High Throughput Screening

In vitro enzymatic assays

• Screening cascade - inhibitors of small molecules

  

• Enzymatic assays

  

  Measurement of substrate reduction (ATP, NAD/NADP, SAM, Acetyl-CoA, peptide, DNA, RNA, etc.) - Down assayMeasurement of products - Up assay

• Enzymatic assay - example 1

  HTRF assay-Homogeneous Time Resolved Fluorescence

  
  Donor=Eu and Tb
  Acceptor=XL665
  Pros：(1)Highly specific, high signal/background ratio；(2) Highly stable fluorescence signal;(3)Homogeneous reaction, high throughput.Cons：(1)Cost for specific antibody is relatively high; (2)Specific antibodies need to be developed for specific target.

• Enzymatic assay - example 2

  ADP-Glo assay

  
  Pros：(1) Highly sensitive；(2) Could detect enzymes other than kinases.

• Enzymatic assay - example 3

  Z’-Lyte assay

  
  Pros：(1)High signal/background ratio, high throughput；(2) Low cost.Cons：(1)Could not detect enzymes with complex reaction mechanism；(2)Compounds with fluorescence itself will be hard to be evaluated；(3)False positive data will occur when testing proteases.

• Enzymatic assay development – key factors

  
  
  

In vitro cellular assays

• Phenotypic assays：Cell proliferation, migration, invasion, apoptosis, necroptosis, autophagy, etc. Functional assays:Downstream signal like protein phosphorylation, second messanger（Ca²⁺, cAMP, IP3, cGMP）.Protein-protein interaction(PPI) in cells:NanoBiT,NanoBRET,BiFC.
  

• Cellular assay development - cytotoxicity test

  1. Cell seeding density Cell viability >80%Seed cells in a plate with different cell densityCulture for 72 to 96 hours，CTG/CCK8 detection of cell viability.Select the density with CTG/CCK8 signal in a linear rangeWindow (Signal/background ratio)>2
  2. Incubation time Select the optimized density and seed cellsIncubate cells/compounds for different time pointsDetect cell viability with CTG/CCK8The time point with a best assay window and data quality will be used for further experimentsWindow (Signal/background ratio) >2
  3. DMSO tolerance Different doses of DMSO were incubated with cells for 72 to 96 hours, cell viability were tested with CTG methodNote: DMSO typically is less than 0.5% in cellular assays
  
  4. Z’ and edge effect QC criteria: Z’>0.4, Window>2Usually edge wells will not be used for long term culture
  
  5. Compound validation Positive compound should be 5-fold within reference data
  
  Plate layout：
  

High throughput screening

• High throughput screening assay flow chart

  1. Assay optimization (Z’, assay window, DMSO tolerance, reference compound test)2. Pilot study with thousands of compounds will be run first to test the assay3.Orthogonal assay will also be set up to eliminate the false positive compounds4. Run HTS with tens of thousands of compounds5. Hits rate usually will be set to 0.1% to 0.5%

• High throughput screening - case study

  PSMA:

  Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed high throughput PSMA enzymatic assays to identify new regulators of PSMA.
  

  Z-factor test and assay window test

  
  384 well plates were used for assay validation, data from four plates showed that Z-factor is higher than 0.5, and assay window is higher than 10-fold.

  HIT identification

  
  Totally 100 x 384 well plates were used for screening of more than 30,000 compounds, and finished in 10 days, hits rate is about 0.8% when cut off was set to 50% inhibition of PSMA activity.

Summary

• Medicilon has strong capability in assay development and compound screening.
  We work closely with partners such as Selleck, MCE and Tocris that provide compound libraries, and we have more than a library of 3000 compounds in-house.
  Our screening capacity could be 200x 384 well/week, or customize it per clients’ needs.

Appendix

• Compound library from MCE (3rd party vendor)

  Library name	Size and Cat.No.	Description
  50K Diversity library	50K  HY-L901	Compounds in this library were selected by dissimilarity search (Tanimoto coefficient of 0.52) to provide a higher variety and broader chemical space coverage.
  UORSY Screening Library	680K  HY-L0103V	UORSY Screening Compounds Library contains about 680,000 compounds. The library is constructed via a polymerization synthesis method that provides a highly diverse chemical structure. More than 85% of the compounds in the library have drug-like physicochemical properties, and more than 35% of the compounds have lead-like properties.
  Bioactive Compound Library Plus	20K  HY-L001P	• Bioactivity and safety confirmed by preclinical research and clinical trials. Some have been approved by FDA. • Widely used in the research focus areas such as Cancer, Stem Cell, Neuronal Signaling, Immunity, and more.
  Peptide library	1.1K  HY-L105	• Includes bioactive peptides, amino acid derivatives, and blocking peptides. • A useful tool for peptide drug discovery and vaccine development.
  10M Virtual Diversity Library	10M  HY-L912V	With MCE's 40,662 BBs, covering around 273 reaction types, more than 40 million molecules were generated. Compounds which comply with Ro5 criteria were selected. Inappropriate chemical structures, such as PAINS motifs and synthetically difficult accessible, were removed. Based on Morgan Fingerprint, molecular clustering analysis was carried out, and molecules close to each clustering center were extracted to form this drug-like and synthesizable diversity library. These selected molecules have 805,822 unique Bemis-Murcko Scaffolds (BMS) with diversified chemical space. This library is highly recommended for AI-based lead discovery, ultra-large virtual screening and novel lead discovery.
  Membrane Receptor-targeted Compound Library	4.2K  HY-L150	• A unique collection of 4333 small molecules targeting variety of membrane receptors. • Targets such as FGFR, TRP Channel, VEGFR, TNF Receptor, Opioid Receptor, 5-HT Receptor, ion channel, etc.
  Fragment Library	22K  HY-L032	• A unique collection of 21965 fragment compounds for traditional lead identification via high-throughput screening (HTS). • A useful tool for the fragment-based approach to drug discovery (FBDD).
  Traditional Chinese Medicine Active Compound Library	2.7K   HY-L065	• A unique collection of 2712 active compounds of Traditional Chinese Medicine for high throughput screening (HTS) and high content screening (HCS). • The compounds in the library contain Saccharides and Glycosides, Phenylpropanoids, Quinones, Flavonoids, Terpenoids and Glycosides, Steroids, Alkaloid, Phenols, Acids and Aldehydes.

  These libraries are available in MCE(MedChemExpress, a 3rd party vendor). We can collaborate with MCE on using them. The one we have in house is Traditional Chinese Medicine Active Compound library.