1. Biochemistry and Chemical Biology

Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors

  1. Lisa Goebel
  2. Tonia Kirschner
  3. Sandra Koska
  4. Amrita Rai
  5. Petra Janning
  6. Stefano Maffini
  7. Helge Vatheuer
  8. Paul Czodrowski
  9. Roger S Goody  Is a corresponding author
  10. Matthias P Müller  Is a corresponding author
  11. Daniel Rauh  Is a corresponding author
  1. Department of Chemistry and Chemical Biology, TU Dortmund University, Germany
  2. Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Germany
  3. Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Germany
  4. Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Germany
https://doi.org/10.7554/eLife.82184
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Cite this article
  1. Lisa Goebel
  2. Tonia Kirschner
  3. Sandra Koska
  4. Amrita Rai
  5. Petra Janning
  6. Stefano Maffini
  7. Helge Vatheuer
  8. Paul Czodrowski
  9. Roger S Goody
  10. Matthias P Müller
  11. Daniel Rauh
(2023)
Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors
eLife 12:e82184.
https://doi.org/10.7554/eLife.82184
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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.

Editor's evaluation

The authors present important 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.

https://doi.org/10.7554/eLife.82184.sa0

Introduction

Ras proteins act as key regulators of many cellular processes by switching between inactive GDP-bound and active GTP-bound states, the latter specifically activating several downstream signalling pathways (Cox et al., 2014). Oncogenic Ras mutations that lead to dysregulation of the switch mechanism are found in about 25% of all human cancers, including three of the most lethal forms (lung, colon, and pancreatic cancer). Among the Ras proteins, KRas is the predominantly mutated isoform (85%), followed by NRas (11%) and HRas (4%), with mutational hotspots at amino acid positions G12, G13, and Q61 (Cox et al., 2014; Hobbs et al., 2016). Although the glycine at position 12 is the most commonly mutated residue, G13 is the second most common mutation (14% of tumors harbor a mutation at this position) and in 6% of these cases, an acquired cysteine is found (Forbes et al., 2015; Visscher et al., 2016). In lung cancer, the prevalence of the G13C mutation is 3%, which is equivalent to approximately 7000 individuals in the US per year (Forbes et al., 2015; Burge and Hobbs, 2022). Because of their prominent role in cancer, Ras oncogenes were identified as attractive targets for cancer therapy since their initial discovery in 1981, but attempts to target Ras have been largely unsuccessful and Ras proteins were long considered undruggable. After decades of failure, new interest has recently arisen from selective targeting of the G12C oncogenic mutant of KRas (Ostrem et al., 2013; Patricelli et al., 2016; Janes et al., 2018; Shin, 2019; Canon et al., 2019; Hong, 2020; Fell et al., 2018; Hallin et al., 2020; Fell, 2020). Inhibitors that bind irreversibly to the G12C mutated cysteine residue within a previously unknown switch-II pocket were originally identified and designed in the Shokat laboratory, and have been further developed within the academic and industrial world to advance candidates into the clinic (clinicaltrials.gov, 2018a; clinicaltrials.gov, 2019a; clinicaltrials.gov, 2018b; clinicaltrials.gov, 2019b; Goebel et al., 2020). In May 2021, the first-in-class KRasG12C inhibitor Sotorasib (Amgen) was approved by the FDA for the treatment of non-small-cell lung cancer (NSCLC), confirming the therapeutic susceptibility of mutant KRas in cancer (Mullard, 2021). A second approach for targeting mutant KRas has been described using nucleotide competitive inhibitors that can covalently bind to KRasG12C (Lim et al., 2014; Xiong et al., 2017). Strategies involving direct competition with nucleotide binding were originally set aside because of the high affinity of GDP/GTP for Ras and high cellular GDP/GTP concentrations. However, the combination of nucleotide competition with covalent binding of the inhibitors to the Ras protein has fuelled new hope. Gray and colleagues developed SML-8-73-1, a GDP derivative harbouring an electrophilic group on the β-phosphate to irreversibly bind to the mutant cysteine at position 12 within the P-loop of Ras (Lim et al., 2014). Unfortunately, the modification of the β-phosphate leads to a dramatic loss of affinity because of the loss of important interactions with the protein and the Mg2+ ion (Müller et al., 2017). In this publication, we demonstrate that GDP/GTP/GppCp analogues with an electrophilic group attached to the ribose interact with the necessary high reversible affinity to KRas and are able to react covalently with KRasG13C.

Results and discussion

Selective covalent modification of KRasG13C by 2’,3’-modified nucleotide analogues

Available structural information about the binding of GDP and GTP toward Ras provides a detailed understanding of the underlying high affinity of nucleotides through multiple reversible interactions. Based on published crystal structures of Ras proteins (Figure 1) and a multiple sequence alignment of the Ras small GTPase superfamily (Figure 1—figure supplement 1, Supplementary file 1), we designed and synthesized guanine nucleotide-based inhibitors with an additional Michael acceptor as a covalent warhead for targeting oncogenic KRas variants harboring cysteines in the P-loop (KRasG12C and KRasG13C). Whereas the G12C mutation has been successfully addressed in the past, KRasG13C is a largely unexplored target in cancer therapy. However, based on pKa calculations, we were able to show that the G13C mutation should also be generally addressable by appropriately positioned Michael acceptors (Supplementary file 2). In contrast to Gray and colleagues, we chose the 2’,3’-OH groups of the ribose for attachment of the warhead since modifications at this position do not significantly alter nucleotide affinity (Figure 1A, B; Eberth et al., 2005). Nucleotide derivatives with different linkers (eda: ethylenediamine, pda: propylenediamine, bda: butylenediamine) were synthesised based on published procedures (Method section), including GDP/GTP/GppCp analogues (R=OH), resulting in the formation of mixed 2’ and 3’-isomers, as well as dGTP analogues (R=H) (Eberth et al., 2005; Cremo et al., 1990). In addition to acrylamide-bearing nucleotides that could potentially bind irreversibly to cysteine containing P-loop mutants via Michael addition, we prepared acetamide derivatives as non-reactive control analogues (Figure 1B). The cysteine light-version of KRas constructs lacking other cysteines (C51S, C80L, C118S) were used for initial MS experiments, which indicated that the acrylamide nucleotide derivatives can selectively react with KRasG13C1-169 (Cys-light), but not with KRasG12C1-169 (Cys-light) (Figure 1C, D, E). The eda linker led to the most efficient covalent protein modification (Figure 1C). This tendency is presumably because of a favourable orientation of the reactive warhead and/or a reduced flexibility. Using dGTP analogues, the rate of covalent protein modification was further increased in the case of the eda derivative, indicating that the linker in the 3’-position of the ribose is superior to the 2’-position with respect to targeting KRasG13C. Although, complete modification of KRasG13C was only observed at elevated pH, significant modification of the protein also occurred at a physiological pH within 24 hr. On incubating KRasG13C1-169 (Cys-light) with the GTP analogues, we observed at intermediate stages a mixture of covalently bound GDP and GTP forms of KRasG13C1-169 (Cys-light), but ultimately the reaction yielded only the diphosphate form, indicating that the nucleotides were still hydrolysed after the covalent reaction and were properly positioned in the active site of KRas (Figure 1—figure supplement 2). The time-resolved labelling of KRasG13C1-169 (Cys-light) with either GDP or GTP derivatives led to comparable covalent protein modification rates (Figure 1—figure supplement 3). Additionally, we tested whether the nucleotides were generally able to compete with their natural counterparts and monitored the reaction also in the presence of equimolar concentrations as well as 10 x and 100 x excess of GDP/GTP (Figure 1—figure supplement 4). This shows that although the reaction is slowed down due to the competing nature of GDP and GTP, the modified nucleotides are able to compete. Upon incubating KRasG13C1-169 (Cys-light) with a non-cleavable GppCp derivative, we also observed covalent modification, but without subsequent hydrolysis of the nucleotide (Figure 1—figure supplement 5). To further investigate the specificity of the reaction towards KRasG13C1-169 (Cys-light), we also tested the wild type protein. For KRasWT1-169, very little unspecific labelling was observed at pH 9.5 compared to the G13C mutant (approximately 3% at pH 9, 8% at pH 9.5, whereas under equivalent conditions, KRasG13C modification was ≥90%; Figure 1E, Figure 1—figure supplement 6), thus showing that the warhead reacts preferentially with the cysteine at position 13, but not other cysteines in KRas nor the additional neighboring cysteine in KRasG12C. In addition, the multiple sequence alignment of the Ras small GTPase superfamily revealed that only about 7% of the GTPase members contain cysteines within the P-loop that might potentially be accessible by our linker design and only 3 contain Cys at the position equivalent to residue 13 in Ras (Arl4a, RheBL1, Rab21). Upon incubating Rab21 with edaGDP, we indeed observed similar modification compared to KRasG13C (Figure 1—figure supplement 7). However, since cross-reactivities of covalently binding molecules is well-known and documented also for example in kinase inhibitors such as osimertinib, which modifies a number of off-target kinases (Finlay et al., 2014), we are confident that this will not generally preclude the usage of the nucleotides described. In fact, Rab21 might be an interesting target itself since knock-down of Rab21 has been reported to have beneficial effects in human glioma cells (Ge et al., 2017).

Figure 1 with 7 supplements see all
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Rational design of nucleotide-based covalent KRasG13C inhibitors.

(A) Structure of KRasWT in the GDP-bound state (grey, P-loop: violet, switch I: red, switch II: blue, NKxD: green, SAK: yellow; PDB 4obe). (B) Model of KRasG13C (model is based on PDB 4obe) showing the distances between the cysteine residue and the OH-groups of the ribose and the general structure of nucleotide derivatives bearing an electrophilic group at the 2’,3’-position of the ribose moiety, showing that position 12 is further remote compared to position 13 and thus explaining the observed specificity of covalent bond formation. (C) Time-dependent analysis of the covalent modification of KRasG13C1-169 (Cys-light) at pH 9.5 using different linker lengths (eda: ethylenediamine, pda: propylenediamine and bda: butylenediamine). (D) Covalent modification of KRasG13C1-169 (Cys-light) proteins at pH 9.5 after 24 hr at room temperature. (E) In contrast, KRasG12C1-169 (Cys-light) mutant was not modified by nucleotide derivatives and for KRasWT1-169 very little unspecific labelling at pH 9.5 was observed compared to the G13C mutant.

In summary, since other cysteines that are located within the P-loop or other regions close to the nucleotide are mostly further remote compared to Cys13 and even the directly neighboring residue at position 12 does not become modified as shown above, suitable design and optimization of the linker will likely allow sufficient specificity towards KRasG13C also in vivo (Figure 1—figure supplement 1, Supplementary file 1).

