FUN_11

Functional characterization of PfHMGB2 and development as a drug target for the treatment of Cerebral Malaria.

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


SSGCID Functional Project Proposal: 
PI/Lead: Bart Staker, Center for Infectious Disease Research
Co-investigators: Catherine Vaquero, Directeur de Recherche, INSERM,CIMI-Paris, Gabriele Varani, University of Washington

Cerebral Malaria (CM) is an acute manifestation of malaria, fatal in 93% of children diagnosed and responsible for up to a quarter of the 500,000 childhood deaths caused by Malaria each year (WHO, 2014).  Recent research has identified brain-stem herniation as the cause of death in CM.  Herniation of the brain stem is the result of intracranial swelling caused by parasitized erythrocyte sequestration and neuroinflammation (Seydel et al., 2015) . Our collaborator, Dr. Catherine Vaquero, CIMI-Paris, has identified the Plasmodium protein HMGB2 as being necessary for inflammatory pathogenesis in rodent models of CM, suggesting that inhibition of the HMGB2 inflammatory pathway could reduce swelling and increase the survival of children first brought to medical facilities with ongoing CM (Briquet et al., 2015). 

The SSGCID recently solved the NMR structure of PfHMGB2 (PDBID:2MRC, PlfaA.00841.a).  PfHMGB2 is one of two High Mobility Group box domain (HMGB) proteins in Plasmodium falciparum. HMGB's are small (100-200 residues) highly conserved and ubiquitous eukaryotic non-histone nuclear proteins which bind to chromosomal DNA (Briquet et al., 2006).  HMGB proteins bind distorted and bent DNA structures non-sequence specifically.  Although HMGB proteins are intracellular DNA binding proteins, they have been identified to play a significant role in inflammatory processes when secreted or released by cell lysis. HMGB proteins do not stimulate pathogenesis alone, but must first bind to extracellular DNA to induce inflammatory responses.  There is precedent for pharmaceutical targeting of these proteins: human HMGB1 (HsHMGB1) has been investigated as a drug target for inflammatory diseases, including rheumatoid arthritis, atherosclerosis and autoimmune diseases. Several different small molecule compounds as well as antibodies have been shown to bind to HsHMGB1 and inhibit inflammatory responses.

Our goal is to discover and later develop a lead inhibitor compound against PfHMGB2 that is effective in reducing brain swelling and increasing the survival of children with Cerebral Malaria. The work proposed in this Functional study is the logical next step (following structure determination) in the pursuit of this goal and will address two major challenges.  1) There are currently no known compounds which bind and inhibit the malarial PfHMGB2 protein.  2) There are currently no methods to screen or validate hit compounds against malarial PfHMGB2. Given the similarities between human HsHMGB1 and PfHMGB2, we hypothesize that compounds shown to bind to HsHMGB1 can be used as starting points to develop PfHMGB2-based assays and inhibitors, which could then be used in future studies for large scale screening efforts to develop Plasmodium-specific compounds.

Specific Aim 1:  Determine if compounds from the FDA approved compounds list bind to PfHMGB2.
This task will be performed in the lab of Plexera, LLC and carried out during Q1.

Soluble PlfaA.00841.a protein will be provided to Plexera, LLC and screened for binding against the Plexera FDA+ library through Surface Plasmon Resonance (SPR) on a fee-for-service basis ($3874). The Plexera FDA+ library includes 1800 molecules that are FDA approved or proven to be bioactive. There is precedent that HMGB proteins bind to molecules within this set, since HsHMGB1 has been shown to bind several approved compounds, including methotrexate and statins.

Specific Aim 2:  Develop NMR based binding assay for validation of hit compounds.
This task will be performed in the lab of Gabriele Varani at University of Washington, and carried out during Q1-Q3.

The PfHMGB2 structure was solved by Nuclear Magnetic Resonance (NMR) spectroscopy in the lab of Dr. Varani. Thus, amino acid residues have assigned resonance spectra. This information can be utilized to determine if, how and where putative hit compounds are interacting specifically with PfHMGB2 by recording two-dimensional spectra and monitoring changes in the protein signal. These experiments will validate the results of the screen and identify molecular characteristics of their interaction with RNA.

