Serine Protease Screening Libraries

Serine proteases are among the most biologically important families of proteins that have been shown to play multifarious roles in various diseases (such as cancer proliferation, autoimmune disorders, and allergy) [1]. While their main function in humans is coordinating digestion, however, serine proteases are also involved in other processes such as inflammation, blood clotting, and the immune system performance in both prokaryotes and eukaryotes (Fig. 2) [2,3].

Serine proteases have a distinctive structure, consisting of two beta-barrel domains that converge at the catalytic active site, and can be further categorized based on their substrate specificity as trypsin-like (positively charged residues Lys/Arg), elastase-like (small hydrophobic residues Ala, Val, Gly) or chymotrypsin-like (large hydrophobic residues Phe/Tyr/Trp). The enzymes are characterized by a catalytic triad of residues (Ser195, His57, and Asp102, chymotrypsin numbering system) that is responsible for amide bond hydrolysis [4].

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

• Serine Protease Focused Library (8,100 compounds)
• Serine Protease Targeted Library (10,300 compounds)

  Our collection of assay-ready screening sets

• Pre-plated Serine Protease Focused Set: 4,400 small molecules, also available as  complementary and non-overlapping subsets of 3,120 and 1,280 compounds
• Pre-plated Serine Protease Targeted Sets: 6,500 drug-like screening with predicted inhibitory activity against selected serine proteases, available in two subsets of 3,940 and 2,560 compounds.
• Pre-plated Serine Protease Diversity Screening Set: 1,920 compounds selected by high structural diversity to maximize chemical space coverage

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.

Figure 1. Classification and therapeutic applications of serine protease inhibitors (SPIs). Picture source: Harish et al. [4].

Serine Protease Focused Library

This Screening Set contains over 8,100 drug-like screening compounds which are small-molecule analogs of known serine proteinase inhibitors with experimentally determined activity (Fig. 2).

To design this library, a 2D fingerprint similarity search method was used. A reference database of 26,000 biologically active compounds from assays related to serine proteases was compiled, utilizing data from patents and literature publications. The Life Chemicals HTS Compound Collection was analyzed for screening compounds similar to those in the reference database, using MDL public keys and a Tanimoto similarity cut-off of 85%.

Key features:

• No reactive or unstable molecules
• Lilly MedChem Rules
• PAINS filter families A, B, and C applied
• A potential target is indicated for each molecule
• Compound cherry-picking is available

The list of targets used for the Library preparation and a number of their potential inhibitors is represented below:

Representative screening compounds from the Serine Protease Focused Library:

F0381-4714

F0657-0678

F0507-0763

F0265-0940

F3309-2142

F5949-0389

F6665-8583

F1494-0015

F6678-9608

F2262-0555

F3385-2857

F1608-0670

F1051-0076

F0528-0558

F2699-0109

F1765-0146

F3398-5110

F1269-0105

Serine Protease Targeted Library

Life Chemicals has designed this receptor- and receptor-ligand complex-based targeted library of over 10,300 structurally diverse small-molecule screening compounds picked out by virtual molecular screening with predicted inhibitory activity against serine proteases:

• Trypsin
• Chymotrypsin
• Subtilisin-like serine protease
• Thrombin
• Factor Xa (FXA)
• Elastase
• Coagulation factor XIa (FXIa)
• Coagulation factor XIIa (FXIIa)

Key target information was obtained based on known X-ray data for complexes with Chymostatin and other serine protease inhibitors. A virtual screening workflow has been set up, using Schrödinger software, while the bound ligand has been extracted from the reference crystal structures. Molecules from a reference set of different selective serine protease inhibitors and the Life Chemicals HTS Compound Collection that passed in-house structural filters were then docked in the enzyme’s active site. As a result, dedicated screening sets were selected for each target protein. The docked target is indicated for each screening compound.

