Serine Protease Screening Libraries

Serine proteases are one of the most biologically important families of proteins that have been shown to play multifarious roles in various diseases (such as cancer proliferation, auto-immune disorders and allergy) [1]. 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 [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 Serine Protease Screening Libraries for 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)

Serine Protease Focused Library by 2D Similarity

The Life Chemicals Serine Protease Focused Library includes 4,400 drug-like screening compounds that are small-molecule analogs of known serine proteinase inhibitors with experimentally determined activity (Fig. 1).

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 Stock HTS Compound Collection was searched for compounds similar to the compounds from the reference database using MDL public keys and Tanimoto similarity cut-off of 85 %. 

Key features:

  • No reactive or unstable molecules
  • Lilly MedChem Rules
  • PAINS filters 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 1. Representative compounds from the Serine Protease Focused Library by 2D Similarity

Serine Protease Targeted Library by Receptor-based Virtual Screening

One of the most significant representatives of serine proteases is a subtilisin-like serine protease, because these extracellular proteases secreted by fungi may function as virulence factors. Such secreted enzymes may play a central role in pathogen establishment [5]. 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 [6].

In this connection, Life Chemicals designed a receptor-based library of potential subtilisin-like serine protease inhibitors. Key target information was obtained based on known X-ray data for complexes with Chymostatin and other serine protease inhibitors (PDB code - 1WVM, 4LVN, etc.).

Virtual screening workflow has been set up using Schrödinger software, while the bound ligand has been extracted from the reference crystal structure. Molecules from a reference set of   different selective serine protease inhibitors and the Life Chemicals HTS Compound Collection were passed through in-house structural filters and then docked in the enzyme active site.

The screening resulted in around 2,000 compounds which were further narrowed down to almost 1,000 based on manual evaluation of the receptor-ligand complexes and compound diversity.

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

Figure 1.  Spatial structure binding site of the complex of subtilisin-like serine protease from Pseudoalteromonas sp. with two 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.