Chemical Genomics – Biol503 Lecture 4 Other Applications of Chemical Genomics.

Chemical Genomics – Biol503

Lecture 4Other 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

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 ProtectionOf 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 (Kd) 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