Representative screening compounds from the Serine Protease Targeted Library:

F6725-0297

F6594-2347

F6806-6963

F6754-6892

F6564-7599

F6808-7771

F6604-1539

F6755-3341

F2758-0236

F6246-3585

F0827-0169

F5031-0108

F1021-1121

F6465-0238

F2135-1573

F3345-0143

Background

Trypsin and chymotrypsin-like serine proteases from family S1 constitute the largest protease group in humans. The prototypes chymotrypsin, trypsin, and elastase represent simple digestive proteases in the gut, where they cleave nearly any protein. Multidomain trypsin-like proteases are key players in the tightly controlled blood coagulation and complement systems, as well as related proteases secreted from diverse immune cells. Membrane-anchored serine proteases play important physiological roles and have emerging functions in cancer [5-7]. But despite the high diversity of all family members, they share the tandem b-barrel architecture of the chymotrypsin-fold in the catalytic domain, conferred by the large differences of eight surface loops, surrounding the active site, including the active-site-containing domain found in the trypsin family members.

The catalytic activity of the serine proteases from the trypsin family is provided by a charge relay system involving an aspartic acid residue hydrogen-bonded to a histidine, which in its turn is hydrogen-bonded to a serine. The sequences in the vicinity of the active site serine and histidine residues are well conserved in this family of proteases [8]. It should be noted that bacterial proteases that belong to the family S2A are quite similar in the regions of the active site residues to follow the same patterns. A partial list of proteases known to belong to the trypsin family is shown below.

Figure 2. Molecular surface of chymotrypsin, trypsin, and elastase with electrostatic potential. Picture source: Goettig, et al. [7].

Trypsin serine protease

Trypsin is a serine protease that is present in the digestive system, mainly in the small intestine. It is involved in the protein digestion cascade along with pepsin and chymotrypsin, hydrolyzing peptides to amino acids. It is particularly substrate-specific, with electrostatic mechanisms allowing it to act virtually exclusively on positively charged arginine and lysine side chains. Deficiency in trypsin is typical to cystic fibrosis patients, where the secretion of an inactive trypsinogen from the pancreas is suppressed. Similar to other serine proteases, trypsin promotes its own production by catalyzing the activation of trypsinogen

Key features

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 1J17, 1QL9, 3AAV, 3AAS, 1PPH, 1FXY
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 1,500 potential trypsin inhibitors.

Figure 4. Spatial structure in the binding site of the complex of trypsin with lead docking molecules of potential trypsin inhibitors (h-bond interactions).

Chymotrypsin serine protease

Chymotrypsin is secreted to the digestive system by the pancreas as inactive precursor chymotrypsinogen. It is one of the enzymes involved in the hydrolysis of proteins, specifically targeting side-chains of aromatic amino acid residues. Conversion of chymotrypsinogen into its active form is facilitated by trypsin.

Key features

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 4H4F, 1BMA
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 1,100 potential chymotrypsin inhibitors

Figure 5. Spatial structure binding site of the complex of chymotrypsin with lead docking molecules.

Subtilisin-like serine protease

Subtilisin-like serine proteaseis one of the most significant representatives of serine proteases, because these extracellular proteases secreted by fungi may function as virulence factors. Such secreted enzymes often play a central role in pathogen establishment. Although subtilisin-like serine proteases are expanded among pathogenic fungi, this group of proteases also is ubiquitous among eukaryotic organisms and presents a new and interesting drug development target.

Key features

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 1WVM, 4LVN
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 1,000 potential subtilisin-like serine protease inhibitors

Figure 6. Spatial structure in the binding site of the complex of subtilisin-like serine protease from Pseudoalteromonas sp. with lead docking molecules.

Thrombin serine protease

Thrombin is a protein involved in catalysis at many steps of the coagulation cascade. It activates fibrinogen, converting it to a form that assembles into insoluble fibrin, and further strengthens the fibrin strands by promoting covalent bonds between lysine and glutamine side chains through the activation of factor XIII. It also activates platelets by cleavage of the N-terminus of protease-activated receptors in their membranes, further contributing to the coagulation process. Furthermore, it is known to be involved in many diseases that are associated with coagulation, hemorrhaging, vasoconstriction, etc.

