Hit-to-Lead Optimization in Drug Discovery


The ultimate goal of hit-to-lead and lead optimization is to identify candidate drugs with the best possible balance of potency, target selectivity, pharmacokinetics and safety, to give drug development programs the maximum chance of success in the clinical development phase. 
Hit-to-lead optimization is a key phase of the drug discovery process. Following high-throughput screening (HTS), which identifies potential drug candidates (hits), the next step is the hit-to-lead process, where researchers strive to refine and improve upon the initial hits, to generate more potent and selective lead compounds. 
The hit-to-lead and lead optimization process require robust biological testing, medicinal chemistry and rigorous safety assessments in order to refine and improve the chemical structures and biological activity of the compounds.  

Fig 1: An overview of key drug discovery phases 

Hit and lead definition – What do ‘hit’ and ‘lead’ mean in drug discovery?

Hit and lead compounds represent key milestones in the drug discovery process. 
A hit compound is a molecule that exhibits activity at the target of interest during an HTS campaign. 
A lead compound is a candidate drug whose structure has been chemically optimized and whose activity has been rigorously tested via biological assays.  
During HTS, millions of compounds may be screened, typically identifying hundreds to thousands of hits. The hit-to-lead and lead optimization process will refine this number down to one or two candidate molecules for clinical development.  

High Throughput Screening in Drug Discovery

HTS is a common screening approach performed in the early phases of a drug development program. Employed for hit identification and validation, HTS harnesses automation and miniaturization to screen libraries of thousands to millions of compounds against the drug target in parallel.  
In an HTS assay, it is possible to screen compound libraries directly against the isolated drug target (biochemical assay), or against more complex in vitro models (cell-based assay). Cell-based screening platforms can provide numerous benefits compared with biochemical assays, allowing researchers to observe the effects of compounds within a more physiologically relevant context, and interrogate activity in addition to target affinity.  
By mimicking the in vivo microenvironment, developers can identify better hit compounds that demonstrate favorable activity.

3D spheroid culture for HTS

With the emergence of 3D cell culture, automated liquid handling and miniaturization technology, cell-based models can now enhance the hit discovery process like never before. In particular, 3D spheroid culture can bring substantial value to the HTS process. Spheroids are standardized multicellular clusters that can be precisely and accurately dispensed into multi-well plates for HTS assays. Spheroids enable the extreme miniaturization of in vitro testing, and are well-suited as an HTS platform, since they are compatible with automated dispensing systems.  

Fig 2: Scaffold-based and ultra-low attachment plate spheroid culture approaches 


In a well-plate format, spheroids can either be seeded into ultra-low attachment plates, or grown in a scaffold/ matrix, in order to prevent them from clumping together. Scaffold-based approaches enable multiple spheroids to be seeded into a single well, and ensure that the distribution of metabolites, nutrients, oxygen and signaling molecules remains stable and standardized throughout the assay. Hydrogels are often employed as a matrix material since they provide more in vivo-like tissue stiffness that can easily be adapted depending on cell type. 
Commercially available 3D culture matrices vary widely in their composition. Although animal-derived matrices are widely available, these regularly display wide batch-to-batch variability, and may contain biological artifacts such as growth factors, that may interfere with assay results. In contrast, GrowDex® hydrogels are made from nanofibrillar cellulose and water, making for a highly consistent product. Opting for an animal-free hydrogel matrix like GrowDex eliminates the risk of animal artifact contamination, ensuring optimal reliability and replicability in HTS results.

Discover GrowDex® hydrogels for high throughput screening 

Hit-to-lead process 

Following HTS is the hit-to-lead phase. This stage aims to refine the large number of hits down into a small number of highly potent and selective lead compounds for optimization. The process usually begins with hit molecules being grouped together based on structural similarity and ranked based on potency, target affinity and ligand efficiency. These groups may undergo preliminary structure-activity relationship (SAR) studies to identify essential molecular elements associated with desirable activity. From this information, developers can identify the most promising hit groups to progress into more intensive testing. 
The optimized hit groups now represent several core compound structures. Through extensive in vitro assays, these candidates undergo lead identification testing to query potency, selectivity and more—to identify the best lead compounds for downstream lead optimization.

Lead Identification in Drug Discovery

Lead identification is the process of selecting the most promising lead compounds from the pool of optimized hits identified during the hit-to-lead phase. The selected leads should exhibit the best balance of desirable properties e.g., favorable activity with minimized off-target effects. 
Various compound parameters are interrogated at this stage, with rigorous biological testing on both in vitro and in vivo systems central to the process. In vitro models are often first utilized, and are well suited to determine activity, selectivity and other key pharmacological properties, e.g., toxicity. While in vivo testing is usually next implemented for drug metabolism and pharmacokinetics (DMPK), plus further safety testing, the vast capabilities of modern 3D cell culture technology are enabling more and more compound parameters to be queried in vitro

Organoid and spheroid models for lead identification 

3D cell culture systems offer substantial advantages over traditional 2D cell cultures in mimicking the in vivo cellular microenvironment. Harnessing 3D models for lead identification provides more physiologically relevant insights into compound behavior, enabling better decision making. 


