Assay for inhibitors of equilibrative or concentrative nucleoside transporters

Patent metadata

Field	Value
Publication number	US-10648012-B2
Application number	US-201414759973-A
Country	US
Kind code	B2
Filing date	Jan 8, 2014
Priority date	Jan 10, 2013
Publication date	May 12, 2020
Grant date	May 12, 2020

Abstract

Methods and systems for identifying inhibitors of Equilibrative Nucleoside Transporters are provided. Methods and systems for identifying inhibitors of Concentrative Nucleoside Transporters are also provided.

First claim

What is claimed is:

1. A method for identifying an agent as an inhibitor of an equilibrative nucleoside transporter (ENT) comprising
contacting a recombinant yeast, in the presence of a cytotoxic nucleoside and in a yeast culture medium with the agent for a first time period under conditions permitting yeast growth,
wherein the cytotoxic nucleoside is present in the yeast growth medium at a level which has been predetermined to be a minimum concentration of cytotoxic nucleoside that causes complete killing of the recombinant yeast,
and wherein the recombinant yeast comprises a deletion of a yeast de novo purine biosynthetic pathway gene in a genome thereof and expresses a heterologous equilibrative nucleoside transporter (ENT), wherein the gene is a fuil gene or Saccharomyces cerevisiae ADE 4; ADE5; ADE7; ADE8; ADE6; ADE1; ADE2; ADE13; ADE16; ADE17; or ADE12 gene:
quantifying the growth of the recombinant yeast in the presence of the agent;
comparing the growth of the recombinant yeast in the presence of the agent to the growth of the recombinant yeast for a second predetermined time period in the absence of the agent; and
identifying the agent as an inhibitor or not of the ENT,
wherein an increased growth in the presence of the agent as compared to in the absence of the agent indicates that the agent is an inhibitor of the ENT, and wherein no growth in the presence of the agent as compared to in the absence of the agent indicates that the agent is not an inhibitor of the ENT.

2. The method of claim 1 , wherein the growth of the recombinant yeast is measured indirectly by quantifying optical density (OD) of the medium containing the recombinant yeast.

3. The method of claim 1 , wherein the agent is a small organic molecule of 2000 daltons or less.

4. The method of claim 1 , further comprising determining if an agent identified by the method as an inhibitor of the ENT inhibits growth of, or kills, a parasite in which the ENT is found in the wild type thereof, comprising contacting the parasite with the agent identified by the method as an inhibitor and determining the growth or viability of the parasite in the presence of the agent, wherein an agent identified by the method as an inhibitor and which inhibits growth of, or kills, the parasite, is identified as an inhibitor of the growth of, or killer of, the parasite.

5. The method of claim 1 , wherein the cytotoxic nucleoside is 5-fluorouridine, 7-deazaadenosine or tubercidin.

6. The method of claim 1 , wherein the yeast is Saccharomyces cervisiae.

7. The method of claim 1 , wherein the heterologous ENT is a parasite ENT, or wherein the heterologous CNT is a parasite CNT.

8. The method of claim 1 , wherein the parasite is a purine auxotroph.

9. The method of claim 1 , wherein the parasite is a Plasmodium sp., Leishmania sp., Trypanasoma sp., Toxoplasma sp., Entamoeba sp., Schistosoma sp., or Cryptosporidium sp.

10. The method of claim 1 , wherein the ENT is Plasmodium falciparum ENT type 1.

Assignees

• Albert Einstein College Medicine Yeshiva Univ
• Albert Einstein College Medicine

Inventors

• Akabas Myles
• Frame Ithiel James
• Deniskin Roman

Classifications

• C12Q1/025 Primary

  for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18) · CPC title

• C12Q1/18

  Testing for antimicrobial activity of a material · CPC title

• C07K14/705 Primary

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C07K14/44

  from protozoa · CPC title

• C07K14/445

  Plasmodium · CPC title