HIV-1 protease (HIV-1-Pr), an enzyme found in the HIV, acts as the catalyst for the cleavage process of peptides

or proteins. The peptide or protein will act as a substrate and bind to the protease to lower the activation energy needed to cleave the peptide bond. A study carried out by Kramer et al. discovered that HIV-1 protease has the potential as the target for AIDS therapy. Upon addition of an inhibitor, HIV-1 protease undergoes frameshift mutation, preventing the cleavage of the Gag polyprotein precursor a precursor necessary for the maturation of the HIV-1 particles. The blockage of this enzyme will then lead to the formation of immature non-infectious virions. Therefore, studies are done intensively in order to find out the chemical structures which are able to inhibit the protease. However, resistance of HIV-1 protease to currently discovered inhibitors has increased drastically. Therefore, there is a need for broad spectrum protease inhibitors that are effective against these new mutants to be identified. This is where in-situ click chemistry comes into play. It enables the acceleration of discovery of innovative substrates. This is done by selfassembly of fragments that covalently bind to form inhibitors to act on the biological target (HIV-1 protease). Click chemistry enables researchers to sample numerous combinations of fragments (building blocks) which act on the HIV-1 protease, but only the best binders are selected and synthesized. The HIV protease inhibitors must fulfil the following properties: First of all, they must be able to attack a broad range of viral genotypes in order to prevent drug resistance from occurring. Next, they must be able to work with other chemical entities used for HIV treatment to create a synergistic effect and also must be compatible with other therapies used to combat opportunistic infections. Besides, in order to achieve the maximum effect, the concentrations of the inhibitors present in the cells and in the circulation must always remain at levels far above the inhibitory constant (Ki). Next, the inhibitor should preferably possess high oral bioavailability and must be able to penetrate the blood-brain barrier (BBB). Previous studies have always emphasized the use of building blocks with a high affinity for the target, as seen by the discovery of highly potent inhibitors of

acetylcholinesterase and carbonic anhydrase. However, the discovery of HIV protease inhibitor is unique in its own way. Researchers were pleasantly surprised to find the HIV protease enzyme which selectively forms components even though it only exhibited weak binding to the target. A special feature of the in situ click chemistry approach is that it relies completely on bio-orthogonal 1,3-dipolar cycloaddition reaction. This reaction involves two reactants, mainly an organic azide and an alkyne.

Both azides and alkynes are known as energetic species, however, these two functional groups reactivity profiles under physiological conditions are quite low. Even though the cycloaddition process involves large thermodynamic driving force but the high kinetic barrier of azides and alkynes hides them effectively. [1] Reaction between them only occurs when they are brought close enough by a biological template or being activated under the presence of catalyst, for instance, copper (I) which involves in the copper-catalyzed azide-alkyne cycloaddition (CuAAC) to form triazole derivatives.

The above diagram shows that the formation of five membered nitrogen heterocycles, 1,2,3-triazoles in which the reaction is highly exergonic. This process can be done in the presence of a biological template (HIV protease) which allows these reactants to be brought into close proximity, allowing the enzyme to selectively form an inhibitor. The triazoles formed can participate in hydrogen bonding actively. Besides, the derivatives also involves in dipole-dipole and -stacking interactions. In a nutshell, the HIV-1-Pr inhibitor is proven to be formed through the approach of in situ chemistry. This concept involves the HIV-1-Pr as a template which greatly enhances the rate of formation of the 1,4-triazole product (inhibitor). The unique characteristic of the building blocks to form the HIV-1-Pr inhibitor which posses lower affinities for the biological target should encourage the use of this concept to other systems in which the structure of high

affinity binders are not known. This is important for the discovery of new protease inhibitors that are able to overcome the drug resistant problems at hand.

Gian Cesare Tron, Tracey Pirali, Richard A. Billington, Pier Luigi Canonico, Giovanni Sorba and Armando A. Genazzani (2007) Click Chemistry Reactions in Medicinal Chemistry: Applications of the 1,3-dipolar Cycloaddition Between Azides and Alkynes. Medicinal Research Reviews, Vol. 28, No. 2, 278 -308 Hartmuth C. Kolb and K. Barry Sharpless (2003) The growing impact of click chemistry on drug discovery. DDT Vol. 8, No. 24