Shuhui
Lim‡
a,
Nicolas
Boyer‡
b,
Nicole
Boo
a,
Chunhui
Huang
b,
Gireedhar
Venkatachalam
a,
Yu-Chi
Angela Juang
a,
Michael
Garrigou
b,
Hung Yi Kristal
Kaan
a,
Ruchia
Duggal
b,
Khong Ming
Peh
a,
Ahmad
Sadruddin
a,
Pooja
Gopal
a,
Tsz Ying
Yuen
c,
Simon
Ng
c,
Srinivasaraghavan
Kannan
c,
Christopher J.
Brown
c,
Chandra S.
Verma
c,
Peter
Orth
d,
Andrea
Peier
d,
Lan
Ge
d,
Xiang
Yu
e,
Bhavana
Bhatt
e,
Feifei
Chen
e,
Erjia
Wang
e,
Nianyu Jason
Li
e,
Raymond J.
Gonzales
e,
Alexander
Stoeck
b,
Brian
Henry
a,
Tomi K.
Sawyer
b,
David P.
Lane
c,
Charles W.
Johannes
*c,
Kaustav
Biswas
*b and
Anthony W.
Partridge
*a
aMSD International, Singapore 138665, Singapore. E-mail: awpartridge@gmail.com
bMerck & Co., Inc., Boston, Massachusetts 02115, USA. E-mail: kaustav.biswas@merck.com
cAgency for Science, Technology and Research (A*STAR), Singapore 138665, Singapore. E-mail: cwjohannes@gmail.com
dMerck & Co., Inc., Kenilworth, New Jersey 07033, USA
eMerck & Co., Inc., West Point, Pennsylvania 19486, USA
First published on 25th November 2021
Macrocyclic peptides have the potential to address intracellular protein–protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure–activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight – the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry – an approach that has yet to succeed in the clinic despite a long history of attempts – carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers – some of the most treatment-resistant human malignancies.
Several research groups have indeed reported high affinity bona fide peptide binders to KRAS that represent valuable starting points for drug discovery.9–12 In particular, Sakamoto et al. used phage display to identify a disulfide cyclized peptide with the sequence Ac-Arg1-Arg2-Arg3-Arg4-Cys5-Pro6-Leu7-Tyr8-Ile9-Ser10-Tyr11-Asp12-Pro13-Val14-Cys15-Arg16-Arg17-Arg18-Arg19-NH2.10 This molecule, termed KRpep-2d (see Fig. 1A), bound KRASG12D with nanomolar affinity in both the GDP and GTP analog loaded states. Alanine-scanning mutagenesis identified Leu7, Ile9, and Asp12 as critical binding residues.13 X-ray crystallography revealed the binding site to be near Switch II, allosterically blocking the interaction of KRAS with the guanine nucleotide exchange factor, SOS1.14 Subsequently we15 and others12 verified this peptide as having high-affinity and stoichiometric binding to KRAS. Although KRpep-2d represents a promising and novel KRAS binder, we concluded that structural modifications were required to render it cell-active. In particular, the disulfide crosslink is not expected to remain intact within the reducing environment of the cytosol. Furthermore, additional medicinal chemistry optimization could address potential peptide stability and permeability deficiencies.
