Cisbio and HTRF®: leadership in assay development and services for drug discovery

Cisbio offers a comprehensive technological platform for GPCR and kinase screening, two of the most important target classes investigated within drug discovery, and a range of custom labeling and assay design and development services. The company also produces a selection of biological reagents and methods used by pharmaceutical and biotechnology companies and contract research organizations. With facilities in France and the U.S. and sales offices in Japan and India, Cisbio meets customer needs in every major pharmaceutical and biotechnology market.

HTRF technology
HTRF combines standard FRET technology with time-resolved measurement of fluorescence and involves the use of specific fluorophores, the fruit of Prof. J.M. Lehn’s work, for which he was awarded a Nobel Prize for Chemistry in 1987. During the FRET process, a specific signal emission is caused by the proximity of two fluorophores, a donor and an acceptor. Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer. Time-resolved measurement allows the signal to be cleared of background fluorescence. HTRF fluorophores allow for the elimination of short-lived background fluorescence while providing sensitivity, stability and reliability.

Due to these long-lived emissions, HTRF provides critical benefits to drug development researchers by limiting assay development time while offering exceptional possibilities for miniaturization and scale-up. HTRF is particularly well-suited to evolving HTS needs and most HTRF assays can be performed under a cell-based mode, which allows for enhanced, more relevant results.

Broad range of products to study important targets

• Cisbio has recognized the need to associate HTRF technology to cell-based formats and to create a new generation of functional tools that can provide information about cell function in a high throughput mode. HTRF Assay Kits benefit from Cisbio’s well-established expertise in immunoassay design, particularly in the areas of GPCR and kinase screening, two of the most important target classes investigated within drug discovery.

GPCR screening
A great deal of Cisbio’s R&D efforts have been committed to developing cAMP assays adapted for GPCR HTS. GPCRs are transmembrane receptors which transmit signals from the outside to the inside of a cell and are involved in every aspect of human physiology, including sleep regulation, blood pressure, mood, food intake, perception of pain, control of cancer growth and immune response. Upon activation, GPCRs carry information within the cell via two major signaling pathways: one results in variations of the cyclic AMP (cAMP) level whereas the other one results in a transient increase of intracellular Ca2+ triggered by inositol (1,4,5) tri-phosphate (IP3).

Cisbio developed the HTRF IP-One Assay, the first cell-based high throughput system that can easily detect inositol(1)phosphate (IP1), one of the major products of the phosphatidyl inositol cascade and which tightly correlates with Gq-coupled activity. IP-One integrates d2, a proprietary small molecule organic dye developed by Cisbio to optimize assay robustness, performance and stability, which was also introduced in three of Cisbio’s cAMP assays due to its advanced properties. IP-One was designed on the same template as cAMP, producing a comprehensive technological platform for Cisbio’s proprietary GPCR screening process.

Kinase screening
Kinases are major targets in drug discovery and of particular interest in oncology research. Cisbio has developed HTRF-based tools for HTS of all kinases, with specific applications for Tyrosine (Tyr) and Serine/Threonine (Ser/Thr) kinases. This kinase screening platform offers customers the ability to use a single technology for both universal and specific kinase assays, saving valuable development time and cost.

HTRF Transcreener™ ADP, developed with BellBrook Labs, is a universal and flexible assay for profiling and HTS of all kinases and ATPases. This homogeneous, non-radioactive solution measures the generation of ADP (Adenosine 5’-diphosphate), which is directly detected by a specific antibody labelled with Eu3+ cryptate and correlates tightly with the amount of phosphorylated substrate. By measuring ADP and not the substrate itself, the assay is compatible with any substrate. The enzyme-free detection mode also provides an alternative with less interference and a lower false positive rate than assays which measure ATP depletion. This is particularly suitable when measuring difficult kinases, such as ones where no specific antibody is available.

HTRF KinEASE™, developed with Millipore (Upstate), is a universal and sensitive assay allowing for profiling and HTS of a large range of Ser/Thr kinases. The overall solution is monoclonal antibody-based, and consists of three different kits which contain the same antibody and one of three defined substrates, S1, S2 or S3. A fourth kit, HTRF KinEASE Discovery, comprises the antibody and all three peptides for any kinase assay development and optimization.

Cisbio also developed a fifth kit that addresses Tyr kinases, HTRF KinEASE TK. Based on the same monoclonal antibody principle as HTRF KinEASE, the kit contains a fourth substrate, S4, and is easy to use for Cisbio’s existing HTRF KinEASE customers. To date, this kinase screening platform has been validated on over 160 kinases.

Inflammation, metabolic diseases and CNS
Cisbio released a number of specified and ready-to-use assays, which also incorporate d2, for the precise quantification of biomarkers such as cGMP, cortisol, PGE2, insulin and cytokines. These are related to important targets in the fields of inflammation, metabolic diseases and CNS-related disorders.

