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. 2022 Sep 26;2(6):617–626. doi: 10.1021/acsbiomedchemau.2c00045

KRAS Inhibitor that Simultaneously Inhibits Nucleotide Exchange Activity and Effector Engagement

Cynthia V Pagba †, Amit K Gupta †, Ali K Naji ∥, Dharini van der Hoeven ∥, Kelly Churion ⊥, Xiaowen Liang †, Jacob Jakubec †, Magnus Hook ⊥, Yan Zuo †, Marisela Martinez de Kraatz †, Jeffrey A Frost †,§, Alemayehu A Gorfe †,‡,*
PMCID: PMC10125367  PMID: 37101428

Abstract

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We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

Keywords: Ras protein, inhibitor, signal transduction, anticancer drug, allosteric inhibition

Introduction

RAS proteins are small GTPases that play crucial roles in cell signaling pathways that regulate cell growth and proliferation. They act as molecular switches by cycling between active GTP-bound and inactive GDP-bound conformational states. This cycle is regulated by GTPase activating proteins (GAPs) and nucleotide exchange factors (GEFs).1 Mutations in RAS disrupt this process and result in constitutive activation, which then leads to uncontrolled cell growth, proliferation, and cancer.2,3 Mutations in KRAS, one of the three most common human RAS proteins, are associated with ∼20% of all cancers, including >90% of pancreatic cancers, and >40% of colon cancers.2,4,5 However, developing direct inhibitors of KRAS has been elusive4,5 partly due to the very high affinity (picomolar)6,7 of KRAS for its endogenous ligands GDP and GTP, which are abundant in the cell and prevent potential drugs from effectively competing for active site inhibition.8−10 Hence, current efforts are directed toward allosteric inhibition. Our previous work,11−14 along with that of others,15−17 identified multiple allosteric sites/pockets that can potentially be targeted for inhibition by small molecules. Several small molecules that target these pockets and inhibit RAS activities have already been reported.18−29 Covalent inhibitors targeting KRAS(G12C) are either in clinical trials (e.g., adagrasib) or recently approved by the FDA (sotorasib).30−32 However, their application is limited to a few cancer types such as small cell lung cancer. Non-covalent allosteric inhibition will be needed to target many important KRAS mutations, including KRAS(G12D), KRAS(G12V), KRAS(G13D), and KRAS(Q61H). These mutations account for >78% of KRAS-associated cancers4,33 and are common in, among others, colon, lung, and pancreatic cancers. It is becoming increasingly clear that development of non-covalent KRAS inhibitors is possible, with the remaining challenges being overcoming problems of weak affinity and pan-RAS activity (e.g., refs (22), (34–36)).

In the current work, we describe the KRAS binding and inhibitory profile of 2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid (ACA-14), which was predicted to target pocket p1 in our previous study combining molecular docking with nuclear magnetic resonance (NMR) experiments.19 We found that ACA-14 binds KRAS(WT) and various oncogenic KRAS mutants, including KRAS(G12D), in the low, single digit micromolar range but does not bind to HRAS or NRAS. The compound binds KRAS in a nucleotide state-independent manner and inhibits KRAS functions in biochemical assays by modulating both the intrinsic and GEF-mediated GDP/GTP exchange rates, as well as by directly disrupting effector binding. We also show that ACA-14 inhibits MAPK signaling in KRAS(G12D)-expressing baby hamster kidney (BHK) cells, inhibits the proliferation of oncogenic KRAS-dependent cell lines, and reduces tumor growth in a KRAS(G12C)-driven MIAPaCa-2 xenograft mouse model. The cellular and in vivo activity profiles suggest a somewhat higher IC50 than suggested by the dissociation constants measured in vitro using truncated (residues 1–169) KRAS constructs, which we show is likely due to the limited cell permeability of the compound.

