Inhibitors of retroviral protease as inducers of reversible insulin resistance in vitro and in vivo
AssigneeWashington University
InventorMike Mueckler
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Abstract
This invention provides novel assays for measuring the metabolic side-effects of antiretroviral protease inhibitors on the Glut4 glucose transporter. The invention also provides improved methods for developing antiretroviral protease drugs, particularly those used to fight HIV infection. The invention further provides novel models of insulin-resistant glucose transport disease states.
Claims28
A method for screening an antiretroviral protease inhibitor for a metabolic side-effect on glucose transport, the method comprising the steps of:
- a) providing a reaction medium comprising one or more glucose transporters in a membrane system through which the glucose transporters transport a glucose compound;
- b) adding to the reaction medium a detectable form of the glucose compound;
- c) adding to the reaction medium the protease inhibitor to be screened;
- d) determining activity of the glucose transporter by measuring an amount of the glucose compound transported by the glucose transporter;
- and
- e) quantifying the metabolic side-effects of the protease inhibitor screened as a function of the activity of the glucose transporter in the presence the protease inhibitor as compared with the activity of the glucose transporter in the absence of the protease inhibitor.
A kit for use in screening protease inhibitors for metabolic side effects on glucose transport, the kit comprising one or more components selected from the group consisting of a reaction vessel, a cell line capable of expressing one or more glucose transporters, a membrane system containing one or more glucose transporters, one or more nucleic acid sequences encoding a glucose transporter, one or more mRNA sequences encoding a glucose transporter for transient expression, a detectable glucose compound, a standardized protease inhibitor, a control glucose transport inhibitor, glucose transport altering substances, control cells, standards for validating the assay, detailed instructions, quality control certifications, disposable labware, and disposable personnel protective items.
A cell-free assay for determining if a selected glucose transporter physically associates with a selected protease inhibitor, the method comprising the steps of:
- a) labeling the glucose transporter or the protease inhibitor, or both, with a detectable label, wherein a measurable feature of the detectable label changes if the glucose transporter physically interacts with the protease inhibitor;
- b) adding the glucose transporter and the protease inhibitor to a reaction medium under conditions enabling physical association, if any, between the glucose transporter and the protease inhibitor;
- and
- c) measuring a change, if any, in the measurable feature of the detectable label, the change being indicative that the glucose transporter and the protease inhibitor physically interact.
A method for screening therapeutic test compounds for their ability to alleviate insulin resistance, the method comprising the steps of:
- a) providing a reaction medium comprising:
- i) cells that produce one or more glucose transporters;
- ii) a quantity of an inhibitor of retroviral protease that reversibly inhibits insulin-dependent glucose transport of the transporter;
- iii) a quantity of insulin;
- and
- iv) a glucose compound;
- b) measuring the amount of the glucose compound transported into the cells under preestablished conditions for a preestablished time period;
- c) adding the test compound to the reaction medium;
- d) measuring the amount of the glucose compound transported into the cells under preestablished conditions for a preestablished time period;
- and
- e) determining the difference between the amount of glucose transported into the cells after addition of the test compound and the amount of glucose transported into the cells before addition of the test compound, an increase in the amount of glucose transported into the cells after addition of the test compound being indicative that the test compound is capable of reversing the inhibition of the insulin-dependent glucose transport caused by the protease inhibitor.
Dependencies are inferred from the claim text.