Specific Aim 3:  Develop DNA competition assay using NMR.
Work to be performed in the lab of Gabriele Varani at University of Washington, carried out during Q2-Q4.

Several small DNA duplex structures have been shown to bind PfHMGB2 and HsHMGB1. We will use NMR to screen for binding of PfHMGB2 to DNA in competition experiments with hit compounds to identify small molecules that do not just bind the protein, but inhibit its function as well. Although this might seem a taller order, the protein has a characteristic L-shape that is adapted to bind to DNA. Molecules that bind in the elbow of the structure have the potential to disrupt its ability to adapt to binding to DNA.

TIMELINE

MILESTONE

Quarter 1

09.2015-11.2015

  • M1: Complete SPR screening

 

Quarter 2

12.2015-02.2016

  • M2. Validate compound binding using NMR

Quarter 3

03.2016-05.2016

  • M3. Complete compound screening using NMR

Quarter 4

06.2016-08.2016

  • M4. Complete competition screening of hits using DNA competitor

 

 

The results from these studies will provide direct evidence that small molecule compounds that bind to PfHMGB2 can block DNA binding and identify the first lead compounds available for future drug discovery efforts on PfHMGB2.  The highest affinity lead compound which binds to PfHMGB2 and competes with DNA will be sent to Dr. Vaquero for preliminary testing in mouse models of CM.  Additionally, these results will allow for larger scale screening of compound libraries for compounds which bind to PfHMGB2 and block DNA binding.

Other ongoing studies: Outside of this Functional Study, there are parallel studies progressing through the normal SSGCID pipeline and funding. At the request of Dr. Vaquero, SSGCID is working on the NMR structures of PfHMGB1 as well as Plasmodium vivax (Pv) PvHMGB1 and PvHMGB2.  The work described in this proposal is outside the scope of the normal SSGCID pipeline and cannot be carried out with existing resources on the SSGCID contract

 

Briquet, S., Boschet, C., Gissot, M., Tissandie, E., Sevilla, E., Franetich, J. F., . . . Vaquero, C. (2006). High-mobility-group box nuclear factors of Plasmodium falciparum. Eukaryot Cell, 5(4), 672-682. doi: 10.1128/EC.5.4.672-682.2006

Briquet, S., Lawson-Hogban, N., Boisson, B., Soares, M. P., Peronet, R., Smith, L., . . . Vaquero, C. (2015). Disruption of parasite hmgb2 gene attenuates Plasmodium berghei ANKA pathogenicity. Infect Immun. doi: 10.1128/IAI.03129-14

Seydel, K. B., Kampondeni, S. D., Valim, C., Potchen, M. J., Milner, D. A., Muwalo, F. W., . . . Taylor, T. E. (2015). Brain swelling and death in children with cerebral malaria. N Engl J Med, 372(12), 1126-1137. doi: 10.1056/NEJMoa1400116

WHO. (2014). World Malaria Report. Geneva, Switzerland: World Health Organization.

FINAL REPORT

Functional Study 11: PfHMGB2 compound screens.

Project lead: Gabriele Varani,  UW NMR
Co-investigator: Bart Staker, CID Research

Project collaborators: Catherine Vaquero, Directeur de Recherche, INSERM,CIMI-Paris. 

Status: Completed

Time-line:

ORIGINAL

TIMELINE

MILESTONE

ACHIEVED

Quarter 1

09.2015-11.2015

  • M1: Complete SPR screening

X

Quarter 2

12.2015-02.2016

  • M2: Validate compound binding using NMR

X (failed)

Quarter 3

03.2016-05.2016

  • M3: Complete compound screening using NMR

X

Quarter 4

06.2016-08.2016

  • M4: Complete competition screening of hits using DNA competitor

 

Summary: As reported in the June 15, 2016 Quarterly Report, and agreed upon with the COTR, this project was completed early (retired) since the proteins either aggregated when mixed with the compounds or failed to produce a change in the NMR spectra, suggesting that the reports of direct binding to the target protein in the initial SPR screens was incorrect.


 

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