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Fig. 1 Replacement of the KRpep-2d's disulfide bridge with a D-Cys5–CH2–L-Cys15 thioacetal linkage results in a redox-stable, high affinity peptide. (A) SPR analysis shows that oxidized (cyclic) but not reduced (linear) KRpep-2d binds with high affinity to GDP-loaded KRASG12D. The chemical structure is shown, with the N- and C-terminal arginine chains and disulfide linker highlighted in red. (B) Redox-stable peptides MP-6483 and MP-4090 display cell-based inhibition of KRAS signaling (pERK and pAKT) in AsPC-1 cells, as assessed by Western blot at 1 hour and 16 hour time-points; non-binding control enantiomeric peptide MP-4956 showed no activity (Left). Sequences for the peptides are shown. (C) MP-6483 and MP-4090 but not the non-binder controls (MP-9657 and MP-9658) display cell-based inhibition (pERK) in AsPC-1 cells, as assessed by Alpha SureFire Ultra Multiplex Phospho/Total ERK1/2 assay (Perkin Elmer) when treated for 1 h (n = 6, black symbols) and 18 h (n = 2, red symbols). The D-Cys5 residue, N- and C-terminal arginine residues, core modifications and thioacetal linker is highlighted in red (D) the same lysates in panel C were also assessed for membrane toxicity, as measured by the CytoTox-ONE™ homogenous membrane integrity assay (Promega). (E) Superimposition of co-crystal structures involving (i) KRpep-2d in complex with KRASG12D (GDP) (PDB ID 5XCO, blue) and (ii) MP-9903, a peptide containing the D-Cys5–CH2–L-Cys15 thioacetal linkage, with KRASG12D (GMPPCP) (PDB ID 7ROV, pink), shows a highly similar KRAS conformation and that binding of MP-9903 involves a cis peptide bond between D-Cys5 and Pro6. For clarity, terminal arginine residues are either transparent or hidden. The D-Cys5 residue, N- and C-termini and thioacetal linker are highlighted in red in the structure and the termini changes to MP-6483 is noted. |
To achieve cell permeability, cationic and hydrophobic residues are often incorporated into peptides targeting intracellular proteins. However, these design features can confound the interpretation of biochemical and functional assays.15 Indeed, some recently reported putative peptidic KRAS inhibitors incorporating these elements were, in fact, false positives.15 Thus, we took extra caution in our studies with the KRpep-2d scaffold since it contains several hydrophobic residues and a total of eight exocyclic arginines. In particular, we applied rigorous controls in the experiments described herein to ensure that the macrocyclic peptides we designed were not only bona fide KRAS binders but also authentic, on-target cell active molecules. These peptides therefore represent a new approach towards blocking KRAS-driven signaling beyond the G12C mutation, an area of high unmet need.
Using KRpep-2d as the starting point, we sought to improve binding affinity, increase proteolytic stability, and impart membrane permeability to advance a molecule capable of blocking cellular KRAS signaling. After exploring different strategies, we successfully replaced the disulfide bond with a thioacetal crosslink involving a D-Cys residue at position 5. X-ray crystallography revealed that peptides using this macrocyclization motif bind to KRAS in a similar manner but with a cis peptide bond between D-Cys5 and Pro6. Replacing the N- and C-terminal arginine residues with their D-amino acid counterparts produced a peptide with weak cellular activity. Introduction of an α-methyl group at Ser10 resulted in a molecule (MP-3995) with prolonged proteolytic stability and cellular blockade of pERK activity in KRASG12D (AsPC-1) cells but was inactive against a KRASWT cancer line (A375) harboring BRAFV600E, a MAPK pathway activating mutation that is downstream of KRAS. On-target cellular activity was further verified using non-binding peptide controls, counter-screens, and target engagement assays. In a panel of cancer cell lines, MP-3995 inhibited proliferation in KRAS dependent lines but not in KRAS independent lines. Despite these favorable attributes, we identified the arginine-rich nature of this series to be a barrier for further development as strong histamine release was observed in an ex vivo assay. Initial attempts to reduce the flanking Arg residues resulted in a loss of membrane permeability and cellular activity. Nevertheless, the peptides described in this report represent a valuable scaffold for the genesis of novel validated inhibitors of KRAS signaling in cancer patients unserved by current successes with covalent G12C inhibition.