Reversible affinities of the nucleotide analogues are comparable to those of the unmodified nucleotides

To evaluate the impact of the attached linker on nucleotide binding, we first determined the affinity and kinetics of the interaction of the nucleotide derivatives with Ras compared to unmodified GDP/GTP that have dissociation constants (KD) in the picomolar range (Table 1). For this purpose, we measured the kinetics of the nucleotide association (kon) in a stopped-flow instrument using competition experiments between mantdGDP (2 μM) and increasing amounts of competing nucleotides (1, 2, and 6 μM; Müller et al., 2017). As shown in Table 1—source data 1, the competitive binding of the competing nucleotide and mantdGDP led to a significant decrease in the fluorescence signal because of smaller amounts of mantdGDP binding to KRas. By fitting the data to a previously described model (Müller et al., 2017), we obtained the corresponding kon values, and those for the nucleotide analogues were comparable to those of the unmodified nucleotide (Table 1—source data 2). To further analyse the ability of the modified nucleotides to compete with GDP/GTP, KRasWT:GDP was mixed with equal amounts of the acetamide derivatives and incubated either for 7 days at room temperature in the absence of EDTA, for 24 hr at 4 °C in the presence of EDTA, or for 1 hr at room temperature in the presence of SOS (guanine nucleotide exchange factor) to increase the rate of nucleotide exchange and to allow the reaction to equilibrate. After this equilibration time and buffer exchange, the Ras proteins were concentrated and the nucleotide state was analysed by isocratic HPLC runs. By integrating the corresponding peaks for GDP and the guanosine nucleotide analogues after distinct time points, we observed that the modified nucleotides can indeed compete with GDP (Table 1—source data 3, Table 1—source data 4). Based on the relative abundance of bound nucleotides determined by the HPLC assay, the dissociation constants for the pda and bda derivatives were calculated to be 8.6±1.3 pM and 9.6±0.5 pM, respectively (overlap with the GDP elution peak prevented accurate determination in the case of the eda-derivative; Method section, Table 1). Thus, the attached linker has very little impact on the reversible interaction and the affinity, an important fact that must be considered for the approach of using nucleotide-competitive inhibitors (Müller et al., 2017). In contrast, SML-8-73-1, a GDP derivative harbouring an electrophilic group on the β-phosphate showed a dramatic loss of reversible affinity (KD = ~140 nM) (Jeganathan et al., 2018).

Table 1
Overview of the calculated kinetic parameters (KD, kon and koff).
KD[pM]kon[µM–1s–1]koff[s–1]
GDP2.54.221.1x10–5
pdaGDP8.6±1.33.342.9x10–5
bdaGDP9.6±0.53.123.0x10–5
SML-8-73-1~140 nM--
Table 1—source data 1

Kinetics of the nucleotide association (kon).

(A) Competitive binding experiments of GDP and mantdGDP; 1 μM KRas and 2 μM mantdGDP was used in the absence (black curve) or in the presence of 1 µM (red), 2 µM (blue) or 6 µM (green) competing nucleotides in a stopped-flow instrument. (B) The binding curves were globally fit to the indicated model using KinTek Explorer to obtain the corresponding association rates (kon). (C, D, E) Competitive binding experiments between mantdGDP and GDP, GTP, or dGTP analogues. Calculated kon values are shown in Table 1—source data 2.

https://cdn.elifesciences.org/articles/82184/elife-82184-table1-data1-v1.zip
Table 1—source data 2

kon calculation.

Overview of kon rate constants obtained from competitive binding experiments with mantdGDP (Table 1—source data 1).

https://cdn.elifesciences.org/articles/82184/elife-82184-table1-data2-v1.zip
Table 1—source data 3

HPLC-based approach for determination of affinities relative to GDP.

(A, B, C) Competitive binding experiments of GDP and the acetyl-derivatives of GDP; 50 μM KRasWT:GDP was mixed with 50 µM of edaGDP, pdaGDP or bdaGDP and incubated for 7 days at room temperature in the absence of EDTA, for 24 hr at 4 °C in the presence of 10 mM EDTA or for 1 hr in the presence of SOS (0.5 µM). After buffer exchange for removal of any unbound nucleotides, the resulting mixtures were analyzed by isocratic HPLC runs. (D) Relative amounts of the nucleotides and GDP bound to KRas were compared. It should be noted that the relative affinities mentioned in the method section and shown in Table 1—source data 4 are calculated from these experiments and represent an average affinity of the 2’ isomers, and that of the 3’-isomer seen to be bound in the X-ray structure is presumably actually higher than this, since it can be seen from Figure 2—figure supplement 1 that there is a preference for one of the isomers, presumably the 3’-isomer, so that this species and the derivatives of dGDP/dGTP must have a very similar affinity to that of GDP/GTP.

https://cdn.elifesciences.org/articles/82184/elife-82184-table1-data3-v1.zip
Table 1—source data 4

KD calculations.

Overview of the calculated KD values of pdaGDP and edaGDP obtained from an HPLC-based approach (Table 1—source data 3); For reference, the KD values of GDP and the nucleotide analogue SML-8-73-1 are also listed.

https://cdn.elifesciences.org/articles/82184/elife-82184-table1-data4-v1.zip

First crystal structures of the KRasG13C mutant

To gain further insight into the binding mode of the covalently bound nucleotides, we solved the first X-ray crystal structure of the oncogenic KRasG13C mutant, in this case with covalently bound edaGDP (PDB 7ok3) and bdaGDP (PDB 7ok4) (Figure 2, Figure 2—figure supplement 1). The overall structures are very similar to the known structure of KRas:GDP (PDB 4obe), and the nucleotide scaffold, as well as the covalent linkage for both nucleotide analogues with cysteine at position 13 are well resolved in the electron density (Figure 2C, E, Figure 2—figure supplement 1). Interestingly, only the 3’-isomer of the nucleotide analogues was observed in the structures, consistent with results of the MS experiments comparing the efficiency of labelling of the dGTP derivative and the mixed isomers of the GTP derivative and showing a faster reaction for dGTP (Figure 1C). Both structures showed that the nucleotides are bound within the active site in a manner that is comparable to non-covalently bound GDP in other structures of Ras, with similar reversible interactions between the protein and the nucleotide and the additional well-resolved covalent link to Cys13. However, both structures lacked the Mg2+ ions in the active site despite a Mg2+ concentration of 2 mM in the Ras solution. The missing Mg2+-ions are probably a result of the crystallization buffer containing NH4F or NaF, leading to precipitation of poorly soluble MgF2, and this has also been observed in other PDB-deposited structures presumably because of similar effects of the reservoir solutions used in the crystallization process (e.g. PDB 4m1o, 4lyf, 4lyh, 4m21, 4m1s, 4m1t, 4m1t, 4m1y) (Ostrem et al., 2013). In both crystal structures, the eda and bda linker are remote from the Mg2+ binding site and do not directly interfere with Mg2+ binding, suggesting that Mg2+ can generally bind. Thus, the structural analysis verified the nucleotide binding pose and the high reversible affinity comparable to the natural nucleotides and will guide design and optimisation of the linker and the warhead in further studies.

Figure 2 with 1 supplement see all
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Crystal structure of KRasG13C covalently locked with either edaGDP or bdaGDP.

(A) Comparison of KRasG13C covalently locked with edaGDP (grey, P-loop: violet, switch I: red, switch II: blue, NKxD: green, SAK: yellow, PDB 7ok3) and KRas:GDP (white, PDB 4obe). (B) Comparison of KRasG13C covalently locked with bdaGDP (grey, P-loop: violet, switch I: red, switch II: blue, NKxD: green, SAK: yellow, PDB 7ok4) and KRas:GDP (white, PDB 4obe). (C) Enlarged view of the nucleotide binding pocket in KRasG13C-edaGDP showing the 2Fo-Fc electron density map (countered at 1.0 σ). (D) Structural superposition of KRas:GDP and the covalently locked G13C mutants. (E) Enlarged view of the nucleotide binding pocket in KRasG13C-bdaGDP showing the 2Fo-Fc electron density map (countered at 1.0 σ).

Figure 2—source data 1

Data collection and refinement statistics for KRasG13C-edaGDP and KRasG13C-bdaGDP.

https://cdn.elifesciences.org/articles/82184/elife-82184-fig2-data1-v1.docx

Inhibition of oncogenic KRasG13C signalling by covalent nucleotide analogues

Finally, we set out to test whether the GDP nucleotide derivatives we are focusing on for cancer therapy were indeed able to inhibit oncogenic signalling by KRasG13C. In a first experiment, we tested and compared the SOS-catalysed nucleotide exchange on Ras. Whereas SOS efficiently catalysed nucleotide exchange on KRasWT, KRasG13C and as a control on KRasG13C:acetyledaGDP, it was unable to do so with the covalently locked G13C-edaGDP mutant, showing that the protein is indeed locked in the inactive state (Figure 3A, Figure 3—figure supplement 1). Interestingly, in these experiments we also observed a drastically increased intrinsic nucleotide exchange rate for KRasG13C compared to KRasWT in the absence of SOS (Figure 3A, blue area), an effect that probably contributes to the increased signalling and oncogenic effect of this mutant, and also this effect is abrogated in the case of covalently locked KRasG13C-edaGDP (Hunter et al., 2015). Similarly, no SOS-catalysed nucleotide exchange was observed in case of covalently modified KRasG13C-edaGppCp, indicating that the protein can also be trapped in the active conformation with the corresponding nucleoside-triphosphates (Figure 3—figure supplement 1B). Thus, the modified GppCp derivatives could also be used as artificial and irreversible activators and generally as tool compounds to further investigate the biological role of KRasG13C in cells. In addition to SOS-catalysed nucleotide exchange, GAP-stimulated GTP hydrolysis was also analysed. While the intrinsic GTP hydrolysis of KRasG13C-edaGTP (t1/2 = 187 min) is comparable to that of KRasWT:GTP (t1/2 = 126 min) and even faster than for the KRasG12C mutant (t1/2 = 300 min) (Li et al., 2021; Figure 3—figure supplement 2), a drastically decreased GAP-stimulated GTP hydrolysis was observed for the G13C mutant as expected (Scheffzek et al., 1997).

Figure 3 with 9 supplements see all
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Cellular evaluation of nucleotide-based covalent inhibitors.

