A novel in-cell ELISA method for screening of compounds inhibiting TRKA phosphorylation, using KM12 cell line harboring TRKA rearrangement.

Tropomyosin-related kinase A (TRKA) fusion was originally detected in colorectal carcinoma that had resulted in expression of the oncogenic chimeric protein TPM3-TRKA. Lately, many more rearrangements in TRK family of kinases generating oncogenic fusion proteins have been identified. These genetic rearrangements usually result in fusion of cytoplasmic kinase domain of TRK to another gene of interest resulting in constitutive kinase activity. Estimation of TRK inhibitor potency in a cellular context is required for drug discovery programs and is measured by receptor phosphorylation levels upon compound administration. However, since a large chunk of the TRK protein is lost in this rearrangement, it's difficult to set up sandwich ELISA for detection of receptor phosphorylation in any cell assay harboring these fusion proteins. In order to address this issue, we developed a novel and robust in-cell ELISA method which quantifies the phosphorylation of TRK kinase (Tyr 674/675) within the KM12 cells. This cell based method is more versatile & economical than conventional ELISA using engineered overexpressing cell line and/or western blot methods. Performance reliability & robustness for the validated assay were determined by %CV and Z factor in assays with reference molecule larotrectinib. This in-cell ELISA method can be used with any TRKA rearranged oncogenic fusion cell type and can be extended to other TRK isoforms as well. We have used this assay to screen novel molecules in KM12 cells and to study pharmacodynamic properties of compounds in TRKA signaling.

Erectogenic and Aphrodisiac Effects of Butea frondosa Koenig ex Roxb. in Rats: Involvement of Enzyme Inhibition.

Butea frondosa Koenig ex Roxb. (BF) is traditionally used to manage male sexual disorders including erectile dysfunction (ED). Methanol extract of BF (bark) inhibited Rho-kinase 2 (ROCK-II) enzyme activity in vitro with an IC50 of 20.29 ± 1.83 µ g/mL. The relaxant effect of methanol extract of BF (MEBF) was studied on phenylephrine precontracted corpus cavernosum smooth muscle (CCSM) isolated from young rats. The effect of MEBF treatment on sexual behaviour of both young (5 month) and aged (24 month) rats was also studied in addition to the influence on smooth muscle, collagen (collagen-I and -III) level in penis, and sperm characteristics of young and aged rats. MEBF relaxed CCSM up to 21.77 ± 2.57% and increased sexual behavior of young and aged rats. This increase in sexual function could be attributed to ROCK-II inhibition and increase in ratio of smooth muscle to collagen level in rat penile tissue. Increased sperm production and decreased defective sperms in young and aged rats corroborate the usefulness of Butea frondosa in male infertility in addition to ED.

Screening for Rho-kinase 2 inhibitory potential of Indian medicinal plants used in management of erectile dysfunction.

THE AIM OF THE STUDY: Activation of Rho-kinase 2 (ROCK-II) results in contraction of corpus cavernosum smooth muscle and ROCK-II inhibitors relax corpus cavernosum in vitro and in vivo hence, plant extracts capable of inhibiting ROCK-II enzyme may be useful in management of erectile dysfunction (ED). The aim of the study was to screen selected Indian medicinal plants, having similar ethnopharmacological use for ROCK-II inhibition. MATERIALS AND METHODS: Some Indian medicinal plants reported as aphrodisiacs in Ayurveda and modern scientific literature were collected, authenticated and extracted. Direct methanol and successive aqueous extracts of these plants were screened for ROCK-II inhibitory activity using HTRF(®)KinEASE™ STK S2 Kit (Cisbio Bioassays). Relaxant effect of potent extract was recorded on isolated rat corpus cavernosum. RESULTS: Methanolic and successive aqueous extracts of 30 plants were screened for ROCK-II inhibition and 15 herbal extracts showed inhibition ranging between 50 and 88% at 50 µg/mL. While IC(50) of Y-27632, a standard ROCK-II inhibitor, was found to be 163.8 ± 1.2 nM. The Methanolic extract of Terminalia chebula (METC) with IC(50) value of 6.09 ± 0.17 µg/mL was found to be most potent and relaxed isolated rat corpus cavernosum significantly (p<0.01). Chebulagic and chebulinic acid of METC were found to inhibit ROCK-II and might be responsible for the inhibitory potential of the extract. The traditional use of plants like Butea frondosa, Syzygium aromaticum, Butea superba, Chlorophytum borivilianum and Mucuna pruriens, as aphrodisiacs and for male sexual disorder (MSD) might be in part due to the ROCK II inhibitory potential of these plants. CONCLUSION: Some of the Indian medicinal plants have ROCK-II inhibitory potential and those deserve further investigation.

Hepatoprotective and antioxidant activity of standardized herbal extracts.

BACKGROUND: Phyllanthus emblica, Camellia sinensis, Mangifera indica, Punica granatum, and Acacia catechu have been shown to possess widespread pharmacological application against multitude of diseases namely cancer, diabetes, liver disorders, and oxidative stress. OBJECTIVE: We evaluated the hepatoprotective activity of the standardized herbal extracts against tert-butyl hydroperoxide (t-BH) induced toxicity and their mechanism of hepatoprotective action in human hepatocarcinoma cells (HepG2 cell line). MATERIALS AND METHODS: The hepatoprotective activity was studied by observing the effect of these herbal extracts on t-BH induced reduction in cell viability of HepG2 cells. In addition, the reducing power of the extracts and their ability to scavenge free radicals were evaluated using two antioxidant assay systems: cell free [oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)] (ABTS)] and cell based [cellular antioxidant activity (CAA)]. RESULTS AND DISCUSSION: The results obtained showed that these extracts possess significant hepatoprotective activity. This may indicate that the plant extracts contain compounds, which can remove toxic metabolites following t-BH induced toxicity. The extracts exhibited significant antioxidant property as evident by the Trolox values and effective scavenging of DPPH and ABTS radicals. The extracts also demonstrated inhibition of AAPH-induced fluorescence in HepG2 cells. These results indicate the ability of the plant extracts to protect the liver cells from chemical-induced damage, which might be correlated to their radical scavenging potential. CONCLUSION: This study demonstrates that these extracts have potential hepatoprotective activity which is mainly attributed to the antioxidant potential, which might occur by reduction of lipid peroxidation and cellular damage.