Compound | Sequence | Linker | KRASG12D GDP TR-FRET EC50 (nM) |
---|---|---|---|
a Reducing conditions (1 mM DTT), see Fig. S2. Lower case letters represent D amino acids. | |||
KRpep-2d | Ac-RRRR-cyclo(CPLYISYDPVC)-NH2 | L-Cys5–L-Cys15 | 1916 (>50![]() |
1 | Ac-K(N3)-RRRR-cyclo(DPLYISYDPV-Dap)-RRRR-NH2 | L-Asp5–L-Dap15 (lactam) | >50![]() |
2 | Ac-K(N3)-RRRR-cyclo(dPLYISYDPV-Dap)-RRRR-NH2 | D-Asp5–L-Dap15 (lactam) | 19![]() |
3 | Ac-K(N3)-RRRR-cyclo(C(methylene)PLYISYDPVC)-RRRR-NH2 | L-Cys5–CH2–L-Cys15 (thioacetal) | 13![]() |
MP-6483 | Ac-K(N3)-RRRR-cyclo(c(methylene)PLYISYDPVC)-RRRR- NH2 | D-Cys5–CH2–L-Cys15 (thioacetal) | 172 (93a) |
4 | Ac-K(N3)-RR-cyclo(c(methylene)PLYISYDPV-hC)-RR-NH2 | D-Cys5–CH2–L-homoCys15 (thioacetal) | 4719 |
Compound | Modification | KRASG12D GDP TR-FRET EC50 (nM) |
---|---|---|
a The D-Cys5 residue and thioacetal linker is highlighted in red in the structure. The N- and C-termini are shown. MP-1687 has the same macrocyclic core as MP-6483, with only two arginine residues at each terminus. | ||
MP-1687 | None | 60 |
5 | Pro6Ala | >48![]() |
6 | Leu7Ala | >48![]() |
7 | Tyr8Ala | 691 |
8 | Ile9Ala | >48![]() |
9 | Ser10Ala | 1072 |
10 | Tyr11Ala | 2430 |
11 | Asp12Ala | >48![]() |
12 | Pro13Ala | 427 |
13 | Val14Ala | 161 |
![]() |
Compound | Peptide sequencea | Changes | KRASG12D GDP TR-FRET EC50 (nM) | AsPC-1 pERK EC50 1 h/18 h (μM) | AsPC-1 LDH EC50 1 h/18 h (μM) | A375 pERK EC50 1 h/18 h (μM) | NanoClick EC50 4 h/18 h (nM) |
---|---|---|---|---|---|---|---|
a Lower case letters represent D-amino acids. NT = not tested. | |||||||
MP-6483 (binder) | Ac-K(N3)-RRRR-cyclo(c(methylene)PLYISYDPVC)-RRRR-NH2 | None | 172.4 | 15.9/>50 | >50/>50 | NT | 2693/636 |
MP-4090 (binder) | Ac-K(N3)-rrrr-cyclo(c(methylene)PLYISYDPVC)-rrrr-NH2 | All D-Arg | 140.7 | 3.6/30.5 | >50/>50 | >50/>50 | 250.5/34.9 |
MP-3995 (binder) | Ac-KN3)-rrrr-cyclo(c(methylene)PLYI-αMeS-YDPVC)-rrrr-NH2 | All D-Arg, Ser10α-Me-Ser | 172.1 | 1.2/1.5 | >50/>50 | >50/>50 | 1777/32.1 |
MP-4956 (non-binder) | Ac-rrrr-cyclo(c(methylene)plyisydpvc)-rrrr-NH2 | All D-peptide | >48![]() |
>50/>50 | >50/>50 | NT/>50 | NT |
MP-9657 (non-binder) | Ac-K(N3)-rrrr-cyclo(c(methylene)PLYiSYDPVC)-rrrr-NH2 | All D-Arg, Ile9D-Ile | >48![]() |
>50/>50 | >50/>50 | NT | 1756/36.2 |
MP-9658 (non-binder) | Ac-K(N3)-rrrr-cyclo(c(methylene)PLYISYdPVC)-rrrr-NH2 | All D-Arg, Asp12D-Asp | >48![]() |
>50/>50 | >50/>50 | NT/>50 | 196.1/31.4 |
We also designed a series of non-binding control peptides including an all-D version of MP-6483 (MP-4956) and peptides with enantiomeric substitutions at critical binding residues, Ile9 to D-Ile (MP-9657) and Asp12 to D-Asp (MP-9658) (Table 3). None of these peptides showed any binding in the TR-FRET assay (EC50 > 48080 nM) nor a capacity to inhibit cellular function (Fig. 1B, C), despite evidence of permeability in the NanoClick assay (Table 3). They also did not induce membrane damage as assessed by the LDH release assay (Fig. 1D). Furthermore, MP-4090 and its non-binding control (MP-9658) were inactive in A375 and SK-MEL-28 cells (Fig. S5A†), counter-screen cell lines harboring BRAFV600E, a mutant that activates the MAPK pathway downstream of KRAS. AZ628 (BRAF inhibitor,18) and U0126 (MEK inhibitor,19), small molecules that inhibit the pathways downstream of KRAS, inhibited MAPK signaling in these BRAF mutant lines in the expected manner (Fig. S5A†). Live-cell imaging using FAM-labelled versions of these macrocyclic peptides confirmed that permeability was comparable between AsPC-1 and A375, but poorer in SK-MEL-28 (Fig. S5B†). In addition, MP-4090 had no effect on upstream EGF receptor activation (Fig. S6A†) and TNFα-stimulated NFκB signaling (a KRAS-independent pathway) (Fig. S6B†) in AsPC-1 cells. This series of control experiments suggested that the cellular KRAS-inhibitory activity measured for MP-4090via inhibition of ERK phosphorylation was indeed on-target.
