Functional Study FUN_14

David Fox III, Senior Manager, Beryllium (Bainbridge Island, WA)
Collaborators: Damian Krysan, University of Rochester (Rochester, NY), Mitchell Mutz, CSO Amplyx Pharmaceuticals (San Diego, CA)

Generation of inhibitors of Acetyl-CoA Synthetase from pathogenic fungi.

Target Background

Acetyl CoA synthetases (ACS) are present in organisms from bacteria to humans and catalyze the conversion of acetate to acetyl-CoA, an essential component of two-carbon metabolism in all forms of life. In many fungi, ACSs are essential sources of the nucleocytoplasmic acetyl CoA that is required for such critical cellular processes as histone acetylation, lipid biosynthesis, and energy metabolism. In contrast, mammals generate this essential pool of acetyl CoA using ATP-citrate lyase in conjunction with the TCA cycle. This dramatic metabolic distinction between yeast and mammals indicates that ACSs represent a novel and attractive antifungal drug target with a very low potential for human toxicity. To date, only three inhibitors of this enzyme have been reported; one of which was discovered by our collaborators at Rochester.  As described in a recent publication in ACS Infectious Diseases, Dr. Krysan has developed a set of assays and tools to evaluate the biochemistry of candidate ACS inhibitors as well as in vivo assays to assess whether new inhibitors are on-target in the cell. Specifically, these assets place us in an excellent position to perform secondary screening assays to evaluate hits derived from fragment-based screening. The target appears to have the potential to be quite broad spectrum. Indeed, unpublished studies by Dr. Krysan using an identified inhibitor of ACS indicates that it is active against nearly every important human fungal pathogen including some for which there are no current medical therapies.  Importantly, Dr. Krysan has the ability to evaluate its activity against C. albicans, C. neoformans, and molds---the most important human fungal pathogens.

Project Status

We have successfully achieved production of full-length and truncation variants for the primary pathogenic fungi that pose a threat to human health, Cryptococcus, Aspergillus and Candida.  Amplyx and the University of Rochester have provided chemical matter, both designed and natural inhibitors of ACS, for use in NMR and DSF screening and crystallization trials.  Crystallization trials in the presence of both natural substrates and products, along with designed inhibitors have yielded three unique structures of Cryptococcus neoformans ACS (CrneC.00629.a) in the presence of: 1) propyl-AMP (PDB ID: 5IFI), 2) propyl-AMP + Coenzyme A, and 3) AMP (latter two structures are in progress).  These highly informative structures have provided insight into the design of inhibitors and validates our approach. In addition, Beryllium has provided collaborators at University of Rochester with purified ACS for activity testing in enzymatic assays for the further development of inhibitors. 

Functional Study Proposal:

Specific Aim 1: Identification of novel Cryptococcus ACS inhibitors:

The primary purpose of the functional study will be to perform fragment library screening of purified ACS by STD-NMR and to identify fragment hits for both elaboration and crystallization. As indicated above, only three inhibitors of any acetyl CoA synthetase have been reported previously. ACS is a member of the Acyl CoA synthetase/nonribosomal peptide synthase/luciferase family of adenylating enzymes.  Other members of this family have been explored as antibacterial targets for the development of drugs against Gram-negative bacteria and M. tuberculosis.  Thus, ACS represents a novel member of a class of druggable enzymes. 

We plan to interrogate Cryptococcus ACS with fragment compound libraries starting with the Fragments of Life (FOL) with an option to expand to additional fragment libraries, together totaling 5,000 fragments. The primary screening assay will be Saturation-Transfer Difference NMR (STD-NMR) spectroscopy.  As the enzyme performs two successive reactions, we will first screen for compounds against the apo protein.  To identify compounds that are competitively or non-competitively binding in the presence of ATP/AMP, we will then add Propyl-AMP to the apo sample, a mimetic of the first-reaction product, acyl-AMP.  The final addition will include Coenzyme A to identify compounds that are competitive or non-competitive with this substrate. Propyl-AMP will be used in place of Acyl-AMP to mimic, but prevent the natural enzymatic reaction, which converts Acyl-AMP + CoA into Acetyl-CoA.    

