Literature review
1.9 Protease structure, function, inhibitory drugs and resistance
1.9.1 Structure
The HIV-1 PR belongs to a family of aspartic proteases that is symmetrically structured to form a homodimer consisting of two identical subunits each comprising 99 amino acids (AAs) (Katoh et al., 1987; Yang et al., 2012). Each PR subunit is composed of a single α-helix and nine β-sheets.
Of these, four β-sheets that run anti-parallel relative to each other form the conserved dimer interface (Todd et al., 1998; Velazquez-Campoy et al., 2003) as shown in Figure 1.6. Structural studies indicate that residues 1–4 and 96–99 in each subunit of the four anti-parallel β-sheets forms the dimer interface of PR (Choudhury et al., 2003). The dimer interface results in the formation of the hydrophobic active site cavity consisting of PR’s catalytic triad D25-T26-G27 from both monomers (Wlodawer et al., 1989; Zhang et al., 2008). More so, the anti-parallel β- sheets restrict access to the substrate cavity by forming two flexible flaps to cover the active site region. In a ligand-free state, PR assumes a semi-open conformation whereas a PR-ligand complex induces a closed conformation. Two models have been proposed for flap opening and closing states (Yang et al., 2012). The first theorizes that a collision complex between the ligand and PR occurs when the flaps are fully opened which then induces a closed conformation as the substrate approaches the active site (Scott and Schiffer, 2000). The second model proposes that the ligand first approaches a semi-open state which then prompts the flaps to fully open as it enters the substrate cavity. The flaps then extend over the substrate to adopt a closed conformation for proteolysis (Collins et al., 1995; Toth and Borics, 2006). While the flaps are indispensable for catalysis of the viral PR, their exact conformational states during cleavage is debatable (Soares et al., 2016).
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Figure 1.6 Structural representation of the HIV PR showing its active site, flap and identical subunits. Image take from Venkatakrishnan et al. (2012).
1.9.2 Function
In nature, the role of the HIV-1 PR is to cleave the nascent polyproteins and is thus essential for viral replication (Adachi et al., 2009). Consistent with studies evaluating other non-viral PR’s, it is recognized that the acid-base mechanism involving two important active site aspartic residues are essential for catalysis to occur (Brik and Wong, 2003). The widely accepted mechanism of catalysis was initially described by Suguna et al. (1987). The authors suggest that of the two aspartic acids, only one is unprotonated. Therefore, the negatively charged aspartic group activates the nucleophilic water located between the aspartic acids which then proceeds to attack the substrate’s carbonyl group to generate an oxyanion tetrahedral intermediate. Finally, the substrate’s amide nitrogen atom is protonated and rearranged to breakdown the intermediate leading to the formation of the hydrolysis products (Figure 1.7).
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Figure 1.7 Chemical mechanism showing the proteolytic cleavage of the hydrolysis products.
Image taken from Brik and Wong (2003).
In Gag, five cleavage sites (CSs) are recognized by PR during hydrolysis as depicted in Table 1.3 below. Although these regions share limited sequence similarity they are similarly structured. As such, it was proposed that the mechanism by which PR cleaves Gag also depends on the structure of the substrate. Moreover, while PR exhibits symmetry within its subunits, the Gag substrates are asymmetrical in size and charge of the AA residues (Prabu-Jeyabalan et al., 2002).
Table 1.3 WT sequences for the HIV-1 subtype C Gag CSs (adapted from De Oliveira et al., 2003).
CLEAVAGE SITES SEQUENCE
Matrix-Capsid VSQNY -- PIVQN
Capsid-p2 KARVL -- AEAMS
p2-Nucleocapsid NTNIM -- MQKSN
Nucleocapsid-p1 ERQAN -- FLGKI
p1-p6 RPGNF -- LQSRP
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As previously mentioned, the analogous structure of the PIs to the Gag CSs make them ideal inhibitors of HIV-1 maturation (Mudgal et al., 2018). As shown in Table 1.1 above, there are 10 FDA approved PIs currently available on market, nine of which are shown in Figure 1.8 below.
In general, the mechanism of inhibition begins when the hydroxyl group of the PI interacts with the carboxyl group of the aspartic active site residues via hydrogen bonding. Consequently, binding to the active site AAs prevents the HIV-1 PR from successfully cleaving Gag. While several attempts have been made to modify the current PIs, none have been as successful as second-generation PIs Lopinavir (LPV) and Darunavir (DRV) which were modified from Ritonavir (RTV) and Amprenavir (APV), respectively (Lv et al., 2015).
In LPV, the 5-thiazolyl and the 2-isopropylthiazolyl of RTV’s P2 and P2' groups are replaced with a phenoxyacetyl and six-membered cyclic urea, respectively. These substitutions have been shown to improve the potency against drug resistant variants (Sham et al., 1998). On the other hand, having been FDA-approved in 2006, DRV is the newest PI on market (Lv et al., 2015). The only difference between DRV and APV is the substitution of tetrahydrofuran with bis- tetrahydrofuran in the P2 group. Consequently, replacing APV’s chemical moiety allows DRV to form more hydrogen bonds with PR. Due to these modifications LPV boosted ritonavir (LPV/r) and DRV boosted ritonavir (DRV/r) have high genetic barriers against the resistant PR variants in comparison to the other PIs (Doherty et al., 2011; Aoki et al., 2018).
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Figure 1.8 Chemical structures of the PIs. Image adapted from Lv et al. (2015).
1.9.4 Drug resistance
Despite their success, mutations arising in the viral PR have been associated with LPV and DRV drug resistance (Weber et al., 2015). In LPV, the most common resistance mutations include V32I, M46I/L, G48V/M, I54V/T/A/L/M, L76V, V82A/T/F/S, I84V and L90M (Lv et al., 2015) whilst V32I, L33F, I47V/A, I50V, I54L/M, L76V and I84V have been clinically associated with DRV resistance (Tremblay, 2008). Generally, resistance to the PIs occur when the viral PR accumulates several primary or major mutations in various regions of the enzyme (Wensing et al., 2010). Major mutations directly affect resistance by altering the structure of the catalytic site.
This results in reduced contact between PR and the inhibitors. In addition, several secondary or minor resistance mutations emerge at a later stage to compensate for the changes induced by the major protease resistance mutations (PRMs) (Budambula et al., 2015). Therefore, this suggests that the way in which PR resists the PIs is dependent on complex evolutionary and resistance dynamics.
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