, thyroid cancer, and in multiple diverse cancers. RET-altered cancers were initially treated with multikinase inhibitors (MKIs). The efficacy of MKIs was modest at the expense of notable toxicities from their off-target activity. Recently, highly potent and RET-specific inhibitors selpercatinib and pralsetinib were successfully translated to the clinic and FDA approved. We summarize the current state-of-the-art therapeutics with preclinical and clinical insights of these novel RET inhibitors, acquired resistance mechanisms, and future outlooks.