Critical Path Generator
The GOT-IT Team is developing a Critical Path Generator tool, which will assist scientist in planning and structuring a target assessment project. The CP Generator will help to arrange important target assessment Blocks into a project-specific path and will provide support in defining Go/NoGo decision criteria. Using a query form, the CP Generator will help identify strengths and weaknesses of a project to invest critical resources in an optimal way.
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On-target or target-related toxicity refers to exaggerated and adverse effects, that result from manipulating the inherent biological function of the target of interest that is different from therapeutic intervention. It's contribution to the failure of translational drug development programs can be significant: In 1999, an evaluation of 150 compounds from 12 pharmaceutical companies showed that the incidence of target-related toxicity in clinical trials was 35% in Phase I, 39% in Phase II and 43% in Phase III . A later analysis by AstraZeneca found that the ratio between target and chemical toxicity was 25% vs. 75% for preclinical failures and 48% vs. 52% for clinical failures . As a consequence, early identification of potential target-related toxicities and increased understanding of the underlying molecular mechanisms can guide the development of safety biomarkers and help derive mitigation strategies to facilitate project progression. The purpose of this Building Block is to identify potential unintended consequences of target modulation and to confirm and characterize a) unavoidable on-target toxicities in a timely manner, b) early Go/NoGo decisions along the Critical Path and c) to facilitate anticipation, monitoring and management of potential clinical adverse events.
Mechanistic approaches to evaluate target-related safety aspects
To identify safety aspects associated with the target of interest, its associated response pathway or the cell or organ expressing the target, the following activities should be considered, depending on project-specific characteristics. These tasks also form the basis for the set of Guiding Questions discussed in Section 2.2:
- Review of human genetic databases: This can reveal known target loss or gain-of-function mutations, implicated in certain genetic diseases, and may suggest heterogeneity in the patient population of interest.
- Published clinical trials or regulatory approval documents: Potential information sources for toxicity related to drugs modifying the target of interest are a) drug approval document databases (e.g. PharmaPendium, Elsevier) and wider pharmaceutical databases (e.g. Pharma and Life Sciences, Thomson Reuters)
- Gene and protein expression analysis: if a target is highly expressed in non-therapy relevant organs, some critical functional role of the target is likely in these tissues. In addition, due to pleiotropic effects, the same target might exert different functions in different organs or at different times during development. It may therefore, be critical to analyse target expression level throughout the human body. In general, it can be assumed that the broader the expression, the higher the risk for adverse events on systemic drug administration.
- Analysis of the target distribution and expression patterns in humans and in preclinical species of choice will identify tissues containing the target and its prevalence. This will support decisions on extrapolation between different species.
- Signaling Pathway analysis: Target-related toxicity can result from involvement of the target in pathways and/or physiological processes that are harmful if modified. If understanding of a target is limited, the analysis of pathway up- or downstream components (or specific cellular functions related to the target) can be highly informative. Through protein-protein interactions, multiple pathways may be activated or inhibited, ultimately leading to adverse effects. Pathway analysis tools: e.g. Ingenuity, Qiagen.
- Target family: Consideration of paralogues, closely-related genes or proteins with conserved domains similar to the target of interest, may help to identify potential 'off-target' safety issues.
- Target knock-down, knock-out or transgenic phenotypes: Modulation of the target expression levels (in a specific organ) and characterization of the model phenotype can be very useful to identify potential target-related adverse events. Importantly, a target knockout may cause embryonic lethality, thereby restricting further experimental assessment. It may also lead to a different phenotype to that induced by pharmacological means, as other functions of the protein, e.g. recruitment of and interaction with other signaling proteins, may be lost. This issue may be addressed with gene knock-in studies in which the wildtype target is replaced by a mutant protein with properties closer to that of pharmacological intervention e.g. kinase dead mutants.
- Tool compound use: Studies with tool molecules can confirm any target-related safety issues observed using genetic approaches. In particular, in vivo studies using tool compounds can help to determine potential target-related liabilities in organs with the highest expression levels.
- Use of inactive enantiomers: Inactive enantiomers of tool compounds are a powerful control to address target-related safety aspects since target-related toxicities will be minimal and any other structure-related toxicity should be comparable to the active isomer.
- Cross-species similarities and species specificity: When choosing model organisms to study target-related safety aspects, the likely cross-species translation of target-related toxicities must be checked. For example, a recent analysis identified 172 human drug target genes that are absent in the rat. Consequently, for these targets, the rat is not an appropriate model (Sidaway, Phenotox).
