What is Meant by Target Validation and How is Target Validation Done?

What is Meant by Target Validation?

Target validation refers to the accumulation of scientific evidence that a target (usually a protein) is modulated (usually inhibited) for therapeutic use. There are several meanings here. Target validation can be chemical or genetic. The former means it can achieve the therapeutic effect by regulating the target protein with chemical substances. In contrast, the latter implies that gene technology such as animal gene knockout or human gene mutation can produce the therapeutic effect. Target confirmation is also divided into clinical and preclinical. Suppose the compound that regulates a target shows efficacy in clinical trials and you start the project again. In that case, it is often called me-too research and development, which is now commercially meaningless. However, gene activation and inactivation mutations like PCSK9 exist, with opposite characteristics (one with high blood fat and one with low blood fat, and the cardiovascular risk is opposite), and there are few other abnormal targets. Therefore, the current target confirmation is mainly preclinical, that is, animal experiment verification. It is almost impossible to confirm a target 100%. For example, the mechanism of statins is still controversial.

Importance of Target Validation

The reproducibility of some target confirmation experiments has been questioned, and the specific data are challenging to estimate. Unofficial reports indicate that this figure exceeds half. This is considered to be the main reason for clinical Phase II failure, with nearly two-thirds of projects having inconsistencies between published and internal data, significantly prolonging the target validation process and leading to project termination in most cases ( Arrowsmith J, Miller P. Trial watch: phase II and phase III attrition rates 2011-2012[J]. Nature Reviews Drug Discovery, 2013,12(8): 569-569.). Several studies have focused on analyzing the reasons for poor reproducibility, such as poor antibody specificity (Baker M. Blame it on the antibodies[J]. Nature, 2015, 521(7552): 274.), cell line identification error, etc.

How is Target Validation Done?

This stage confirms that the selected target's modulation will elicit the expected biological effect and be relevant to the clinical treatment. Commonly used methods include gene mutation, gene knockout, antibodies, drug-resistant mutations, and siRNA. It is worth noting that 51% of the failures in the second phase of clinical trials were due to insufficient drug efficacy, and attributed many of them to the lack of early target confirmation work. The loss of massive investment in the later stage followed the failure of this stage.

Disease Association

The clinical target validation platform (https://www.targetvalidation.org/) integrates the relationship between potential drug targets and diseases. Its central idea is to discover the relationship between targets and conditions from various data types. DisGeNET (http://www.disgenet.org/web/DisGeNET/menu/home) is a drug discovery platform that integrates gene-disease relationship information, and its data sources are open-source databases and literature.

Chemical Probe

A chemical probe is a known small molecule targeting a specific target, capable of exerting the expected biological effect but lacking in particular properties so that it cannot be used as a drug. It is important to select high-quality probes for target confirmation to avoid misleading results. While chemical probes may be considered primarily to provide evidence of on-target effects and clinical therapeutic effects, they are also crucial in addressing the potential toxicity risks of targets.

It is worth noting that the chemical probe is not a potential drug candidate, and its most critical property is selectivity to the proprietary compound and its metabolites. For chemical probes, the requirement of oral bioavailability is not necessary, and understanding the pharmacokinetic properties of probes can provide insight into their PK/PD relationship. However, not all chemical probes have been studied thoroughly, and more information will be discovered as time goes by. Therefore, a database of chemical probes has been developed for information queries (http://www.chemicalprobes.org).

Figure 1. Comparing the different requirements of chemical probes and drugs 

Gene Knockout

Gene knockout is a genetic technique that infers the function of a gene by disabling a specific gene in an organism and comparing the difference between the standard group and the knockout group. However, the knockout of a single gene can impact the development of an entire organism, so techniques for knocking out a gene in a specific tissue or time-specific knockout have been developed.

Figure 2. The principle of CRISPR-Cas9 knockout-mediated antibody specificity verification 

Small interfering RNA (siRNA)

siRNA is a double-stranded RNA that plays a role in the pathway of RNA interference. As a sequence of complementary nucleotides, siRNA can interfere with the expression of a specific gene, thereby effectively knocking down the target gene. It should be noted, however, that genes with incomplete complementarity may also be downregulated by siRNA.

Studying the regulatory effects of targets in healthy cells allows the assessment of mechanism-based toxic effects. Due to pleiotropy, the same target may have different functions in different organ systems or at other time points during development and adulthood. Data obtained from genetic defects in knockout mice and humans can provide further hints about potential adverse effects.

Figure 3. The mechanism of action of siRNA 

Trim-Away

Trim-Away technology is another method for depleting endogenous proteins, which are drastically degraded in mammalian cells without prior modification of the genome or mRNA. Trim-Away utilizes cellular protein degradation machinery to remove unmodified native proteins in just a few minutes.