The World Health Organization estimates that, since 1981, 65 to 113 (average 85.6) million people have been infected with HIV, 32.9 to 51.3 (average 40.4) million people have died due to HIV related complications, and 33.1 to 45.7 (average 39) million people were living with HIV at the end of 2022 [1]. There are two main types of HIV, the more virulent and infectious type HIV-1 (particularly subtype M) which accounts for almost 90% of global cases, and the less transmissible and less prevalent HIV-2 [2, 3]. Many efforts were made to eradicate this deadly pandemic, chief of which were antiviral medication. There are currently 24 unique FDA approved drugs for treating HIV infections [4]. However, these drugs suffer from major drawbacks such as toxic adverse reactions, drug interactions, poor CNS penetrability, low oral bioavailability due to high CYP450 metabolism and possibly the biggest factor; drug resistance due to virus mutation [5, 6].
This study aims to design novel potent antiretroviral drugs that overcome these drawbacks using a molecular modelling approach.
HIV-1 Protease:
Similar to other retroviruses, HIV Protease is one of the three main enzymes necessary for viral replication alongside Integrase and Reverse Transcriptase. It plays a crucial role in producing mature virulent virions [6]. It functions via proteolysis of Gag and Gag-Pol precursor polypeptides to produce the structural components of infectious virions [6–8]. Hence, a major class of antiretroviral medications (ART) used to treat HIV infections are HIV Protease inhibitors (PIs).
HIV-1 Protease is a homodimer of two identical − 99 amino acid - chains [9]. Two aspartate residues ASP25 and ASP125 (one from each monomer) form the main catalytic active site [6, 9]. There are three main regions in the protease structure: the active site, the flexible “flaps”, and the dimer interface [9]. Protease crystal structure is shown in Fig. 1 (PDB: 2IEN) [10].
Figure 1: 3D structure of HIV-1 Protease complexed with inhibitor Darunavir (PDB: 2IEN)
Protease Inhibitors (PIs):
PIs function through competitive inhibition of HIV protease by binding to active site residues [11]. Several PIs have been approved for treating HIV infections such as Atazanavir and Darunavir (Fig. 2). Inhibitor placement in the active site and closing of the flaps renders the protease unable to process its substrates [9]. The hydroxyl group of the inhibitor binds to the catalytic ASP25 and ASP125 residues. Inhibitors also interact with other adjacent residues, namely Gly27, Asp29, Asp30, and Gly48 [12]. However, drug-resistance mutations decrease inhibitor-protein affinity through multiple mechanisms, such as changing active site shape which reduces hydrophobic interactions with the inhibitor, or introducing bulkier residues to the active site which increases steric hindrance [5, 6, 12]. Moreover, drug resistance could be attributed to limited effective drug concentrations in viral reservoirs such as the central nervous system due to low blood brain barrier penetration [6]. In addition to this, some PIs exhibit serious adverse effects including diarrhea, hyperlipidemia, nephrolithiasis, and hepatitis [6, 13].
Figure 2: Chemical structure of FDA approved Protease Inhibitors
Design rationale:
In order to overcome the aforementioned challenges in designing potent PIs with favorable pharmacokinetic profiles, and that are less affected by HIV mutations, several strategies were implemented. Promoting strong interactions with protease backbone in addition to the catalytic ASP25 and ASP125 residues was shown to be a major strategy in overcoming HIV drug resistance [14, 15]. As such, this study will take into account not only binding affinity, but also the number and strength of favorable interactions between the potential novel inhibitors and protein backbone. The approved drug Darunavir - a second generation PI - was selected as the basis for screening and as positive control, as it exhibits strong bonding with the catalytic site as well as protease backbone [14] (Fig. 3). This led to darunavir having high potency against known PI-resistant HIV strains [14, 16]. However, darunavir was shown to be implicated in liver toxicity ranging from transient asymptomatic aminotransferase serum elevation to acute and severe hepatitis [13, 17]. This study will aim to circumvent this toxicity through in-silico ADMET studies on the new inhibitors while focusing heavily on hepatotoxicity. As previously mentioned, most PIs have low BBB penetrability, causing low CNS drug concentrations, that of which could act as viral reservoirs [5]. As such, blood brain barrier penetration will be a major part of ADMET studies to insure good CNS reachability. Additionally, oral drug likeness will be tested using Lipinski’s Rule of Five and Veber Rule [18–21] to insure good oral pharmacokinetic properties.
Figure 3: A: 3D representation of darunavir docked into HIV-1 protease active site. B: 2D representation of darunavir interactions with active site residues