Assessment of Cell Cycle Inhibitors by Flow Cytometry

Abstract

The main approach used by large pharmaceutical companies for the design of a new drug is based on identifying an initial specific biological target that has been adequately validated. Typically biochemical or cellular assays are used by applying high-throughput screening methods (HTS) to identify hits (HitID) from large chemical libraries in the case of new chemical entities (NCEs) in order to identifying viable parent compounds with pharmacological characteristics. These compounds are starting points for chemical expansions which result in structure activities relationships (SARs). In the next phase (H2L) identification of a lead compound is the endpoint. Leads are already more drug-like and are expected to have certain activities in a number of biological assays, including cellular assay and hints of in vivo efficacy linked to pharmadynamic biomarker modulation. The optimization of a lead compound (LO phase) are typically properties such as potency and selectivity or activity in animal models, pharmacokinetics (e.g. oral bioavailability). In this phase an initial assessment of toxicological findings and a more systematic investigation in different animal models (e.g. xenograft or transgenic animal models) for demonstrating the therapeutic efficacy performed and ideally result in the selection of the final candidate for potential clinical development (Bleicher et al., 2003). In the complex process of drug discovery, particularly for inhibitors of the cell cycle, there are two phases in which flow cytometry (FCM) gives major contributions. Firstly, in target validation, in order to demonstrate that inhibition of a specific target determines alteration of the cell cycle, secondly in the usage of cell-based assays in order to characterize compounds to demonstrate that the modulations observed are in line with the expected mechanism of action. FCM is a major readout the key analysis for studying mechanism of action of drugs that affect proliferation since it is rapid, precise, can be automated with adherent or nonadherent cells. In this review we want to point out technical and strategic aspects in the cytometric field important for new targeted therapies. In the case where the modulation of a target or treatment with a compound produced, a disturbance in the cell cycle is needed, monoparametric DNA analysis is recommended, focusing on speed and throughput of samples by automation. Where, instead, the mechanism of action of a drug is the focus of the study, two-parametric analysis such as 5-bromo-deoxyuridine (BrdU) or 5-ethyl-deoxyuridine