Key features

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 1ETT, 1ETS, 1ETR
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 1,300 potential thrombin inhibitors

Figure 7. Spatial structure in the binding site of the complex of thrombin with lead docking molecules.

Factor Xa serine protease

Factor Xa serine protease (FXA) is an activated form of the coagulation enzyme, factor X. It produces thrombin by cleaving two sites in prothrombin, an action that is further promoted by binding to the activated factor F, which forms the prothrombinase complex. This protease is one of the most common targets in anticoagulant therapies that attempt to inhibit its activity using small molecules such as heparin, and warfarin.

Key features

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 1EZQ, 1XKB and 1XKA
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 1,600 potential FXA inhibitors

Figure 8. Spatial structure in the binding site of the complex of Factor Xa serine protease with lead docking molecules.

Elastase serine protease

Elastase is a serine protease produced by the pancreas that catalyzes the cleavage of carboxyl groups present in small hydrophobic amino acids, such as glycine, alanine, and valine. Its primary role is the breakdown of elastin, a protein that imparts elasticity to connective tissue. There are up to eight genes that encode elastase or elastase-like enzymes, four of which are classified as chymotrypsin-like. Activation of elastase-type serine protease can lead to acute pancreatitis, chronic inflammatory lung diseases, and cancer.

Key features

• Method: high-throughput virtual screening (docking)
• X-Ray data used: 2V0B
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 500 potential elastase inhibitors

Figure 9. Spatial structure in the binding site of the complex of the elastase with lead docking molecules.

Coagulation factor XIa

Coagulation factor XI (FXI) is a serine protease involved in the intrinsic blood coagulation pathway [10]. Its primary role is to activate factor IX [11-12], contributing to thrombin generation and blood clot formation. FXI also plays a regulatory role in inflammation and vascular permeability [13]. While essential for thrombosis, it has a limited role in normal hemostasis [14], making it an attractive drug target. Inhibiting FXI or its active form, FXIa, has potential therapeutic benefits in preventing and treating thrombotic disorders, including stroke, venous thromboembolism, atrial fibrillation, and sepsis-associated coagulopathy, with a reduced risk of major bleeding [15].

Key features:

• Method: structure-based virtual screening using Phase (receptor-ligand complex)
• X-Ray data used: 6TS5, 6TS4
• Filters used: QikProp properties and descriptors
• Number of compounds selected: 2,900

Figure 10. Compound F2780-0350 (violet) in the binding site of coagulation factor XIa protease domain with active site inhibitor (gray) complex (PhaseScreenScore = 2.413).

Coagulation factor XIIa

Factor XIIa (FXIIa), also known as Hageman factor, is a serine protease derived from the zymogen Factor XII upon contact with negatively charged surfaces [16]. FXIIa plays a central role in the intrinsic coagulation cascade and the kallikrein-kinin system (KKS), promoting thrombin generation and bradykinin release [17]. While it is not essential for normal hemostasis, FXIIa contributes to pathological thrombosis and inflammation [18]. It is implicated in diseases such as hereditary angioedema (HAE) [19], sepsis, and thrombosis associated with blood-contacting medical devices [16]. Due to its selective role in pathological coagulation without affecting bleeding, FXIIa is considered a promising therapeutic target.

Key features:

• Method: structure-based virtual screening using Phase (receptor-ligand complex)
• X-Ray data used: 6B77
• Filters used: QikProp properties and descriptors
• Number of compounds selected: 1,000

Figure 11. Compound F6801-8122 (white) in the binding site of coagulation factor XIIa protease domain with active site inhibitor (pink) complex (PhaseScreenScore = 2.143).

Reference:

1. Soualmia F, Amri C. Serine protease inhibitors to treat inflammation: a patent review (2011-2016). Expert Opin Ther Pat. 2018;28(2):93-110. 

2. Burzynski LC, Humphry M, Pyrillou K, et al. The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin. Immunity. 2019;50(4):1033-1042.e6. 