To stabilize the peptide against proteolysis, we synthesized analogs with Tyr8 and Ile9 modifications including α-methylation and backbone homologation, however all were inactive in the TR-FRET assay (data not shown). We then expanded the α-methyl scan to additional residues and tested the resulting peptides for binding and stability (Table 4). Due to challenges with detecting highly cationic 8-Arg peptides on MS instrumentation, we carried out this study on the tetra-arginine parent peptide MP-1687, described in Table 2 previously. α-Methylation was tolerated at positions 10 (Ser, 17) and 13 (Pro, 20) without any change in binding affinity when compared to MP-1687, with a modest 4-fold loss observed at position 14 (Val, 21). In contrast, potency loss was observed at all other positions, ranging from 30-fold at Tyr11 to 800-fold at positions 8 or 15. Position 5 and 9 were not investigated. Comparison of the HeLa cell homogenate half-lives of the α-methylated analogs to the parent MP-1687 showed improvements ranging from >10-fold for position 8 to >4-fold for position 10 and >3-fold for position 13 modifications. Considering both the potency and stability data, the α-methyl-Ser10 mutation was selected for inclusion in future designs. Incorporating the α-methyl-Ser10 modification into our previous lead sequence (MP-4090) led to MP-3995, a peptide with sustained pERK inhibition with EC50 values of 1.2 and 1.5 μM at 1 and 18 hours, respectively (Table 3), eliminating the 8-fold cell potency loss seen with MP-4090 at 18 h vs. 1 h. This peptide also did not induce membrane damage as assessed by the LDH release assay and did not inhibit pathway signaling in the RAS-independent A375 cell line with the BRAFV600E mutation (Table 3). The key steps in SAR evolution from KRpep-2d to MP-3995 is depicted in Fig. 2C.
Compound | Modification | KRASG12D GDP TR-FRET EC50 (nM) | HeLa t1/2 (min) |
---|---|---|---|
MP-1687 | None | 60 | 37 |
14 | α-Me-Pro6 | 10![]() |
142 |
15 | α-Me-Leu7 | 11![]() |
114 |
16 | α-Me-Phe8 | 46![]() |
>373 |
17 | α-Me-Ser10 | 62 | 154 |
18 | α-Me-Tyr11 | 1800 | 71 |
19 | α-Me-Asp12 | 3705 | 41 |
20 | α-Me-Pro13 | 67 | 134 |
21 | α-Me-Val14 | 222 | NA |
22 | α-Me-Cys15 | 48![]() |
120 |
![]() | ||
Fig. 3 KRpep-2d peptide analogs have dual inhibitory mechanisms. (A) MP-3995 and MP-9903 potently inhibited SOS-mediated nucleotide exchange. The D-Cys5 residue, N- and C-termini, core modifications and thioacetal linker are highlighted in red in the structure. (B) MP-6483, MP-4090, MP-3995, and MP-9903 showed a superior capacity to block the KRAS-RBD PPI compared to KRpep-2d, whereas the non-binder control (MP-4956) had no activity. (C) MP-6483 and MP-1687 blocked the co-immunoprecipitation of b-RAF (left panel) and c-RAF (right panel) with KRASG12D. Alanine mutant peptide library analogs of MP-1687 (Table S1†) demonstrated that KRAS binding affinity correlated well with disruption of the PPI. (D) MP-3995 blocked phospho-ERK signaling in cells expressing either NanoLuc-KRASG12C or NanoLuc-KRASG12C/A59G whereas AMG 510 only inhibited NanoLuc-KRASG12C, non-binders (MP-4956 and MP-9658) had no activity. |
Compound | Sequencea | KRASG12D GDP TR-FRET EC50 (nM) | AsPC-1 pERK EC50 1 h/18 h (μM) | # of Arg | rMCD histamine release threshold (μM) | NanoClick EC50 4 h/18 h (nM) |
