Hit Identification in Drug Discovery

High-quality hit compounds are small molecules, peptides or biologics which suffice several criteria:

• The compounds chemical structure and purity have been validated.
• The hit compound has confirmed activity on the biological target.
• The interaction with the target is potent and triggers a biological response in a dose dependent manner.
• The compound exhibits desirable characteristics such as efficacy, selectivity, and safety profile. 
• The compound should be an attractive starting point for lead optimization including physico-chemical and ADME properties. 
• The compound should not be covered by intellectual property rights.

If all these criteria are not met, the compound must be characterized as a challenging hit. This can potentially make the hit-to-lead process and further steps of the drug discovery workflow more complex and ultimately failed projects. 

Early stage drug discovery can either start with a known ligand originating from academic literature, natural products or previous campaigns. Alternatively, de novo hit identification can be applied to identify novel chemical modulators. A range of different methods are employed for hit ID, which all focus on the screening of compound collections. Each method comes with strengths and weaknesses and below are the most promising methods summarized. 

High-Throughput screening (HTS) is a cornerstone in modern drug discovery and has since the 1990s been the primary method for identifying potential hits. HTS involves testing large libraries of compounds against a biological target to identify those who exhibit activity. The process of HTS typically allows for a compound library (stored in multi-well plates) against a recombinantly expressed target protein or enzyme. Compounds showing the highest read-out such as fluorescence or luminescence can then quickly be identified, isolated and validated. 

However, HTS is often associated with high cost, as libraries needs to be acquired or synthesized and typically a robotic setup is utilized to handle the compound collection. Further, the compound collection has most often been synthesized in parallel which potentially hampers the diversity. False positives are also often identified from HTS which may arise from nonselective chemical reactions. Finally, either a biochemical or cellular assay, which generates a readout need to be developed. Where this is typically easy for common drug targets such as kinases and other enzymes, it can be more challenging for non-standard targets such as protein-protein interactions. 

With the exponentially growing capabilities of computational tools, in silico methods such as virtual screening has emerged as a powerful tool for hit ID. Virtual screening can be utilized to predict a small molecule interaction with a target protein before screening of physical compounds occurs. 

While it might seem appealing to screen a virtual library to reduce the number of compounds needed for physical screening, several challenges must be overcome to achieve high quality hit compounds. First of all, high-quality structural data is necessary for the target protein as this will be essential for the subsequent docking studies. For example, a low-resolution crystal structure could potentially yield false positives and a lot of wasted resources.

DNA-Encoded Libraries (DELs) have emerged as powerful ways to generate compound collections ranging from a few millions to billions of compounds. Typically, a purified protein target is immobilized onto a solid support, which is then incubated with the DEL. Washing steps allow for the removal of non-binding library members and subsequent elution will isolate binding compounds which can be identified by PCR amplification followed by next generation sequencing (NGS). 

One challenge with DELs arises from the split-and-pool synthesis where the small molecule is synthesized at one end of the DNA strand and the encoding DNA is ligated at the other end. This allows for truncations to appear leading to a high rate of false positives of resynthesized hits. Vipergen utilizes the YoctoReactor® to synthesize libraries, which ensures that non-reacted building blocks fall out of the library during purification allowing for high fidelity of the library and a low false positives rate during screenings.1

Vipergen has developed binder trap enrichment (BTE) technology allowing us to screen without the need for immobilization of the target enzyme.2 Furthermore, Vipergen can now perform the screening in live cells using cellular binder trap enrichment (cBTE) overcoming the issue of expressing recombinant protein.3 This also allows for screening against proteins situated in membranes, which traditionally can be difficult to express recombinantly.

Fragment based screening (FBS) has emerged as an alternative tool to traditional HTS. By screening and identifying small chemical fragments, subsequent work can focus on linking several fragments together to more complex structures with high affinity. This technique called fragment merging can hereby take simple substructures and merge these to form high affinity compounds. Where the initial screening is usually easy, the subsequent merging can be a time-consuming challenge, which often relies on structural data of the target. 

Hits identified from one of the above screening campaigns first needs to be validated. Compounds with apparent activity need to be isolated in high purity and tested in the primary assay. Here false positives arising from impurities (often seen in HTS), fluorescence or aggregation can be removed. Following this filtering of false positives, a range of secondary assays needs to be employed to show the desired biological activity. A typical set of assays which could be employed is the following:

• Flagging of Pan Assay Interference Compounds (PAINS), compounds with low solubility and compounds which have been observed as hits in prior screens.
• Counter screens against targets where the compounds should not be active and cytotoxicity assays.
• Orthogonal assays which serve to confirm target engagement. 

The range of assays which can be employed to validate a hit range from biophysical methods such as NMR and thermal shift assays to showing target engagement in cells. Ultimately, more disease relevant assays should be investigated to show a mode of action of the identified hit. Besides hits identified from the primary screening hits can be optimized through structure activity relationship (SAR) studies.

After a validated hit has been discovered, the next step in the drug discovery process is to refine the hits into more lead structures which ultimately have optimized the following:

• Potency
• Selectivity
• Absorption
• Metabolic stability
• Safety profile
• Solubility

This is typically achieved through SAR studies, which involves modifying the small molecules to enhance biological activity. A set of secondary assays is used to get information, which can be used to exclude certain hits:

• Counter screens
• Bioavailability assays
• Toxicity assays
• Metabolism studies

The overall goal of the process is to develop a lead compound that shows strong potential for preclinical development. This step is essential in early drug development, as it ensures that only the most promising candidates move forward to the preclinical stage, where their safety and effectiveness are rigorously evaluated.

The hit-to-lead process significantly accelerates drug discovery by narrowing down a vast number of initial hits to a manageable number of viable preclinical candidates, streamlining the path to new therapies. Successful optimization during this stage can ultimately lead to a breakthrough drug that improves patient outcomes.

[1] A Yoctoliter-Scale DNA Reactor for Small-Molecule Evolution, J. Am. Chem. Soc. 2009, 131, 1322-1327

[2] Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule Library, Med. Chem. Commun. 2016, 7, 1332-1339

[3] Screening of DNA-Encoded Small Molecule Libraries inside a Living Cell, J. Am. Chem. Soc. 2021, 143, 7, 2751–2756