3. Gorbacheva LR, Kiseleva EV, Savinkova IG, Strukova SM. A New Concept of Action of Hemostatic Proteases on Inflammation, Neurotoxicity, and Tissue Regeneration. Biochemistry. 2017;82(7):778-790. 

4. Harish BS, Uppuluri KB. Microbial serine protease inhibitors and their therapeutic applications. Int J Biol Macromol. 2018;107(Pt B):1373-1387. 

5. Pannkuk EL, Risch TS, Savary BJ. Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans. PLoS One. 2015;10(3):e0120508.

6. Arnesen JA, Małagocka J, Gryganskyi A, et al. Early Diverging Insect-Pathogenic Fungi of the Order Entomophthorales Possess Diverse and Unique Subtilisin-Like Serine Proteases. G3 (Bethesda). 2018;8(10):3311-3319.

7. Goettig, P., Brandstetter, H., & Magdolen, V. Surface loops of trypsin-like serine proteases as determinants of function. Biochimie. 2019. doi:10.1016/j.biochi.2019.09.004.

8. Brenner S. The molecular evolution of genes and proteins: a tale of two serines. Nature. 1988 Aug 11;334(6182):528-30.

9. Rawlings ND, Barrett AJ. Families of serine peptidases. Methods Enzymol. 1994;244:19-61. doi: 10.1016/0076-6879(94)44004-2.

10. Tsutsui S, Yoshimura A, Iwakuma Y, Nakamura O. Discovery of Teleost Plasma Kallikrein/Coagulation Factor XI-Like Gene from Channel Catfish (Ictalurus punctatus) and the Evidence that the Protein Encoded by it Acts as a Lectin. J Mol Evol. 2023;91(4):536-551. doi:10.1007/s00239-023-10113-4

11. Li C, Voos KM, Pathak M, et al. Plasma kallikrein structure reveals apple domain disc rotated conformation compared to factor XI. J Thromb Haemost. 2019;17(5):759-770. doi:10.1111/jth.14418

12. Shearin S, Venkateswarlu D. Structural insights into the activation of blood coagulation factor XI zymogen by thrombin: A computational molecular dynamics study. Biophys Chem. 2022;281:106737. doi:10.1016/j.bpc.2021.106737

13. Puy C, Moellmer SA, Pang J, et al. Coagulation factor XI regulates endothelial cell permeability and barrier function in vitro and in vivo. Blood. 2024;144(17):1821-1833. doi:10.1182/blood.2023022257

14. He X, Zhang J, Du Y, et al. BJTJ-1837, a novel FXI activation-blocking antibody. Res Pract Thromb Haemost. 2023;7(2):100067. Published 2023 Feb 7. doi:10.1016/j.rpth.2023.100067

15. Lorthiois E, Roache J, Barnes-Seeman D, et al. Structure-Based Design and Preclinical Characterization of Selective and Orally Bioavailable Factor XIa Inhibitors: Demonstrating the Power of an Integrated S1 Protease Family Approach. J Med Chem. 2020;63(15):8088-8113. doi:10.1021/acs.jmedchem.0c00279

16. Walvekar VA, Ramesh K, Kannan M, Kini RM, Sivaraman J, Mok YK. Scaffold stability and P14' residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4. Biosci Rep. 2022;42(5):BSR20220421. doi:10.1042/BSR20220421

17. Shamanaev A, Litvak M, Gailani D. Recent advances in factor XII structure and function. Curr Opin Hematol. 2022;29(5):233-243. doi:10.1097/MOH.0000000000000727

18. Xu P, Zhang Y, Guo J, et al. A single-domain antibody targeting factor XII inhibits both thrombosis and inflammation. Nat Commun. 2024;15(1):7898. Published 2024 Sep 12. doi:10.1038/s41467-024-51745-4

19. Clermont AC, Murugesan N, Edwards HJ, et al. Oral FXIIa inhibitor KV998086 suppresses FXIIa and single-chain FXII-mediated kallikrein kinin system activation. Front Pharmacol. 2023;14:1287487. Published 2023 Dec 19. doi:10.3389/fphar.2023.1287487