---|---|---|---|---|---|---|
a Lower case letters represent D-amino acids. | ||||||
MP-4090 | Ac-K(N3)-rrrr-cyclo(c(methylene)PLYISYDPVC)-rrrr-NH2 | 140.7 | 3.6/30.8 | 8 | ≥0.14 | 251/35 |
MP-6483 | Ac-K(N3)-RRRR-cyclo(c(methylene)PLYISYDPVC)-RRRR-NH2 | 172.4 | 15.9/>50 | 8 | ≥0.14 | 2693/636 |
23 | Ac-K(N3)-RRR-cyclo(c(methylene)PLYISYDPVC)-RRR-NH2 | 75.8 | >50/>50 | 6 | ≥0.14 | 3314/>10![]() |
MP-1687 | Ac-K(N3)-RR-cyclo(c(methylene)PLYISYDPVC)- RR-NH2 | 59.4 | >50/>50 | 4 | ≥3.7 | 4552/4711 |
MP-9903 | Ac-K(N3)-R-cyclo(c(methylene)PLYISYDPVC)-R-NH2 | 138.6 | >50/>50 | 2 | >100 | 6939/6828 |
24 | Ac-K(N3)-cyclo(c(methylene)PLYISYDPVC)-R-NH2 | 187.6 | >50/>50 | 1 | >100 | >10![]() |
25 | Ac-K(N3)-cyclo(c(methylene)PLYISYDPVC)-NH2 | 409.5 | >50/>50 | 0 | >100 | >10![]() |
The discovery of KRpep-2d, a macrocyclic peptide with validated high-affinity binding, represented an attractive starting point for the identification of a non-G12C KRAS inhibitor. However, we recognized that the disulfide-mediated macrocyclization strategy represented a barrier to cellular activity since it would likely be rapidly reduced leading to the non-binding linear form in the intracellular compartment. Indeed, in our hands, this molecule had no effect on cellular KRAS signaling and exhibited a very short half-life in cell homogenate. Most attempts to replace the disulfide bond with a non-reducible linkage resulted in peptides with forfeited KRAS binding. However, by extending our search to include variation of stereochemical centers, we discovered that the linkage of a D-Cys5 residue to Cys15 through a thioacetal bridge resulted in a redox stable, high affinity binder. Next, replacing the proteolytically unstable N- and C-terminal tetra-arginine tails with their enantiomeric counterparts, resulted in a peptide with cellular activity. Addressing a metabolic soft spot within the macrocycle through a Ser10 to α-methyl-Ser substitution further improved metabolic stability and consequently sustained cellular activity (Table 3, Fig. 2C). Such constrained building blocks can force peptides into their biologically active conformations, while often providing remarkable resistance to enzymatic degradation.
Studies by us and others10–13 suggest that the KRpep-2d peptides series might inhibit KRAS signaling in at least two distinct ways, by directly blocking the interaction with KRAS effectors (e.g., RAF) as well as by indirectly preventing these interactions by blocking the conversion of the GDP (off) state to the GTP (on) state. Both activities may indeed be contributing to cellular KRAS inhibition. Dual inhibition of mutant KRAS signalling is attractive, especially considering the observation that cancer cells can reactivate the MAPK pathway to resist G12C covalent inhibitors,25 molecules that trap the protein in the GDP state.21 Initial studies suggest that such compensatory mechanisms may not occur with KRpep-2d family members. First, pathway up-regulation with EGF stimulation did not alter the potency of MP-4090 (Fig. S9†). In addition, biasing KRAS protein to the GTP (on) state with the G12C/A59G double mutant prevented pERK inhibition with AMG 510 (sotorasib) but not MP-3995 (Fig. 3D), suggesting that this peptide can continue to prevent mutant KRAS signaling even when the protein is pushed into the active state.