Materials and Methods

Materials and detailed methods are provided in the Supporting Information. These include thermal shift assay (TSA), isothermal titration calorimetry (ITC), and fluorescence-based techniques such as microscale thermophoresis (MST) in purified systems for the determination of the binding affinity of ACA-14 to wild type (WT) and some of the most prevalent oncogenic KRAS mutants including KRAS(G12D), KRAS(G12V), KRAS(G13D), KRAS(G12C), and KRAS(Q61H). Fluorescence-based techniques have also been used to biochemically characterize the inhibitory profile of the compound via the determination of the rates of intrinsic and SOS1 (a RAS GEF)-mediated nucleotide exchange and release reactions in KRAS(WT), and the interactions of KRAS(WT) with the RAS binding domain (RBD) of c-Raf. Additional experiments include cell-based pull-down and immunoblotting assays to examine the ability of ACA-14 to inhibit KRAS(G12D)–RafRBD interactions and KRAS(G12D)-mediated signaling through the MAPK pathway, as well as cell proliferation assays to examine the impact of the ligand on the growth of a variety of cancer cell lineages. The compound has also been tested in an in vivo setting using a xenograft mouse tumor model, as detailed in the Supporting Information.

Results and Discussion

Figure 1A displays the chemical structure of compound ACA-14 (cLogP = 3.35) that we have previously identified as a KRAS binder based on combined computational and NMR analyses (named compound V14 in ref (19)). However, the previous work did not go beyond the detection of ACA-14 binding to KRAS. We have now conducted an extensive structure/activity relationship analysis and summarize our findings as follows.

Figure 1.

Figure 1

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(A) Chemical structures of ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid). (B) Structure of GTP-KRAS(G12D)-derived from MD simulations, depicting pocket p1 occupied by ACA-14 (green sticks and sphere). Structural regions near p1 involved in nucleotide binding and undergoing major conformational changes during GDP/GTP exchange are labeled: P-loop (orange, residues 10–17), switch I (purple, residues 25–40), and switch II (yellow, residues 60–74). Selected KRAS residues that are predicted to interact with the ligand are highlighted. (C,D) Detailed interactions of ACA-14 (green sticks) with KRAS residues are shown in surface representation colored by electrostatic potential (C) or in a stick model (D).

Predicted Binding Pose of ACA-14 at the p1 Pocket of KRAS

A common target for ACA-14 and another series of previously reported compounds21 was pocket p1. The predicted binding pose of ACA-14 and KRAS residues involved in interactions with the ligand are shown in Figure 1B–D. Among these are a carboxyl oxygen from the benzoic acid moiety of ACA-14 being within a H-bonding distance (1.8 Å) from the amino group of K5, and the NH substituent that is 1.9 Å away from the side-chain carboxyl oxygen of E37 at the effector-binding loop. The phenyl ring of the ligand is 3.9 Å away from the side chains of V7 and L56, suggesting van der Waals (vdW) contacts between them. Moreover, the carbonyl group near the phenylcyclopropyl moiety is within a H-bonding distance (2.3 Å) from Q70 while the cyclopropyl ring is within a vdW distance from Y71. These residues are part of switch 2, which is crucial in structural rearrangements that accompany nucleotide exchange.

ACA-14 Binds to Active and Inactive WT and Oncogenic KRAS Mutants with Low Micromolar Affinity

We used two orthogonal assays in purified systems to assess whether ACA-14 directly interacts with KRAS: TSA and intrinsic fluorescence (IF). TSA is commonly used to monitor protein–ligand complex formation based on the principle that ligand binding alters the thermal stability [i.e., the melting temperature (Tm)] of the protein.37,38 The TSA profiles, as shown in Figure S1, indicate shifts in the Tm of GNP (GTP-analogue)-bound WT and selected KRAS mutants in the presence of 50 μM ACA-14, suggesting structural changes induced by ligand binding (Figure S1A–C). Interestingly, there is no measurable change in the Tm of NRAS and HRAS, which are close homologues of KRAS, when mixed with the same concentration of the compound (Figure S1D,E). This shows that ACA-14 interacts with KRAS in an isoform-specific manner. We also detected direct binding of ACA-14 to KRAS by taking advantage of the weak IF of the compound. Figure S2 displays concentration-dependent changes in the IF of the compound upon mixing varying concentrations of ACA-14 with fixed concentrations of KRAS(WT) or KRAS(G12D). While the weak fluorescence precludes quantitative detection of Kds using IF, the TSA and IF results together establish the direct binding of ACA-14 to both GTP- and GDP-bound KRAS(WT), KRAS(G12D), and KRAS(Q61H) but not to HRAS(WT) and NRAS(WT).

