Targeting the α4–α5 dimerization interface of K-RAS inhibits tumor formation in vivo

Khan, Imran; Spencer-Smith, Russell; O’Bryan, John P.

doi:10.1038/s41388-018-0636-y

Cited by 54 publications

(49 citation statements)

References 26 publications

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“…The α4 and α5 helices, which are distal from the switch regions, have been suggested to drive dimer formation as the vast majority of active Ras crystal structures dimerise via these helices 43 . Inhibition of this interface using a monobody, a small synthetic protein based on a fibronectin type III domain, inhibited oncogenic Ras signalling in cell culture and slowed tumour growth and progression in vivo in a nude mouse model bearing KRas mutant tumours 43,44 …”

Section: Ras Familymentioning

confidence: 99%

Therapeutic peptides targeting the Ras superfamily

2020

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The Ras superfamily of small GTPases are master regulators of numerous essential processes within the cell, so that when they malfunction, cancer and many other diseases can result. For example, activating Ras mutations are present in approximately 20% of human cancers. As such, they are key therapeutic targets, yet more than three decades of intensive research efforts have failed to produce effective Ras inhibitors in the clinic. This is, in part, due to their relatively smooth surfaces which are difficult to target through traditional drug discovery methods using small molecules. Peptides offer a solution to this issue as they occupy larger surface areas on their targets and therefore offer exquisite selectivity and affinity. However, their use in the past has been limited to extracellular targets due to delivery issues. Recent advances in peptide macrocyclisation, modifications and delivery methods have ignited increased interest in the use of these highly effective biologics for intracellular targets. This review will cover progress made in the development of peptides targeting small GTPases to treat a wide range of diseases.

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Section: Ras Familymentioning

confidence: 99%

Therapeutic peptides targeting the Ras superfamily

2020

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“…For example, fibronectin-derived monobodies inhibit signaling by disrupting the dimerization of H and KRAS at the plasma membrane (11). Interfering with Ras dimerization limits the growth of xenografts that inducibly express the monobody (12). An alternative strategy for inhibiting Ras signaling with engineered proteins is to prevent the recruitment of downstream effectors or block the activation of Ras by GEFs.…”

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confidence: 99%

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

Wiechmann

Maisonneuve

Grebbin

et al. 2020

Journal of Biological Chemistry

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The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

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“…A class of binders based on ankyrin repeat proteins ( Guillard et al., 2017 ) can bind and neutralize specific nucleotide loading states of RAS proteins ( Guillard et al., 2017 ). Similarly, a fibronectin type III domain-based RAS binding monobody ( Khan et al., 2019b ; Koide et al., 1998 ; Spencer-Smith et al., 2017 , 2019 ) was shown to bind and inhibit the dimerization of both K- and H-RAS, and the overexpression of this monobody was shown to suppress tumor growth in mice ( Khan et al., 2019b ). Besides inhibition, RAS degradation offers another alternative approach at inhibiting RAS function to target RAS-dependent cancer cells.…”

Section: Discussionmentioning

confidence: 99%

Targeting Endogenous K-RAS for Degradation through the Affinity-Directed Protein Missile System

Röth

Macartney

Konopacka

et al. 2020

Cell Chemical Biology

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Summary K-RAS is known as the most frequently mutated oncogene. However, the development of conventional K-RAS inhibitors has been extremely challenging, with a mutation-specific inhibitor reaching clinical trials only recently. Targeted proteolysis has emerged as a new modality in drug discovery to tackle undruggable targets. Our laboratory has developed a system for targeted proteolysis using peptidic high-affinity binders, called “AdPROM.” Here, we used CRISPR/Cas9 technology to knock in a GFP tag on the native K-RAS gene in A549 adenocarcinoma (A549 GFPKRAS ) cells and constructed AdPROMs containing high-affinity GFP or H/K-RAS binders. Expression of GFP-targeting AdPROM in A549 GFPKRAS led to robust proteasomal degradation of endogenous GFP-K-RAS, while expression of anti-HRAS-targeting AdPROM in different cell lines resulted in the degradation of both GFP-tagged and untagged K-RAS, and untagged H-RAS. Our findings imply that endogenous RAS proteins can be targeted for proteolysis, supporting the idea of an alternative therapeutic approach to these undruggable targets.

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Targeting the α4–α5 dimerization interface of K-RAS inhibits tumor formation in vivo

Cited by 54 publications

References 26 publications

Therapeutic peptides targeting the Ras superfamily

Therapeutic peptides targeting the Ras superfamily

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

Targeting Endogenous K-RAS for Degradation through the Affinity-Directed Protein Missile System

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