Pharmacological modulation of oncogenic Ras by natural products and their derivatives: renewed hope in the discovery of novel anti-Ras drugs

Oncogenic rat sarcoma (Ras) is linked to the most fatal cancers such as those of the pancreas, colon, and lung. Decades of research to discover an efficacious drug that can block oncogenic Ras signaling have yielded disappointing results; thus, Ras was considered “undruggable” until recently. Inhibitors that directly target Ras by binding to previously undiscovered pockets have been recently identified. Some of these molecules are either isolated from natural products or derived from natural compounds. In this review, we described the potential of these compounds and other inhibitors of Ras signaling in drugging Ras. We highlighted the modes of action of these compounds in suppressing signaling pathways activated by oncogenic Ras, such as mitogen-activated protein kinase (MAPK) signaling and the phosphoinositide-3-kinase (PI3K) pathways. The anti-Ras strategy of these compounds can be categorized into four main types: inhibition of Ras–effector interaction, interference of Ras membrane association, prevention of Ras–guanosine triphosphate (GTP) formation, and downregulation of Ras proteins. Another promising strategy that must be validated experimentally is enhancement of the intrinsic Ras–guanosine triphosphatase (GTPase) activity by small chemical entities. Among the inhibitors of Ras signaling that were reported thus far, salirasib and TLN-4601 have been tested for their clinical efficacy. Although both compounds passed phase I trials, they failed in their respective phase II trials. Therefore, new compounds of natural origin with relevant clinical activity against Ras-driven malignancies are urgently needed. Apart from salirasib and TLN-4601, some other compounds with a proven inhibitory effect on Ras signaling include derivatives of salirasib, sulindac, polyamine, andrographolide, lipstatin, levoglucosenone, rasfonin, and quercetin

Advances and challenges in developing andrographolide and its analogues as cancer therapeutic agents

Andrographolide (AGP), a naturally occurring bioactive compound, has been investigated as a lead compound in cancer drug development. Its multidimensional therapeutic effects have raised interest among medicinal chemists, which has led to extensive structural modification of the compound, resulting in analogues with improved pharmacological and pharmaceutical properties. Nevertheless, the analogues with the improved properties need to be rigorously studied to identify drug-like lead compounds. We scrutinised articles published from 2012 to 2018, to objectively provide opinions on the mechanisms of action of AGP and its analogues, as well as their potential as viable anticancer drugs. Preclinical and clinical data, along with the extensive medicinal chemistry efforts, indicate the compounds are potential anticancer agents with specific value in treating recalcitrant cancers such as pancreatic and lung cancers

In silico and saturation transfer difference NMR approaches to unravel the binding mode of an andrographolide derivative to K-Ras oncoprotein

Background: Andrographolide and its benzylidene derivatives, SRJ09 and SRJ23, potentially bind oncogenic K-Ras to exert anticancer activity. Their molecular interactions with K-Ras oncoproteins that lead to effective biological activity are of major interest. Methods & results: In silico docking and molecular dynamics simulation were performed using Glide and Desmond, respectively; while saturation transfer difference NMR was performed using GDP-bound K-RasG12V. SRJ23 was found to bind strongly and selectively to K-RasG12V, by anchoring to a binding pocket (namely p2) principally via hydrogen bond and hydrophobic interactions. The saturation transfer difference NMR analysis revealed the proximity of protons of functional moieties in SRJ23 to K-RasG12V, suggesting positive binding. Conclusion: SRJ23 binds strongly and interacts stably with K-RasG12V to exhibit its inhibitory activity

Abrogation of oncogenic k-ras function by andrographolide derivatives via in silico, in vitro, and in vivo approaches