We used MST and ITC for a more quantitative assessment of the ligand–KRAS interactions. Representative titration/binding curves from both ITC and MST are shown in Figures 2 and S3, respectively, and Table 1 summarizes equilibrium dissociation constants derived from those curves. One can see that, first, MST and ITC yielded comparable Kd values for GDP-bound KRAS(WT) and KRAS(G12D), which provides confidence in the reliability of the MST data. Second, the combined MST and ITC results make clear that ACA-14 binds to both GDP-bound and GNP-bound forms of WT and most KRAS mutants, with the exception of GDP/GNP-KRAS(G12V) and GNP-KRAS(G13D), for which the MST data were noisy. An enthalpy-dominated (Figure 2) binding with Kd ≈ 1.0–50 μM (Table 1) was measured for the GDP-KRAS proteins. An enthalpy-dominated interaction of ACA-14 and KRAS is consistent with the substantial number of predicted hydrogen bonding and vdW interactions (Figure 1). The MST binding curves for GNP-bound KRAS(WT), KRAS(G12D), and KRAS(Q61H) suggest two transition events (Figure S3B,C) and dissociation constants (Table 1): a high affinity binding event with Kd ≈ 50–400 nM and a lower affinity binding event with Kd ≈ 3–10 μM. Preliminary ITC measurements also suggest the existence of an entropy-dominated, high-affinity binding event in GNP-bound KRAS, but the data were too noisy for quantitative analysis (not shown). A two-step transition in MST binding curves may suggest ligand binding at two different sites, which is possible because KRAS harbors multiple ligand-binding sites. For example, ACA-14 may bind KRAS at pockets p1 and p2 with different affinities. Alternatively, different isomers of ACA-14 may bind at the same pocket with different affinities. Resolving these issues by solving co-crystal structures, for example, would allow for a more complete structure–activity relationship study focused on identifying features that favor selective binding to activated KRAS. The current work, however, focuses on the weaker binding mode that is shared by the active and inactive forms of KRAS because it is more consistent with data from functional assays discussed in subsequent sections. Binding in the low micromolar range is also consistent with the docking results, which predicted binding free energies in the range of −6.5 to −7.5 kcal/mol at pocket p1.

Figure 2.

Figure 2

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ITC profiles of ACA-14 with (A) GDP-KRAS(WT), (B) GDP-KRAS(G12C), (C) GDP-KRAS(Q61H), and (D) GDP-KRAS(G13D) obtained using Malvern’s MicroCal PEAQ-ITC.

Table 1. Summary of Equilibrium Dissociation Constant (Kd) Values Derived from MST and ITCa.

  Kd (μM) from MST or (ITC)
KRAS GDP-bound GNP-bound
WT 4.5 ± 1.5 (1.1 ± 1.6) 0.05 ± 0.03; 10.0 ± 1.7b
G12D >50 (45.9) 0.35 ± 0.08; 2.5 ± 0.4b
G12C (11.8 ± 5.49) 0.34 ± 0.07
Q61H (1.04 ± 0.63) 0.02 ± 0.01; 3.5 ± 1.5b
G12V ND ND
G13D (34.1) ND
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a

ND = not determined (data were noisy).

b

The two Kds suggest the possibility of two binding sites with different affinities.

The combined data from TSA, IF, MST, and ITC demonstrate that ACA-14 is likely a pan-KRAS binder and interacts with both the active and inactive forms of WT and most of the prevalent oncogenic KRAS mutants with a single-to-double digit micromolar binding affinity. Still, there is up to five-fold difference in Kd among the various mutants in the GDP state [e.g., compare KRAS(Q61H) and KRAS(G12D) in Table 1] and up to a 10-fold difference among those in the GTP state (G12C vs WT; Table 1). This may be explained by the significant differences among KRAS mutants in dynamics, particularly at the flexible switch regions that undergo conformational changes during nucleotide exchange and GTP hydrolysis,39−43 or by variations in the physicochemical properties of pocket p1 among oncogenic KRAS mutants.19 Similarly, GDP-bound and GNP-bound KRAS differ at switch 1 and switch 2.40,44−46 Such structural changes may result in the rearrangement of residues proximal to ligand-binding sites p1 or p2.

