Abnormalities of the MAPK pathway play vital roles in cancer initiation and progression. RAS GTPases that are key upstream mediators of the pathway are mutated in 30% of human cancers. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed as potential targeted therapies against the RAS-driven cancers. The current study reports on the optimization of the PCAIs and the determination of their mechanisms of action in KRAS-mutant cancer cells. They display ClogP values ranging from 3.01 to 6.35, suppressing the viabilities of KRAS-mutant MDA-MB-231, A549, MIA PaCa-2, and NCI-H1299 cells in 2D and 3D cultures with EC50 values of 2.2 to 6.8, 2.2 to 7.6, 2.3 to 6.5 and 5.0 to 14 µM, respectively. When A549 cells were treated with the PCAIs, NSL-YHJ-2-27, for 48 h, no significant difference was observed in the levels of total or phosphorylated B- and C-Raf proteins. However, at 5 µM, it stimulated the phosphorylation of MEK1/2, ERK1/2, and p90RSK by 84%, 59%, and 160%, respectively, relative to controls. A non-farnesylated analog, NSL-YHJ-2-62, did not elicit similar effects. These data reveal that effects on the RAS-MAPK signaling axis most likely contribute to the anticancer effects of the PCAIs, possibly through the proapoptotic isoforms of p90RSK. The PCAIs may thus have the potential to serve the unmet therapeutic needs of patients with aberrant hyperactive G-protein signaling.

Finding effective therapies against cancers driven by mutant and/or overexpressed hyperactive G-proteins remains an area of active research. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) are agents that mimic the essential posttranslational modifications of G-proteins. It is hypothesized that PCAIs work as anticancer agents by disrupting polyisoprenylation-dependent functional interactions of the G-Proteins. This study tested this hypothesis by determining the effect of the PCAIs on the levels of RAS and related monomeric G-proteins. Following 48 h exposure, we found significant decreases in the levels of KRAS, RHOA, RAC1, and CDC42 ranging within 20-66% after NSL-YHJ-2-27 (5 μM) treatment in all four cell lines tested, A549, NCI-H1299, MDA-MB-231, and MDA-MB-468. However, no significant difference was observed on the G-protein, RAB5A. Interestingly, 38 and 44% decreases in the levels of the farnesylated and acylated NRAS were observed in the two breast cancer cell lines, MDA-MB-231, and MDA-MB-468, respectively, while HRAS levels showed a 36% decrease only in MDA-MB-468 cells. Moreover, after PCAIs treatment, migration, and invasion of A549 cells were inhibited by 72 and 70%, respectively while the levels of vinculin and fascin dropped by 33 and 43%, respectively. These findings implicate the potential role of PCAIs as anticancer agents through their direct interaction with monomeric G-proteins.

: Aberrant activation of monomeric G-protein signaling pathways drives some of the most aggressive cancers. Suppressing these hyperactivities has been the focus of efforts to obtain targeted therapies. Polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in various cancers. Its inhibition induces the death of cancer cells that harbor the constitutively active K-Ras proteins. Furthermore, the viability of cancer cells driven by factors upstream of KRas, such as overexpressed growth factors and their receptors or the mutationally-activated receptors is also susceptible to PMPMEase inhibition. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) were thus designed to target cancers with hyperactive signaling pathways involving the G-proteins. The PCAIs were however poor inhibitors of PMPMEase, with Ki values ranging from 3.7 to 20 µM. On the other hand, they inhibited cell viability, proliferation, colony formation, induced apoptosis in cells with mutant K-Ras and inhibited cell migration and invasion with EC50 values of 1 to 3 M. HUVEC tube formation was inhibited at submicromolar concentrations through their disruption of actin filament organization. At the molecular level, the PCAIs at 2 to 5 M depleted monomeric G-proteins such as K-Ras, RhoA, Cdc42 and Rac1. The PCAIs also deplete vinculin and fascin that are involved in actin organization and function while disrupting vinculin punctates in the process. These demonstrate a polyisoprenylation-dependent mechanism that explains the observed PCAIs’ inhibition of the proliferative, invasive and angiogenic processes that promote both tumor growth and metastasis.

Cancers driven by mutant and/or overexpressed hyperactive G-proteins are resistant to current therapies. Finding effective therapies targeting such cancers remains an area of active research. Attempts at directly targeting the essential G-protein polyisoprenylation posttranslational modifications (PTMs) have met significant efficacy and toxicity challenges. The polyisoprenylated cysteinyl amide inhibitors (PCAIs) are a novel class of compounds designed around the Ras PTMs that have been shown to be very effective against various cancer biology phenomena. The design strategy incorporated the farnesyl tail, substituted amide bioisostere of the endogenous ester and a linker with a terminal ionizable group on the α-amino of the polyisoprenylated cysteine. This study is aimed at improving the potency and bioavailability of the PCAIs through synthetic optimization to mitigate the hydrophobicity of the pharmacophore. We report on newly synthesized PCAIs that are significantly less hydrophobic, their effectiveness in 2D and 3D cancer culture and the molecular mechanisms driving their anticancer effects. The new PCAIs were designed to have shorter linkers by coupling 4-methylpiperazine-1-yl aliphatic acids with L-S-trans,trans-farnesyl cysteine methyl ester to give the intermediate, 4-methylpiperazine-1-yl acyl-L-S-trans,trans-farnesyl cysteine methyl ester. The methyl esters of the intermediates were hydrolyzed with base and the resulting carboxylic acids coupled with cycloalkyl amines to afford the final products. The new PCAIs displayed Clog P values from 3.01 to 6.35. They suppressed the viabilities of MDA-MB-231, MIA PaCa-2 and NCI-H1299 cells that harbor mutant Ras with 48 h EC50 values from 2.2 to 6.8, 2.3 to 6.5 and 5.0 to 14 µM, respectively. The PCAIs with the free α-amino on the cysteine displayed a significant loss in potency with EC50 values ranging from 22 to over 50 µM for the different cell lines. Control analogs lacking the polyisoprenyl moiety had no effect at inhibiting the viabilities of the cells even at 50 µM. Treatment of MDA-MB-231 and NCI-H1299 3D spheroids with the most potent PCAIs suppressed their viabilities at 5 µM and 20 µM, respectively. Moreover, it was observed that ERK1/2 phosphorylation increased by 6, 33, and 121 % following treatment of NCI-H1299 cells for 24 h with 1, 2, and 5 µM of NSL-BA-055, respectively. However, after 48 h of treatment, 1 and 2 µM of NSL-BA-055 showed significant increase by 147 and 266 %, respectively, while the 5 µM showed a 20 % decrease of phospho-ERK1/2 relative to controls. The data reveal that (1) the more polar PCAIs maintained potency, (2) the three appendages on the cysteine of the PCAIs all contribute to potency and (3) the Ras-signaling axis is involved in the anticancer effects. Citation Format: Nada Tawfeeq, Yonghao Jin, Nazarius S. Lamango. Synthetic optimization of polyisoprenylated cysteinyl amide inhibitors to target cancers with hyperactive G-proteins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2591.