Home page > en > Facilities > Screening Platform Marseille-Luminy (PCML)

Screening Platform Marseille-Luminy (PCML)

Thursday 1 December 2011, by Karine ALVAREZ, Karine BARRAL, Bruno CANARD, Cécilia EYDOUX, Jean-Claude GUILLEMOT, Barbara SELISKO

Available translations [English] [français]


The screening platform Marseille-Luminy (PCML) is involved in antiviral research and belongs to the Antiviral Drug Design Platform (AD2P). A collaborative network is on track since 2005 between the screening platform Marseille-Luminy (PCML-JC Guillemot), the screening platform on viruses Marseille-Timone (PCVMT-G.Querat) and the Interactions, Dynamics and Drug Design platform (INT-3D-X. Morelli). (for further information, see : www. ad2p….)

JPEG - 142.4 kb

Next : Activities on PCML

Activities on PCML

PCML is involved in many fields of antiviral research and drug design :

Generation and/or management of library

JPEG - 20 kb

About 60,000 molecules from various origins are in stock:

  • Prestwick commercial library (880)
  • National cancer institute diversity library (NCI)(2000)
  • ChembridgeTM "Diversity Set" library (30,000)
  • The “Chimiothèque Nationale” (21,000) divided into :
    • Strasbourg National Library (5000)
    • Curie Institute Library (8000)
    • Gif-sur-Yvette ICSN chemicals (3000) and natural extracts library (5000)
    • Caen National Library (400)
  • Active sightTM fragment based library (360)

Compounds developed in-house by our team of medicinal chemists (400) or molecules from collaborative studies (2000) are regularly added to this collection.

JPEG - 115.2 kb
Fig 1 : “Mother plates” of a library

Compounds are stored at minus -20°C in 96-well sealed "mother plates" (Fig.1), in 100% DMSO at minus 20°C. "Daughter plates" or duplicates of “mother plates” are filled automatically on a Biomek NX (Beckman) pipetting robot equipped with a 96-channel head (Fig.2).

JPEG - 126 kb
Fig 2 : Biomek NX automation workstation

Enzymatic screening

Compounds can be evaluated for their potential inhibitory activity on purified viral enzymes. These include, but are not limited to:

  • RNA-dependant RNA-polymerases (Flaviviridae, Picornaviridae, Caliciviridae, Nidovirales)
  • Proteases (Flaviviridae, Togaviridae, Alphavirus)
  • NTPases (Flaviviridae, Picornaviridae, Calicivirdae)

And also, but not yet adapted to high throughput :

  • mRNA cap methyltransferases (Flavivirus, Coronavirus)
  • Endoribonucleases (Nidovirus)
  • RNA triphosphatases (Flavivirus)
  • RNA or NTP binding activities (Flaviviridae, Picornaviridae, Caliciviridae, Nidovirus)
  • Poly(ADP)ribose binding activities (Nidovirales, Alphavirus)
  • Helicases (Flaviviridae, Caliciviridae)

Radioactive assays are conducted in a dedicated specific area (P2 laboratory) allowing us to work with 3H et 32P radioelements (Fig.3).

JPEG - 124.8 kb
Fig 3 : Restrictive P2 area to experiment with radioactivity

Radioactive read-outs are carried out by a Wallac MicroBeta® Trilux (Perkin-Elmer) (Fig.4) or a Fujifilm phosphoimager (Life science) (Fig.5).

JPEG - 125.2 kb
Fig 4 : WallacMicroBeta® Trilux (Perkin Elmer)
JPEG - 91 kb
Fig 5 : Phosphoimager Fujifilm (Life science)

In order to perform high throughput testing, assays are robotized using both a Biomek 3000 pipetting robot (mono-channel and 8-channel head) and a Biomek NX (96-channel head, also adapted to 384 screening) (Video 1 and 2).


How do we screen a chemical compound or natural extract library ?
On available assays, a first round of screening is made at 50 µM (or 50 µg/ml in case of natural extracts) (1 well/compound, 80 compounds/plate), which allows us to analyze up to 240 molecules/day in triplicates.
Preliminary plate preparation together with final data analysis usually requires 2 extra days.
The “hit” molecules with an inhibitory activity higher than 70-80% are selected.
Automated "cherry-picking" of hits and the second round screening at 10 µM (or 10 µg/ml) in triplicates is also a matter of a few days.

Third experiment series (confirmation) is usually run with freshly diluted compounds from the original powders, and followed by an IC50 determination. Depending on the results of the screening, the biochemical characterization of the “hits” is pursued.