- Biomarker: Developing robust safety biomarkers is key for assessing target-related safety aspects and the success of translational drug discovery programs
- Immunomodulatory targets: To study capacity to combat an infection, host resistance assays can be performed, in which mice are infected with a relevant pathogen
- Risk-benefit ratio: Can risk be controlled by excluding particular patient subpopulations? The risk profile could support modulation of a drug target in certain diseases and patient groups but not in others. Toxicities may restrict the patient population and identify exclusions for certain patient groups (Patient stratification)
- Organotypic quality: models to address safety aspects should not only consider the species but also the organotypic quality of the model. For instance, a human primary hepatocyte model is considered to be more organotypically relevant than data from a human cell line such as HepG2 cells.
- Differential expression in samples representing human disease versus healthy controls is an additional parameter contributing to an early assessment of putative target related adverse events.
 Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, Lilly P, Sanders J, Sipes G, Bracken W, Dorato M, van Deun K, Smith P, Berger B, Heller A.
Concordance of the toxicity of pharmaceuticals in humans and in animals.
Regulatory Toxicology and Pharmacology (2000)
Safety and toxicity
 Cook D, Brown D, Alexander R, March R, Morgan P, Satterthwaite G, Pangalos MN. Lessons learned from the fate of AstraZeneca's drug pipeline: a five-dimensional framework. Nature Reviews Drug Discovery (2014) Target identification and validation & Drug discovery and development PMID: 24833294
For anti-bacterial research (and other therapeutic areas) drug discovery approaches have typically been divided into two classes: target-based drug discovery (TDD) and phenotypic drug discovery (PDD): The target-based strategy is based on the identification and validation of a molecular target before lead discovery starts. In contrast, phenotypic screening can be described as the process that identifies chemical matter that induce desired phenotypic changes (i.e. antibacterial activity) in cells or organisms. This PDD approach does not usually require prior knowledge about disease pathophysiology or the compound's mode of action against a specific molecular target.
The retrospective identification (target deconvolution) and validation of the target underlying the observed phenotypic responses can, however, greatly facilitate subsequent target-specific enhancement of pharmacological properties, allowing efficient structure–activity relationship studies to be carried out in chemical optimization programs. In addition, bacterial-selectivity as well as target-based side effects and safety (see Assessment Block - Target-Related Safety) can be addressed, thereby potentially reducing later-stage 'attrition'.
Hence, if target deconvolution and validation are critical steps for specific drug discovery projects, the following points may need to be considered.
Mechanistic approaches/considerations to evaluate antibiotic targets
- Antibacterial target properties: a) essential for growth and survival, b) amenable to chemical inhibition, c) accessible to the inhibitor, d) distinct from related mammalian molecules, e) present in a spectrum of bacteria, f) low resistance potential and g) lack of target-based cross-resistance.
- Targeted gene modification: to validate a gene product as an attractive target, the transient or permanent abrogation of function of a specific gene product should result in a loss of bacterial viability. Target mutations can be used to investigate the resistance-associated mechanism (mechanism of action) by transferring resistant mutations identified back to the parental strain.
- Downregulation of the target expression level should reduce the Minimum Inhibitory Concentration (MIC) of a tool compound if the target is essential/responsible for the antibacterial activity.
- Overexpression of a target should raise the MIC from compound binding, lowering its effective intracellular concentration. However, note that another MIC-determining target may also contribute to growth inhibition.
- Conditional expression systems enable simultaneous assessment of multiple conditional mutants and regulation of target gene expression in response to a stimulus. Infection models based on conditional expression systems help to clarify whether a target is required for initiating or maintaining an infection (see #8).
- Tool compound use: The chemical inhibition/modulation of the target should lead to growth inhibition or death, provided that a general or good SAR between target/enzyme inhibition and the MIC exists. However, this does not show causality and linking whole-cell activity to inhibition of the enzyme remains critical for target validation (see #7).
- As a specificity control, target mutants are useful, preventing interaction with the compound, with reduced compound inhibition of the altered target. With single-targets, mutations can be identified by their isolation and mapping of mutations, which increase the MIC significantly. An alternative option is replacing the target gene with a known insensitive version.
- In vivo differences between chemical compounds and genetic knockout: a KO removes all target functions, incl. enzymatic activity and scaffolding, whereas 100% inhibition with small molecules is unlikely. Generally, an inhibitor is given for an established infection, whereas a KO model also addresses initiation of an infection. Given that downstream intracellular pathways may be triggered when bacteria enter a host, a potential target may only be relevant during initial stages of an infection and be dispensable for maintenance. Here, conditional silencing of target expression in established infection can be helpful.
