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Targeted programs represent a good majority of drug discovery efforts. They allow for a convenient rationalization of the biological events leading to pathologies and the modulation of such pathological events. They are based on the hypothesis that the modulation of the target is of therapeutic benefit. As a consequence, the key tool for the identification of modulators is a biochemical assay where the activity of the target is measured.

At IRBM, we have developed, optimized and run several types of biochemical assays to support drug discovery efforts. They can be divided in two major areas:

  • Enzymatic assays
  • Binding assays

In both cases, the integrated environment that IRBM offers has the potential to generate better and more relevant readouts to the desired compound mode of action. We can produce and validate customized proteins and reagents, such as small molecules and peptide probes.

We highly value developing and running enzymatic assays, constantly monitoring:

  • Enzyme purity and identity to avoid picking up unspecific activities
  • Enzyme kinetics to ensure robust evaluation and comparison of compound activities (e.g. Km determination)
  • Continuous quality control of the assay outcome
  • Robust data analysis with no manual intervention and full traceability of raw data

In addition to purity, identity, quality control and data integrity, for binding assays we particularly focus on:

  • The validation of the relevance of the in vitro binding event(s) for the subsequent functional effect
  • The optimization of the binding condition for an optimized evaluation and comparison of binding disruptors

Biochemical assays in drug discovery are very often accompanied by higher throughputs. With our extensive experience in high-throughput screening and early drug discovery efforts, we have a strong understanding of the key determinants to align biochemical assays and throughput. They are:

  • Assay miniaturization to high density formats (up to 1536 wells/plate)
  • Assay metrics (Z’, robust Z’, dynamic range, signal to background) optimization to achieve robust assays
  • Microplate related issues and solutions.

Finally, we employ state of the art biochemical assays for in vitro ADME that are required for compound profiling and selection toward pre-clinical candidates. We include CYP inhibition assays, hERG binding assay and other standard ADME assays.

Additional description of ADME assays

Approaches of Screening Modulatory Effects of Xenobiotics on Cytochrome
P450 (CYP) Enzymes

Cytochrome P450s (CYPs) constitute a superfamily of isoforms that play an important role in the oxidative metabolism of drugs. Each CYP isoform possesses a characteristic broad spectrum of catalytic activities of substrates. Whenever 2 or more drugs are administered concurrently, the possibility of drug interactions exists.

The ability of a single CYP to metabolize multiple substrates is responsible for a large number of documented drug interactions associated with CYP inhibition. In addition, drug interactions can also occur as a result of the induction of several human CYPs following long term drug treatment. From the viewpoint of drug therapy, to avoid potential drug-drug interactions, it is desirable to develop a new drug candidate that is not a potent CYP inhibitor or inducer and the metabolism of which is not readily inhibited by other drugs.

In reality, drug interaction by mutual inhibition between drugs is almost inevitable, because CYP-mediated metabolism represents a major route of elimination of many drugs, which can compete for the same CYP enzyme. The clinical significance of a metabolic drug interaction depends on the magnitude of the change in the concentration of active species (parent drug and/or active metabolites) at the site of pharmacological action and the therapeutic index of the drug. The smaller the difference between toxic and effective concentration, the greater the likelihood that a drug interaction will have serious clinical consequences.

Thus, careful evaluation of potential drug interactions of a new drug candidate during the early stage of drug development is essential.

Although the criteria for potency are project and isoform-specific, potent inhibition is considered unfavourable and may preclude the development of a compound. For that reason, compounds can be categorised into the classification bands reported in the Table below.

 

 

Luminescence-based assays, in 384-well plate format, are suitable for CYP screening during early Drug Discovery. These assays are an effective, cheap, and highly sensitive method for enzyme screening.

On the other hand, *LC-MS or LC-MS/MS (liquid chromatography tandem mass spectrometry) is a more powerful, analytical approach to use in the later stages of drug development.

Induction of CYP enzymes by drugs can give rise to significant clinical drug interactions. Induction of the major hepatic CYP3A4/5 enzyme is assessed in cultured human hepatocytes.

Due to large inter-individual variability both in the expression of drug-metabolizing CYP and in the extent to which inducers can induce CYP enzymes in humans, experiments are conducted at least in 3 different donors of human hepatocytes. The assays are in 24-well plate format with the possibility to use both single and pooled donors.

In vitro Toxicity Tools:

  • hERG inhibition assay

hERG (human Ether-a-go-go-Related Gene) is a gene encoding alpha subunit of potassium channel. This channel is responsible for the repolarizing current in cardiac cycle. The inhibition of the channel or mutations may cause the adverse condition called long QT syndrome. In the past few years, several drugs have been withdrawn from the market due to their interaction with certain ion channels that may cause life-threatening arrhythmias and sudden cardiac death.

Invitrogen’s Predictor™ hERG Fluorescence Polarization Assay provides valuable information about possible binding of test compounds to hERG channel followed by QT prolongation on echocardiogram (ECG). Although manual patch-clamp testing is the gold standard for this purpose, biochemical hERG assay allows checking multiple compounds at the earliest stages of drug discovery. Screening compounds for hazardous side effects on hERG channel early in the drug development process is likely to cut the drug development costs.

The assay is performed in 384-well format. The results are expressed as % of inhibition and an IC50 is determined in case of a significant hERG channel inhibition is observed.

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