ACA-14 Modulates Nucleotide Exchange and Disrupts Effector Binding

To examine the potential effects of ACA-14 on KRAS functions, we used fluorescent-based biochemical assays to monitor the rates of nucleotide dissociation or exchange reactions of KRAS, as well as interaction of KRAS with the RBD of its effector c-Raf. We found that ACA-14 modulates nucleotide exchange (Figure 3) but not GDP release/dissociation (Figure S4). Specifically, ACA-14 reduced the rates of both intrinsic and SOS-mediated GDP/GTP exchange reactions in a dose-dependent manner, with EC50 of 3.8 ± 0.6 and 2.1 ± 0.4 μM, respectively (Figure 3A,B). These EC50 values are remarkably close to the Kd of ACA-14 for KRAS(WT), as assessed by MST and ITC (Table 1), providing additional evidence for direct binding. The lack of effect on the rate of intrinsic or SOS-mediated GDP release (Figure S4) suggests that, while able to bind to the GDP state, ACA-14 may not directly affect the conformation of residues involved in GDP dissociation and/or SOS binding (see, e.g., refs (27)(47), and (48) for how SOS interacts with GDP-RAS). Its effect on GDP/GTP exchange may therefore be associated with ACA-14 sterically or allosterically hindering GTP loading, altering the structure of the nucleotide-free KRAS/SOS complex,49 or interfering with SOS binding to activated KRAS(WT) by altering the orientation of residues in the switch 2 region.27,47

Figure 3.

Figure 3

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Effect of ACA-14 on the intrinsic (A) and SOS-mediated (B) nucleotide exchange rates determined by monitoring the change in fluorescence intensity increase in BGTP upon binding to KRAS. Ligand concentrations: 0 (black circle), 1.56 (red square), 3.12 (green triangle), 6.25 (blue inverted triangle), 12 (purple left-sided triangle), 25 (gray right-sided triangle), and 50 μM (light-orange diamond). Fluorescence intensity (I) was normalized using the formula:27Inormalized = (Iraw – I0)/(Imax – I0) where Iraw is the fluorescence intensity at a given time, I0 is the intensity at 60 s, and Imax is the maximum fluorescence (of KRAS alone) at the end of the experiment. Linear (A) or single exponential (B) fits are superimposed as solid lines. Inset: calculated rates (×10–4 s–1) as a function of ligand concentrations; EC50 values derived by fitting the curves are shown. KRAS, BGTP, and SOS concentrations were 0.5 μM each, and the buffer (pH 7.5) contained 25 mM Tris Cl, 50 mM NaCl, and 10 mM MgCl2.

To evaluate the effect of ACA-14 on effector binding, we measured the affinity of BODIPY-GTP-γ-S (BGTP)-bound KRAS(WT) to RBD using fluorescence polarization (FP) assay (Figure 4A). The dissociation constant for the interaction of BGTP-KRAS and RBD (Kd = 1.15 ± 0.09 μM) increased by a factor of ∼5 (Kd = 6.14 ± 1.86 μM) in the presence of 20 μM ACA-14. This result is consistent with data from pull-down assay using BHK cells expressing GFP-tagged KRAS(G12D) (Figure 4B–D). The level of GFP-tagged GTP-KRAS(G12D) and endogenous RAS pulled down by the c-Raf RBD was significantly reduced when lysates were treated with 10 μM or more of the ligand. These results strongly suggest that ACA-14 inhibits the binding of KRAS to its effector c-Raf, possibly by blocking KRAS residues involved in c-Raf binding (such as the effector-interacting E37,50 see Figure 1D). Together, our analyses using purified proteins and cell lysates demonstrate that ACA-14 modulates KRAS functions by both decelerating the rate of nucleotide exchange and impeding interaction with effector proteins. The ability of ACA-14 to bind both the active and inactive forms of KRAS likely explains its dual action. We propose that such broad-acting KRAS inhibitors can have major therapeutic value upon further optimization because they have the potential to simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

Figure 4.

