GDP-bound human KRAS4b G12C mutant bound to its inhibitor molecule AMG 510

Amanda Harris '25 and Sofiia Shyroka '25

Contents:

I. Introduction

KRAS (Kirsten rat sarcoma viral oncogene homolog) is a small GTPase protein that plays a central role in regulating cell growth and survival. KRAS is one of the most frequently mutated oncogenes in pancreatic, colorectal, and lung cancers. Overall, mutations in KRAS are considered to cause up to 1/5 of all human malignancies.

Like other proteins from the RAS-GTPase family, KRAS is a binary molecular switch inactive in its form and active when GTP-bound. SOS protein, a guanine nucleotide exchange factor, facilitates the exchange of GDP for GTP, making KRAS active. Active KRAS interacts with other effector proteins, such as RAF, PI3K, and mTORC2, passing the proliferation signals downstream, activating the transcription of genes that ulitimately promote cell cycle progression and proliferation. Wild-type KRAS is capable of cleaving the GTP itself, returning to its inactive state. KRAS mutations at positions 12 and 13 impair the protein's intristic GTPase activity, locking KRAS its active form and, therefore, making it tumorigenic. KRAS protein with a relatively common (observed in ~17.7% of all colorectal and non-small cell lung cancers) G12C mutation is presented here, along with the AMG510 small-molecule drug that, upon binding, induces an allosterric change in KRAS and locks the mutant in its GDP-bound form, preventing further signal transduction. AMG510 is highly G12C mutant-specific due to the nature of its binding addressed later in this tutorial, and was the first FDA-approved treatment targeting specifically KRAS. KRAS

Figure 1: KRAS signaling pathway and its role in activation of cell survival and proliferation mechanisms (Zhu et al., 2021).

II. General Structure

KRAS protein has two isoforms: KRAS4a and KRAS4b, depending on the splicing of (Janakiraman et al., 2010). KRAS4b is considered the most abundant isoform in human cells and comprises of 188 amino acids. Its structure primarily consists of : a G domain and a hypervariable region (HVR) at the C-terminal domain. is a flexible, unstructured domain responsible for anchoring KRAS to the lipid bilayer and engaging in flexible conformational changes necessary for effector binding. Since HVR is hypervariable, however, it is difficult to define from a crystal structure of KRAS on its own and is, therefore, missing 19 residues in this tutorial. On the example of a structure of protein aquired via X-ray crystallography, we can see that KRAS HVR ends on a (CVIM) which allows for a post-translational modification called farnesylation (attachement of a 15-carbon farnesyl lipid group to a protein's C-terminus), facilitating KRAS attachment to the bilayer (Winzker et al., 2020). Farnesyl attaches to the in the CaaX sequence.

Scheme

Figure 2: Schematic representation of wild-type KRAS secondary structure (Pantsar et al., 2010).

The presented of KRAS G12C encompases N-terminal , five alpha , six beta , -loop, and flexible Switch- and Switch- regions. Switches I and II undergo conformational change upon GTP binding and act as binding interfaces for other proteins. In addition, a is a cricial player in KRAS GTPase function as it is neccesary for high-affinity nucleotide binding and GTP hydrolysis.

III. GTP binding site

GTP binds KRAS in a formed by the Switch I and Switch II and the phosphate-binding (P) loop. Here, a GDP-bound, conformation of Switches I and II is presented. In the this pocket adopts a flexible, "" confirmation, ready for interactions with downstream effectors and the GTPase-activator proteins (GAPs) that significantly accelerate the GTP hydrolysis.

In most known GTP-bound KRAS structures, Switch II is and, therefore, difficult to define, yet this region is critical for the catalytic activity. For example, Q61 is known to play a key role in positioning a water molecule for nucleophilic attack on the GTP gamma phosphate. . Upon hydrolysis, Switch II folds over the GTP, positioning Q61 in the active site (Fink et al., 2024; not shown).

Mutations that impair KRAS' intrinsic GTPase activity or alter its interactions with GAPs leave KRAS in a constitutively active form. Examples include mutations at (, , and ). Residue is located within the Switch I region and is crucial in determining the positioning of neighboring residues involved in KRAS binding and GTP hydrolysis. In mutant KRAS, the introduction of cysteine at this site is used as a therapeutic vulnerability for AMG510 drug to bind KRAS covalently. In the absence of the drug, interacts with GDP via : one with the beta phosphate group of GDP and one contact. The tumorigenecity of the G12C mutation is believed to be caused by the introduction of the polar thiol (-SH) group in the position of WT nonpolar glycine which significantly affects GTP binding, favouring the active form (not shown).

The hydroxyl group of also plays a crucial role in the facilitation of GTP hydrolysis (Bunda et al., 2014). When GDP is bound, points away from the nucleotide, interacting with Tyr40, forming hydrogen bonds between the two hydroxyl groups. When GTP is bound, on the other hand, undergoes a significant conformational change, allowing for appropriate positioning of Tyr32 along with other key catalytic residues, including Thr 35 () and the catalytic (Fink et al., 2024). Mutations at Tyr32 have been shown to significantly impair KRAS' intrinsic hydrolysis rate, highlighting its importance in the protein's GTPase function (Buhrman et al., 2010).

IV. G12C Mutation

Intriguingly, most KRAS-driven cancers are caused by missense mutations in very specific sites: G12, G13, and G61. Glycines in positions 12 and 13 are located in the and the P-loop of KRAS, a region responsible for GTP binding and proper hydrolysis. G61 is positioned in the Switch II part, which is essential for interacting with effector proteins. Since all three of these hotspots are exposed on the surface of KRAS, mutations in these sites lead to significant changes in protein-NTP and protein-protein interactions. This KRAS mutation causes a rotation in the histidine-95 side chains which leaves His95, Tyr96, and Gln99 in a compromised position that results in a that is hidden between the cysteine-12 side chain and the Switch II. Interactions with the pocket have negative impacts on oral bioavailability and overall decrease flux and clearance rates. The ARS-1620 scaffold can help inhibit the formation of the cryptic pocket, however, when tested on rodents, it had low bioavailability and very high clearance. Thus, ARS-1620 was soon ruled out as a KRAS inhibitor for in vivo systems. AMG510 was discovered because the new angle of the His95 side chain creates a less stable hydrogen bond with N1 of the GDP-bound unit. This lack of stability causes a significant loss in functional activity and strongly blocks the N1 position.


V. References

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