Anti-microbial lead discovery

Stage No. Compounds No. Wells Parallel testing
Screening 150,000 600,000 Four screening campaigns against pathogens
Confirmation 1,630 13,000 Profiling on four strains at two concentrations
Potency determination 1,600 96,000 Serial dilutions for six assays

In an ongoing collaboration, 150,000 compounds from the Exquiron compound library served as starting point for an anti-microbial hit discovery campaign against various pathogens in parallel. Exquiron provided assay-ready plates to the Client, initially for screening, and then for confirmation of active compounds.

Exquiron then acquired solid compound, and upon solubilization and generation of serial dilutions for MIC determination and cytotoxicity evaluation, potency and selectivity profiles for library compounds were determined at the Client. Structural clustering of validated hits by Exquiron’s computational chemistry team permitted identification of active chemotypes, which were further complemented by a search for analogues in a database of more than 5 Mio commercially available compounds. Identified compounds were purchased as solid stock, or resynthesized, solubilized and provided as dilution series to the Client for activity, selectivity and cytotoxicity determination.

Currently, the Client has initiated profiling activities on a broad set of in vitro and in vivo assays, fully supported by our compound management expertise, to further prioritize and select hit series for progression to lead status.

Multivariate analysis of cell phenotype, viability, and cytokine secretion of human PBMCs



Recent successes of immunomodulatory approaches for the treatment of serious diseases such as cancer have generated a significant growth in efforts aimed at the discovery of novel therapeutics in this area. Immunomodulatory agents are expected to affect interactions among cells and signaling molecules involved in regulating the immune system. Therefore, hit discovery campaigns need to deliver profiles of the impact of compounds on these complex interactions.

Human peripheral mononuclear cells (PBMCs) are a primary source of various immune cells – including but not limited to NK-cells, B-cells, and T-cells of various subtypes and stages of differentiation. PBMCs are an excellent model system for studying effects of potential drugs on the immune system, as many of the modulatory effects of compound treatment can be recapitulated. In this complex cell mixture, activation or suppression of the immune response (immunomodulation) is often seen in concert with coordinated cytokine secretion patterns.
T-cells can be activated by treatment with phytohemagglutinin (PHA), which will trigger proliferation of the T-cell population as well as modulate the cytokine secretion profile of a PBMC culture as a whole. T-cells are identified by the surface marker CD3. A specific subtype, cytotoxic T-cells (CTL), will also express CD8. Compounds that alter the ability of PHA-stimulated T-cells to proliferate or secrete certain cytokines might be candidate immune-modulatory compounds for further investigation.

Screening worklow
PBMCs were batch-labeled with the MultiCyt® FL4 Cell Proliferation Dye before plating into 384-well plates containing compounds from a SAR expansion selection based on known immunomodulatory substances. Each plate also included four reference substances as dilution series (Resveratrol, Verapamil, Dexamethasone and Mitomycin C). Following plating of cells, PHA was added and cells were incubated for 3 days under appropriate tissue culture conditions. After incubation was complete, 10 µL aliquots were stamped from each treatment plate into a multiplex of immunophenotyping antibodies (anti-CD3-FITC and anti-CD8-PE) and the MultiCyt FL3 Membrane Integrity Dye. A second stamp of 3 µL from the same motherplate was used for QBeads detection of IL-17f, IL-6, and TNF. Each plate was read on the iQue Screener immediately after staining, without wash steps. Each 384-well plate took about 25 minutes to read (Fig A, courtesy of IntelliCyt Corp.)

Data analysis
Among all the data generated by the iQue Screener, eleven parameters were extracted based on their biological significance.
Data were normalized plate-wise to the PHA-activated control cell population, using a modified z score transformation, and activity profiles were generated.

Calculation of the Euclidian distance between profiles and subsequent similarity search against the profiles of the reference substances allowed the identification of compounds displaying specific phenotypes (Fig B: Cpds1-4 induce dexamethasone-like, Cpds 5-9 verapamil-like phenotypes).
Through clustering and subsequent visual inspection of the activity profiles, compounds eliciting new phenotypes (i.e. phenotypes not covered by the controls or reference substances) were identified as well. Examples for compounds inducing TNF secretion are shown in Fig C.

Index Population Parameter
1 Total cells % proliferated cells
2 % viable cells
3 Cytotoxic T-cells
% cytotoxic T-cells (CTL)
4 % viable CTL
5 % proliferated CTL
6 Other T-cells
% non-cytotoxic T-cells (nCTL)
7 % viable nCTL
8 % proliferated nCTL
9 Secreted cytokines IL-6 (median FL2-H)
10 TNF (median FL2-H)
11 IL-17f (median FL2-H)

High-throughput, multiplex screening of compounds on primary cells generates information-rich multivariate compound activity profiles that can be used for identifying or prioritizing potential therapeutics candidates.
Application of advanced data mining techniques to these profiles allows for the rapid identification of compounds with activity similar to reference substances (potentially bridging the gap between phenotype and mechanism of action), but also identifies compounds eliciting new, potentially interesting phenotypes.