- Pathway analysis: pathways involving targets for antibacterials are generally essential for bacterial viability and often related to macromolecular synthesis. Disruption of the pathway (or reaction) should significantly impair bacterial survival, for instance, under disease-relevant conditions. The target-relevant pathway can be determined by effects on macromolecular synthesis (MMS) - for example using tracers for DNA, RNA, protein, cell wall, and fatty acid/lipid synthesis, assessing the dose response relationship for tool and control compounds. If a macromolecular synthesis pathway is affected specifically, incorporation of precursors of pathway end products should be inhibited preferentially. Inhibition of all pathways within a narrow concentration range indicates an off-target effect, e.g. membrane lysis or energy poisoning.
- Safety: Do close human target homologs exist? Many targets are common (similar) in most bacteria strains, so not only bacterial populations that cause disease but also beneficial bacterial populations may be affected by pharmacologic intervention.
- Permeability and efflux properties: Not only target properties, but also permeability and efflux characteristics of various bacterial strains, especially Gram-negative bacteria, are critical determinants of antibacterial activity. Combination with efflux inhibitors may be useful to prevent bacterial transporters pumping the drug back out of the organism.
- Resistance: Bacteria display various modes of resistance and target-related resistance mechanisms include target modification to alter drug-binding and emergence of alternate mechanisms to circumvent target functions. Thus, a single enzyme as target may increase the likelihood of rapid resistance selection. In this case, the frequency of single-step spontaneous resistance should be monitored as those mutations would be present in a large infectious load, competing with susceptible siblings.
- Target Location and Expression: For Gram-negative bacteria, extracellular or periplasmic targets are preferable as compounds enter through the outer membrane and efflux and penetration of the cytoplasmic membrane are issues. If the target enzyme is known to be present or essential in only specific species, then lack of activity of a compound against other species is supportive evidence of the specificity of action.
- Demonstration of phenotypes expected to be associated with inhibition of the target: These phenotypes include morphological changes, stress responses or specific promoter induction.
In the context of the GOT-IT guidelines, Innovation is defined as a scientific breakthrough in basic research or discovery and its transforming into a marketable product that can be successfully launched into highly regulated markets. The key question is how to position validated targets favorable for the large-scale (external) investment required to develop new therapeutic products.
Thus, this Building Block deals with strategic options for managing and protecting IP associated with validated targets, the competitor landscape, and medical as well as commercial needs - with the aim to generate valuable data for business and commercialization plans, attractive licensing propositions for commercial partners as well as for potential investors.
For a successful translational target assessment project, some of the decision-making needs to be led by drug discovery concerns, e.g. intellectual property. Therefore - rather than trying to draw a line between target validation and drug discovery in the pharmaceutical industry - the following aspects should be considered when bridging the translational gap between where university research often ends, and commercial drug development begins.
Evaluating the innovation potential of the target of interest
Differentiation (current standard of care analysis)
A rationale needs to be provided for how modulation of target has potential to drive differentiation over current standard of care. Ideally, a target is either a) totally novel or b) addressed using novel technology that promises to be advantageous over previous approaches, e.g. allosteric modulators vs catalytic site inhibitors. An assessment of the current standard of care also implies that competitors are identified in the generic and proprietary domains.
As a form of intellectual property, a patent gives its owner specific property rights (for a certain period of time) and may include claims that meet relevant patentability requirements, such as novelty, usefulness, and non-obviousness.
Several points need to be considered when protecting/patenting drug targets:
- The lack of novel chemical compounds or biologics rules out the possibility of obtaining a 'composition of matter' patent, a very strong form of patent protection.
- Patent application against the drug target usually contain two major types of claim:
- 'Reach-through' claims: the use of an agonist/antagonist of target x to treat disease y.
- 'Screening/assay' claims: a method of screening for molecules that agonize/antagonize target x.
- 'Reach-through' claims have limited commercial value, however, as US and European courts have generally ruled against patents which claim the use of any possible hypothetical compound/antibodies against the target of interest.
- 'Screening assay' claims are more likely to be granted as a novel screening assay, developed by the patent applicant, can be included in the patent to exemplify the claims.
- Information about novel compounds, peptides and antibodies can be added into the patent before the end of the first year - even if these molecules do not exist or are not available a the time of filing the patent.