(A) GEF-catalysed nucleotide exchange of KRasWT:GDP, KRasG13C:GDP and KRasG13C-edaGDP (step 1) which were mixed with an excess of mantdGDP (step 2) and subsequently with 0.25 µM (red curve) or 0.5 µM (black curve) of SOS (step 3). The intrinsic nucleotide exchange is depicted in the blue box whereas the SOS-catalysed nucleotide exchange is shown in the grey box. (B) Western blot analysis after electroporation of indicated amounts of full-length KRasWT, KRasG13C, and KRasG13C-edaGDP into HeLa cells. (C) Quantification of protein levels from western blots was performed using Empiria Studio (Li-Cor). The mean fold change was plotted against increasing amounts of protein. Error bars indicate the standard deviation for each measurement (n=3). One-way ANOVA was performed using GraphPad Prism. The uncropped western blots are available as source data (Goebel et al_Source_data_file_WB.xlsx).

Figure 3—source data 1

Original uncropped Western Blots for data in Figure 3.

https://cdn.elifesciences.org/articles/82184/elife-82184-fig3-data1-v1.zip

In addition to the effects of GEFs and GAPs, we also investigated whether the covalent modification with edaGDP was sufficient to preclude effector binding. For this, we performed pull-down experiments with GST-tagged RafRBD: Whereas KRasG13C:GDP did not bind to the RafRBD, it was activated in the presence of GppNHp alone or in combination with SOS and was pulled down by RafRBD. This finding again highlights the self-activating nature of this Ras mutant, even in the absence of a stimulus (SOS). In contrast, covalently locked KRasG13C-edaGDP was neither in the presence of GppNHp nor of additional SOS able to bind to the RafRBD (Figure 3—figure supplement 3).

Finally, after the detailed in vitro characterization of the nucleotide analogues, the next experiment was to investigate whether oncogenic signalling could also be inhibited in vivo. Since the nucleotides are unable to cross the cell membrane without loss of the phosphate groups and full labelling of the G13C mutant was only achieved at relatively high pH values within 24 hr, we used electroporation to deliver recombinant full-length KRasWT, KRasG13C and KRasG13C-edaGDP into human cells. Importantly, we confirmed that the covalently locked protein can still be fully farnesylated in vitro (Figure 3—figure supplement 4) and the covalent modification of full-length KRasG13C was further validated through MS/MS‐analysis, which revealed selective labelling of the targeted cysteine residue at position 13 (Figure 3—figure supplement 5). Upon electroporation into HeLa cells, a concentration-dependent increase in abundance of KRas was observed for all variants, indicating successful delivery into cells (Figure 3B, C, Figure 3—figure supplements 6 and 7). However, whereas we observed a concentration-dependent activation of the downstream signalling and upregulation of pcRaf, pAkt, pErk, and pS6 upon delivery of KRasG13C, the covalently locked variant was unable to induce these effects and, similarly to KRasWT, no increase in downstream signalling was observed (Figure 3B, C, Figure 3—figure supplements 6 and 7). In addition, upon electroporation of non-covalently modified KRasG13C:acetyledaGDP or covalently modified KRasG13C-edaGppCP, activation of the Ras pathway comparable to KRasG13C was observed, showing that covalent modification is essential for inhibition of oncogenic signalling and that artificial activation can be induced using non-hydrolysable GTP derivatives, which potentially adds further possibilities of using these nucleotide analogues to study Ras biology (Figure 3—figure supplements 8 and 9). Thus, our cellular data provide an additional proof-of-concept for the use of nucleotide-based covalent inhibitors and activators in KRasG13C-driven cancer.

Conclusion

In summary, we have successfully developed nucleotide-based covalent inhibitors of oncogenic KRasG13C, a variant of KRas that is largely unexplored as a target even though it is a frequently observed mutation in cancer (Visscher et al., 2016). Thorough biochemical and structural characterization revealed that the nucleotide analogues designed and synthesized in this publication have similar affinities towards Ras compared to their natural counterparts. Since we are currently unable to directly test the nucleotides in cells, we instead extensively tested them in vitro regarding their ability to covalently lock KRasG13C in the inactive state and to effectively inhibit oncogenic signaling. We could show that the nucleotides prohibit (SOS-mediated) nucleotide exchange and lead to an effective interference in the induction of oncogenic effects by KRasG13C in living cells. Thus, after the first successful examples of inhibition of KRasG12C by Shokat and colleagues, our study breaks ground to effectively inhibit another important oncogenic variant of Ras by using small molecules.

Further optimization of the nucleotides to overcome the limitations regarding reactivity at physiological pH, as well as cell-permeability, are necessary and currently ongoing. In this respect, we are focusing on various linker designs based on the determined structure of the adduct, including cyclic linkers, to fine-tune the warhead’s orientation and reactivity towards the cysteine at position 13. Additionally, protective esterification of the diphosphate moiety is currently being investigated to deliver prodrugs of the nucleotides into cells, which subsequently become activated by unspecific esterases (Meier, 2017; Mehellou et al., 2018). We are also investigating whether appropriate modification of the linker length and the electrophilic warhead would also make KRasG12C or other relevant mutants (e.g. HRasG12S in Costello syndrome) (Gripp and Lin, 2012) a possible target in a similar approach.

Methods

Sequence alignment

A multiple sequence alignment of the G domain Ras superfamily members was performed using (UniProt Consortium, 2021). The uniport accession codes that were used for the sequence alignment of the GTPases can be taken from Supplementary file 1.

pKa calculations

For the protein pKa prediction, a program from OpenEye based on the Zap finite difference Poisson–Boltzmann solver was used. Regarding partial charges of the protein, Delphi radii and CHARMM36 All-Hydrogen partial charges were utilized. The linker was removed in the bda/edaGDP structures. The am1bccsym method was used to assign appropriate partial charges to the ligands. An inner dielectric of 10, an ionic strength of 0.05 M and ionization (i.e. pH) of 7.5 was applied. For cysteine, a reference pKa of 8.6 was used. All hydrogen atoms were modeled explicitly, and except for the orientations of the OH and SH protons, which were sampled in 10° steps, the rest of the structure was static. Optimizing ionization state and SH orientation was achieved by applying ten million Monte Carlo steps (Supplementary file 2; Word and Nicholls, 2011).

Synthesis of nucleotide-based covalent inhibitors

Synthesis of the nucleotide analogues was carried out according to the protocol established by Cremo et al. and described by Eberth et al., 2005; Cremo et al., 1990. A strong cation exchanger (Ion exchanger I, Merck, Darmstadt, Germany) was used to prepare the tributylammonium salts of the nucleotides. The commercially available sodium salt of the nucleotide (0.5 mmol) was dissolved in 3 mL ddH2O and was applied on the column pre-equilibrated with pyridine/H2O (1:1). Nucleotide elution was achieved using methanol/H2O (1:1) and the eluate was dripped into 1 mL TBA. After monitoring the nucleotide elution by spotting samples onto a TLC plate with fluorescent indicator and removing of the methanol/H2O solution the mixture was dried by repeated rotary evaporation from dry DMF (3x20 mL).

Preparation of 5´-phosphorylimidazolidate 2’,3’-O-carbonate of nucleotides

The remaining solid was dissolved in 20 mL dry DMF and CDI (2.5 mmol) was added under argon atmosphere. After stirring overnight at 4 °C to form the carbonate the reaction was quenched by the addition of absolute methanol (150 µL; Scheme 1).

Scheme 1
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Preparation of 5´-phosphorylimidazolidate 2’,3’-O-carbonate of nucleotides.

HRMS (ESI-MS)

nucleotidecalculated[M-H]-found
GDP518.0232C14H14O11N7P2518.0220
GTP597.9895C14H15O14N7P3597.9869
dGTP650.0315C17H19O13N9P3650.0304
GppCp*545.9828C12H15O14N5P3545.9829
‍*in case of GppCp, the cyclic carbonate was observed with a free terminal phosphate group.

Preparation of the respective 2’,3’-O-carbamates of nucleotides

The primary amine (eda, pda or bda; 2.5 mmol) dissolved in 5 mL dry DMF was slowly added to the carbonate mixture to prepare the phosphoramidate derivative. The resulting precipitate was recovered by centrifugation (10,000 rpm, 10 min) and washed three times with DMF (Scheme 2).

Scheme 2
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Preparation of the respective 2’,3’-O-carbamates of nucleotides.

LCMS (ESI-MS)

nucleotidelinkercalculated[M-H]-found
GDPeda578.1C16H22N9O11P2578.1
pda592.1C17H24N9O11P2592.1
bda606.1C18H26N9O11P2606.1
GTPeda658.1C16H23N9O14P3658.0
pda672.1C17H25N9O14P3672.1
bda686.1C18H27N9O14P3686.1
dGTPeda634.1C15H27N9O13P3634.1
pda662.1C17H31N9O13P3662.1
bda690.2C19H35N9O13P3690.2
GppCpeda648.1C16H29N9O13P3648.1

Cleavage of the phosphoramidate

The solid was dissolved in 20 mL ddH2O and the pH was adjusted to 1.5 with 0.25 M hydrochloric acid to hydrolyse the phosphoramidate. After stirring at 4 °C for 1–3 d the mixture was then raised to pH 7.5 using 0.25 M NaOH. The nucleotides were purified at 4 °C on a Q-Sepharose column (column volume: 130 mL) preequilibrated with 50 mM triethylammonium bicarbonate buffer (pH 7.6) and eluted by a linear gradient of 50 mM – 1 M TEAB over 600 min with a flow rate of 1 mL/min. The nucleotide containing fractions were analysed by HPLC 50 mM KPi pH 6.6, 10 mM TBAB, 16% ACN; column: ProntoSIL 120–5 C18-AQ, Bischoff, Germany and were lyophilized several times from ddH2O to remove the buffer (Scheme 3).

Scheme 3
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Cleavage of the phosphoramidate.

HRMS (ESI-MS)

nucleotidelinkercalculated[M-H]-foundpurity (%)yield (%)
GDPeda528.0651C13H20O12N7P2528.06549038
pda542.0802C14H22O12N7P2542.0809>9565
bda556.0964C15H24O12N7P2556.0960>9529
GTPeda608.0314C13H21O15N7P3608.02898956
pda662.0471C14H23O15N7P3662.04718783
bda636.0621C15H25N7O15P3636.06039467
dGTPeda592.0359C13H21O14N7P3592.03498423
pda606.0516C14H23O14N7P3606.05219311
bda620.0672C15H25N7O14P3620.06639034
GppCpeda606.0516C14H23N7O14P3606.051389-

Insertion of the Michael acceptor or non-reactive acetamido derivative

The nucleotide based covalent inhibitors were prepared by dissolving eda, pda or bda nucleotides in a small amount of tetraborate buffer (100 mM, pH 8.5) and adding N-acryloxysuccinimide (1 eq.) dissolved in 25 µL DMSO. The reaction mixture was stirred for 24 h at room temperature and the reaction progress was monitored using HPLC. The covalent nucleotide analogues were purified using Q-Sepharose as described above and were stored at –20 °C as concentrated solutions (~100 mM) in 200 mM HEPES (pH 7.5; Scheme 4).