Table 1: Key differences between organoid and spheroid models 


3D organoid models can be extremely valuable during the lead identification phase. An organoid is a simplified 3D model of an organ or tissue that mimics the key functional, structural and biological complexity of the tissue. Organoids can be derived from primary patient tissues or genetically engineered to accurately model specific disease states. This allows researchers to study drug responses in the context of the target disease.  
By encompassing multiple cell types into a 3D structure, organoids empower developers to make more accurate predictions of in vivo toxicity earlier on in the discovery pipeline. Liver organoids are a highly popular choice for compound safety testing during the lead identification phase, since they can exhibit metabolic functions like drug metabolism.  
Liver organoids can also be maintained in culture for extended periods, opening the door to chronic exposure studies that mimic prolonged drug treatment courses in vivo. GrowDex® hydrogels can support functional primary hepatocytes, liver organoids and other cell lines for up to 35 days, maintaining an optimal growth environment for long-term exposure studies.  

Explore the benefits of GrowDex® for liver organoid culture 

What is the aim of lead optimization?

Following the lead identification phase, several of the most promising lead compound structures are selected for optimization. Medicinal chemistry and confirmatory testing are central to much of the lead optimization process. By modifying the chemical structure of the lead compounds, it’s possible to improve their potency, selectivity, pharmacokinetics and safety. The challenge is to characterize and maintain favorable molecular properties while addressing any structural issues related to toxicity or side effects. 
Any structural molecular changes need to be extensively tested via in vitro and in vivo biological assays. As well as the obvious drug characteristics, additional traits, such as genotoxicity, metabolic stability and in vivo behavior, may be investigated. Another key goal of lead optimization is to identify an optimal dose range, including considerations such as the preferred route of administration (oral, intravenous, etc.), and the development of a drug formulation that ensures long-term stability and reliable delivery of the drug. 

Drug Candidate Selection 

This is the final stage of preclinical drug development. In this phase, a handful of promising optimized lead molecules must now be reduced to just one drug candidate to take forward into clinical testing and development. All data and information gathered up until this point— representing years of work—must now be reviewed to inform this vital decision. 
Drug candidate profiles may be constructed, outlining all drug characteristics and data for a particular lead. Engagement with regulatory bodies will be active during this time, who will outline their expectations and requirements for the clinical development phases. Once the all-important selection has been made, an investigational new drug (IND) application for the selected candidate must be submitted to the regulatory authorities for approval.

Hit-to-lead services

From compound screening to lead optimization, hit-to-lead service providers are a popular choice for developers looking to outsource elements of the drug discovery and preclinical development process. Hit-to-lead service companies heavily rely upon automation and miniaturization to perform screening and biological testing at scale.  
Miniaturized organoid and spheroid models are swiftly becoming essential for processes such as HTS and biological testing, providing biological and disease relevance for improved efficiency in lead identification. Achieving 3D cell models at the scale and level of miniaturization required for hit-to-lead service providers can be challenging; however, automation is imperative for realising this challenge by ensuring reproducibility and scalability. 
Some 3D culture hydrogels are a barrier to successful automation – many must be cooled to keep them in liquid form and possible to dispense. In contrast, GrowDex hydrogels can be handled in room temperature, making them excellent for high throughput assays and handling with pipetting robots or dispensers. GrowDex hydrogels have been proven successful in automated dispensing up to 1536 well-plates, a favorable choice for hit-to-lead service providers.  

Better models, better leads

We have highlighted the important role that biological assays play throughout the preclinical drug discovery process, from HTS to lead optimization. Thanks to the emergence of 3D cell culture, and particularly organoid and spheroid culture, developers can now extract rich data earlier on in the drug discovery pipeline. With the ability to create and miniaturize disease- relevant models, lead candidate selection can be informed like never before.

Moreover, the use of disease-relevant 3D cell models not only enriches the information available about potential drug candidates, but also accelerates the research and development process. By identifying the most promising leads earlier, pharmaceutical companies can streamline their pipeline, reduce costs, and focus resources on the most promising leads.

Thus, lead candidate selection is more informed and efficient than ever before, offering significant cost and time advantages.

Find out how GrowDex® could elevate your hit-to-lead and lead optimization process


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