HTS:  High Throughput Screening

Summary. 
High-throughput screening (HTS) originally referred to an automated strategy to identify specific small-molecule drug candidates among a large library of different small molecules.  HTS assays typically employ a micro-titer plate format (e.g., 96- or 384-well plates), robotic sample dispensing and automatic fluorescence/luminescence read-out.  In G protein-coupled receptor (GPCR) research, HTS can be used to find ligands or drug candidates that bind to a specific receptor target.  HTS methodologies have evolved over time to include cell-based assays in which binding reactions are detected indirectly by monitoring downstream signal outputs, such as increases in intracellular calcium or second messenger concentrations.  The Sakmar Laboratory employs a variety of HTS platforms and novel screening technologies to study GPCR signaling, mainly focusing on chemokine receptors.

Recent Developments. 
The Sakmar Laboratory has a proven track record of HTS assay development and is a major user of the Rockefeller University HTS Resource Center (HTSRC).  In one established assay of GPCR activation, fluorescent calcium-sensing dyes are used to measure changes in intracellular calcium concentration as a function of time.  The Sakmar Laboratory was one of the first laboratories to use this method to study the pharmacology of chemokine receptors. 
Another assay called the SEAP (secreted alkaline phosphatase) assay measures intracellular cAMP levels, which are regulated by inhibitory G proteins linked to chemokine receptors.  One recent innovation involves the development of a useful amber codon suppression system to carry out unnatural amino acid mutagenesis (UAAM) of GPCRs heterologously expressed in mammalian cells in culture.  The UAAM method facilitates the introduction of fluorescent probes into expressed engineered receptors.  The Sakmar Laboratory is also developing high-throughput and low volume assays for detecting GPCR activation using lanthanide-based resonance energy transfer (L-RET) principles.

Chemokine Receptors. 
In addition to these HTS technology applications, the Sakmar Laboratory has several ongoing research projects that require the use of the HTS methods.  For example, the Sakmar Laboratory is involved in an on-going research collaboration with the International Aids Vaccine Initiative (IAVI) to develop Rhesus monkey CCR5 expressing stable cell lines, which will be used as tools in the development of an AIDS vaccine.  The development of stable cell lines requires careful functional characterization of the expressed receptor using HTS methods.