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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 dedicated Screening Sets of potential serine protease modulators to facilitate small-molecule high-throughput screening in serine protease-related drug discovery projects:

  1. Serine Protease Focused Library by 2D Similarity (4,400 compounds)
  2. Serine Protease Targeted Library by Receptor-based Virtual Screening (1,000 compounds)

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.

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

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

Serine Protease Focused Library by 2D Similarity

This Screening Set contains over 4,400 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 collected, using the data available from patents and literature publications. Life Chemicals HTS Compound Collection was analyzed for screening compounds similar to the compounds from the reference database, referring to the MDL public keys and Tanimoto similarity cut-off of 85 %. 

Key features:

  • No reactive or unstable molecules
  • Lilly MedChem Rules
  • PAINS filter families A, B, 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:

  • ATP-dependent Clp protease proteolytic subunit - 1673
  • Cathepsin G - 1
  • Coagulation factor X, XII - 62
  • Dipeptidyl peptidase IV, VIII, IX, II, X, XII - 1482
  • Enteropeptidase - 25
  • Epoxide hydratase - 153
  • Epoxide hydrolase - 19
  • Hepatitis C virus NS3 protease/helicase - 22
  • Hepatitis C virus polyprotein - 8
  • Kallikrein 7 - 391
  • Leukocyte elastase - 290
  • Leukocyte proteinase 3 - 4
  • Matriptase - 21
  • Membrane-bound transcription factor site-1 protease - 2
  • Plasma kallikrein - 2
  • Plasminogen - 3
  • Prolyl endopeptidase - 4
  • Seprase - 3
  • Subtilisin-like serine protease - 26
  • Thrombin - 70
  • Tripeptidyl aminopeptidase - 33
  • Trypsin - 153
  • Tryptase beta-1 - 1
  • Urokinase-type plasminogen activator - 15
  • Serine protease hepsin - 7

Representative compounds from the Serine Protease Focused Library by 2D Similarity

Figure 2. Representative compounds from the Serine Protease Focused Library by 2D Similarity

Serine Protease Targeted Library by Receptor-based Virtual Screening

Life Chemicals has designed a Receptor-based Targeted Library of over 6,500 structurally-diverse small-molecule screening compounds picked out by virtual molecular screening with predicted inhibitory activity against serine proteases:

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.

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 that are secreted from diverse immune cells. Membrane-anchored serine proteases fulfill important physiological tasks with emerging roles 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 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.

  • Acrosin.
  • Blood coagulation factors VII, IX, X, XI and XII, thrombin, plasminogen, and protein C.
  • Cathepsin G.
  • Chymotrypsins.
  • Complement components C1r, C1s, C2, and complement factors B, D and I.
  • Complement-activating component of RA-reactive factor.
  • Cytotoxic cell proteases (granzymes A to H).
  • Duodenase I.
  • Elastases 1,2,3A,3B (protease E), medullasin.
  • Enterokinase (enteropeptidase).
  • Hepatocyte growth factor activator.
  • Hepsin.
  • Glandular (tissue) kallikreins (including EGF-binding protein types A, B, and C, NGF-gamma chain, gamma-renin, prostate specific antigen (PSA) and tonin).
  • Plasma kallikrein.
  • Mast cell proteases (MCP) 1 (chymase) to 8.
  • Myeloblastin (proteinase 3) (Wegener's autoantigen).
  • Plasminogen activators (urokinase-type, and tissue-type).
  • Trypsins I, II, III, and IV.
  • Tryptases.

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

Figure 3. 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 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.

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

 

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

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

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

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

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. 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

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

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

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

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

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

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

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.
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