FUN_16

Functional Study FUN_16

PI/Lead:  Ron Quinn, Eskitis Institute for Drug Discovery, Griffith University, Australia.

SSGCID Lead:  Garry W. Buchko, Pacific Northwest National Laboratory.

 

Bioaffinity Mass Spectrometry Small Molecule Screening of Mycobacterium Targets


Since its inception, SSGCID has determined the structure for 181 Mycobacterium proteins.   Among this list of potential drug targets are ten proteins with no known function and 41 with annotated “putative” or “probably” function.  We propose to use a new high throughput protocol to screen a library of small molecules against these targets to assist/confirm the biological function of these “unknown” and “putative” proteins.    


Bioaffinity Mass Spectrometry Screening (BMSS) is a novel screening approach using electrospray ionization Fourier Transform Mass Spectrometry (ESI-FTMS) under physiological condition as a detection means. High resolution, high mass accuracy measurements coupled with soft ionization techniques to preserve the integrity of complexes allows for the direct observation of ligand-protein complexes. Fourier transform mass spectrometry (FTMS) can detect a non-covalent protein-ligand binding interaction. This is a top-down method that detects intact proteins in the native or folded state. The output is a spectrum containing native protein and ligand-bound protein. The difference between the mass-to-charge ratio (Δm/z) for the unbound protein and the ligand-bound protein complex ions multiplied by the charge state (z) directly afford the molecular weight of the bound ligand.  Upon finding “hits” it is then possible to bin the compounds into strong/medium/weak binders by adjusting the data collection parameters.


The assay will be performed using a natural product fragment compound library of 452 fractions that show anti-TB activity.  This activity against Mycobacterium tuberculosis H37Rv was selected from 202,983 compounds in the Nature Bank fraction library (AstraZeneca (Bangalore)).  Pools of eight compounds will be incubated with protein for one hour at room temperature. When a noncovalent complex forms, the molecular weight of the binding compound can be deduced from the spectrum.  To date our group has identified 26 drug-like, low MW natural products that bind to nine M. tuberculosis proteins, including Rv0577 and Rv2763c, proteins provided by SSGCID for our initial “proof-of-principle” efforts.

  
Specific Aim #1:  BMSS screen 48 “unknown” or “putative” Mycobacterial proteins.

Work to be performed at the Eskitis Institute for Drug Discovery.

 

Ron Quinn and his team at the Eskitis Institute for Drug Discovery at Griffith University in Australia will screen one target/week for 48 weeks (one year).  Protein will be provided by SSGCID (largely frozen stock currently under storage).

 

Specific Aim #2:  Co-crystallization screening.

Work to be performed at Beryllium.

 

Compounds observed to bind tightly to their targets will be supplied by the Eskitis Institute for Drug Discovery to Beryllium for co-crystallization screening.   These screens will enter the normal SSGCID crystallization pipeline.  

 

Specific Aim #3:  Chemical shift perturbation studies to map ligand-binding surface.

Work to be performed at PNNL.

 

Chemical shift perturbation studies will be performed on compounds (supplied by Eskitis) that bind to targets whose structure have been determined using NMR methods.  If necessary, accurate binding constants will be determined using Isothermal Titration Calorimetry.  

 

The results of these efforts will be made publicly available through reports and peer-reviewed publications. Identified chemical probes will provide a better understanding of the function of these “unknown” and “putative” proteins and useful starting points for drug discovery

 

 

TIMELINE

MILESTONE

Quarter 1

 

  • Screen 12 targets

 

Quarter 2

 

  • Screen 12 targets
  • Co-crystal screens or NMR perturbation studies.

Quarter 3

 

  • Screen 12 targets
  • Co-crystal screens or NMR perturbation studies.

Quarter 4

 

  • Screen 12 targets
  • Co-crystal screen or NMR perturbation studies.
  • ITC.
  • Reports/publications.

Featured Structures

Overlay of 3 Bartonella BID domains

Bartonella BID


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