“In the past, thousands of proteins were considered undruggable. This meant that previous efforts to develop a drug against them failed. Today, the combination of novel chemical modalities and advanced technical approaches has resulted in new clinical candidates for previously undruggable targets.”

Nuska Tschammer, Senior Manager Scientific & Technological Leadership at CRELUX, a WuXi AppTec company

  

Examples of Challenging Targets

  

Key Characteristics of Challenging Targets

• Lack of catalytic active sites 
• Presence of metal ions 
• Lipophilicity of residues
• Featureless binding sites
• Need for adaptive  conformational changes

  

Addressing Formerly Undruggable Targets with These Screening Methods...

Affinity Selection Mass Spectrometry (AS-MS) Screen

Definition: A method to assess the binding of the compound to soluble protein target.

Screened Molecules: ~300K.

Advantages: 

• Ultra-high throughput
• Also applicable for solubilized membrane proteins
• Depending on their solubility, low-affinity compounds (〜100 μM) can still be detected

Limitations:

• Low-affinity binders hard to detect due to high off-rates
• Protein-ligand complexes may be fully or partially distorted in the process

  

DNA Encoded Libraries (DEL)

Definition: In DEL, compounds are individually coupled to DNA tags, which are used as amplifiable identification barcodes.

Screened Molecules: 80B+.

Advantages: 

• Several screenings for different targets can run in parallel
• Billions of compounds can be screened in a small format inside an Eppendorf tube
• Low protein requirements

Limitations:

• Resynthesis of hits without the DNA linker required
• Limited synthesis possibilities due to aqueous chemistry
• Requires deep sequencing to detect lower frequency hits

  

High Throughput Screen (HTS)

Definition: A method for the identification of active compounds or biologics that modulate a particular biomolecular pathway.

Screened Molecules: ~300K.

Advantages: 

• Information about the activity of the compound
• Biochemical, automated microtiter plate assay
• Compounds can be screened in intact cells for phenotypic/functional responses

Limitations:

• Only compounds with a strong affinity (< 10 μM) are identified

• Problems screening more difficult targets, such as protein-protein interactions

• Usually requires known binding site or activity

  

Microscale Thermophoresis (MST) Fragment Screen

Definition: A technology for detecting the movement of fluorescent molecules in temperature gradients.

Screened Molecules: 3,5K+.

Advantages: 

• Low protein consumption
• Applicable to solubilized membrane proteins
• High sensitivity

Limitations:

• Requires fluorophore labeling

• Requires strong intrinsic fluorescence of the target

  

Nuclear Magnetic Resonance (NMR) Fragment Screen

Definition: Ligand-observed NMR confirms ligand binding to unlabeled protein Protein-observed NMR monitors changes in the protein signal upon ligand binding.

Screened Molecules: ~1,5K

Advantages: 

• Suited for studying protein-fragment interactions

• Unlabeled ligand & protein in every experiment

• Can derive structural information

Limitations:

• Requires large amounts of isotopically labeled protein

• Large library screening is challenging

• Highly sensitive to false-positives

  

Surface Plasmon Resonance (SPR) Fragment Screen

Definition: An optical biosensor that measures the interactions between immobilized molecules and molecules in solution.

Screened Molecules: 3,5K+

Advantages: 

• Low protein consumption

• High sensitivity

• Accurate affinity & kinetics measurements

Limitations:

• Immobilization can cause inactivation of low-stability proteins

• Signal may be affected by the solvent effect

  

Virtual High Throughput Screening (vHTS)

Definition: A computational method to screen in silico collections of compound libraries to identify the binders for a given target.

Screened Molecules: ~200M

Advantages: 

• Screening can be performed without physically existing compounds

• Saves time compared to HTS

• Make-on-demand libraries facilitate access to compounds

Limitations:

• Requires structural information on the target or its homologs

• Access to compounds is not always possible

• False positives & false negatives possible

  

X-ray Crystallography Fragment Screen

Definition: A method that uses the fragments’ crystal structure to determine its binding mode.

Screened Molecules: ~1K

Advantages: 

• Structural information at atomic resolution

• Fine mapping of fragment binding site

• Can even identify weakly-binding fragments

Limitations:

• Requires large quantities of homogeneous protein

• No affinity information

• Relatively low throughput

  

How WuXi Leveraged Its Expertise to Find The Right Screening Method for STING

The STING Pathway

STimulator of INterferon Genes or STING plays a crucial role in the innate immune system and was long perceived as a difficult and almost undruggable target.

 In 2010, for instance, a Phase III clinical trial testing the promising STING-targeting compound DMXAA flatlined. It turned out that DMXAA targeted only mouse STING protein and could not activate human STING, a crucial factor that had been overlooked from the preclinical phase through Phase II.

 As a pattern recognition receptor, STING plays a part in the body’s response to pathogens, tumors, and DNA in the cytoplasm. When activated, STING triggers the production of Type I interferons and pro-inflammatory cytokines, which can be used in the fight against cancer.

  

   

Screening Methods Used…

MST and SPR were chosen due to their high sensitivity, low protein consumption, and short turnaround time. The hits were identified and confirmed within three weeks.

• MST & SPR used to screen 2600+ molecules in WuXi CRELUX’s fragment library to identify novel STING-binding chemical scaffolds
• X-ray crystallography used to determine fragment binding sites and their orientation in the STING fragment binding pocket

  

Results

  

Conclusion

The screening methods employed helped the WuXi team at CRELUX to understand the key interactions between the fragments and the protein.

Fragment screens are well suited for gaining knowledge on the druggability of a particular target or binding site. Therefore, the high fragment hit rate determined by the WuXi team is a promising starting point for the development of a high-affinity, small-molecule ligand.

Furthermore, the team identified novel scaffolds among the fragments, which will now undergo a first round of in silico growing. The process will be followed by hit-to-lead optimization and take place within the WuXi AppTec units.

  

Sources

WuXi AppTec, Journal of Chemistry & Biology, FEBS Letters, Progress in Biophysics and Molecular Biology, Journal of Biomolecular Screening, The International Journal of Student Research, Journal of The American Society for Mass Spectrometry, Bioinformation, ACS Medicinal Chemistry Letters, Cell Chemical Biology, Small Molecule Targets In Immuno-Oncology

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Text: Larissa Warneck

Design and images: Elena Resko

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