Scheme 4
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Insertion of the Michael acceptor or non-reactive acetamido derivative.

HRMS (ESI-MS)

Acrylamideslinkercalculated[M-H]-foundpurity (%)yield (%)
GDPeda582.0751C16H22N7O13P2582.0741>9557
pda596.0907C17H24N7O13P2596.09078765
bda610.1064C18H26N7O13P2610.1062>9578
GTPeda662.0414C16H23N7O16P3662.03949354
pda676.0571C17H25N7O16P3676.0551>9563
bda690.0727C18H27N7O16P3690.07058675
dGTPeda646.0465C16H23N7O15P3646.04529363
pda660.0621C17H25N7O15P3660.06119081
bda674.0778C18H27N7O15P3674.07719374
GppCpeda660.0621C17H27N7O16P3660.06197915*
Acetylamideslinkercalculated[M-H]-foundpurity (%)yield (%)
GDPeda570.0751C15H22N7O13P2570.0734>9557
pda584.0907C16H24N7O13P2584.08899051
bda598.1064C17H26N7O13P2598.10427973
GTPeda650.0414C15H23N7O16P3650.0392>9523
pda664.0571C16H25N7O16P3664.05539440
bda678.0727C17H27N7O16P3678.07009331
*in case of GppCp, the total yield was calculated over 4 steps.‍

Protein expression and purification

KRasWT1-169 and KRasG13C1-169 Cys-light (C51S C80L C118S) were expressed in BL21 (DE3) E. coli whereas the full-length KRasWT and KRasG13C were expressed in BL21 (DE3) RIL E. coli at 37 °C. Protein expression was induced at A600 nm of 0.5 by addition of 0.2–0.3 mM isopropyl-b-D-thiogalactoside (IPTG), and growth was continued at 19 °C overnight. The bacteria were collected by centrifugation and the obtained pellet resuspended in Ni-NTA buffer (KRas1-169: 50 mM Tris pH 8.0, 250 mM NaCl, 40 mM imidazole, 4 mM MgCl2, 10 µM GDP, 1 mM dithiothreitol (DTT), and 5% glycerol; full-length KRas: 50 mM HEPES pH 7.2, 500 mM LiCl, 2 mM MgCl2, 10 µM GDP, 2 mM β-mercaptoethanol (βME)). The cells were lysed with a microfluidizer, and after addition of protease inhibitor cocktail (Roche complete EDTA free) and 1% CHAPS (w/v) stirring was continued for 1 hr at 4 °C. The lysate was cleared by centrifugation (35,000 x g, 1 h) and the supernatant was loaded onto a Ni-affinity chromatography column (Qiagen Ni-NTA Superflow, 20 mL) pre-equilibrated with Ni-NTA buffer. KRas1-169 proteins were eluted with a linear gradient of imidazole buffer (40 mM – 500 mM), whereas the full-length KRas proteins were collected with a stepwise elution (2, 5, 10, 20, 30, 50, and 100% of Ni-NTA-buffer containing 500 mM imidazole). For cleavage of the N-terminal hexahistidine-tag, TEV protease was added to the pooled elution fractions and dialyzed overnight into dialysis buffer at 4 °C (KRas1-169: 25 mM Tris pH 8.0, 100 mM NaCl, 4 mM MgCl2, 10 µM GDP, 1 mM DTT, and 5% glycerol; full-length KRas: 20 mM HEPES pH 7.2, 200 mM NaCl, 2 mM MgCl2, 10 µM GDP, 2 mM βME and 5% glycerol). The cleaved protein was then applied to a reverse Ni-affinity chromatography column and finally purified by size-exclusion chromatography (GE HiLoad 16/60 Superdex 75 pg) in a final buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl2, 10 µM GDP, 1 mM TCEP, and 5% glycerol. GST-tagged RafRBD was purified as described previously by affinity chromatography and subsequent size exclusion chromatography in a final buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1 mM TCEP, and 5% glycerol (Herrmann et al., 1995).

Nucleotide exchange

A total of 50 µM Ras protein was incubated with a 10-fold excess of nucleotides in 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5% glycerol, and 10 mM EDTA for 3 hr at 4 °C. Nucleotide exchange was terminated by the addition of 20 mM MgCl2 and Ras proteins were washed using centrifugal filter devices to remove any unbound nucleotide. Nucleotide exchange was controlled by isocratic HPLC runs.

Covalent modification of proteins

To analyse the amount of covalent modification of KRasG13C1-169, 50 µM Ras protein was incubated with a 10-fold excess of the acryl-bearing nucleotides in 100 mM CHES (pH 9.5), 50 mM NaCl, 1 mM TCEP, and 1 mM EDTA. After incubation at room temperature for the appropriate time, the modification of the protein was controlled by ESI-MS. For covalent modification of full-length KRasG13C, a nucleotide exchange with a 10-fold excess of the acryl-bearing nucleotides at pH 7.5 was first performed following incubation at room temperature for 24 h at pH 9.5 for covalent protein modification. Covalent protein modification was controlled by ESI-MS. The MS spectra were recorded on a VelosPro IonTrap (Thermo Scientific) with an EC 50/3 Nucleodur C18 1.8 μm column (Macherey and Nagel) and a gradient of the mobile phase A (0.1% formic acid in water) to B (0.1% formic acid in acetonitrile).

Stopped-flow experiments

The association kinetics of the nucleotide analogues were analyzed with a SX-20 stopped-flow instrument (Applied Photophysics) at 25 °C in a buffer consisting of 25 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM MgCl2, and 0.5 mM TCEP. 1 µM of nucleotide-free KRas (Müller et al., 2017) in one syringe was mixed rapidly with 2 µM mantdGDP in the other. In subsequent experiments, the second syringe contained competing nucleotides (1, 2, and 6 µM) in addition to 2 µM mantdGDP. The resulting progress curves were globally fit using KinTek Explorer to obtain the corresponding association rate constants as previously described (Table 1—source data 1, Table 1—source data 2 Johnson et al., 2009).

HPLC-based approach for determination of affinities relative to GDP

The relative affinities of the nucleotide analogues were measured using an HPLC-based approach. 50 µM of KRasWT1-169:GDP was mixed with 50 µM of the acetamide derivatives and incubated either for 7 days at room temperature in the absence of EDTA, for 24 hr at 4 °C in the presence of 10 mM EDTA, or for 1 hr at room temperature in the presence of SOS in a buffer consisting of 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 1 mM TCEP and 5% glycerol. After incubation, the Ras proteins were washed five times with buffer (15 mL) using centrifugal filter devices to remove any unbound nucleotide, concentrated and analysed by isocratic HPLC runs. The resulting curves were analysed by integrating the corresponding peaks for GDP and the guanosine nucleotide analogues after distinct time points using the Agilent ChemStation Software (Table 1—source data 3, Table 1—source data 4).

Calculation of the KD values of the nucleotide analogues

Based on the percentage distribution determined by the HPLC assay, the relative association constants (KrelA) for pdaGDP and bdaGDP could subsequently be calculated. In contrast, the superposition of the GDP signal prevented an exact determination of the relative association constant for edaGDP. KrelA values of 0.34 and 0.28 relative to GDP were determined for pdaGDP and bdaGDP, respectively. Finally, considering the KD value of 2.5 pM for GDP described by Jeganathan et al. the corresponding dissociation constant KD values could be determined, which are shown in Table 1—source data 4; Jeganathan et al., 2018.

(1a) Ras+GDPkGDP→Ras:GDP
(1b) Ras+aGDPkaGDP→Ras:aGDP

By rearranging the equilibrium reactions listed in (Equation 1a) and (Equation 1b), the following relationships are obtained for KGDP (Equation 2a) and KaGDP (Equation 2b):

(2a) KGDP=Ras:GDPRasGDP
(2b) KaGDP=Ras:aGDPRasaGDP

Assuming that the concentration for aGDP used in the HPLC assay is 50 µM, the following GDP concentrations for pdaGDP (Equation 3a) and bdaGDP (Equation 3b) can be determined at time t=0 hr using the percentages determined in Table 1—source data 3:

(3a) GDPtotal=%GDP%pdaGDP=37%63%×50μM=29.4μM
(3b) GDPtotal=%GDP%bdaGDP=43%57%×50μM=37.7μM

The concentrations of the components listed in equation (Equation 2b) can be calculated as follows for pdaGDP (Equation 4a, Equation 4b, Equation 4c, Equation 4d) and bdaGDP (Equation 5a, Equation 5b, Equation 5c, Equation 5d) by considering the total GDP concentrations calculated previously:

(4a) [Ras:pdaGDP]=47100×29.4μM=13.8μM
(4b) [Ras:GDP]=53100×29.4μM=15.6μM
(4c) [pdaGDP]=50μM−13.8μM=36.2μM
(4d) [GDP]=29.4μM−15.6μM=13.8μM
(5a) [Ras:bdaGDP]=39.5100×37.7μM=14.9μM
(5b) [Ras:GDP]=60.5100×37.7μM=22.8μM
(5c) [bdaGDP]=50μM−14.9μM=35.1μM
(5d) [GDP]=37.7μM−22.8μM=14.9μM

The relative association constant KrelA can be calculated by the following formula (Equation 6):

(6) KrelA=KaGDPKGDP=Ras:aGDP×GDPaGDP×Ras:GDP

By inserting the concentrations of each component determined from (Equation 4a, Equation 4b, Equation 4c, Equation 4d) and (Equation 5a, Equation 5b, Equation 5c, Equation 5d) into (Equation 6), the relative association constants KrelA for pdaGDP (Equation 7a) and bdaGDP (Equation 7b) can be calculated:

(7a) KrelA=KpdaGDPKGDP=13.8μM×13.8μM36.2μM×15.6μM=0.34
(7b) KrelA=KbdaGDPKGDP=14.9μM×14.9μM35.1μM×22.8μM=0.28

Based on the relative association constants KrelA, the KA values of the nucleotide analogues can be calculated by multiplication with the association constant of GDP:

(8) KA=KAGDP×KrelA

The KD value of 2.5 pM described by Jeganathan et al., 2018 was used as the reference value for GDP. By substituting the reference value into KA = 1/KD, a KA value for GDP of 0.4 pM–1 was determined. Accordingly, the KA values for pdaGDP (Equation 9a) and bdaGDP (Equation 9b) are as follows:

(9a) KApdaGDP=0.4pM-1×0.34=0.136pM-1
(9b) KAbdaGDP=0.4pM-1×0.28=0.112pM-1

By converting the determined KA values into the corresponding KD values using KD = 1/KA, the following dissociation constants could be determined for pdaGDP (Equation 10a) and bdaGDP (Equation 10b):