A key foundation for the identification of bona fide, functionally active peptides targeting intracellular proteins is the application of stringent experimental controls, both chemical and biological in nature. This is important as cationic and hydrophobic elements that are often highly prevalent in cell penetrant peptides can lead to false positive cellular read-outs through cell membrane disruption. Indeed, this has been shown specifically for peptides that have been incorrectly reported to have on-target cellular activity against KRAS.15 Recently, it was demonstrated that these features can also lead to phospholipidosis, at least when present in small molecules.26 Thus, we applied a host of chemical and biological controls in our studies. For the former, we made a series of control peptides designed to be as similar as possible to the cell active molecules but devoid of KRAS binding. This was accomplished through stereo-inversion of all or key binding amino acids. The observation that these peptides were indeed non-binders but also inactive in our pERK assay suggested that the cell activity of our lead peptides (MP-4090 and MP-3995) was due to the consequences of KRAS binding. A series of biological controls gave us further confidence that these peptides had bona fide cellular activity. Indeed, the absence of LDH release and off-target activity in an irrelevant signaling pathway was consistent with the on-target nature of this series. As well, the lack of pERK and proliferation inhibition activities in a KRAS independent line (A375) further suggested that off-target mechanisms were not involved. In addition, evidence of direct target engagement was provided by peptide-induced KRAS thermal stabilization in a CETSA® assay and through the capacity of these peptides to displace GFP–RBD–CRD protein from the cell membrane. In both cases, non-binding control peptides gave negative results, as expected. Overall, given the propensity of macrocyclic peptides for both biochemical and cellular false-positive signals,15 we advocate that such studies routinely apply an array of biological and chemical controls. A relevant counterexample to our work is that published by Sakamoto et al.11 This work described an interesting set of amino acid substitutions (including non-natural residues) and disulfide replacements (including bicyclic designs) to the KRpep-2d scaffold. Although the studies were extensive, we found the results difficult to interpret since there was a paucity of both biological and chemical controls. In particular, the peptides were not tested for membrane disruption activity which could artificially influence pERK readouts due to the leakage of cellular contents. In addition, other relevant counter-screens were not applied – such as those involving KRAS independent lines (e.g., HEK293 and A375). The work we reported here highlighted the lack of cell permeability in the original KRpep-2d scaffold which we successfully engineered and validated using cell permeability readouts like imaging and our internally developed NanoClick assay.16 It was not clear if KS-58 was cell permeable based on the reported data. In addition, no evidence for cellular target engagement was provided and cell proliferation assays were conducted at a single, relatively high (30 μM), concentration, rather than in a dose dependent manner. The reported in vivo study was also particularly challenging to interpret since the xenografts did not grow from day 5 to day 29 in the control group. Despite these shortcomings, KS-58 may indeed represent important improvements to the original peptide and should be considered for future efforts aimed at advancing the KRpep-2d family of peptides towards the clinic.
The reliance on poly-arginine sequences at the N- and C-termini make it challenging to progress the KRpep-2d analogs studied here toward the clinic. Indeed, ex vivo mast cell degranulation studies confirmed a strong correlation between the number of arginine residues and histamine release, a phenomenon that can lead to serious consequences in vivo. Unfortunately, reducing the number of arginines also correlated with a loss of cell permeability and subsequent cellular activity, therefore confounding their pipeline progression.
Four decades of KRAS research has started to bear fruit with the 2021 approval of the first direct inhibitor of a specific KRAS mutation, G12C. The search for efficacious inhibitors of all other mutant KRAS-driven cancers continues. Despite the structural liabilities identified by us, the peptides described herein represent valuable templates for achieving in vivo activity against KRAS-driven, non-G12C cancers. These efforts would focus on identification of sequence variants that achieve cell entry without dependence on arginine-rich sequences. Our efforts directed towards such objectives will be the subject of future communications.
Overall, systematic studies reported here made key advances on this peptide series and used rigorous controls to validate the potential of blocking mutant-KRAS function in cancer cells via binding to this unique epitope. As such, novel avenues are open for impacting KRAS-mediated cancers beyond the recent successes with covalent G12C modulation.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1sc05187c |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2021 |