Figure 4

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ACA-14 disrupts KRAS-Raf interaction (A–D) and inhibits MAPK signaling (E–H). (A) Effect of ACA-14 on the interaction of varying concentrations of GST-RBD of Raf with 0.5 μM BGTP-γ-S-KRAS in the absence (red; Kd = 6.14 ± 1.86 μM) and presence of 20 μM ACA-14 (1.15 ± 0.09). The dissociation constant (Kd) was derived from the quadratic ligand-binding equation55,56Inline graphic, where P1 and P2 are polarization of free and RBD-bound KRAS, respectively, c is the total concentration of KRAS, and x is the total concentration of RBD. (B–D) Representative western blots, including loading controls total GFP-KRAS(G12D) and total endogenous RAS (B), level of GFP-tagged GTP-KRAS(G12D) (C), and endogenous RAS (D) pulled-down by GST-RBD after treatment of BHK cell lysates ectopically expressing GFP-tagged KRAS(G12D) with increasing concentrations of ACA-14. (E) Representative western blots of whole cell lysates from BHK cells expressing GFP-tagged KRAS(G12D) treated for 3 h with vehicle (DMSO) or the indicated concentrations of ACA-14 under serum-starved condition. Vinculin was used as a loading control. (F–H) Quantitation of the levels of phosphorylated c-Raf (p-cRaf) (F), ERK (p-ERK) (G), and AKT (p-AKT) (H). (C,D,F–H) Values are normalized against DMSO control and averaged from three independent experiments. Statistical significance was calculated between vehicle (DMSO) and compound-treated samples using unpaired Student’s t test.

ACA-14 Inhibits Signaling through the MAPK Pathway and the Proliferation of Cancer Cells Expressing Mutant KRAS

We used cell signaling and proliferation assays to check whether the inhibitory activities of ACA-14 observed in the biophysical and pulldown assays described above translated into inhibitory activities in the cell. In the signaling assays, BHK cells expressing KRAS(G12D) were treated with compound or DMSO control, and the levels of phosphorylated KRAS effectors were monitored by immunoblotting. Figure 4E–H shows that ACA-14 significantly reduced the levels of phosphorylated c-Raf (p-cRaf) and ERK (p-ERK) but had little effect on p-AKT levels in the concentration range tested. This suggests that ACA-14 selectively inhibits KRAS signaling through the MAPK pathway, consistent with its effect on the interaction of KRAS(WT) with RafRBD (Figure 4A–D).

We then examined the inhibitory effects of ACA-14 on cell proliferation using selected colon and pancreatic cancer cell lines (Figure 5A). Up to 100 μM of ACA-14 had no effect on the proliferation of the KRAS-independent BxPC-3 [pancreatic adenocarcinoma cells expressing KRAS(WT)], CaCO-2 [a colon cancer cell harboring KRAS(WT)], or HPNE [normal, immortalized pancreatic duct cells with KRAS(WT)]. On the other hand, treatment with 25–100 μM of ACA-14 significantly inhibited the proliferation of pancreatic cancer cells that express mutant KRAS, including KRAS(G12C) (MIAPaCa-2), KRAS(G12R) (MOH), and KRAS(G12D) (PANC-1). These are all KRAS-dependent cell lines (see, e.g., ref (51) and references therein). We obtained similar results for the colon cancer cell lines SW116 and SW948 (Figure 5A), which harbor KRAS(G12A) and KRAS(Q61L), respectively.52 The higher concentration (>20 μM) required to inhibit MAPK signaling (Figure 4) or the proliferation of cancer cells expressing various mutants of KRAS (Figure 5A) may be due to the weak cell permeability of the ligand. This is supported by data from initial permeability tests in a MDR1-MDCK cell monolayer system (Table 2), which show that the rate of permeability of ACA-14 into the cell is about six-fold less than that for the control ligand propranolol, which has high cell permeability. Table 2 also shows that the efflux ratio of ACA-14 (0.72 nm/s) is comparable to that of propranolol (0.52 nm/s) but differs from amprenavir by 24-fold. Amprenavir is a known substrate of the efflux pump P-glycoprotein (Pg-P) while propranolol is not (note: efflux ratio >2.0 indicates that a ligand is a Pg-P substrate). This result suggests that once in the cell, ACA-14 is not readily expelled and thus likely has a long half-life and good bioavailability. Indeed, we found that ACA-14 exhibits a very low rate of intrinsic clearance (<0.3 mL/min/g) and has a long half-life in mouse, rat, and human microsomes (>180 min). Interestingly, this activity profile mirrors that of the previously reported KRAS(G12D) inhibitor, BI-2852 (Kessler, et al.20), which binds with high affinity (∼700 nM) and inhibits SOS and Raf with 500–700 nM IC50in vitro but exhibits low μM IC50 against cancer cell lines harboring mutant KRAS.

