HIV Protease

HIV history
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HIV Protease Microplate Kinetic Assay

The HIV retrovirus has infected more than 40 million people worldwide and has caused one of the worlds most challenging chronic pandemics. In the early 90’s, and over the course of the last 17 years, many viral proteins associated with HIV have been characterized and exploited as potential drug targets. One of the earliest and most successful class of drugs to treat HIV, targets the HIV-1 Protease, a virus encoded enzyme that catalyzes the proteolytic processing of a large precursor protein into an array of smaller, but functional, HIV proteins that assemble into mature and infectious HIV particles. Because the protease is so crucial to the formation of infectious virions, the inhibition of its activity has proven to extend the life spans of HIV infected individuals, as well as reduce viral load, and increase CD4 white cells counts.

As a class, the drugs represented by the suite of HIV-1 Protease inhibitors that are currently prescribed, when combined with other antiviral treatment options in cocktails, have been quite successful in their outcome. However, due to the fact that, to date, the HIV-1 Virus is the fastest mutating virus to infect humans, the current medications are becoming increasingly less effective over a growing percentage of the infected population.

Over 400 basic patterns of mutations that render some form of resistance, have accumulated, and have been characterized in Stanford’s HIV-1 Drug Resistance Database. The mutations occurring in the HIV-1 Protease are classified as either primary mutations, or secondary in nature. Primary mutations give rise to drug resistance to a protease inhibitor whether or not they are in combination with any other mutations. In contrast, secondary mutations are accessory, in that they by themselves do not cause any substantial resistance to any drug, but cause enhanced resistance when they occur in combination with other primary mutation(s). Therefore, next generation Protease inhibitors must not only be able to effectively curb the propagation of the virus, but also combat the drug resistant forms of the protease. The following innovative enzymes represent a new research tool for researchers who seek to aid in solving this challenge.

HIV Proteases
Traditionally, the HIV-1 Protease is known to be highly unstable due to the effects of autoproteolysis, cysteine oxidation, and dimer instability. The conventional approach to stabilizing the HIV-1 Protease has been to introduce a series of artificial (non-physiologic) point mutations into the enzyme (the most common stabilizing mutation being Q7K).

We offer the WT form of the protease with a Q7K mutation (H6789), and now the WT form of the protease with an L63P mutation (H1415), as well as 4 of the most challenging drug resistant mutants as catalogued in the Stanford HIV Drug Resistance Database. H1040, H1540, H1290, and H1165 have been engineered to include the most commonly occurring background polymorphism (L63P) as measured in the treated HIV population. This polymorphism is accessory in that it normally occurs with other key point mutations that add to the drug resistance profile of the protease. In a real sense, the L63P mutation can be considered to be the newest "wild type" sequence, as the virus has mutated since its discovery due to protease inhibitors being introduced into the population.

HIV Substrate
The substrate H0790 is specifically designed to fluoresce (Ex: 320, Em: 420) when the bond occurring between Nle and Tyr is hydrolyzed by the action of the protease. This substrate is highly specific for the substrate recognition requirements of the HIV-1 Protease, as well as the drug resistant mutants, and is therefore a perfect counterpart for all of the HIV-1 Proteases described above. The enzyme activity in these preparations, as measured on this substrate, is entirely due to the activity of the HIV-1 Protease dimer occurring in solution. Experiments demonstrate that the activities of these preparations are completely eliminated by mutating the active site (D25N) of the protease, which proves this defining quality-control characteristic.

HIV Protease Microplate Kinetic Assay
Kinetic Assay Procedure
Due to the enhanced stability and activity of these enzymes, more meaningful and dynamic data are generated in experiments during the course of: drug discovery (high throughput screening, secondary screening, etc), kinetic analysis, Ki/IC50 determination, and substrate specificity experiments. The benefits of conducting research on the physiologically occurring genetic sequence of a stable and active HIV-1 Protease are substantial.

• Stable
• Catalytically efficient
• Wide dynamic range
• High signal noise

These factors allow a time-persistent activity curve to be achieved. The graph below demonstrates the substantial increase in data-generating power associated with enhanced activity and reagent stability.

Substrate Kinetic Activity Data
FRET Substrate: Sequence: Abz-Ala-Arg-Val-Nle-Tyr(NO2)-Glu-Ala-Nle-NH2
Ex: 320nm Em: 420nm
Units of HIV-1 Protease: 25 (2.5 µl of Enzyme in 100 µl Reaction)
Substrate Concentration: 100 µM
Temperature: 37 °C
Reaction Volume: 100 µL

HIV Protease Microplate Kinetic Assay Procedure
Equipment and Materials Required

• Fluorescent Microplate Reader (Ex: 320nm. Em: 420nm)
• Black Opaque Flat Bottom 96 Well Microplates (plates used for fluorescence)
• Dilution buffer-100 mM Sodium Acetate (S8750), 1M NaCl (S9888), 1mM EDTA (E9884), 1mM DTT (D0632) (add right before use (step 2), and 10% Glycerol (G7893), pH 5.5
• Fluorescent HIV Substrate (H0790): 1mM Stock prepared in 20% Acetonitrile, 80% H20 (substrate stock solution) -Sequence: Abz-Ala-Arg-Val-Nle-Tyr(NO2)-Glu-Ala-Nle-NH2
• Incubator set at 37 °C

Procedure

1. Thaw appropriate amount of HIV-1 Protease at room temperature (see step 3).
2. Add fresh DTT to the activity buffer to a final concentration of 1 mM DTT.
3. Add HIV-1 protease into dilution Buffer at a ratio of at least 10 Units of HIV protease to 50 µl of dilution buffer. This will be referred to as the stock solution. If performing assay in 100 µl Total Volume, 50 µl of stock solution will be needed for each reaction. Note: HIV-1 protease is a dimer and diluting it below 10 Units per 50 µl of dilution buffer can substantially lower its activity via dissociation.
4. Aliquot 50 µl of the stock solution into the appropriate # wells in the 96 well microplate.
5. If conducting an inhibition assay, add the appropriate inhibitors to the corresponding wells.
6. Pre-incubate the 96 well plate in a 37 °C incubator for 5 minutes to allow temperature equilibrium. If conducting an inhibition assay, also allow ample time for inhibitor to bind to the protease.
7. Prepare a Substrate Working Solution by diluting 1 mM substrate stock 1:5 into dilution buffer. 50 µl of Substrate Working Solution is required for each microwell.
8. Remove plate from incubator and pipette 50 µl of Substrate Working Solution into each well. The total reaction volume is now 100 µL. This starts the reaction. Note: Final substrate concentration is 100 µM.
9. Read in fluorescent microplate reader at Ex: 320nm, Em:420nm at appropriate temperature. Note: 37 °C works well in these assays with a 2 second shake between each read. Using a time between reads of 1 min over the course of 30 minutes to 1 hour is typical. Assay will also perform well at room temperature.

Note: This procedure is for informational purposes. For a current copy of Sigma's quality control procedure contact our Technical Service Department.

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