Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors
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eLife assessment
The authors present interesting information regarding the possibility of targeting the oncogenic K-Ras(G13C) mutant with nucleotide competitors. The experiments represent a solid support of the claims and show that this approach can work despite concerns about the high affinity of GTP and its high cellular concentration. These results will be of high interest for all working in the Ras field and in targeting oncogenes with small molecules. A weakness of the manuscript is the lack of direct physiological insights.
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Abstract
Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.
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eLife assessment
The authors present interesting information regarding the possibility of targeting the oncogenic K-Ras(G13C) mutant with nucleotide competitors. The experiments represent a solid support of the claims and show that this approach can work despite concerns about the high affinity of GTP and its high cellular concentration. These results will be of high interest for all working in the Ras field and in targeting oncogenes with small molecules. A weakness of the manuscript is the lack of direct physiological insights.
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Reviewer #1 (Public Review):
Ras is the first discovered oncogene and KRAS is the most frequently mutated isoform. Recent studies led to the development of mutation specific inhibitors, especially against the KRASG12C mutant. However, unfortunately the patients treated with Adagrasib or others develop resistance due to further gain of function mutations and amplification of KRASG12C allele apart from mutations in the downstream signaling components. One of the oldest approaches to target Rho GTPases like RAS is to compete with the nucleotide binding of RAS and it has for a long time remained difficult owing to the picomolar affinity for GTD/GDP. Gray and colleagues in 2014 tried to overcome these issues by employing GDP derivatives that can undergo covalent reaction with disease specific mutations but Muller etal reported in their …
Reviewer #1 (Public Review):
Ras is the first discovered oncogene and KRAS is the most frequently mutated isoform. Recent studies led to the development of mutation specific inhibitors, especially against the KRASG12C mutant. However, unfortunately the patients treated with Adagrasib or others develop resistance due to further gain of function mutations and amplification of KRASG12C allele apart from mutations in the downstream signaling components. One of the oldest approaches to target Rho GTPases like RAS is to compete with the nucleotide binding of RAS and it has for a long time remained difficult owing to the picomolar affinity for GTD/GDP. Gray and colleagues in 2014 tried to overcome these issues by employing GDP derivatives that can undergo covalent reaction with disease specific mutations but Muller etal reported in their previous work (Sci.reports 2017) that the issue with these derivatives was with the loss of reversible affinities for beta modified derivatives for RAS of atleast 10000 fold compared to GDP and GTP. Here the authors present novel GDP derivatives different from Gray and colleagues and demonstrate that they could lock KRASG13C covalently, another important mutant of KRAS in an inactive form with a multiple set of biochemical, structural and cellular assays.
However, the issue is a lack of evidence to demonstrate "target engagement" in cells and these derivatives need to be developed further as they cannot pass through cell membranes. The complete covalent modification of the compound is achieved at very high pH. Also its not clear if addition of edaGDP would disrupt KRASG13C and effector interaction directly.
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Reviewer #2 (Public Review):
The authors have demonstrated a covalent strategy to target the oncogenic K-Ras(G13C) mutation, which is found in about 3,000 cancer patients in the US each year. G13C is a major contributor to G13 mutations, the next hotspot mutation after codon 12. Moreover, there is no approved therapy for G13 mutations and no published inhibitors of any KRAS G13 mutant proteins, making this a particularly important contribution to the rapidly expanding repertoire of RAS inhibitors. A striking difference in comparison to G12 mutations, mutations occurring at Codon 13 exhibit impaired pM-nucleotide binding affinity of K-Ras. This weaker nucleotide affinity offered the authors the opportunity to develop a nucleotide based inhibitor of a RAS protein. With the high nucleophilicity of cysteine mutation, G13C the authors set …
Reviewer #2 (Public Review):
The authors have demonstrated a covalent strategy to target the oncogenic K-Ras(G13C) mutation, which is found in about 3,000 cancer patients in the US each year. G13C is a major contributor to G13 mutations, the next hotspot mutation after codon 12. Moreover, there is no approved therapy for G13 mutations and no published inhibitors of any KRAS G13 mutant proteins, making this a particularly important contribution to the rapidly expanding repertoire of RAS inhibitors. A striking difference in comparison to G12 mutations, mutations occurring at Codon 13 exhibit impaired pM-nucleotide binding affinity of K-Ras. This weaker nucleotide affinity offered the authors the opportunity to develop a nucleotide based inhibitor of a RAS protein. With the high nucleophilicity of cysteine mutation, G13C the authors set out to target this mutant oncogene.