We plan to perform orthogonal biophysical binding assays on select STD-NMR hits to assist in rank ordering for chemical elaboration. Through resources available at Beryllium and Rochester, biophysical assays may include one or more of the following: 1) Surface Plasmon Resonance (SPR), 2) Isothermal calorimetry (ITC), and/or Differential Scanning Fluorimetry. Purified pathogenic fungal enzymes (Cryptococcus, Aspergillus, Candida, and Coccidioides) will be compared to the commercially available S. cerevisiae enzyme. Rochester will determine if strongly binding fragments (apparent KB < 50 µM) are able to inhibit ACS activity directly using in vitro ACS inhibition assays, and in vivo antifungal activity and on-target biological assays. This will allow us to rapidly prioritize fragments for further development. We have previously established an optimized co-crystallization system for Cryptococcus ACS1 with compounds.  Therefore, at Beryllium, we plan to co-crystallize and/or soak compounds identified with Cryptococcus ACS1.  Crystal structures generated will provide the structural information for initiation of structure-based drug design efforts.  

Specific Aim 2: Chemical elaboration of ACS inhibitors:

Since Rochester has a currently active program of structure-activity work, we are in a position to take high priority fragments and apply both computational chemistry and molecular modeling to synthesize candidate inhibitors. These candidates can then be evaluated using in vitro ACS inhibition assays; antifungal activity assays; and on-target cell biological assays that we are currently using to evaluate derivatives of the scaffold based on the previously identified inhibitor. Compounds of interest will then be placed into crystallization trials at Beryllium to provide structural feedback required for compound elaboration.  While NMR screening and evaluation of fragment hits are being conducted in Specific Aim 1, Rochester and Beryllium will evaluate both natural and designed inhibitors of ACS1 through techniques and methods discussed above.   

Future Impact of Study:

ACS represents a completely novel antifungal target that has been initially developed in Dr. Krysan’s laboratory.  The structural data from the crystallography that has emerged from the work at SSGCID/Beryllium is a huge boon to on-going efforts to rationally and systematically develop inhibitors of this promising target.  Dr. Krysan at Rochester is preparing an R01 renewal application that will focus on achieving these goals.  The fragment-based screen proposed herein will provide invaluable leads for the design of scaffolds and focused activity-based screens for new inhibitors.  To date, only three structural classes of antifungal drugs have been developed during the era of anti-infective research; the studies proposed herein could facilitate the development of a novel addition to that meager armamentarium. We project approximately 0.5 FTE (20 hours/wk for one year) at Beryllium for this research. Rochester will provide separate funding for activities outlined at Rochester. 




Quarter 1


  • STD-NMR fragment screen (Beryllium, Aim 1)
  • Synthesis of ACS inhibitors (Rochester, Aim 2)
  • Co-crystallization of ACS inhibitors designed at Rochester (Beryllium, Aim 2)

Quarter 2


  • Biophysical screening of fragment hits (Beryllium/Rochester, Aim 1)
  • Enzymatic and biological assays of screening hits (Rochester, Aim 1)
  • Co-crystallization of screening hits (Beryllium, Aim 1)

Quarter 3


  • Design and synthesis of fragment derivatives (Rochester, Aim 2)
  • Confirm activity of derivatives using enzymatic and biological assays (Rochester, Aim 2)
  • Co-crystallization of screening hits (Beryllium, Aim 1)

Quarter 4


  • Co-crystallization of designed compound derivatives (Beryllium, Aim 2)
  • Submit manuscript detailing fragment screening, characterization and first round of chemical elaboration, provide supporting RO1 material (Beryllium/Rochester).


Featured Structures

Overlay of 3 Bartonella BID domains

Bartonella BID

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