Martin A. Carrasco

UNLABELLED: KRAS mutations are the most common genetic abnormalities in cancer, but the distribution of specific mutations across cancers and the differential responses of patients with specific KRAS mutations in therapeutic clinical trials suggest that different KRAS mutations have unique biochemical behaviors. To further explain these high-level clinical differences and to explore potential therapeutic strategies for specific KRAS isoforms, we characterized the most common KRAS mutants biochemically for substrate binding kinetics, intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities, and interactions with the RAS effector, RAF kinase. Of note, KRAS G13D shows rapid nucleotide exchange kinetics compared with other mutants analyzed. This property can be explained by changes in the electrostatic charge distribution of the active site induced by the G13D mutation as shown by X-ray crystallography. High-resolution X-ray structures are also provided for the GDP-bound forms of KRAS G12V, G12R, and Q61L and reveal additional insight. Overall, the structural data and measurements, obtained herein, indicate that measurable biochemical properties provide clues for identifying KRAS-driven tumors that preferentially signal through RAF. IMPLICATIONS: Biochemical profiling and subclassification of KRAS-driven cancers will enable the rational selection of therapies targeting specific KRAS isoforms or specific RAS effectors.

We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling.

Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.

Supercooling ability is a critical component among the suite of adaptations contributing to subzero temperature-tolerance of insects, whether they follow freeze-tolerance or freeze-avoidance strategies. Supercooling points (SCP, nucleation temperature, or crystallization temperature) of insects and other terrestrial arthropods range tremendously, from −2 °C to −100 °C or lower. Supercooling is affected by a number of factors, including the volume and water content of the organism, and the ability of the body surface to prevent inoculative freezing by external ice. However, the topics of this review, ice nucleators and antifreeze proteins, are often of critical importance. Antifreezes can be both small-molecular-mass solutes, such as polyhydroxyl alcohols that depress the freezing point of water on a strictly colligative basis, and high-molecular-mass molecules such as antifreeze proteins that suppress freezing by a non-colligative mechanism. Freeze-tolerant species often exhibit high SCPs (above −10 °C) and have selected for extracellular ice nucleators, while freeze-avoiding insects generally have selected against ice nucleators and for antifreezes, allowing them to supercool below ambient temperatures to which they are exposed over the winter. This review will attempt to provide a broad update on ice nucleators, antifreeze proteins and related adaptations in insects and other arthropods, primarily from the standpoint of how they function in organisms to promote winter survival.