Targeting small GTPases: emerging grasps on previously untamable targets, pioneered by KRAS

Abstract

Small GTPases including Ras, Rho, Rab, Arf, and Ran are omnipresent molecular switches in regulating key cellular functions. Their dysregulation is a therapeutic target for tumors, neurodegeneration, cardiomyopathies, and infection. However, small GTPases have been historically recognized as "undruggable". Targeting KRAS, one of the most frequently mutated oncogenes, has only come into reality in the last decade due to the development of breakthrough strategies such as fragment-based screening, covalent ligands, macromolecule inhibitors, and PROTACs. Two KRAS^G12C covalent inhibitors have obtained accelerated approval for treating KRAS^G12C mutant lung cancer, and allele-specific hotspot mutations on G12D/S/R have been demonstrated as viable targets. New methods of targeting KRAS are quickly evolving, including transcription, immunogenic neoepitopes, and combinatory targeting with immunotherapy. Nevertheless, the vast majority of small GTPases and hotspot mutations remain elusive, and clinical resistance to G12C inhibitors poses new challenges. In this article, we summarize diversified biological functions, shared structural properties, and complex regulatory mechanisms of small GTPases and their relationships with human diseases. Furthermore, we review the status of drug discovery for targeting small GTPases and the most recent strategic progress focused on targeting KRAS. The discovery of new regulatory mechanisms and development of targeting approaches will together promote drug discovery for small GTPases.

Fig. 1

Overview of small GTPases biology. Phylogenetic tree (a), and five conserved boxes (G1–G5) (b, d), of Ras, Rho, Rab, Arf and Ran families. c Molecular switch diagram: GEF-mediated GDP-GTP exchange for activation, GAP-mediated GTP hydrolysis for deactivation, GDI stabilizes the GDP-bound form of Rho and Rab proteins, and the GTP-bound small GTPases interact with effector proteins. e Structural difference of Switch regions between GDP-state (PDB: 4LPK, light purple) and GTP-bound state (PDB: 6GOD, pink). Conformational ensembles of Switch II (f), and Switch I (g), regions of HRAS are generated from NMR structures (PDB: 1CRP and 2LCF)

Fig. 2

Cellular signaling coordinated by Ras and Rho subfamilies. a Ras-mediated signaling pathways include RAS-RAF-MEK-ERK, RAS-PI3K-AKT, RAS-RALGDS-RAL, RAS-PLCε, RAS-RASSF, the crosstalk between Ras, and other small GTPases including CDC42, RAC1 and RAP1, RHEB, and RAL are highlighted by red color. b Cytoskeletal dynamics are regulated by Rho, Rac and CDC42 through their main downstream signaling nodes such as ROCK, mDIA1, LIMK, PAK, WRC, and N-WASP. This figure was created with BioRender.com

Fig. 3

Cellular trafficking coordinated by Rab, Arf and Ran subfamilies. a Multiple vesicle trafficking steps are coordinated by Rab proteins: (1) ER to Golgi by RAB1, (2) Golgi to plasma membrane by RAB8, (3) Secretory vesicles and granules trafficking between Golgi and plasma membrane by RAB3, RAB26, RAB27 and RAB37, (4) Golgi to melanosome by RAB32 and RAB38 for biogenesis of melanosome, (5) Golgi to ER by RAB2, RAB6, RAB22, RAB33 and RAB40, (6) GLUT4 vesicle trafficking to plasma membrane by RAB8, RAB10 and RAB14, (7-9) Plasma membrane to early endosome (RAB5), then to late endosome (RAB7) and finally to lysosome (RAB7), (10-11) Early endosome to recycling endosome (RAB4), then to plasma membrane (RAB11), (12) Early phagosome to late phagosome by by RAB5, RAB14 and RAB22, (13) Late phagosome to lysosome by RAB7, (14-15) Autophagosome budding from plasma membrane by RAB24 and RAB33 and trafficking to lysosome by RAB7, (16) Lipid droplet biogenesis and homeostasis mediated by RAB18, (17) Mitochondrial fission mediated by RAB32, (18) Endocytosis by RAB5 and RAB21, (19) Ciliogenesis by RAB8, RAB17 and RAB23. b Vesicle trafficking and cytoskeletal dynamics are regulated by Arf proteins including ARF, ARL and SAR. c, Nucleocytoplasmic cargo transport through NPC is controlled by RAN. This figure was created with BioRender.com

Fig. 4

Small GTPases in human diseases. Distribution of mutations or aberrant expression of small GTPases across human diseases including cancers and neurologic diseases. This figure was created with BioRender.com

Fig. 5

Direct targeting of small GTPases by small molecules. a G12C-specific inhibitors bind to SII-P with the covalent warheads, other ligands specifically bind to SII-P of KRAS hotspot mutants (G12D/S/R). b Compounds bind to SI/II-P of WT KRAS and different mutants

Fig. 6

Direct targeting of small GTPases by macromolecules and emerging paradigm. a Cyclic peptides and staple peptides bind to KRAS and RALB. b Protein ligands interact with switch regions or allosteric lobe of KRAS. c Newly-developed targeting strategies and drug screening methods

Fig. 7

Strategies for indirectly targeting RAS and emerging approaches for targeting oncogenic RAS with immunotherapy. a Targeting key post-transcriptional modifications of RAS protein as well as upstream and downstream factors of RAS signaling have been proved as promising strategies for interfering with RAS activity and exhibiting anti-tumor effects in RAS mutant-driven tumors. Inhibitors targeting the key factors controlling RAS post-translational modifications and membrane trafficking (FTase, PDE6δ), upstream mediators (RTK receptors, SHP2, SOS1/2), and downstream effectors (RAF, MEK, ERK, CDK4/6, PI3K, AKT, mTOR1, AURKA, PLK) are listed. FDA-approved drugs are highlighted in blue, others are still under preclinical or clinical study. b Combination of KRAS^G12C inhibitors with immune checkpoint blockade and development of bispecific antibodies with T cell engager targeting neoepitopes derived from KRAS^G12C inhibitor-mediated covalent modification of KRAS^G12C. This figure was created with BioRender.com

Fig. 8

Intrinsic, adaptive, and acquired resistance to KRAS^G12C inhibitors. Resistance of KRAS^G12C inhibitors can be classified as (a) intrinsic, (b) adaptive, and (c) acquired resistance. a Lineage specific signaling between different tumor types, concurrent gene mutations with KRAS oncogenes, atypical GAP dependency/sensitivity have been reported to drive intrinsic resistance to KRAS^G12C inhibitors. b ERK signaling and PI3K/AKT signaling rebound driven by strong reactivation of different nodes such as RTK, SOS/SHP2, wild type RAS, or KRAS^G12C is the main cause of adaptive resistance to KRAS^G12C inhibitors. c Acquired genetic alterations in RTK, NRAS, HRAS, RAF, MEK, loss of function mutation of GAPs such as NF1, secondary mutations of KRAS^G12C including G13, Q61, R68, H95, Y96, and A146, as well as enhanced tumor plasticity including EMT and histologic transformation are associated with the acquired resistance to KRAS^G12C inhibitors. This figure was created with BioRender.com