On new enzymes (external source), a feasibility phase is followed by a development and automation of the assay. The knowledge acquired on previous assays is a strong asset for the success of these developments.

In case the enzyme also is not yet available, tests could be requested at the Cloning and Expression Screening Module of the Structural Genomics Platform of AFMB.

Synthesis facilities and hit to lead optimization

One of our strengths is the presence of a team of medicinal chemists within the AFMB, headed by Dr. K. Alvarez, involved in the development and monitoring of various projects of the PCML. The chemists are involved in analyzing the results, the classification of "hits" and from there, in selecting families of molecules to identify analogues to synthesize as well as in the long process "hit-to-lead” optimization. "
In order to improve the pharmacological profile of the "hits" and / or to improve the activity, specificity, solubility, toxicity, we realize the synthesis of several series of analogues.
In order to address the chemical synthesis of ’improved’ compounds, we chose modern and innovative techniques based on parallel synthesis. We have chemical synthesis (Multi-Buchi reactor Syncore, Microwave Biotage Initiator) and parallel purification devices (Biotage SP4). All of these tools allows us to synthesize targeted compound libraries.
The devices for synthesis and purification are completed by tools to analyze the purity of the generated compounds by HPLC (Waters) and HPLC / MS (ThermoFisher). Both devices can also check / monitor the chemical and / or enzymatic stability of the molecules tested on the screening platform.

JPEG - 9.5 kb
Fig 6 : Multi-reactor Buchi Syncore
JPEG - 7.4 kb
Fig 7 : Micro-waves Initiator Biotage
JPEG - 4.7 kb
Fig 8 : HPLC/MS (Thermofisher)

In cellulo screening

Our activities are based not only on enzymatic screening but also on cellular screening to determinate the cytotoxicity and the inhibitory potential of chemical compounds against viral replicon systems.
- Evaluation of cytotoxicity of chemical compounds can be done with viability assays on two cellular types (BHK-21 and COS-7 cells) and TC50 can be determined.
- Identification of inhibitors against viral replicon systems use replicon-EGFP cell-lines

In the same way as an enzymatic screening, chemical molecules are tested to evaluate their ability to inhibit the viral replicon system developed at the PCML (Fig 9)

PNG - 261.9 kb
Fig 9 : Screening of chemical compounds on a cells-based replicon model
Fig 6. Screening of chemical compounds on a cells-based replicon model : We established a stable cell lines in which non-structural proteins are able to replicate independently as a virus and express PAC-GFP (puromycin N acetyl transferase – green fluorescent protein) protein fusion. The levels of fluorescence are in accordance with the replicon activity and report of the inhibitor potential of chemical compounds

All these assays are based on fluorescence, using a Tecan Safire2 fluorometer (Fig 10).

JPEG - 128.5 kb
Fig 10 : Tecan Safire2 fluorometer

LIMS and data management

Upon the creation of the platform, our aim was to provide a system for managing and tracing experiments done by the platform. To reach this goal, we co-developed a LIMS (Laboratory Information Management System), in partnership with the Modul-Bio company (
The main activity of Modul-Bio is to develop tools for monitoring, control and management of LIMS. This partnership led to the creation of a LIMS suitable for screening and adapted to our specific needs, which met later commercial success and which is used by other academic laboratories and companies.
The LIMS provides solutions for managing screening results through the single management of several databases: the libraries of compounds (plates / visualization / tracking / research), the libraries of viruses (strains, phylogeny, etc.) and screening campaigns (tracking / search results / quality of campaigns).
The integration of new experimental tools to the LIMS is possible through a modular software, a development program and a customized controller. The LIMS has evolved and is a link between the three platforms forming the AD2P.

JPEG - 106.5 kb
Fig 11 : LISM database at the PCML

Each compound of each library is referenced in the database, related to its chemical and biological characteristics. Each plate is bar-coded and both plates and experimental data are recorded in our LIMS database (Fig 11).

Identification of inhibitors against protein-protein interaction in situ and in vitro.

We have developed screening methods to determine the inhibitory potential of chemical compounds against the interaction of protein partners of interest.

PNG - 101.6 kb
Fig.12 : Strategy of Bioluminescence Resonance Energy Transfert to screen chemical compounds
We use the BRET technology to target protein partners, and identified specific inhibitors

Protein-protein interactions can be investigated applying a BRET (Bioluminescence Resonance Energy Transfer) assay in living cells and in lysates. BRET technology is based on luminescence/fluorescence energy transfer detected with a PolarStar Omega device (BMG Labtech) (Fig 13).

