Question

There is a new strain of HIV that has an atypical enzyme structure. As such, none of the current inhibitors are effective against the HIV protease [Essential Biochemistry (p206, 3rd edition); (p198, 4th edition)]. You are charged with developing a new inhibitor for this new HIV strain with different protease.

1. Describe how you can design an inhibitor for this HIV protease.

2. How can you test the inhibitor for biologic effect?

Answer 1

1. Describe how you can design an inhibitor for this HIV protease.

#### HIV protease  is a retroviral aspartyl protease (retropepsin) which is an enzyme involved with peptide bond hydrolysis in retroviruses, that is essential for the life-cycle of HIV, the retrovirus that causes AIDS.

### HIV protease cleaves newly synthesized polyproteins (namely, Gag and Gag-Pol) at nine cleavage sites to create the mature protein components of an HIV virion, the infectious form of a virus outside of the host cell. Without effective HIV protease, HIV virions remain uninfectious.

###The human immunodeficiency virus (HIV) uses protease in the final stages of its reproduction (replication) process

The third class of antiretroviral drugs developed against HIV were the protease inhibitors. These work far back in the life cycle of HIV, after host cell integration but before budding. These drugs affect the enzyme protease, which is used to cut up the HIV protein to be packaged into virions.

When the cell produces HIV proteins, the raw material is in a long connected string. The enzyme protease acts as a "scissor" to cut up the string into the protein for each virion. Protease inhibitors prevent protease from doing this. They resemble pieces of the protein string that protease usually cuts. This disrupts the cutting process, which prevents the chain from being cut into small pieces, which prevents HIV from making copies of itself.

******designing an inhibitor for this HIV protease.

The discovery of human immunodeficiency virus (HIV) protease inhibitors and their utilization in highly active antiretroviral therapy have been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS).

However, despite the successes in disease management and the decrease of HIV/AIDS-related mortality, several drawbacks continue to hamper first-generation protease inhibitor therapies. The rapid emergence of drug resistance has become the most urgent concern because it renders current treatments ineffective and therefore compels the scientific community to continue efforts in the design of inhibitors that can efficiently combat drug resistance.

The present line of research focuses on the presumption that an inhibitor that can maximize interactions in the HIV-1 protease active site, particularly with the enzyme backbone atoms, will likely retain these interactions with mutant enzymes.

structure-based design of HIV PIs specifically targeting the protein backbone has led to exceedingly potent inhibitors with superb resistance profiles.

for designing an inhibitor we have to introduce templates, particulary nonpeptidic conformationally constrained P₂ ligands that would efficiently mimic peptide binding in the S₂ subsite of the protease and provide enhanced bioavailability to the inhibitor. Cyclic ether derived ligands appeared as privileged structural features and allowed us to obtain a series of potent PIs. Following the structure-based design approach, develope  a high-affinity 3(R),3a(R),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) ligand that maximizes hydrogen bonding and hyrophobic interactions in the protease S₂ subsite. Combination of this ligand with a range of different isosteres led to a series of exceedingly potent inhibitors.

Darunavir, initially TMC-114, which combines the bis-THF ligand with a sulfonamide isostere, directly resulted from this line of research. This inhibitor displayed unprecedented enzyme inhibitory potency (K_i = 16 pM) and antiviral activity (IC₉₀ = 4.1 nM). Most importantly, it consistently retained is potency against highly drug-resistant HIV strains. Darunavirʼs IC₅₀ remained in the low nanomolar range against highly mutated HIV strains that displayed resistance to most available PIs.

detailed crystal structure analyses of darunavir-bound protease complexes clearly demonstrated extensive hydrogen bonding between the inhibitor and the protease backbone. Most strikingly, these analyses provided ample evidence of the unique contribution of the bis-THF as a P₂-ligand. With numerous hydrogen bonds, bis-THF was shown to closely and tightly bind to the backbone atoms of the S₂ subsite of the protease. Such tight interactions were consistently observed with mutant proteases and might therefore account for the unusually high resistance profile of darunavir. Optimization attempts of the backbone binding in other subsites of the enzyme, through rational modifications of the isostere or tailor made P₂ ligands, led to equally impressive inhibitors with excellent resistance profiles.