New Scaffolds of Proteasome Inhibitors: Boosting Anticancer Potential by Exploiting the Synergy of In Silico and In Vitro Methodologies.

Cancer is a complex multifactorial disease whose pathophysiology involves multiple metabolic pathways, including the ubiquitin-proteasome system, for which several proteasome inhibitors have already been approved for clinical use. However, the resistance to existing therapies and the occurrence of severe adverse effects is still a concern. The purpose of this study was the discovery of novel scaffolds of proteasome inhibitors with anticancer activity, aiming to overcome the limitations of the existing proteasome inhibitors. Thus, a structure-based virtual screening protocol was developed using the structure of the human 20S proteasome, and 246 compounds from virtual databases were selected for in vitro evaluation, namely proteasome inhibition assays and cell viability assays. Compound 4 (JHG58) was shortlisted as the best hit compound based on its potential in terms of proteasome inhibitory activity and its ability to induce cell death (both with IC50 values in the low micromolar range). Molecular docking studies revealed that compound 4 interacts with key residues, namely with the catalytic Thr1, Ala20, Thr21, Lys33, and Asp125 at the chymotrypsin-like catalytic active site. The hit compound is a good candidate for additional optimization through a hit-to-lead campaign.

Imaging probes for non-invasive tumoral detection and functional monitoring of cancer multidrug resistance.

Aim: Several cationic radiotracers originally developed as myocardial perfusion agents have shown potential for both early detection of cancer and non-invasive monitoring of multiple drug resistance (MDR) by single photon emission computed tomography. We have introduced two cationic complexes, 99mTc-DMEOP [di-methoxy-tris-pyrazolyl-99mTc-(CO)3] and 99mTc-TMEOP [tri-methoxy-tris-pyrazolyl-99mTc-(CO)3], which showed excellent preclinical results as cardiac imaging probes, namely a persistent heart uptake with rapid blood and liver clearance. This study aimed at the evaluation of their usefulness for tumoral detection and functional assessment of MDR. Methods: The uptake and efflux kinetics of 99mTc-DMEOP and 99mTc-TMEOP were evaluated in human prostate, lung, and breast cancer cell lines, including drug-resistant cell lines that are known to overexpress the MDR P-glycoprotein (Pgp). The effects of MDR modulators were also studied. In vivo studies were performed in xenografted tumor models, and the MDR phenotype of the tumors was confirmed by Western blot. Results: The uptake kinetics of both complexes in human cancer cell lines is comparable with the one found for 99mTc-Sestamibi, increasing over time. The uptake of 99mTc-TMEOP is greatly reduced in cells overexpressing Pgp and increased in the presence of a Pgp modulator. In nude mice, the tumor uptake of 99mTc-TMEOP was higher in the MCF-7 xenografts compared with the MCF7 Pgp tumors. Conclusion: The uptake kinetics of both complexes in human cancer cell lines is comparable with the one found for 99mTc-Sestamibi, increasing over time. The uptake of 99mTc-TMEOP is greatly reduced in cells overexpressing Pgp, and increased in the presence of a Pgp modulator. In nude mice, the tumor uptake of 99mTc-TMEOP was higher in the MCF-7 xenografts compared with the MCF7 Pgp tumors.

High resolution mass spectrometry-based methodologies for identification of Etravirine bioactivation to reactive metabolites: In vitro and in vivo approaches.

Drug bioactivation to reactive metabolites capable of covalent adduct formation with bionucleophiles is a major cause of drug-induced adverse reactions. Therefore, elucidation of reactive metabolites is essential to unravel the toxicity mechanisms induced by drugs and thereby identify patient subgroups at higher risk. Etravirine (ETR) was the first second-generation Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) to be approved, as a therapeutic option for HIV-infected patients who developed resistance to the first-generation NNRTIs. Additionally, ETR came into market aiming to overcome some adverse effects associated with the previously used efavirenz (neurotoxicity) and nevirapine (hepatotoxicity) therapies. Nonetheless, post-marketing reports of severe ETR-induced skin rash and hypersensitivity reactions have prompted the U.S. FDA to issue a safety alert on ETR. Taking into consideration that ETR usage may increase in the near future, due to the possible use of the drug for coinfection with malaria and HIV, the development of reliable prognostic tools for early risk/benefit estimations is urgent. In the current study, high resolution mass spectrometry-based methodologies were integrated with MS3 experiments for the identification of reactive ETR metabolites/adducts: 1) in vitro incubation of the drug with human and rat liver S9 fractions in the presence of Phase I and II co-factors, including glutathione, as a trapping bionucleophile; and 2) in vivo, using urine samples from HIV-infected patients on ETR therapy. We obtained evidence for multiple bioactivation pathways leading to the formation of covalent adducts with glutathione and N-acetyl-L-cysteine. These results suggest that similar reactions may occur with cysteine residues of proteins, supporting a role for ETR bioactivation in the onset of the toxic effects elicited by the drug. Additionally, ETR metabolites stemming from amine oxidation, with potential toxicological significance, were identified in vitro and in vivo. Also noteworthy is the fact that new metabolic conjugation pathways of glucuronide metabolites were demonstrated for the first time, raising questions about their potential toxicological implications. In conclusion, these results represent not only a contribution towards the elucidation of new metabolic pathways of drugs in general but also an important step towards the elucidation of potentially toxic ETR pathways, whose understanding may be crucial for reliable risk/benefit estimations of ETR-based regimens.