Project Update: 19 January 2019

Dear Backers,

Thirteen compound analogues of a scaffold from a lead hit were purchased. The same compounds were shipped to my collaborator at New York University, School of Medicine where she will perform biological testing.

There were a couple of changes to the project that I want to mention. Originally we were going to test both glucose kinases (i.e. hexokinase and glucokinase) in Trypanosoma cruzi parasites. However, we decided to not work with the T. cruzi hexokinase anymore for this particular project. Instead, we already expressed and purified T. cruzi glucokinase and we are in the process of expressing and purifying Homo sapiens hexokinase IV (HsHxKIV). The first two photos below show the over-expression work we have recently completed in the lab for HsHxKIV. This change was made to see how well a compound could inhibit TcGlcK vs. HsHxKIV in selectivity. The final change made in the project was that in addition to observing the T. cruzi biological testing, we will also look at how well compounds work in the biological testing of Leishmania parasites, particularly the species L. amazonensis. Leishmania protozoa are another trypanosomatid parasite but cause severe skin infections. This parasite also contains a glucokinase that has a 44% overall sequence identity to that of TcGlcK (see the 3rd photo).

We should hear back in a month or so from now on the biological testing versus both parasites. For enzyme assays, once the HsHxKIV is purified, we will run the primary screens using TcGlcK and HsHxKIV. This will take place in early February.

Yours Truly,

Dr. Edward D'Antonio

 

This is a 5-mL culture of E. coli that over-expresses the enzyme H. sapiens hexokinase IV.

 

Protein over-expression time point to inoculate flasks containing 500 mL of E. coli media using 5-mL E. coli starter cultures.

 

Structure-based sequence alignment of L. braziliensis glucokinase (PDB entry 6EDI; Buechner et al., 2019) vs. T. cruzi glucokinase (PDB entry 5BRD; D'Antonio et al., 2015). The overall sequence identity is 44% based on this alignment.

References

Buechner, G. S., Millington, M. E., Perry, K., and D'Antonio, E. L. (2019) The crystal structure of glucokinase from Leishmania braziliensis. Mol. Biochem. Parasitol. 227, 47-52. DOI: 10.1016/j.molbiopara.2018.12.002.

D'Antonio, E. L., Deinema, M. S., Kearns, S. P., Frey, T. A., Tanghe, S., Perry, K. Roy, T. A., Gracz, H. S., Rodriguez, A., and D'Antonio, J. (2015) Structure-based approach to the identification of a novel group of selective glucosamine analogue inhibitors of Trypanosoma cruzi glucokinase. Mol. Biochem. Parasitol. 204, 64-76. DOI: 10.1016/j.molbiopara.2015.12.004