The authors developed several covalent molecules derived from GDP/GTP, the natural substrate of K-Ras's nucleotide binding pocket, interestingly, not through the oligophosphate chain (explored by Gray and co-workers in an earlier report) but the 2,3-diol of the ribose. This turned out to be a judicious choice for targeting G13C because of the closer proximity to the 2',3' rather than the phosphates. Previous work by Gray et. al. used the phosphate attachment point for the electrophile but this compromised binding affinity overall-whereas the relatively tolerant modifications at 2',3' led to higher affinity electrophilic ligands. This change led to much tighter binders and effective covalent modifiers through C13. With two co-crystal structures resolved, the authors unambiguously showed the covalent cross-linking between artificial G-nucleotides and K-Ras(G13C).
It is not surprising that one of the major limitations of these GDP-based competitive ligands suffer from permeability issues. GDP or GTP analogs made in this study were not permeable through plasma membrane. The authors nicely worked around these limitations by delivering the fully modified proteins to the cells and measured cell signaling effects. Through electroporation the authors demonstrated the covalent adduct to be able to inhibit downstream signaling by compare introduction of K-Ras WT or K-Ras(G13C) or K-Ras(G13C) covalent adduct.
A number of very intriguing aspects of the covalent adduct were noted which should guide others in the field, including that the adduct with eda-GTP could get hydrolysed to eda-GDP after the covalent modification of the protein--furthermore GAP stimulation of this adduct still occurred. By use of a non-hydrolyzable form of GTP (CP) this could be prevented and could be a very useful method for preventing hydrolysis after introduction in cells--an application Goody and coworkers applied to a previous covalent base adduct.
Overall, the manuscript addresses an important problem relating to whether covalent small molecules can engage K-Ras(G13C) and provided two timely co-crystal structures for future research and development.
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Reviewer #3 (Public Review):
Ras mutations are found in almost 25 percent of cancer patients. It has been difficult to directly target Ras proteins due to the lack of druggable pockets on the surface of the protein and the extremely high binding affinity of nucleotides to Ras proteins. Recently a mutant specific irreversible drug that targets the mutation G12C has been FDA approved. This drug binds to a shallow pocket on the surface of Ras and attacks the G12C mutation irreversibly. Another approach is to compete with the nucleotides bound to Ras. An attempt to generate nucleotide competitors that can take advantage of the G12C mutant has been proposed. Nevertheless, these published competitors had much lower affinities compared to endogenous nucleotides which would hinder the covalent modification in the presence of other nucleotides.
T…
Reviewer #3 (Public Review):
Ras mutations are found in almost 25 percent of cancer patients. It has been difficult to directly target Ras proteins due to the lack of druggable pockets on the surface of the protein and the extremely high binding affinity of nucleotides to Ras proteins. Recently a mutant specific irreversible drug that targets the mutation G12C has been FDA approved. This drug binds to a shallow pocket on the surface of Ras and attacks the G12C mutation irreversibly. Another approach is to compete with the nucleotides bound to Ras. An attempt to generate nucleotide competitors that can take advantage of the G12C mutant has been proposed. Nevertheless, these published competitors had much lower affinities compared to endogenous nucleotides which would hinder the covalent modification in the presence of other nucleotides.
To overcome this, the authors propose to introduce a warhead in the ribose ring. Indeed, this modification did not affect the reversible binding affinity of these nucleotides to Ras wild type, in comparison to GDP and GTP. This finding represents a new opportunity to target G13C ras by competing with the nucleotides in cells. The authors support their claims with the appropriate in vitro experiments. Nevertheless, these experiments were performed at non physiologically high pH (9.5) and those compounds were not able to cross the cellular membrane. Thus, it is too early to draw conclusions regarding the appropriateness of the approach and whether it will prove successful in cells or if it will have medical application.
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