(10a) KDpdaGDP=10.136pM-1=7.4pM
(10b) KDbdaGDP=10.112pM-1=8.9pM

Crystallization and structure determination

KRasG13C1-169 Cys-light (C51S C80L C118S) was covalently modified by incubating 100 µM KRas with a 10-fold excess of the acryl-bearing nucleotide in 100 mM CHES (pH 9.5), 50 mM NaCl, 1 mM TCEP, and 1 mM EDTA at room temperature for 24 hr. Modification of the protein was monitored by ESI-MS and terminated by the addition of 20 mM MgCl2. The protein was purified by size-exclusion chromatography (GE HiLoad 16/60 Superdex 75 pg) in a final buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1 mM TCEP, and 5% glycerol, and subsequently concentrated to 67 mg/mL. To identify the initial crystallization conditions, commercially available protein crystallization screens (JCSG Core I –IV Suites, PEGs and PACT) were used. Using a TTP labtech Mosquito LCP crystal liquid-handling robot, 100 nL of protein solution was mixed with 100 nL reservoir solution in 96-well plates, and crystals were grown using the hanging-drop method at 20 °C. After 1 day of incubation, one successful crystallization condition was obtained for KRasG13C-edaGDP (0.2 M (NH4)F, 20% PEG3350) and KRasG13C-bdaGDP (0.2 M NaF, 20% PEG3350), and the crystals were cryoprotected in mother liquor and flash cooled in liquid nitrogen. The data sets were collected at the PXII X10SA beamline of the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) and indexed and scaled using XDS (Kabsch, 2010). The crystal structures were solved by molecular replacement with PHASER using PDB 4obe as a template (Read, 2001). The manual modification of the molecule of the asymmetric unit was performed using the program COOT (Emsley and Cowtan, 2004), and with the help of the Dundee PRODRG server (Schüttelkopf and van Aalten, 2004), the inhibitor topology file was generated. For multiple cycles of refinement, PHENIX.refine (Adams et al., 2010) was employed, the final structure was evaluated by the PDB_REDOserver (Joosten et al., 2014) and crystal structures were visualized using PyMOL. Data collection, structure refinement statistics, and further details for data collection are provided in Figure 2—figure supplement 1.

Guanine nucleotide-exchange factor assay

SOS-catalysed nucleotide exchange was monitored at 25 °C in a FluoroMax-3 spectrofluorometer (excitation at 360 nm, emission at 440 nm) in 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM MgCl2, and 1 mM TCEP. 5 µM KRas1-169 was mixed with 10 µM mantdGDP and subsequently with different concentrations of SOS (0.25 µM and 0.5 µM) (Figure 3A; Figure 3—figure supplement 1).

GAP-stimulated GTP hydrolysis

First, for KRasWT1-169, nucleotide exchange with GTP was performed at pH 7.5, and for KRasG13C1-169, covalent modification with a 10-fold excess of acryl-edaGTP at pH 9.5 was done, both in the presence of 50 mM EDTA to block intrinsic GTP hydrolysis. After incubation, the Ras proteins were washed five times with buffer (20 mM HEPES (pH 7.5), 100 mM NaCl, and 1 mM TCEP) using centrifugal filter devices to remove any unbound nucleotide. Nucleotide exchange of KRasWT1-169 was controlled by isocratic HPLC runs, and covalent modification of KRasG13C1-169 was verified by ESI-MS. GTP hydrolysis of Ras proteins was initiated by addition of 2 mM MgCl2 in the absence or presence of Ras-GAP (1:1000 for KRasWT1-169:GTP and 1:1 for KRasG13C1-169-edaGTP). At defined time points (0, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min), samples were taken and immediately snap-frozen in liquid nitrogen. For KRasWT1-169:GTP, samples were thawed and denatured for 5 min at 95 °C. Samples were centrifuged at 14,000 rpm for 10 min at 4 °C and subsequently analysed by isocratic HPLC runs. For KRasG13C1-169-edaGTP, samples were centrifuged and analysed by ESI-MS. The relative amount of each nucleotide was determined by integrating the area of the GTP and GDP peaks using Origin (Figure 3—figure supplement 2; Eberth and Ahmadian, 2009).

Effector binding (pull-down experiments)

Pull-down experiments were performed in 20 mM HEPES pH 7.5, 50 mM NaCl and 2 mM MgCl2. 10 µg KRasG13C:GDP or the covalently modified KRasG13C-edaGDP and 20 µg GST-tagged cRafRBD (amino acids 51–131) were incubated with/ without 100 µM GppNHp and with/ without 1 µg SOS over night at room temperature. Afterwards, 50 µL glutathione magnetic beads were added to each sample for 30 min. After washing the beads with 500 µL buffer, the beads were settled with a magnet and the supernatant was carefully removed. The beads were resuspended in 50 µL of 4xSDS-loading buffer and visualized via SDS-PAGE (Figure 3—figure supplement 3).

In vitro farnesylation

50 µM of full-length KRas protein was mixed with 250 µM farnesyl pyrophosphate (FPP) and 10 µM farnesyltransferase (FTase). After incubation at room temperature for 1 hr the mixture was centrifugated at 14,000 rpm for 10 min at 4 °C and analyzed via ESI-MS (Figure 3—figure supplement 4).

Competition assay

To analyze the amount of covalent modification of KRasG13C1-169 (Cys-light) with the acryl-bearing nucleotide analogue (edaGDP) under competitive conditions with GDP and GTP, four different conditions were tested (buffer: 100 mM CHES pH 9.5., 50 mM NaCl, 1 mM TCEP, 2 mM MgCl2, 5% glycerol). 5 µM KRasG13C (Cys-light) was incubated (1) in the presence of 36 µM edaGDP with and without SOS, (2) in the presence of 305 µM edaGDP, 36 µM GDP and 305 µM GTP with and without SOS, (3) in the presence of 36 µM edaGDP, 36 µM GDP and 305 µM GTP with and without SOS and (4) 36 µM edaGDP, 360 µM GDP, and 3050 µM GTP with and without SOS. After incubation for 1, 2, 3, 4, 5, 22, and 24 hr at room temperature, the covalent protein modification was controlled by ESI-MS.

MS/MS analysis

For MS/MS analysis, the samples were dissolved in 100 mM TEAB and incubated for 1 hr at 55 °C in the presence of 10 mM TCEP. 17 mM iodoacetamide was added and the samples were incubated in the dark at room temperature for 30 min. Samples were precipitated by adding pre-chilled acetone and stored overnight at –20 °C. After drying the pellets, Trypsin (Roche) was added and the samples were digested at 37 °C with 300 rpm shaking overnight. The digestion was quenched by the addition of 2% TFA and a stage tip purification (Rappsilber et al., 2007) was performed, samples were evaporated to dryness and stored at –20 °C until MS/MS analysis. For nanoHPLC-MS/MS analysis samples were dissolved in 20 μL of 0.1% TFA in water and 3 μL were injected onto an UltiMateTM 3000 RSLCnano system (ThermoFisher Scientific, Germany) online coupled to a Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer equipped with a nanospray source (Nanospray Flex Ion Source, Thermo Scientific). All solvents were LC-MS grade. Samples were injected onto a pre-column cartridge (5 μm, 100 Å, 300 μm ID * 5 mm, Dionex, Germany) using 0.1% TFA in water as eluent with a flow rate of 30 μL/min. Desalting was performed for 5 min with eluent flow to waste followed by back-flushing of the sample during the whole analysis from the pre-column to the PepMap100 RSLC C18 nano-HPLC column (2 μm, 100 Å, 75 μm ID ×50 cm, nanoViper, Dionex, Germany) using a linear gradient starting with 95% solvent A (water containing 0.1% formic acid) / 5% solvent B (acetonitrile containing 0.1% formic acid) and increasing to 30% solvent B in 90 min using a flow rate of 300 nL/min. Afterwards, the column was washed (two steps 60 and 95% solvent B) and re-equilibrated to starting conditions. The nanoHPLC was online coupled to the Quadrupole-Orbitrap Mass Spectrometer using a standard coated SilicaTip (ID 20 μm, Tip-ID 10 μM, New Objective, Woburn, MA, USA). Mass range of m/z 300–1,650 was acquired with a resolution of 70000 for a full scan, followed by up to 10 high energy collision dissociation (HCD) MS / MS scans of the most intense at least doubly charged ions using a resolution of 17500 and a NCE energy of 25%. Data evaluation was performed using MaxQuant software (Cox and Mann, 2008) (v.1.6.3.4) including the Andromeda search algorithm (Cox et al., 2011) and searching the KRas sequence together with a database containing typical contaminants like keratins, trypsin etc., which is included in the MaxQuant software. The search was performed for full enzymatic trypsin cleavages allowing two miscleavages. For database search oxidation of methionine and N-terminal acetylation of proteins, carbamidomethylation of cysteines, and artificial modification of cysteines were defined as variable modifications. The mass accuracy for full mass spectra was set to 20 ppm (first search) and 4.5 ppm (second search), respectively and for MS/MS spectra to 20 ppm. The false discovery rates for peptide and protein identification were set to 1%. For further analysis, the peptide intensities of KRas were compared for modified and unmodified KRas. (Figure 3—figure supplement 5).

Cell culture

HeLa cells were obtained from the American Type Culture Collection (ATCC) and were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (PAN-Biotech) and 1% penicillin/streptomycin (Gibco). Cells were cultured in a humidified incubator at 37 °C in the presence of 5% CO2. The identity of the cells was validated by STR profiling and the cells were tested negative for mycoplasma contamination.

Electroporation

Electroporation of full-length KRas constructs (KRasWT, KRasG13C, KRasG13C-edaGDP, KRasG13C:acetyledaGDP and KRasG13C-edaGppCp) was performed based on the protocol described by Alex et al., 2019 using the Neon Transfection System Kit (Thermo Fisher). For electroporation, 3 million cells per experiment were harvested by trypsinization, washed with PBS, and resuspended in 85 µL of the electroporation buffer R (Thermo Fisher). Increasing amounts of recombinant protein samples for each construct (0, 50, 100, 200 and 300 µg) were diluted 1:1 in buffer R followed by the addition of 30 µL of this protein master mix to the cell suspension. This cellular slurry was loaded into a 100 mL Neon Pipette Tip (Thermo Fisher) and electroporated with 2x35 ms pulses at 1000 V. After electroporation, the cells were washed twice with PBS (15 mL) to remove non-internalized extracellular protein and the cell pellet was resuspended in 2 mL complete growth media. Cells were transferred into six-well tissue culture plates (Sarstedt) and incubated for 24 hr at 37 °C and 5% CO2 in a humidified incubator for recovery before being processed for western blotting analysis.