Figure 5.

Figure 5

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Effect of ACA-14 on the proliferation of pancreatic cells and tumor growth. (A) Effect of ACA-14 on the proliferation of pancreatic (BxPC-3, HPNE, MIAPaCa-2, MOH, and Panc-1) and colon (CaCO-2, SW948, and SW1116) cancer cell lines. (B) Effect of ACA-14 on MIAPaCa-2 subcutaneous tumor growth. ACA-14 was administered intraperitoneally at 5 mg/kg, diluted in corn oil. Controls are an equal volume of diluent (DMSO) added to corn oil alone. Tumor sizes were measured using electronic calipers, and tumor volumes were calculated using the formula volume = (length × width2)/2. Shown are the average tumor volumes for control (n = 8) and ACA-14-treated mice (n = 7). Errors are standard error of the mean (SEM). (C) Volume of tumor on day 20 of the experiment. (D) Weights of DMSO and ACA-14-treated mice. Mice were weighed once per week. Errors are SEM. (E) Representative western blots for p-ERK, total ERK, and GAPDH in DMSO (lanes 1 to 4) and ACA-14 (lanes 5 to 7)-treated tumors. (F) Quantification of p-ERK western blots in DMSO (n = 8) and ACA-14 (n = 7)-treated tumors. Errors are SEM * = p<0.05.

Table 2. Permeability and Efflux of ACA-14 in a MDR1-MDCK Cell Monolayer Systema.

  ACA-14 amprenavir propranolol
Papparent, A > B (nm/s) 54.25 16.52 312.08
Papparent, B > A (nm/s) 35.14 170.98 150.01
Papparent efflux ratio (ER) 0.65 10.35 0.48
Pexact, A > B (nm/s) 53.55 14.36 399.79
Pexact, B > A (nm/s) 38.33 243.25 207.15
Pexact efflux ratio 0.72 16.93 0.52
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a

This experiment was conducted by Alliance Pharma as paid service. Propranolol and amprenavir are used, respectively, as a non P-gp-dependent high transcellular permeability marker and a positive control for P-gp efflux activity. Papparent is the membrane permeability index of the compound (donor) determined by considering only two-compartment system (donor and receiver). Pexact corrects for the volume occupied by the cells in the donor side of the membrane.

ACA-14 Inhibits Tumor Growth

To assess the anti-tumor efficacy of ACA-14, we tested its ability to inhibit MIAPaCa-2 xenograft tumor growth. MIAPaCa-2 cells harbor homozygous KRAS(G12C) mutations and require oncogenic KRAS for tumor growth.53,54 Nude mice were injected subcutaneously in the hind flank with MIAPaCa-2 cells. After 3 days, tumor volumes were measured, and the mice were separated into two treatment groups with equal mean tumor volumes. Mice were then treated 5 days per week with intraperitoneal injections of either ACA-14 (5 mg/kg) or vehicle (corn oil). Tumor volumes were measured twice per week with electronic calipers. Mice weights were measured once per week. When control tumors reached an average of 2 cm3, all mice were euthanized. We observed that ACA-14 treatment caused a small but statistically significant reduction in tumor growth that was maintained until day 20 (Figure 5B,C). After that time, a trend toward reduced tumor growth was maintained, but statistical significance was lost due to variability in tumor sizes. Importantly, ACA-14 appeared to be well tolerated as it did not affect weight gain in treated mice (Figure 5D). We also measured ERK activation in tumor lysates as readout for KRAS inhibition. We observed that ACA-14 treatment caused a small but significant reduction in ERK activation within tumors (Figure 5E,F). These data indicate that ACA-14 is well tolerated in mice and is capable of inhibiting tumor growth in vivo.

