Chemical Genomics – Biol503 Lecture 4 Other Applications of Chemical Genomics

Chemical Screening by Mass Spectrometry

Chemical Screen Assays Many chemical screening assays rely on fluorescent strategies to report on enzymatic activities However, the need for labels may be a detriment due to: Labels can compromise activity of substrates Some enzymatic assays can not be adapted to fluorescent labels Fluorescent properties of small molecules in the libraries can lead to false positive signals

MS Methods Mass Spectrometry based methods avoid the need for labels in analyzing the products of enzymatic reactions because they report on the mass of the substrate, an intrinsic property of every molecule Min et al. described a class of surfaces that both simplifies sample preparation and gives clear and easily interpretable peaks in MS They combine self-assembled monolayers (SAMs) with MALDI-TOF mass spectrometry to discover inhibitors of anthrax lethal factor

Anthrax virulence factors Bacillus anthracis secrete three major virulence factors: Protective antigen, it binds to a TEM-8 cell- surface receptor of the host cell and mediates delivery of edema factor and lethal factor into the cytosol Edema factor, an ademylate cyclase that causes edema Lethal factor, a protease that cleaves N terminus of mitogen-activated protein kinase kinase (MAPKK or MEK1)

Anthrax lethal factor Lethal factor Source: Min et al Nat Biotech 2004

Strategy and Results

Dose response curve for compound DS-998 Source: Min et al Nat Biotech 2004

Chemical Genomics coupled with MS Source: Min et al Nat Biotech 2004 Cellular Protection Of DS-998

Chemical Screening using Microfluidics

Microfluidics Microfluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale. Quake Group at Stanford developed a microfluidic platform for measuring binding constants by using mechanical trapping of molecular interactions (MITOMI)

Microfluidics The Quake Group studied RNA binding by the HCV transmembrane protein NS4B It was shown that NS4B binds RNA and that this binding is specific for the 3’ terminus of the negative strand of the viral genome with a dissociation constant (K d ) of 3.4 nM. A high throughput microfluidic screen of a compound library identified 18 inhibitors of binding of RNA by NS4B.

Maekkl and Quaje, Science 2007 MITOMI (Mechanical Trapping of Molecular Interactions)

Chemical Genomics coupled with microfluidics Source: Einav et al Nat Biotech 2008

Chemical Genomics coupled with microfluidics Source: Einav et al Nat Biotech 2008