Western blot analysis

After recovery of the electroporation, cells were washed twice with ice-cold PBS and lysed in 100 µL of phosphatase and protease inhibitor containing RIPA buffer (Cell Signaling Technology). Cells were incubated on ice for 30 min and then harvested by scraping followed by centrifugation at 14,000 rpm for 10 min at 4 °C. Protein concentrations were determined using the Pierce BCA protein assay (Thermo) following the manufacturer’s recommended procedure. Equal amounts of protein (10 µg) were analyzed by SDS-PAGE and transferred to Immobilon-FL PVDF membranes (Merck Millipore) using Pierce 1-step transfer buffer (Thermo) and the Pierce Power Blotter (Thermo). Membranes were washed with ddH2O for 5 min, blocked with OdysseyBlocking Buffer TBS (Li-Cor) for 1 hr at room temperature and then incubated with primary antibodies diluted in OdysseyBlocking Buffer TBS overnight at 4 °C with gentle agitation. KRas (Sigma Aldrich, SAB1404011-100UG), pcRafS338 (CST, 9427), tAkt1 (CST, 2938), pAktS473 (CST, 4060), tErk (CST, 4696), pErkT202/Y204 (CST, 4370), pS6S235/236 (CST, 4858), and β-actin (CST, 4970/Sigma-Aldrich, A5441) antibodies were used to detect the individual proteins. After primary antibody incubation, membranes were washed three times with TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4) for 5 min before being incubated with secondary antibodies (anti-mouse IgG (H+L) (DyLight 680 Conjugate) (CST, 5470) / anti-rabbit IgG (H+L) (DyLight 800 4 X PEG Conjugate) (CST, 5151)) diluted in OdysseyBlocking Buffer TBS for 1 hr at room temperature with gentle agitation. After secondary antibody incubation, the membranes were washed three times for 5 min with TBS-T and then scanned using an OdysseyCLx imaging system (Li-Cor). Quantification of protein levels from western blots was performed using Empiria Studio (Li-Cor) (Figure 3B, C, Figure 3—figure supplements 6–9).

Data availability

Diffraction data have been deposited in PDB under the accession code 7ok3 and 7ok4.

The following data sets were generated
    1. Goebel L
    2. Mueller MP
    3. Rauh D
    (2022) RCSB Protein Data Bank
    ID 7OK4. Crystal Structure of KRasG13C in Complex with Nucleotide-based covalent Inhibitor bdaGDP.
    https://www.rcsb.org/structure/7OK4
    1. Goebel L
    2. Mueller MP
    3. Rauh D
    (2022) RCSB Protein Data Bank
    ID 7OK3. Crystal Structure of KRasG13C in Complex with Nucleotide-based covalent Inhibitor edaGDP.
    https://www.rcsb.org/structure/7OK3

References

    1. Emsley P
    2. Cowtan K
    (2004) Coot: model-building tools for molecular graphics
    Acta Crystallographica. Section D, Biological Crystallography 60:2126–2132.
    https://doi.org/10.1107/S0907444904019158
    1. Kabsch W
    (2010) XDS
    Acta Crystallographica. Section D, Biological Crystallography 66:125–132.
    https://doi.org/10.1107/S0907444909047337

Decision letter

  1. Volker Dötsch
    Senior and Reviewing Editor; Goethe University, Germany

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Volker Dötsch as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Can the edaGDP derivative competes with 1 mM of GDP and GTP in covalently modifying KRAS G13C and the wild type KRAS (well to a lesser extent)?I see the authors also employed Cys light version of RAS like the Shokat group in some of their assays and perhaps wild type protein is probably better here as well as anyway they are performing MS analysis.

2) Related: K-Ras wildtype was labeled by eda/pda/bdaGDP to a much lower extent (Figure 1D, E). however, K-Ras(G12C) Cyslight was not labeled by these GDP analogs at all. Do the authors have any MS2 data to identify the site of modification in the case of K-Ras WT? There is growing interest in covalent adducts to RAS, so identifying the nearest residues which can be targeted (especially non-Cys residues) maybe of assistance to others.

3) The paper will immensely benefit from discussing weaknesses. a): The complete modification of KRASG13C happens in high pH (9.5!!) which is of a significant concern. Do the authors envision a solution to the pH that is required for the covalent modification to take place. b): Do the authors see hope of designing something that can cross the membranes. c): These nucleotides will have the possibility to bind to other small GTPases or enzymes how is that going to affect this strategy. In particular: Other Ras superfamily member GTPases naturally have residue C13 equivalent 13 in Ras, such as Arl4a, RheBL1, Rab21 mentioned here. Have the authors tested the reactivity of edaGDP or analogs in these recombinant proteins? Is it possible that these compounds can be converted by NKDs to the GTP form?

4) There is no data to demonstrate that KRASG13C-effector binding is directly disrupted by eda GDP or others in vitro or in cells. Can addition of edGDP disrupt KRASG13C-effector interaction esp at lower pH?

5) Both structures lacked Mg2+ and is this the reason for the downstream effects?

6) It is important for the authors to convincingly demonstrate that their approach is better than the current state of the art esp. when there are options like SOS inhibitors and pan RAS inhibitors with good oral bioavailability which didn't exhibit overt toxicity and present a exploitable therapeutic window.

7) Related: I don't see an SOS mediated exchange using those compounds. The authors only show that SOS does not work on modified KRas. I find it difficult to deduce the time and the efficiency of covalently binding to KRasg13C in the presence of other nucleotides and in the presence of SOS. What kind of concentrations and how long will it take if you have in one cuvette physiological amounts of GTP/GDP and your compounds and Ras and SOS. This experiment would improve the manuscript significantly.

8) Perhaps the authors can already test if their derivatives would still work on patients who has gained resistance mutations like Y96C, H95Dor R68S with KRASG13C.

9) Please include the total protein blots for all the phospho-proteins tested in all the westerns. pS338 is not a reliable marker for checking the activation of CRAF. There are instances where CRAF is 338 phosphorylated but not activated. Ideally the authors should look at MEK phosphorylation. I appreciate the effects at pERK1/2.

10) One should also be careful with the general cytotoxicity independent of KRASG13C as shown with SML-8-3-1 especially when they have to be employed at higher concentrations for reasonable modification of KRASG13C. Please note that this allele can be amplified in patients.

11) The authors need to show target engagement in cellular systems directly. As of now, at least one could try to do electroporation into KRASG13C expressing cells and check if the modified protein is dimerizing with the endogenous version and has implications in the downstream signaling.

Reviewer #1 (Recommendations for the authors):

Ras is the first discovered oncogene and KRAS is 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 similar ones 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 is to compete with the nucleotide binding of RAS and it has for a long time remained difficult owing to the picomolar affinity of RAS for GTD/GDP. Gray and colleagues tried to overcome these issues by employing GDP derivatives that can undergo covalent reaction with disease specific mutations but the authors reported in their previous work that the issue with these derivatives was with the loss of reversible affinities for β modified derivatives for RAS of at least by 10000 fold compared GDP and GTP. Here the authors present novel GDP derivatives different from Gray and colleagues and demonstrate that they could lock KRASG13C, another important mutant of KRAS in GDP bound form with a multiple set of biochemical, structural and cellular assays. While I find the study scientifically interesting I have several concerns that diminish my enthusiasm for publication in eLife. As such the manuscript is more appropriate for a journal with focus on structural /Chemical biology.

1) Can the edaGDP derivative competes with 1 mM of GDP and GTP in covalently modifying KRAS G13C and the wild type KRAS (well to a lesser extent)? I see the authors also employed Cys light version of RAS like the Shokat group in some of their assays and perhaps wild type protein is probably better here as well as anyway they are performing MS analysis.

2) The complete modification of KRASG13C happens in high pH (9.5!!) which is of significant concern.

3) Gray and colleagues performed the "caging " approach to allow their derivatives for passive cellular uptake. They did show that these modified GDP derivatives inhibit atleast signalling and binding to RAS effectors in cells. As of now the authors electroporate KRAS protein into HeLa cells to evaluate their effects. They claim to be working on ways to protect esterification for cellular entry. We need such development and proof of principle experiments that this can at least be a "prodrug" that can be further developed. I would like to see assays performed in KRASG13C allele addicted cell lines. First, I'm also surprised that the WT-KRAS didn't activate MAPK signaling as WT protein should be GTP bound and thus have basal activity while purified or when introduced to cells, especially in excess.

4) There is no data to demonstrate that KRASG13C-effector binding is directly disrupted by eda GDP or others in vitro or in cells. Can addition of edGDP disrupt KRASG13C-effector interaction esp at lower pH?

5) Both structures lacked Mg2+ and is this the reason for the downstream effects?

6) It is important for the authors to convincingly demonstrate that their approach is better than the current state of the art esp. when there are options like SOS inhibitors and pan RAS inhibitors with good oral bioavailability which didn't exhibit overt toxicity and present a exploitable therapeutic window.

7) Perhaps the authors can already test if their derivatives would still work on patients who has gained resistance mutations like Y96C, H95Dor R68S with KRASG13C.

8) Please include the total protein blots for all the phospho-proteins tested in all the westerns. pS338 is not a reliable marker for checking the activation of CRAF. There are instances where CRAF is 338 phosphorylated but not activated. Ideally the authors should look at MEK phosphorylation. I appreciate the effects at pERK1/2.

9) One should also be careful with the general cytotoxicity independent of KRASG13C as shown with SML-8-3-1 especially when they have to be employed at higher concentrations for reasonable modification of KRASG13C. Please note that this allele can be amplified in patients.

10) The authors need to show target engagement in cellular systems directly. As of now, at least one could try to do electroporation into KRASG13C expressing cells and check if the modified protein is dimerizing with the endogenous version and has implications in the downstream signaling.

Reviewer #2 (Recommendations for the authors):

Suggestions and questions for the authors:

1. K-Ras wildtype was labeled by eda/pda/bdaGDP to a much lower extent (Figure 1D, E). however, K-Ras(G12C) Cyslight was not labeled by these GDP analogs at all. Do the authors have any MS2 data to identify the site of modification in the case of K-Ras WT? There is growing interest in covalent adducts to RAS, so identifying the nearest residues which can be targeted (especially non-Cys residues) maybe of assistance to others.

2. Other Ras superfamily member GTPases naturally have residue C13 equivalent 13 in Ras, such as Arl4a, RheBL1, Rab21 mentioned here. Have the authors tested the reactivity of edaGDP or analogs in these recombinant proteins?

Reviewer #3 (Recommendations for the authors):

1 – The introduction would be improved if the authors mention the prevalence of G13C mutations. How much does it represent of all mutant Ras and if it is found in a specific cancer.

