No abstract

Cancer Stem Cells (CSCs) constitute a subpopulation at the top of the tumor cell hierarchy that contributes to tumor heterogeneity and is uniquely capable of seeding new tumors. Because of their biological properties, CSCs have been pointed out as therapeutic targets for the development of new therapies against breast cancer. The identification of drugs that selectively target breast CSCs requires a clear understanding of their biological functions and the experimental methods to evaluate such hallmarks. Herein, we review the methods to study breast CSCs properties and discuss their value in the preclinical evaluation of CSC-targeting drugs.

Here, we show the utility of the fluorescent biosensor hCaM-M124C-mBBr in detecting and determining the affinity of serotonin (5-HT). We obtained a Kd of 5-HT (0.71 μm) for the first time, the same order of magnitude as most anti-CaM drugs. This data can contribute to understanding the direct and indirect modulation of CaM on its binding proteins when the 5-HT concentration varies in different tissues or explain some of the side effects of anti-CaM drugs. On the other hand, molecular modeling tools help the rational design of biosensors and adequately complement the experimental results. For example, the docking study indicates that 5-HT binds at the same site as chlorpromazine (site 1) with a theoretical Ki of 2.84 μM; while the molecular dynamics simulations indicate a stability of the CaM–5-HT complex with a theoretical ΔG of −4.85 kcal mol−1, where the enthalpy contribution is greater. Thus, the combination of biotechnology and bioinformatics helps in the design and construction of more robust biosensors.

In the present study, we reported the interactions at the molecular level of a series of compounds called Bisindolylmaleimide, as potential inhibitors of the calmodulin protein. Bisindolylmaleimide compounds are drug prototypes derived from Staurosporine, an alkaloid with activity for cancer treatment. Bisindolylmaleimide compounds II, IV, VII, X, and XI, are proposed and reported as possible inhibitors of calmodulin protein for the first time. For the above, a biotechnological device was used (fluorescent biosensor hCaM M124C-mBBr) to directly determine binding parameters experimentally (Kd and stoichiometry) of these compounds, and molecular modeling tools (Docking, Molecular Dynamics, and Chemoinformatic Analysis) to carry out the theoretical studies and complement the experimental data. The results indicate that this compound binds to calmodulin with a Kd between 193–248 nM, an order of magnitude lower than most classic inhibitors. On the other hand, the theoretical studies support the experimental results, obtaining an acceptable correlation between the ΔGExperimental and ΔGTheoretical (r2 = 0.703) and providing us with complementary molecular details of the interaction between the calmodulin protein and the Bisindolylmaleimide series. Chemoinformatic analyzes bring certainty to Bisindolylmaleimide compounds to address clinical steps in drug development. Thus, these results make these compounds attractive to be considered as possible prototypes of new calmodulin protein inhibitors.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.