JPEG - 111.7 kb
Fig 13 : PolarStar Omega (BMG Labtech)
(Fluorescence and luminescence measurement)

Retour : Introduction | Suite : What can PCML do for you ?

What can PCML do for you ?

Our services are designed to be as modular as possible allowing investigators to choose from several options. The PCML offers its work experience and know-how to :

  • Screening compounds on viral proteins
    • Screening against viral enzymes and biochemical characterization of hits
    • In cellulo screening and cytotoxicity determination
    • Miniaturization of assays for HTS screening
  • Providing chemical libraries
    • General in-house chemical libraries
    • Selection of oriented chemical libraries
    • Synthesis of oriented chemical libraries and hit-to-lead optimization
  • Development of new technologies
    • In situ BRET
    • Fluorescent assays
    • Development of HTRF assays …

It is possible to either screen the inhibitory activity of our compounds on an enzyme of interest, or screen external compounds on our in-house enzymes.
All collaboration opportunities will be readily evaluated, please send your proposals to the head of the platform:
Pr Jean-Claude Guillemot (jean-claude.guillemot

Retour : Activities on PCML | Suite : The Team

The Team

Plateform Head
- Pr Jean-Claude GUILLEMOT

Scientific board
- Pr Jean-Claude GUILLEMOT
- Dr Karine ALVAREZ
- Dr Bruno CANARD

Operating staff
- Dr Barbara SELISKO
- Dr Cécilia EYDOUX
- Dr Karine BARRAL

Cécilia EYDOUX

Retour : What can PCML do for you ? | Suite : Collaborations


List of completed and ongoing collaborative and service projects

  • 2011 Service for REGA Institute Leuven / Belgium (Project SILVER)
    • Providing a chemical library (ChemBridge)
  • 2011 Service for AFMB “Structural disorder and molecular recognition” research group
    • Development and miniaturization of an HTRF assay
  • 2011 Service for REGA Institute Leuven / Belgium
    • Determination of IC50 values of potential inhibitors on dengue virus (Flaviviridae) polymerase
  • 2011 Collaboration with Bombay College Of Pharmacy, Mumbai
    • Screening of potential inhibitor molecules from Bombay College on four in-house methyltransferases (Coronaviridae, Flaviviridae, human)
  • 2010 to date Collaborations within FP7 European Project SILVER (Small-molecule Inhibitor Leads Versus emerging and neglected RNA viruses)
    • Characterization of several inhibitory molecules on Picornaviridae polymerases (specificity, Ki values, mode of inhibition)
    • Characterization of inhibitory molecules on dengue virus polymerase
  • 2007 to 2011 Collaborations with Institut de Chimie des Substances Naturelles (ICSN), Faculté de Pharmacie UMR 8076 Université Paris-sud and LIVE-UNCEA Université de Nouvelle Calédonie
    • Screening of natural extracts and fractions (Bioguided fractionation)
    • Discovery of inhibitors of dengue virus polymerase
    • Biochemical characterization of inhibitors on dengue virus polymerase and their specificity using other virus polymerases
    • Determination of cytotoxicity and inhibitory potency on a viral replicon system
  • 2007 to 2010 Collaboration with Institut Curie Paris, Institut de chimie des substances naturelles (ICSN) and School of Biotechnology and Pharmacy, Strasbourg within ANR Dengue
    • Screening of “chimiothèque nationale”
    • Discovery of inhibitors of dengue virus polymerase
    • Biochemical characterization of inhibitors on dengue virus polymerase and their specificity using other virus polymerases
    • Determination of cytotoxicity and inhibitory potency on a viral replicon system
  • 2006 to 2009 Collaborations within FP6 European Project VIZIER (Comparative Structural genomics of Viral Enzymes involved in Replication)
    • Discovery of an inhibitor of dengue virus nucleoside-2’O methyltransferase
    • Screen of molecules on Coxsackie virus B3 polymerase (Picornaviridae)
  • 2008 Collaboration with the Centre for Infectious Disease Research, School of Chemistry and Molecular Biosciences, University of Queensland
    • Test of the inhibitory effect of anti-West-Nile virus NS5 antibodies on polymerase activity
  • 2008 Service for Vivalis
    • Screening on dengue virus polymerase and determination of IC50 values of interesting molecules.

Retour : Team members - Suite : Publications


Previous : Isothermal Titration Calorimetry
Next : Multi-Angle Light Scattering coupled to Size-Exclusion Chromatography (MALS/SEC)

© AFMB UMR7257