Conclusions

In this work, we describe the small molecule ACA-14 as a lead compound for the development of therapies that directly act on KRAS to inhibit its abnormal signaling activities. We have shown that the compound binds to KRAS in a nucleotide-independent manner with affinities in the single-digit micromolar range, likely targeting a pocket formed by residues from the three N-terminal beta-strands and the switch regions of KRAS, or between helices 2 and 3. These sites have been previously characterized as robust targets for small-molecule binding. Using a variety of biophysical and cell biological assays, including FP, signaling, and pull-down assays, we have shown that ACA-14 impedes the interaction of KRAS with the RBD of its effector c-Raf and reduces the rates of intrinsic and GEF-mediated exchange of GDP to GTP. As a result, the ligand inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS, as assessed by monitoring the levels of phosphorylated c-Raf and ERK. It also inhibits the growth of pancreatic and colon cancer cells harboring mutant but not WT KRAS and attenuates tumor growth in a xenograft mouse tumor model. Compared to its effects in biochemical assays outside the cell, the inhibitory activities of ACA-14 in the cell required the use of slightly higher concentrations. One reason for this could be a smaller effective concentration in the cell due to limited cell permeability. This is supported by results from permeability assays, which suggest that, while the ligand is not a Pg-P substrate, there is room for improvement regarding its cell permeability. Since it is amenable to a range of chemical modifications to address these issues, we propose ACA-14 as a starting point for developing leads toward dual-acting KRAS inhibitors that target a wide variety of cancers driven by G12, G13, and Q61 KRAS mutations.

Acknowledgments

We are grateful to Drs. John Hancock and Yong Zhou (UTHealth) for illuminating discussions, Xiaodong Cheng (UTHealth) for making his NanoTemper MST instrument freely available to us, and Carla Mattos (Northeastern University) and John Putkey (UTHealth) for GNP-KRAS samples. This work was supported by grants from the Cancer Prevention and Research Institute of Texas (CPRIT grant numbers DP150093 and RP190366). J.A.F. and Y.Z. are supported by the Department of Defense Breast Cancer Research Program (grant number W81XWH-20-1-0004). A.K.G. was supported by a postdoctoral training fellowship from the Keck Center Computational Cancer Biology Training Program (CCBTP) of the Gulf Coast Consortia (CPRIT grant no. RP170593).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomedchemau.2c00045.

  • Detailed methodologies, TSA, IF of ACA-14 with and without KRAS, MST assay, and effect of ACA-14 ligand on the intrinsic and SOS-mediated nucleotide release rates (PDF)

Author Contributions

CRediT: Cynthia V Pagba conceptualization (supporting), data curation (lead), formal analysis (equal), methodology (lead), software (lead), writing-original draft (equal), writing-review & editing (equal); Amit K Gupta conceptualization (supporting), data curation (supporting), formal analysis (supporting), methodology (supporting), software (supporting), writing-original draft (supporting), writing-review & editing (supporting); Ali K Naji methodology (supporting), software (supporting); Dharini van der Hoeven methodology (supporting), software (supporting); Kelly Churion methodology (supporting), software (supporting); Xiaowen Liang methodology (supporting), software (supporting), writing-original draft (supporting); Jacob Jakubec methodology (supporting); Magnus Hook formal analysis (supporting), methodology (supporting); Yan Zuo methodology (supporting), software (supporting), writing-original draft (supporting), writing-review & editing (supporting); Marisela Martinez de Kraatz methodology (supporting); Jeffrey A Frost formal analysis (supporting), methodology (supporting), writing-original draft (supporting), writing-review & editing (supporting); Alemayehu A. Gorfe conceptualization (lead), formal analysis (equal), funding acquisition (lead), investigation (lead), methodology (lead), project administration (lead), resources (lead), supervision (lead), writing-original draft (lead), writing-review & editing (equal).

The authors declare no competing financial interest.

Supplementary Material

bg2c00045_si_001.pdf (610.9KB, pdf)

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