Toolbox reagents
HTRF Toolbox Reagents comprise a comprehensive list of cryptate and XL665 conjugates against different tags, such as GST-6HIS, immunoglobulins as well as conjugated Streptavidin. These reagents are the ideal tool for probing a wide variety of molecular interactions. Over the past ten years, many studies have proven the flexibility of this toolbox to address complex cellular mechanisms ranging from nuclear receptor activity to polyubiquitination, as well as heparanase or protease activity.

HTRF Kinase Toolbox Reagents are recognized as one of the most powerful technologies for kinase screening. Protein kinases, of which phosphorylate protein or peptide substrates are a large family, are enzymes that are involved in intracellular signaling, cell proliferation, differentiation and apoptosis.

Cisbio’s HTRF kinase toolbox reagents are built around a common cassette system involving the specific anti-phosphoresidue antibody labeled with Eu3+ cryptate on one side and on the other, an XL665 conjugate such as Sa-XLent! or anti-GST-XL665, available from the affinity system reagent or the anti-tag toolboxes, respectively. The HTRF kinase toolbox consists of Eu3+-cryptate labeled antibodies for studying both Ser/Thr and Tyr kinases.

Commitment to assay development, custom services and technical support
In addition to a comprehensive line of HTRF-based, ready-to-use assays and toolbox reagents, Cisbio provides custom assay design, development, labeling and on-site support and technical training that enable customers to run HTRF screens more efficiently.

Cisbio’s highly-trained technical team serves as a time-saving resource, answering customer questions and providing on-site support. Dedicated scientific consultants partner with customers for the custom conversion of heterogeneous, radiometric and other conventional immunoassays to an optimized HTS format, and ensure that this transition runs smoothly with a dedicated assay transfer and training program. Cisbio also develops custom HTRF assays for new targets, for example:  protein-protein interactions, protein modifications, immunoassays for the detection of cell secreted markers, protein-DNA/RNA interactions, enzyme activities and receptor ligand interaction.

New assay development can require labeling of specific components, and Cisbio offers custom labeling services for antibodies, peptides, oligonucleotides and other biomolecules using conjugates including cryptate, XL665, d2, biotin or DNP. Its laboratories use both customer-supplied materials and reagents from independent vendors, and are optimized to handle small scale to industrial processing sizes.

To complement these services, Cisbio trains customers on HTRF technology theory and its applications in drug discovery, running HTRF cell-based assays and labeling biomolecules for HTRF.

User-focused event
As part of its ongoing commitment to providing the highest quality technical support to HTRF customers, Cisbio established its annual “HTRF in Drug Discovery Symposium” in 2005 as a forum for exchanging ideas on drug screening and the latest in HTRF innovation. The symposium assembles experts in compound screening and lead optimization, therapeutic area leaders and scientists to discuss HTRF and its applications in drug screening, target discovery and mechanistic.

The symposium addresses the major technical challenges in GPCR and kinase research in an objective, scientific environment. The conference features keynote speeches addressing groundbreaking developments in screening methodologies and a number of in-depth scientific sessions and roundtables held with drug discovery experts who use HTRF. These address how HTRF improves all research phases for new active molecules, in particular when applied to the fields of GPCR and kinase screening, and provides a forum for discussing how HTRF benefits technological evolution in other target areas having similar needs. Scientific support is a key element to assay implementation, and the symposium also enables HTRF users to exchange technical information and network with like-minded experts and Cisbio’s scientific team.

The 2008 “HTRF in Drug Discovery Symposium” will be held in Avignon, France September 24th – 26th.

Partnerships impacting drug discovery
Cisbio has embarked on a number of partnerships and customer-based programs with research laboratories and companies from around the globe. These collaborations combine HTRF technology with R&D innovation to provide new tools and technologies for customers.

Long-standing partnerships are with well-known instrument makers with HTRF-compatible readers (such as Tecan, BioTek Instruments, BMG Labtech, Molecular Devices, Beckman Coulter and Furuno), industrial experts (such as Millipore and BellBrook), as well as leading academic institutes (such as IGF in Montpellier, France).

Cisbio’s commitment to customers and anticipating their needs is also reflected in its recent partnership with Lumiphore, Inc., an early-stage biotechnology company with proprietary lanthanide technology. Cisbio is developing a second generation of HTRF assay platforms for GPCR and kinase screening incorporating HTRF and Lumiphore’s Lumi4™ compounds that has the potential to bring additional benefits to the drug discovery community. Lumi4 compounds bring excellent photophysical properties, increased sensitivity and stability to TR-FRET assays for HTS applications and the growing homogeneous assay market. Cisbio has the exclusive rights to apply this technology to its R&D initiatives in TR-FRET-based assays for drug discovery.

Cisbio and HTRF in brief:
Technology that can apply to all phases of drug screening
Proven robustness and reliability that enables cell-based assays
Product portfolio covering a broad range of targets
Recognized technical support for assay implementation and customized services

HTRF® is a registered trademark of Cisbio
KinEASE™ is a trademark of Millipore (Upstate)
Transcreener™ is a trademark of BellBrook Labs
Lumi4™ is a trademark of Lumiphore