Collaborative hit discovery project with SAMDI Tech



Exquiron and SAMDI Tech worked jointly on a Client’s hit finding campaign in which two related enzymes (Enz1 and Enz2) were targeted. In a duplexed assay format, the two distinct peptide substrates (S1 and S2) and products (P1 and P2) were detected and quantified by the SAMDI label-free assay technology (Figure A, courtesy of SAMDI Tech, Inc.). Being a mass spectrometry-based platform, SAMDI allows multi-analyte analysis without the need for antibodies, fluorophores, or radioactivity. Exquiron provided assay-ready compound plates, SAMDI Tech developed and optimized the assays, performed HTS using its platform, and provided data files to Exquiron for data evaluation. Exquiron compiled data and determined hits using specific criteria, and provided characterization of selected compounds in a counter-screening assay with fluorescence readout.

In the assay set up phase, Exquiron first provided uniformity plates and serial dilutions of a reference inhibitor for potency determination on both targets. Next, 10,000 compounds were provided at two different concentrations for a pilot screen to determine compound concentration for HTS.

After determining an appropriate screening concentration of 10 µM, the remaining 240,000 compounds from the Exquiron collection were plated and shipped to SAMDI Tech in less than three weeks. The HTS was completed in four weeks, including data analysis and hit calling. The assays were very robust with average Z’ of 0.82, and only one plate (from 740 plates) failed the Z’ quality criterion of >0.6 (Figure B). Given the low plate failure rate, compounds from this plate were repeated in the hit confirmation phase.

For activity confirmation of putative hits, 6,600 compounds were cherry-picked in duplicates and were provided to SAMDI Tech within one week of hit calling decision. The same compound set was picked and tested in parallel in a counter assay performed at Exquiron.

Finally, 692 confirmed active compounds were picked and formatted as serial dilutions in quadruplicates, and provided to SAMDI Tech for potency determination. In parallel, the same set of compounds was tested in the counter assay at Exquiron, and purity was determined by LCMS analysis. The entire data analysis and curve fitting was carried out at Exquiron, with IC50 values, graphical overlay plots and results of purity determination provided 4 weeks after initiation of this phase.

A summary of the project with phases and timelines is shown in the table.

Project Phase No. Compound Plates No. Compounds No. Data Points Average Z' Turnaround (Weeks)*
Assay Development 18 - - N/A 3
Pilot Screen 60 (2x 30) 10,000 40,000 0.80 3
HTS 740 240,000 480,000 0.82 7
Hit Confirmation 76 (2x 38) 6,600 26,400 0.84 (enzymes)
0.90 (counter)
IC50 Determination 132 (3x 34) 692 41,520 0.79 (enzymes)
0.89 (counter)

*All timelines include preparation and shipment of assay-ready compound plates to SAMDI Tech in the United States.

SAMDI Label free screening technology





The self-assembled monolayer matrix assisted desorption ionization (SAMDI) technology relies on high density biochip arrays on microplates for immobilization of substrates and products of enzyme reactions, combined with MALDI-TOF mass spectrometry. All steps of the process are compatible with automated liquid handling in 384- and 1536-well format.

Assay volumes and throughput
Assay volumes can be as low as 1 µl, both with reactions in solution, or with assay components immobilized on 384-well or 1536-well microtiter plates. Together with fast readout times, this renders the process amenable to high throughput, enabling processing of >10,000 samples per day. Assay development fully considers enzyme kinetics to warrant robustness (Z’ routinely ~ 0.8) while maintaining sensitivity.

Application to histone methyltransferase PRMT1 enzymatic reaction
Shown in the figure is the application of the SAMDI technology to the detection of peptide methylation products through the histone methyltransferase PRMT1. After the enzymatic reaction, the peptidic analytes are attached to the monolayer on the gold surface and analyzed through MALDI-TOF mass spectrometry. The relative abundance of substrate, mono- and di-methylated products can be quantified and the PRMT1 enzyme kinetic can be followed over time (image courtesy of SAMDI Tech, Inc.)

Application examples
Enzyme families Substrate types Surface compatibility Capture chemistry
Methyltransferases Peptides Enzymes Thiol-maleimide
De-methylases Proteins Cell lysates Click, azide-alkyne
Acetyltransferases DNA/RNA Detergents NHS-Amine
De-acetylases Antibodies Salts Biotin-Streptavidin
Kinases Small molecules Organics Electrostatic interaction
Phosphatases Custom chemistries
Ubiquitin ligases
and many more…