Importantly, a patent cannot be obtained if an invention was previously known or used by others, or published anywhere in the world, including poster presentations and grant abstracts/applications (if published).
A distinction can be made between 3 main types of IP-related searches:
- Patentability search: The purpose of a patentability search is to help decide whether a concept (e.g. target modulation) is unique over what already exists ("prior art"). A patentability search, however, does not provide any assurances on whether it would be safe to e.g. use certain assay technology to identify modulators against a target for the treatment of specified diseases.
- Freedom to Operate (FTO) analysis: FTO is the ability to proceed with a particular activity, such as development of an assay to identify modulator for the target, without infringing the intellectual property (IP) or tangible property (TP) rights of third parties. The focus of an FTO search will center around the patent claims with the goal to see if the new concept omits at least one element of each independent claim in the patents retrieved in the search.
- Patent validity search: The purpose of a patent validity search is to gauge the validity of claims in an already granted patent by searching for prior art publications referencing the claimed elements. Typically, a validity search is directed to finding new prior art that was not cited during the course of the patent-pending application process.
For all these searches, it is critical to understand that patent applications will not be available prior to publication, and so their contents remain unknown for a period of 18 months after the earliest effective filing date.
The target needs a unique selling point over established and emerging therapies, especially compared to similar modalities on the target or in the same pathway. The assessment of the competitive environment is therefore closely linked to the current standard of care analysis and the therapeutic indication: The task is to identify patient needs not yet met by competitors for specific indications and to identify a sub-set of patients who might benefit most from the target modulation. Any patient segmentation will necessarily restricts the size of the market, but, importantly, also accentuates the potential differentiation from the competitors' approach.
Unmet medical needs
The only way to uncover the unmet clinical need is to clearly define the area of clinical interest at the beginning of the project :
The scale of unmet medical need can be assessed by the following factors a) mortality vs morbidity, b) number of patients, c) current treatment (disease modifying or symptomatic) and d) acceptability of current and proposed new treatment (side effects, pricing, dosing, mode of administration).
All target validation approaches should, whenever possible, also incorporate available tool compounds or established drug treatments. As an example, a new drug target in cancer should not only be validated by examination of its effect on tumour weight in either transgenic or knockout mice but it should also be evaluated if the effect is still observed on top of treatment with established anticancer drugs.
In addition, it might be necessary to broaden the therapeutic use by looking for additional indications in which the target might also play a role. This allows for broadening of the therapeutic landscape of a target if, in later phases of development, difficulties arise for the anticipated indication.
Evaluation of commercial needs
Commercial needs are often linked to a reasonable market size and the range of patients available for therapy. Although in many cases, the disease subdivisions are of sufficient size to encourage commercial investment, there can be a major challenge in enabling the prosecution of targeted therapies for rare diseases with small patient populations. Performing high-throughput screens to generate library 'hits' and even drug-like 'leads' can increase the commercial value of early stage biological discovery and the chance of partnering with industry or even creating new spin-out companies based on these discoveries.
Data Quality and Robustness
Several steps within the drug discovery value chain are well controlled by GxP-based quality control (e.g. GLP toxicology). However, these same standards cannot be applied to the non-regulated, preclinical areas of drug discovery and target validation. There is a need for a specialized set of quality guidelines that specifically focus on study design, unbiased conduct, statistical analysis and transparent reporting. Only then can research findings, obtained in either academic or industrial laboratories, form a strong basis for successful drug discovery projects.
The GOT-IT guidelines will facilitate the practical implementation of "confirmatory research" standards in academic target validation. This includes the implementation of crucial processes like blinding and randomization, appropriate statistical power analyses, primary endpoint definitions, etc. to increase the internal validity of experiments.
Multiple independent replicates and preclinical trials via multi-centre studies as well as several orthogonal technologies provide higher confidence and converging evidence in therapeutic relevance of a target.
Quality of tools and reagents
A major requirement for validating experimental results is that researchers routinely question reagent purity, authenticate cell lines, validate antibodies and animal models, and include appropriate controls when assessing the design, execution, and the outcome of an experiment.
An experimental record should provide the amount of information and level of detail to permit peers to reconstruct and/or repeat the experiment based on the information provided and compare outcomes. The description of an experiment should be clear and unambiguous. The ability to find the source of data (raw and analyzed) that is presented in a report or other presentation and a contemporaneous description of the experiment in which the data were generated. Thus, it is critical to ensure that experimental records are attributable, legible, contemporaneous, original, accurate and complete.