Cysteine Protease Screening Libraries

Cysteine proteases are one of the most biologically important clusters of proteins involved in a variety of cellular pathways. They participate in multiple processes, among which there are immune invasion, parasite invasion, parasite egress, hydrolysis, extracellular matrix turnover, that makes them promising drug targets for various diseases (e.g., AIDS, cancer, cardiovascular and inflammatory diseases, thrombosis, respiratory disease, pancreatitis, and neurological disorders) [1-2].

Being divided into 14 superfamilies, they are structurally different, however, sharing a common mechanism of action. Their unique role in protein degradation determines a broad distribution of cysteine proteases. It also implies a variability of active sites as a result of the target folding specificity. Cysteine proteases are often targeted with compounds containing electrophilic warheads (such as epoxides or vinyl sulfones) that specifically interact with catalytic residues in the active center to mimic the peptide substrates of the target enzyme. [3]

Life Chemicals has designed two dedicated Screening Sets of potential cysteine protease modulators to facilitate small-molecule high-throughput screening in cysteine protease-related drug discovery projects:

• Cysteine Protease Focused Library by 2D Similarity
• Cysteine Protease Targeted Library by Receptor-based Virtual Screening

The compound selection can be customized based on your requirements, cherry picking is available.

Please, contact us at orders@lifechemicals.com for any additional information and price quotations.

For a pre plated set based on this Screening Library, please explore our Pre-plated Focused Libraries.

Cysteine Protease Focused Library by 2D Similarity

In total, this Screening Set comprises almost 9,200 drug-like screening compounds with potential inhibitory activity against cysteine proteases (Fig. 1-2):

To design this Library, a 2D fingerprint similarity search method was used. A reference database of over 15,000 biologically active compounds from assays related to cysteine proteases was compiled, using the data available from patents and literature publications. The Life Chemicals HTS Compound Collection was searched for small-molecule analogs of these reported cysteine proteinase inhibitors with experimentally determined activity by means of MDL public keys and the Tanimoto similarity more than 85 %. It should be noted that PAINS filters were not applied for this Library preparation, as they would remove a substantial part of both reference molecules and analogous screening compounds due to the nature of cysteine protease-targeted drugs. Reactive compounds were eliminated with in-house MedChem filters.

Figure 1. Compound analogues from the Cysteine Protease Focused Library, which are similar to known cysteine protease inhibitors. 

Cysteine Protease Targeted Library by Receptor-based Virtual Screening

This Screening Compound Library consists of around 5,300 structurally-diverse screening molecules picked out by virtual molecular screening against the following cysteine protease targets:

• Cathepsin K (CatK)
• SARS Papain-like Protease
• SARS Main Protease

Cathepsin K (CatK) Inhibitor Set

Cathepsin K (CatK) is one of the most potent proteases in the lysosomal cysteine protease family, with main function to mediate bone resorption. Currently, CatK is among the most attractive protease targets for anti-osteoporosis drug discovery and development. Moreover, cathepsins and other cysteine proteases may become good targets for major diseases such as arthritis, osteoporosis, AIDS, immune-related diseases, atherosclerosis, cancer, and for a wide variety of parasitic diseases, such as malaria, amebiasis, chagas disease, leishmaniasis, or African sleeping sickness.

At Life Chemicals, we have designed a proprietary Screening Set of over 1,200 drug-like screening compounds with potential cathepsin K inhibitory activity picked out by virtual molecular screening.

Molecules from a reference set of reported selective cathepsin K protease inhibitors and the Life Chemicals HTS Compound Collection were passed through in-house structural filters and then docked in the enzyme active site using the Glide from Schrödinger Suite, while the bound ligand has been extracted from the reference crystal structures. A set of constraints was defined to improve docking result quality.

Key features

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 2ATO, 1NLJ, 1VSN, 1YK8
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 1,200

Figure 2. Spatial structure binding site of the complex of Cathepsin K with lead docking molecule

SARS Papain-like Protease Inhibitor Set

Papain-like protease (PLpro) is a cysteine protease, playing a crucial role in the life cycle of RNA viruses (including coronaviruses). PLpro facilitates virus replication by hydrolyzing peptide bonds in viral and cellular substrates. PLpro is utilized as a drug target in different coronaviruses, including SARS, MERS, and HCV.

The docking-based virtual screening was carried out on the basis of the crystal structure of the SARS CoV-2 papain-like protease in a complex with peptide inhibitor VIR250. Each molecule from the Life Chemicals HTS Compound Collection was docked into the PLpro catalytic site with 6 intermolecular hydrogen bond constraints set, with three of them being of essential importance. Rotated groups in the active site were allowed where necessary. Application of MedChem filters allowed a selection of 1,700 drug-like screening compounds (Fig. 4). 

Key features

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 6WUU
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 1,700

Figure 3. Spatial structure binding site of the complex of SARS Papain-like Protease with lead docking molecule F5824-0042

SARS Main Protease Inhibitor Set

The 2019-nCoV protease active site binders were selected with a docking-based virtual screening of the Life Chemicals HTS Compound Collection against the main protease of 2019-nCoV in complex with an inhibitor N3 (Fig. 4). No docking constraints have been used to allow the docking algorithm to explore as many ligands’ positions and orientations as possible. No Ro5 constraints were applied to avoid filtering out any peptide-mimicking compounds. Any molecules containing toxicophore/reactive groups were excluded to finally result in obtaining 2,300 screeningcompounds.

Key features

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 6LU7
• Constraints: no
• Filters used: toxic, reactive
• Number of compounds selected: 2,300

Figure 4. Spatial structure binding site of the complex of 2019-nCoV main protease with lead docking molecule F1259-0053

References:

1. Turk B. Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 5:785-799, 2006.
2. Verma, S.; Dixit, R.; Pandey, K. C. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets. Front. Pharmacol., 25, 2016, 7 (APR), 107. https://doi.org/10.3389/fphar.2016.00107.
3. L. Kaysser. Built to bind: Biosynthetic strategies for the formation of small-molecule protease inhibitors. Nat. Prod. Rep., vol. 36, no. 12, pp. 1654–1686, 2019, doi: 10.1039/c8np00095f.
4. Fournier J, Chen K, Mailyan AK, Jackson JJ, Buckman BO, Emayan K, Yuan S, Rajagopalan R, Misialek S, Adler M, Blaesse M, Griessner A, Zakarian A. Total Synthesis of Covalent Cysteine Protease Inhibitor N-Desmethyl Thalassospiramide C and Crystallographic Evidence for Its Mode of Action. Org Lett. 2019 Jan 18;21(2):508-512. doi: 10.1021/acs.orglett.8b03821.
5. Costa TF, Lima AP. Natural cysteine protease inhibitors in protozoa: Fifteen years of the chagasin family. Biochimie. 2016 Mar;122:197-207. doi: 10.1016/j.biochi.2015.11.002.
6. Rustgi S, Boex-Fontvieille E, Reinbothe C, von Wettstein D, Reinbothe S. The complex world of plant protease inhibitors: Insights into a Kunitz-type cysteine protease inhibitor of Arabidopsis thaliana. Commun Integr Biol. 2017 Dec 14;11(1):e1368599. doi: 10.1080/19420889.2017.1368599.
7. Lee H, Shin EA, Lee JH, Ahn D, Kim CG, Kim JH, Kim SH. Caspase inhibitors: a review of recently patented compounds (2013-2015). Expert Opin Ther Pat. 2018 Jan;28(1):47-59. doi: 10.1080/13543776.2017.1378426.