GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf

Zhang, Ziyang; Gao, Rong; Hu, Qi; Peacock, Hayden; Peacock, D. Matthew; Dai, Shizhong; Shokat, Kevan M.; Suga, Hiroaki

doi:10.1021/acscentsci.0c00514

Cited by 79 publications

(78 citation statements)

References 47 publications

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“…X-ray crystallography revealed the binding site to be near Switch II, allosterically blocking the interaction of KRAS with the guanine nucleotide exchange factor, SOS1 [14]. Subsequently we [15] and others [12] verified this peptide as having high-affinity and stoichiometric binding to KRAS. Although KRpep-2d represents a promising and novel KRAS binder, we concluded that structural modifications were required to render it cell-active.…”

Section: Introductionmentioning

confidence: 66%

“…Several research groups have indeed reported high affinity bona fide peptide binders to KRAS that represent valuable starting points for drug discovery [9][10][11][12]. In particular, Sakamoto et al used phage display to identify a disulfide cyclized peptide with the sequence Ac-Arg1-Arg2-Arg3-Arg4-Cys5-Pro6-Leu7-Tyr8-Ile9-Ser10-Tyr11-Asp12-Pro13-Val14-Cys15-Arg16-Arg17-Arg18-Arg19-NH2 [10].…”

Section: Introductionmentioning

confidence: 99%

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Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Lim¹,

Boyer

Boo³

et al. 2021

Preprint

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RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations and picomolar affinities for KRAS and hence is challenging to target. The recent FDA approval of sotorasib, a covalent modifier of the KRASG12C mutant protein, has clinically validated KRAS as an oncology target. However, traditional challenges remain for targeting the more common KRAS mutations such as G12D and G12V. As an alternative approach, we investigated the superior binding capacity of macrocyclic peptides to identify KRAS inhibitory molecules. We focused on the recently reported disulfide-cyclized arginine-rich peptide KRpep-2d, discovered through phage display and previously independently confirmed by us as a bona fide KRAS binder. To mitigate intracellular disulfide reduction and loss of binding, we identified an alternate cyclization motif by inverting the configuration of Cys5 and linking it to Cys15 through a thioacetal bridge. The corresponding peptide bound KRAS through cis isomerization of the peptide bond between D-Cys5 and Pro6 as observed through x-ray crystallography. Prototypic molecules displayed measurable cellular inhibition of RAS signaling without membrane lysis and counter-screen off-target activity. An analogue containing azido-lysine confirmed the cell penetrant nature of this molecule using our recently reported NanoClick assay. To improve cellular activity, we sought to improve proteolytic stability. Structure-activity relationship studies identified key scaffold residues critical for binding and revealed that replacement of N- and C- terminal arginine residues with D-arginines and introduction of an α- methyl moiety at Ser10 resulted in a molecule with improved proteolytic stability as indicated by its persistence in whole cell homogenate. The resulting peptide MP- 3995 had improved and sustained cell-based efficacy. On-target activity was validated through confirmation of target engagement, lack of signaling in irrelevant pathways, and use of non- binding control peptides. Mechanism of action studies suggested that peptide binding to both GDP- and GTP-states of KRAS may contribute to cellular activity. Although validated as bona fide and on-target inhibitors of cell based KRAS signaling, this series is unlikely to advance to the clinic in its current form due to its arginine-dependent cell entry mechanism. Indeed, we showed a strong correlation between net positive charge and histamine release in an ex vivo assay making this series challenging to study in vivo. Nonetheless, these molecules provide valuable templates for further medicinal chemistry efforts aimed at leveraging this unique inhibitory binding site on KRAS.

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Section: Introductionmentioning

confidence: 66%

Section: Introductionmentioning

confidence: 99%

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Lim¹,

Boyer

Boo³

et al. 2021

Preprint

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“…Cyclic peptides are increasingly leading to the discovery of novel binding surfaces to modulate protein function and elucidate protein dynamics, as recently demonstrated for lysine-specific demethylase one and a missense-mutated K-Ras(G12D)-Raf interaction ( 40 , 41 ). In the present study, ipglycermide orchestrates a novel bidomain clamp-like property assisted by metal-ion chelation to achieve potent binding affinity across iPGM species orthologs.…”

Section: Discussionmentioning

confidence: 99%

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Wiedmann

Dranchak

Aitha

et al. 2021

Journal of Biological Chemistry

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“… 42 , whose mechanism was to block RAS-effector interaction in vitro . Since then, many peptide inhibitors with the same mechanism have been discovered, such as compound 12 43 , KD2 44 , Cyclorasin 9A5 45 , etc. In addition, peptide inhibitors such as HBS3 discovered by Patgiri et al.…”

Section: Discussionmentioning

confidence: 99%

Apoptosis-inducing activity of synthetic hydrocarbon-stapled peptides in H358 cancer cells expressing KRASG12C

Zhao

et al. 2021

Acta Pharmaceutica Sinica B

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Lung cancers are the leading cause of cancer deaths worldwide and pose a grave threat to human life and health. Non-small cell lung cancer (NSCLC) is the most frequent malignancy occupying 80% of all lung cancer subtypes. Except for other mutations ( e.g ., KRAS G12V/D ) that are also vital for the occurrence, KRAS G12C gene mutation is a significant driving force of NSCLC, with a prevalence of approximately 14% of all NSCLC patients. However, there are only a few therapeutic drugs targeting KRAS G12C mutations currently. Here, we synthesized hydrocarbon-stapled peptide 3 that was much shorter and more stable with modest KRAS G12C binding affinity and the same anti-tumor effect based on the α -helical peptide mimic SAH-SOS1 A . The stapled peptide 3 effectively induced G2/M arrest and apoptosis, inhibiting cell growth in KRAS-mutated lung cancer cells via disrupting the KRAS-mediated RAF/MEK/ERK signaling, which was verified from the perspective of genomics and proteomics. Peptide 3 also exhibited strong anti-trypsin and anti-chymotrypsin abilities, as well as good plasma stability and human liver microsomal metabolic stability. Overall, peptide 3 retains the equivalent anti-tumor activity of SAH-SOS1 A but with improved stability and affinity, superior to SAH-SOS1 A . Our work offers a structural optimization approach of KRAS G12C peptide inhibitors for cancer therapy.

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GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf

Cited by 79 publications

References 47 publications

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Apoptosis-inducing activity of synthetic hydrocarbon-stapled peptides in H358 cancer cells expressing KRASG12C

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