2 – The paper will immensely benefit from discussing the weakness …do the authors envision a solution to the pH that is required for the covalent modification to take place. Do the authors see hope of designing something that can cross the membranes. These nucleotides will have the possibility to bind to other small GTPases or enzymes how is that going to affect this strategy. is it possible that these compounds can be converted by NKDs to the GTP form?

3 – Experimentally: I don't see an SOS mediated exchange using those compounds. The authors only show that SOS does not work on modified KRas. I find it difficult to deduce the time and the efficiency of covalently binding to KRasg13C in the presence of other nucleotides and in the presence of SOS. What kind of concentrations and how long will it take if you have in one cuvette physiological amounts of GTP/GDP and your compounds and Ras and SOS. This experiment would improve the manuscript significantly.

https://doi.org/10.7554/eLife.82184.sa1

Author response

Essential revisions:

1) Can the edaGDP derivative competes with 1 mM of GDP and GTP in covalently modifying KRAS G13C and the wild type KRAS (well to a lesser extent)?I see the authors also employed Cys light version of RAS like the Shokat group in some of their assays and perhaps wild type protein is probably better here as well as anyway they are performing MS analysis.

Since we see only very minor modification of KRasWT even after 24 h at pH 9.5 (please see our answer and discussion to question 3 below), we have performed the experiments for KRasG13C (Cys light) below with equimolar concentrations of the competing nucleotides as well as 10x and 100x excess. As expected, the presence of other competing nucleotides slows down the reaction, but modification nevertheless still occurs. In fact, the object and reasoning of modifying the nucleotides at the 2’,3’-positions was to obtain nucleotides with comparable reversible affinities compared to the natural analogues as discussed in the manuscript. However, as stated throughout the manuscript, further improvement of the reactivity (kinact) is necessary to finally achieve reaction rates that are compatible with in vivo use.

We would also like to stress the point that the Cys-light version of KRas is the standard model protein used to develop covalent inhibitors and was used by other labs as well (as already stated by the reviewer) 1.

2) Related: K-Ras wildtype was labeled by eda/pda/bdaGDP to a much lower extent (Figure 1D, E). however, K-Ras(G12C) Cyslight was not labeled by these GDP analogs at all. Do the authors have any MS2 data to identify the site of modification in the case of K-Ras WT? There is growing interest in covalent adducts to RAS, so identifying the nearest residues which can be targeted (especially non-Cys residues) maybe of assistance to others.

This is an excellent point and we have extensively tried to find the additional sites of modification in K‑RasWT using both the full-length protein as well as a truncated protein without the C-terminus (residues 1-169). Unfortunately, we have not been able to identify additional sites, which – considering the very low extent of modification and the difficulties we had previously in detecting the site of modification in KRasG13C by MS/MS experiments (Figure 3 —figure supplement 5) – is not unexpected.

3) The paper will immensely benefit from discussing weaknesses. a): The complete modification of KRASG13C happens in high pH (9.5!!) which is of a significant concern. Do the authors envision a solution to the pH that is required for the covalent modification to take place. b): Do the authors see hope of designing something that can cross the membranes. c): These nucleotides will have the possibility to bind to other small GTPases or enzymes how is that going to affect this strategy. In particular: Other Ras superfamily member GTPases naturally have residue C13 equivalent 13 in Ras, such as Arl4a, RheBL1, Rab21 mentioned here. Have the authors tested the reactivity of edaGDP or analogs in these recombinant proteins? Is it possible that these compounds can be converted by NKDs to the GTP form?

We have expanded the discussion our approach and we have modified the last paragraph of the conclusions accordingly. The paragraph now reads:

“Further optimization of the nucleotides to overcome the limitations regarding reactivity at physiological pH, as well as cell-permeability, are necessary and currently ongoing. In this respect, we are focusing on various linker designs based on the determined structure of the adduct, including cyclic linkers, to fine-tune the warhead's orientation and reactivity towards the cysteine at position 13. Additionally, protective esterification of the diphosphate moiety is currently being investigated to deliver prodrugs of the nucleotides into cells, which subsequently become activated by unspecific esterases.32,33 We are also investigating whether appropriate modification of the linker length and the electrophilic warhead would also make KRasG12C or other relevant mutants (e.g., HRasG12S in Costello syndrome)34 a possible target in a similar approach.”

Additional, we would like to stress:

a) Protein modification does NOT ONLY happen at pH 9.5, but as we clearly show it does also happen at pH 7.5 (~14% after 24h), pH 8 (~37% after 24h), pH 8.5 (~57% after 24h) and pH 9 (~90% after 24h) in the case of our best compound edaGDP, whereas KRasWT is only modified to a very small extend at pH > 9 (~3% at pH 9, ~8% at pH 9.5 after 24h), thus showing a rather good overall selectivity (Figure 1—figure supplement 5). For preparative labeling of KRasG13C (for subsequent crystallization, electroporation etc.), we chose pH 9.5 to ensure full modification as quickly as possible, and it will be undoubtedly necessary to increase the reactivity at physiological pH, however we are confident that we are on a very good track as pointed out above. In order to clarify this point, we have changed the manuscript accordingly (page 5-6).

b) Optimization of the nucleotide analogs with regard to cell permeability by synthesizing the corresponding prodrug derivatives, in which the negative charge of the phosphates is masked by esterification and which have already been described2-4, is currently being investigated.

c) Regarding the specificity: We cannot say anything for certain at this point and until we have cell-penetrable compounds. However, the problem regarding specificity is a well-known issue for covalently binding inhibitors also e.g. in the kinase field and this is also known and documented for the marketed drug osimertinib5.

Additionally, we have tested the reactivity of the nucleotides towards Rab21 (the only protein that is currently available to us with a cysteine at a position analogous to that in KRasG13C) and we see modification comparable to KRasG13C as expected. However, we currently cannot say what effect this might have in the context of inhibiting growth of KRasG13C dependent cancer cells or if this might have a negative (or potentially even positive) effect on this treatment option. Interestingly, knock-down of Rab21 has been shown to have positive effects in the context of human glioma cells 6.

With respect to the last question (i.e. whether NKDs could potentially phosphorylate the diphosphate to the triphosphate form), we cannot say anything for certain at this point. However, even if this was the case and the nucleotides were phosphorylated within the cells, the intrinsic (and possibly GAP stimulated) hydrolysis will ultimately lead to the diphosphate-form.

4) There is no data to demonstrate that KRASG13C-effector binding is directly disrupted by eda GDP or others in vitro or in cells. Can addition of edGDP disrupt KRASG13C-effector interaction esp at lower pH?

We thank the reviewer for this suggestion and have now included direct evidence that the effector interaction is not possible for Ras-edaGDP. For this, we have performed pulldown experiments with GST-tagged RafRBD. The corresponding experiments have now been included in the manuscript (page 9, lines 206-213).

5) Both structures lacked Mg2+ and is this the reason for the downstream effects?

As we have stated in the manuscript, the lack of Mg2+-ions is, in all likelihood, a result of the crystallization conditions containing fluoride in both cases. This presumably leads to precipitation of poorly soluble MgF2. Additionally, we systematically and thoroughly measured the nucleotide affinities for the modified nucleotides in comparison to GDP/GTP, and these clearly show that affinities are essentially unchanged in case of the modified nucleotides, which is incompatible with significantly altered Mg2+-affinities. Additionally, we also measured the rate of intrinsic GTP-hydrolysis for KRasG13C-edaGTP, which is similar to the wild-type protein and also incompatible with a significantly altered Mg2+-affinity.

Nonetheless, we have also measured the Mg2+-affinity (Author response image 1), and we could not observe a difference between KRasWT and KRasG13C. These measurements were carried out as described previously7. Briefly, 1 μM KRas:mantdGDP or KRasG13C:mantdGDP were titrated against increasing concentrations of MgCl2 (1, 2.5, 5, 7.5, 10, 15, 20, and 25 μM) and the resulting curves were analysed by plotting the relative fluorescence intensity versus MgCl2 concentration.

Author response image 1
Download asset Open asset
Mg2+ affinity assay.

(A, B) Fluorescence titration of KRasWT:mantdGDP or KRasG13C:mantdGDP with increasing concentrations of Mg2+ (1, 2.5, 5, 7.5, 10, 15, 20, and 25 μM); (C) The resulting curves were analysed by plotting the relative fluorescence intensity vs. MgCl2 concentration.

6) It is important for the authors to convincingly demonstrate that their approach is better than the current state of the art esp. when there are options like SOS inhibitors and pan RAS inhibitors with good oral bioavailability which didn't exhibit overt toxicity and present a exploitable therapeutic window.

We respectfully disagree that options are available, like SOS inhibitors and panRas inhibitors, since none of these has proven effective in clinical settings. It is correct that there are several studies as indicated by the reviewers (e.g. BI-1701963 and others), but at least until one approach has proven to be effective in patients, we feel that additional ideas and approaches are urgently needed (including our approach on using these covalently binding nucleotides). It is highly likely that even if an effective compound were available for treatment, upcoming resistance mutations would necessitate additional means of targeting Ras.

Additionally and importantly, the approaches mentioned above are not specific and do not allow direct targeting of a specific mutant of Ras (e.g. KRas G13C, which has NOT been addressed at all until now). We, therefore, feel that also under consideration of the current knowledge and available inhibitors, our study is an important additional contribution to the field, just like the studies on pan-Ras inhibitors, SOS-inhibitors, RasG12C inhibitors or PDEdelta inhibitors.

7) Related: I don't see an SOS mediated exchange using those compounds. The authors only show that SOS does not work on modified KRas. I find it difficult to deduce the time and the efficiency of covalently binding to KRasg13C in the presence of other nucleotides and in the presence of SOS. What kind of concentrations and how long will it take if you have in one cuvette physiological amounts of GTP/GDP and your compounds and Ras and SOS. This experiment would improve the manuscript significantly.

See point 1, corresponding experiments have been added to the manuscript.

8) Perhaps the authors can already test if their derivatives would still work on patients who has gained resistance mutations like Y96C, H95Dor R68S with KRASG13C.

It would indeed be interesting to test the effect of additional mutations, however, this is beyond the scope of this work.

Additionally, the reported mutations at position 96, 95 and 68 are all located within the switch-II pocket (and not the nucleotide-binding region) and are reported as a result of treatment with switch-II-pocket inhibitors that target the RasG12C mutation8, 9. Thus, it is highly likely that these will not have an effect on nucleotide binding, and most likely other resistance mutations will be of relevance upon using nucleotide-competitive inhibitors described in this publication. Possibly, the high conservation of the nucleotide-binding pocket could eventually even be an advantage regarding emerging resistances because only very few mutations might be generally tolerated, but this hypothesis will have to be tested and challenged in the future.