The rat sarcoma (Ras) proteins are small guanosine triphosphatases (GTPases) that act as molecular switches in major signalling pathways involved in cell proliferation, differentiation, and survival, such as mitogenactivated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades. Ras exists in three isoforms – K-Ras, H-Ras, and N-Ras. Approximately 30% of all human cancers harbour Ras mutations, with the most frequently mutated isoform being K-Ras, which exclusively appears in pancreatic ductal adenocarcinoma (PDAC). Mutated K-Ras proteins are constitutively active with the GTPase activity being compromised. Oncogenic K-Ras is currently a valuable oncology target and its inhibition represents an important therapeutic strategy. Recent in silico study has revealed a direct binding of andrographolide (AGP) and its benzylidene derivatives, SRJ09 and SRJ23, to K-Ras oncoprotein, which abrogated its function and downstream MAPK signalling. The present study aims to investigate the potential of AGP derivatives as anti-Ras therapeutics through in silico, in vitro, and in vivo approaches. The anticancer potential of SRJ09 and SRJ23 has been welldemonstrated in the human colon (HCT-116) and prostate (PC-3) cancer cells, respectively. These two cell lines have been made resistant to the compounds previously and were used in the present study to examine the altered gene profile in relation to the expression of regulatory genes involved in the compounds’ anticancer activity using microarray analysis. Regulatory genes associated with autophagy and apoptotic processes, such as ATG12 and HMOX1, as well as MAPK and PI3K pathways, such as FGF19 and SPRY2 that play major roles in promoting cell growth and survival, were found to be altered. New benzylidene derivatives have been previously synthesised using SRJ09 and SRJ23 as parent compounds, yielding SRS compounds. In the present investigation, the most druggable binding pocket on K-Ras mutants namely p2 was revealed through in silico simulations. SRJ23 and SRS157 were found to bind via intermolecular hydrogen bonding to this pocket. The anti-PDAC activity of selected AGP derivatives (SRJ23, SRJ09, SRS07, and SRS157) and their mechanisms of action were elucidated in vitro. SRJ23 and SRS157 were shown to perform differently particularly in terms of activity on Erk, a crucial signalling protein in the K-Ras-associated MAPK cascade. Its activation was unanticipatedly enhanced by SRJ23 and significantly suppressed by SRS157 upon 24-h treatment of the compounds. SRS07 presented as a superior anti-PDAC agent by promoting oxidative stress, possibly through enhancement of Akt activation in the K-Ras-mediated PI3K pathway. A simple pharmacokinetic study performed in BALB/c mice at a single dose of 100 mg/kg SRJ23 revealed that the compound achieved a maximum plasma concentration of 18.8 μM after 30 min of administration, with long halflife (4.28 h) and mean residence time (6.30 h). Subsequent in vivo antitumour study reported that 100 mg/kg SRS157 delayed the doubling of tumour growth in the PDAC-xenograft nude mouse model more effectively than SRJ23 at the same dose. In conclusion, the outcomes of the present study provide a strong indication of the potential of AGP derivatives, which specifically target the oncogenic K-Ras and abrogate its function, as promising clinical antipancreatic cancer candidates

Microarray-based identification of differentially expressed genes associated with andrographolide derivatives-induced resistance in colon and prostate cancer cell lines

Chemoresistance poses a major hurdle to cancer treatments. Andrographolide-derived SRJ09 and SRJ23 were reported to exhibit potent, selective inhibitory activities against colon and prostate cancer cells, respectively. In this study, previously developed resistant colon (HCT-116rst09) and prostate (PC-3rst23) cancer cell lines were used to elucidate the molecular mechanisms contributing to chemoresistance. Cytotoxic effects of SRJ09 and SRJ23 on both parental and resistant cells were investigated. Cell cycle distributions in HCT-116rst09 cells following SRJ09 treatment were analysed using flow cytometry. Whole-genome microarray analysis was performed on both parental and resistant cells to obtain differential gene expression profiles. Microarray data were subjected to protein-protein interaction network, functional enrichment, and pathway analyses. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the changes in expression levels of selected genes. Besides morphological changes, HCT-116rst09 cells showed 7.0-fold resistance to SRJ09 while PC-3rst23 cells displayed a 5.5-fold resistance to SRJ23, as compared with their respective parental cells. G0/G1-phase cell cycle arrest was observed in HCT-116rst09 cells upon SRJ09 treatment. Collectively, 77 and 21 genes were found differentially modulated in HCT-116rst09 and PC-3rst23 cells, respectively. Subsequent bioinformatics analysis revealed several genes associated with FGFR4 and PI3K pathways, and cancer stemness, were chemoresistance mediators in HCT-116rst09 cells. RT-PCR confirmed the HMOX1 upregulation and ATG12 downregulation protected the PC-3rst23 cells from SRJ23 cytotoxicity. In conclusion, acquired chemoresistance to SRJ09 and SRJ23 in colon and prostate cancer cells, respectively, could be attributed to the alterations in the expression of genes such as those related to PI3K and autophagy pathways

Nanoplate-based digital PCR for highly sensitive pork DNA detection targeting multi-copy nuclear and mitochondrial genes

The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a ‘rain effect’ were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive ‘rain effect’ was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no ‘rain effect’. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05 (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated