FUN_05

FUN_05: Use the SSGCID solved structure of Brucella melitensis methionine-tRNA-synthetase (MetRS) to find inhibitors for potential drug development for brucellosis.

 

THE PROJECT PROPOSAL IS PRESENTED HERE AND THE FINAL REPORT IS PRESENTED BELOW.

 

SGCID Functional Project Proposal:  PI,  Wesley C. Van Voorhis MD PhD, Co-investigators: Stephen M. Boyle PhD, Prof Emeritus and Director, Center for Molecular Medicine and Infectious Diseases, Virginia Tech, Blacksburg, VA;  Erkang Fan PhD, University of WA (UW) Dept Biochem; Kayode Ojo PhD, UW Dept Med; Thomas Edwards PhD, Emerald Bio.


Brucellosis remains a very hard infection to treat, requiring at least two antibiotics and multiple months of therapy.  New antibiotics are needed for Brucella and other Gram-negative Rod (GNR) bacteria that can treat these infections which have become increasingly resistant to available antibiotics.  Methionine-tRNA-synthetase (MetRS) is known to be a good target for bacteria, particularly when there is only one version present in the genome, such as Brucella spp. which have only one type 1 MetRS(1) gene. The MetRS1 proteins have one zinc binding domain whereas MetRS2 have two and generally inhibitors directed to MetRS1 do not inhibit MetRS2.  Within SSGCID, we have solved the structure of Brucella melltensis MetRS1 (PDB 4DLP, BrabA.10201.a.A1).   Prof. Erkang Fan of Univ. of WA Biochemistry Department has developed a panel of about 60 MetRS1 inhibitors for a NIH-funded project with Trypanosoma brucei MetRS1.  We plan to screen these inhibitors against BmMetRS1 by thermal melt, then solve structure of the best inhibitors by co-crystallography, then use the structures of these inhibitors to improve the potency and drug-like properties of the inhibitors.  At the same time, the inhibitors will be tested for their activity against E. coli and E. coli with the EcMetRS replaced with BmMetRS1 gene for complementation.

Specific Aim 1:  Screen BmMetRS1 protein with Dr. Fan’s MetRS1 inhibitors.
Work to be performed in the lab of Kayode Ojo PhD at UW, carried out during Q(s) Q1-Q2.

The screening will be performed first by thermal melt, then by enzyme inhibition.  We predict many of these inhibitors will cause thermal shifts in melting of BmMetRS1 and will inhibit BmMetRS1 activity, and we will use these studies to prioritize studies for Aim 2.

Specific Aim 2: Co-crystallize BmMetRS1 protein.
Work to be performed in the lab of Thomas Edwards at Emerald, carried out during Q(s) Q1-Q4. 

The co-crystallization will be performed with the best MetRS1 inhibitor(s) identified by thermal melt and enzyme inhibition assays. The co-crystal structures will allow ideas for improving potency of molecules while optimizing drug-like properties for Aim 3. Once Aim 3 is underway, co-crystallization with improved compounds will likely occur during Q4.  

Specific Aim 3: Use medicinal chemistry to optimize hits-to-leads.
Work to be performed in the lab of Erkang Fan PhD at UW, carried out during Q(s) Q2-Q4.  

The work will improve inhibition of BmMetRS1 and at the same time improve drug-like properties of hits.  In addition to testing these inhibitors in Aims 1&4, we will employ in vitro and in vivo PK/ADME/Toxicology assays to optimize their drug-like properties.

Specific Aim 4: Perform mutant growth inhibition studies.
Work to be performed in the lab of Kayode Ojo at UW (E. coli  in vivo testing) and in the lab of Steven Boyle, Virginia Tech, carried out during Q(s) Q2-4.

Test the compounds against E. coli wild type strain (wt) and E. coli with native EcMetRS deleted and replaced by BmMetRS1.  Our prediction will be that the latter strain, transfected with BmMetRS1, but not the wt E. coli strain will be sensitive to growth inhibition and death by the inhibitors. We will also send inhibitors for testing for growth inhibition and killing of Brucella spp.  (Dr. Boyle’s lab).   

In the end, the structure of  SSGCID structure for Brucella melltensis MetRS1 (PDB 4DLP, BrabA.10201.a.A1) will be used to initiate a structure based drug development utilizing inhibitors and co-crystallography to mprove and optimize drug-like inhibitors as potential antibiotics with activity for brucellosis and other bacteria with type 1 MetRS1. We will not complete all 4 Aims in 12 months, but the results will be competitive to apply for external funding to continue this project and to prepare a publication for submission to a peer-reviewed journal.  We estimate 2080 hours (1 annual FTE) effort, in aggregate will be required, over a period of 12 months, to perform the experiments described above.  This work cannot be carried out with existing resources on the SSGCID contract. The results of this research will be published and will facilitate a starting point for structure-based drug development (SBDD) for Brucella and other Gram-negative bacteria.

FINAL REPORT

Functional Study 5 (FUN_05): Use the SSGCID solved structure of Brucella melitensis methionine-tRNA-synthetase (BrMetRS) to find inhibitors for potential drug development for brucellosis.

Project lead: Wes Van Voorhis,  UW PPG

Project collaborators: Stephen M. Boyle, Center for Molecular Medicine and Infectious Diseases, Virginia Tech, Blacksburg, VA; Erkang Fan, Department of Biochemistry, and Kayode Ojo, Department of Medicine, University of Washington, Seattle, WA.

Status: Completed

Time-line:

ORIGINAL

TIMELINE

MILESTONE

REVISED   MILESTONE

ACHIEVED

Quarter 1

09.2013-11.2013

  • Screen existing   inhibitors for binding to BrMetRS and develop enzyme assay
  • Co-crystallize   initial inhibitor(s) with BrMetRS

 

(X)

Quarter 2

12.2013-02.2014

  • Construction of E. coli expressing BrMetRS for   screening
  • Begin MedChem of   initial inhibitor of BrMetRS

 

(X)

Quarter 3

03.2014-05.2014

  • Complete med chem   to increase potency of BrMetRS inhibitors
  • Test compounds from   med chem in E. coli BmMetRS
  • Construction of E. coli expressing BmMetRS for   screening
  • Begin MedChem of   initial inhibitor of BmMetRS
  • Complete MedChem to   increase potency of BmMetRS inhibitors
  • Test compounds from   MedChem in E. coli BmMetRS

(X)

Quarter 4

06.2014-08.2014

  • Co-crystallization   of increased potency compound from Q2-Q3 MedChem efforts
  • Test compounds for   inhibition of Brucella and E. coli BmMetRS growth
  • Complete   construction of E. coli expressing BmMetRS for screening
  • Test compounds for   inhibition of Brucella and E. coli-BmMetRS growth
  • Complete analysis   of fragment screen

(X)

 

Summary: Brucellosis remains a very hard infection to treat, requiring at least two antibiotics and multiple months of therapy.  Thus, new antibiotics are needed for Brucella and other Gram-negative Rod (GNR) bacteria that can treat these infections, which have become increasingly resistant to available antibiotics.  Methionine-tRNA-synthetase (MetRS) is known to be a good target for bacteria, particularly when there is for bacteria such as Brucella that have only one type 1 MetRS(1) gene. MetRS1 proteins have one zinc binding domain, whereas MetRS2 have two, and generally inhibitors directed to MetRS1 do not inhibit MetRS2.  Within SSGCID, we solved the structure (PDB: 4DLP) of Brucella melltensis MetRS1 (BrabA.10201.a.A1) and Erkang Fan of Univ. of WA Biochemistry Department had developed a panel of >90 MetRS1 inhibitors for a NIH-funded project with Trypanosoma brucei MetRS1.  This FUN project planned to piggy-back on his work and use these inhibitors to develop an assay, find the best inhibitors, and initiate an structure-based antibiotic development campaign for  new drugs against Brucella.  As detailed below, we completed two of the four Specific Aims, while another Aim was partially completed.  Drs Fang and Buckner will continue this project independently and are applying for funding from the NIH and other sources.  The co-crystal structures generated by SSGCID will inform their synthetic program, as they seek to retain the low nM potency of the lead compounds, while improving their PK properties. 

Specific Aim 1:  Screen BmMetRS1 protein with Dr. Fan’s MetRS1 inhibitors.

Completed during Quarter 1 in the labs of Erkang Fan and Kayode Ojo at UW, with help from Ranae Ranade in Fred Buckner’s lab at UW.  The screening was performed first by thermal melt, then by enzyme inhibition.  Dr. Ojo developed an efficient KinaseGlo (ATP consumption) enzyme assay; Dr. Fan screened over 90 compounds from his MetRS inhibitor library and three enzyme inhibitors were detected from the thermal melt shifting-compounds (1312, 1415, and 1433).  Because of limitations of the ATP depletion assay, Ranae Ranade developed a radiolabeled Met assay for BmMetRS1, which showed lower IC50 values for all three compounds.

Specific Aim 2:  Co-crystallize BmMetRS1 protein.

Completed during Quarters 1 & 2, in the lab of Thomas Edwards at Beryllium, with the work performed by David Dranow.  The co-crystallization was successfully performed with three MetRS1 inhibitor(s) identified by thermal melt and enzyme inhibition assays. 

Specific Aim 3:  Use medicinal chemistry to optimize hits-to-leads.

Not completed:  Unfortunately, the structures of 1312, 1415, and 1433 bound to BmMetRS1 revealed no clear pathway toward further optimization for more potency, as as the major barrier was poor pharmacokinetics (rapid metabolism) of the compounds  Since we were unable to predict compounds with a better scaffold to over-come this problem, Beryllium performed a fragment screen using NMR in order to acquire new leads for MedChem. The primary screen was completed during Quarter 4 and a follow-up screen is queued for confirmation of the top 24 fragments and determination of their position by NMR and/or co-crystallization.  The results will be released to Drs Buckner and Fang so they can continue new scaffold development in order to find compounds with better PK.

Specific Aim 4:  Perform mutant growth inhibition studies.

Partially completed:  We initially planned to test the compounds against an E. coli strain with native EcMetRS deleted and replaced by BmMetRS1, since we predicted that this strain (but not WT E. coli) would be sensitive to growth inhibition by inhibitors of BmMetRS1.  However, despite 100s of hours of work by Drs Hillesland and Ojo at UW, they were unable to complete the construction of the replacement vector and transfect it into E. coli. Therefore, it was decided to directly test the three compounds for activity against Brucella melitensis during Quarters 3 and 4, in collaboration with Stephen Boyle’s laboratory at Virginia Tech. All three compounds demonstrated low micromolar activity against B. melitensis in the two assays performed.

 

 

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