9) Please include the total protein blots for all the phospho-proteins tested in all the westerns. pS338 is not a reliable marker for checking the activation of CRAF. There are instances where CRAF is 338 phosphorylated but not activated. Ideally the authors should look at MEK phosphorylation. I appreciate the effects at pERK1/2.

The total protein blots for all the phospho-proteins tested in the western blots were already present in the original submission (Goebel et al_Source_data_file_WB.xlsx). Please let us know if anything else is missing.

We are aware that there are findings that phosphorylation at Ser-338 can be independent of c-Raf activity10. However, our Western blots clearly and unambiguously indicate a correlation between phospho-cRaf, phospho-pErk and the KRas concentration, i.e. a correlation between Ras, Raf and Erk activity (which is undoubtedly a good marker). Thus, we see no point in doing these additional experiments for MEK or c-Raf (Tyr340/341) activation that would require disproportionate additional effort and time for something that has already been clearly answered.

10) One should also be careful with the general cytotoxicity independent of KRASG13C as shown with SML-8-3-1 especially when they have to be employed at higher concentrations for reasonable modification of KRASG13C. Please note that this allele can be amplified in patients.

We agree that toxicity will need to be evaluated at a later point in time. Actually, in the design of the nucleotides, we purposely did not use chloroacetamides (as in SML-8-73-1), but rather less reactive and biocompatible acrylamides, since these are well known and have been shown to be tolerated in many approved drugs(e.g. osimertinib, afatinib to name just two clinically relevant examples of covalent drugs).

11) The authors need to show target engagement in cellular systems directly. As of now, at least one could try to do electroporation into KRASG13C expressing cells and check if the modified protein is dimerizing with the endogenous version and has implications in the downstream signaling.

The reviewer brings up an excellent point and we have indeed tried to do the experiment as suggested and probed extensively for target engagement in a cellular context by electroporating the modified nucleotides into cells. However, presumably because of the relatively low reactivity at physiological pH and undefined (probably low) concentrations of the nucleotides in cells upon electroporation, we could not observe an effect. Additionally, available KRas G13C cell lines such as H1355 (BRCA2: p.Gly2044Asp (homozygous), KRAS: p.Gly13Cys (heterozygous), TP53: p.Glu285Lys (homozygous)) and H1734 (ATM: p.Gln65Pro (heterozygous), KRAS: p.Gly13Cys (heterozygous), RB1: p.Ser127Ile (homozygous), TP53: p.Arg273Leu (homozygous)) are heterozygous for the G13C mutation and also generally contain additional mutations that complicate any conclusions drawn from the respective experiments (e.g. can any effect be expected at all against the background of the additional mutations and what percentage of modification of KRasG13C is required for this?). We are, therefore, currently working on establishing a homozygous Ba/F3 cell line that is dependent on the KRasG13C mutation that will be better suited for these experiments, but although we have spent some effort on this already, it has not been successful yet. For these reasons, we used the assay as described in the current manuscript and electroporated the already modified protein.

As stated above, we are also working on protected nucleotides by esterification of the phosphates that allow passage of the cell membrane. However, the synthesis of these nucleotides containing modifications at both the phosphates and the ribose is far beyond the scope of the current manuscript.

In addition to the comments we have addressed above, we would like to add a few more points:

Reviewer 1 asked why there is no upregulation observed in the cellular electroporation experiments for KRasWT, since it should be GTP-bound after purification (question #3). In our hands however, KRasWT is GDP-bound after purification as can be clearly seen in isocratic HPLC analyses and thus, no effect is to be expected. Even KRasG13C is GDP-bound after the purification (all purification buffers contain 10µM GDP), however the increased intrinsic nucleotide exchange rate (shown in Figure 3A) of this mutant even in the absence of any stimulus presumably leads to the activation observed in cells.

Reviewer 3 suggested to elaborate on the significance of the G13C mutation within the introduction. We have added a short paragraph to the introduction:

“Whereas position 12 is the most commonly mutated residue, G13 is the second most common mutation (14% of tumors harbor a mutation at this position) and in 6% of these cases, an acquired cysteine is found.11, 12 In lung cancer, the prevalence of the G13C mutation is 3%, which is equivalent to approximately 7000 individuals in the US per year.11, 13”

References

[1] Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A., and Shokat, K. M. (2013) K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions, Nature 503, 548-+.

[2] Mehellou, Y., Rattan, H. S., and Balzarini, J. (2018) The ProTide Prodrug Technology: From the Concept to the Clinic, Journal of medicinal chemistry 61, 2211-2226.

[3] Meier, C. (2017) Nucleoside diphosphate and triphosphate prodrugs – An unsolvable task?, Antivir Chem Chemother 25, 69-82.

[4] Meier, C., Meerbach, A., and Balzarini, J. (2004) Cyclosal-pronucleotides--development of first and second generation chemical trojan horses for antiviral chemotherapy, Front Biosci 9, 873-890.

[5] Finlay, M. R., Anderton, M., Ashton, S., Ballard, P., Bethel, P. A., Box, M. R., Bradbury, R. H., Brown, S. J., Butterworth, S., Campbell, A., Chorley, C., Colclough, N., Cross, D. A., Currie, G. S., Grist, M., Hassall, L., Hill, G. B., James, D., James, M., Kemmitt, P., Klinowska, T., Lamont, G., Lamont, S. G., Martin, N., McFarland, H. L., Mellor, M. J., Orme, J. P., Perkins, D., Perkins, P., Richmond, G., Smith, P., Ward, R. A., Waring, M. J., Whittaker, D., Wells, S., and Wrigley, G. L. (2014) Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor, Journal of medicinal chemistry 57, 8249-8267.

[6] Ge, J., Chen, Q., Liu, B., Wang, L., Zhang, S., and Ji, B. (2017) Knockdown of Rab21 inhibits proliferation and induces apoptosis in human glioma cells, Cell Mol Biol Lett 22, 30.

[7] John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., and Goody, R. S. (1990) Kinetics of interaction of nucleotides with nucleotide-free H-ras p21, Biochemistry 29, 6058-6065.

[8] Awad, M. M., Liu, S., Rybkin, II, Arbour, K. C., Dilly, J., Zhu, V. W., Johnson, M. L., Heist, R. S., Patil, T., Riely, G. J., Jacobson, J. O., Yang, X., Persky, N. S., Root, D. E., Lowder, K. E., Feng, H., Zhang, S. S., Haigis, K. M., Hung, Y. P., Sholl, L. M., Wolpin, B. M., Wiese, J., Christiansen, J., Lee, J., Schrock, A. B., Lim, L. P., Garg, K., Li, M., Engstrom, L. D., Waters, L., Lawson, J. D., Olson, P., Lito, P., Ou, S. I., Christensen, J. G., Janne, P. A., and Aguirre, A. J. (2021) Acquired Resistance to KRAS(G12C) Inhibition in Cancer, N Engl J Med 384, 2382-2393.

[9] Goebel, L., Müller, M. P., Goody, R. S., and Rauh, D. (2020) KRasG12C inhibitors in clinical trials: a short historical perspective, RSC Medicinal Chemistry.

[10] Oehrl, W., Rubio, I., and Wetzker, R. (2003) Serine 338 phosphorylation is dispensable for activation of c-Raf1, J Biol Chem 278, 17819-17826.

[11] Forbes, S. A., Beare, D., Gunasekaran, P., Leung, K., Bindal, N., Boutselakis, H., Ding, M., Bamford, S., Cole, C., Ward, S., Kok, C. Y., Jia, M., De, T., Teague, J. W., Stratton, M. R., McDermott, U., and Campbell, P. J. (2015) COSMIC: exploring the world's knowledge of somatic mutations in human cancer, Nucleic Acids Res 43, D805-811.

[12] Visscher, M., Arkin, M. R., and Dansen, T. B. (2016) Covalent targeting of acquired cysteines in cancer, Curr Opin Chem Biol 30, 61-67.

[13] Burge, R. A., and Hobbs, G. A. (2022) Not all RAS mutations are equal: A detailed review of the functional diversity of RAS hot spot mutations, Advances in cancer research 153, 29-61.

https://doi.org/10.7554/eLife.82184.sa2

Article and author information

Author details

  1. Lisa Goebel

    Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
    Contribution
    Validation, Investigation, Visualization, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3477-1228

  2. Tonia Kirschner

    Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  3. Sandra Koska

    Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  4. Amrita Rai

    Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Contribution
    Investigation
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    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5471-5250

  5. Petra Janning

    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Contribution
    Investigation
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    No competing interests declared

  6. Stefano Maffini

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
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    Resources, Methodology
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    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6380-6560

  7. Helge Vatheuer

    Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  8. Paul Czodrowski

    Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
    Contribution
    Supervision, Methodology
    Competing interests
    No competing interests declared

  9. Roger S Goody

    Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing
    For correspondence
    roger.goody@mpi-dortmund.mpg.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0772-0444

  10. Matthias P Müller

    Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing
    For correspondence
    matthias3.mueller@tu-dortmund.de
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    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1529-8933

  11. Daniel Rauh

    Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
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    Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing
    For correspondence
    daniel.rauh@tu-dortmund.de
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    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1970-7642

Funding

Deutsche Forschungsgemeinschaft (RA 1055/5-1)

  • Roger S Goody

Deutsche Forschungsgemeinschaft (GO 284/10-1)

  • Daniel Rauh

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was co-funded by the Deutsche Forschungsgemeinschaft (DFG; RA 1055/5–1 and GO 284/10–1) and the Drug Discovery Hub Dortmund (DDHD). We thank Nathalie Bleimling, Andreas Arndt and Paul Siebers for invaluable technical assistance as well as Prof. Christian Herrmann for providing the plasmid for c-RafRBD. We acknowledge the Paul Scherrer Institut, Villigen, Switzerland for provision of synchrotron radiation beamtime at beamline X10SA of the SLS and would like to thank the staff of the SLS for assistance.

Senior and Reviewing Editor

  1. Volker Dötsch, Goethe University, Germany

Version history

  1. Preprint posted: July 25, 2022 (view preprint)
  2. Received: July 26, 2022
  3. Accepted: March 3, 2023
  4. Version of Record published: March 27, 2023 (version 1)
  5. Version of Record updated: April 12, 2023 (version 2)

Copyright

© 2023, Goebel et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Lisa Goebel
  2. Tonia Kirschner
  3. Sandra Koska
  4. Amrita Rai
  5. Petra Janning
  6. Stefano Maffini
  7. Helge Vatheuer
  8. Paul Czodrowski
  9. Roger S Goody
  10. Matthias P Müller
  11. Daniel Rauh
(2023)
Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors
eLife 12:e82184.
https://doi.org/10.7554/eLife.82184

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