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    • The IN gene of the

    2022-03-10

    The IN gene of the HIV isolate from Papua (CRF01_AE isolate) contains genetic polymorphisms (Fig. 1A) that encode for variant amino acids in the IN protein, based on the Stanford HIVdb mutation interpretation database (Fig. 1B). These variations slightly changed the three-dimensional structure of IN. Wild-type IN contains a longer helix domain as compared to variant IN (Fig. 2). However, this structural change had a minimal to no effect on IN stability (Table 1). This result may be due to the fact that the amino Acarbose substitutions are not located in the conserved central core domain of IN (Asp64, Asp116, and Glu152) [16]. Molecular docking analysis indicated that the ALLINIs, EVG, and RAL did not much change the binding affinity to the wildtype or variant IN (Table 2). These three INIs can bind in the wild-type and variant IN(Fig. 3). IN residues 125, 128, 170, 171, and 173 are positioned to make contact with INIs; substitutions at these amino acid positions likely disrupt the inhibitor-mediated interface directly [7]. IN resistance to ALLINI is dependent on the A128T polymorphism [17], which was not observed in this study. ALLINI bound to residues Glu170 and His171 of both the wild-type and variant IN. ALLINI also interacts with ALA98 and A125 of variant IN via a halogen bond and a pi-alkyl interaction, respectively (Fig. 4a). A study conducted by Lu et al. demonstrated that halogen bonding plays an important role in inhibitor recognition and binding to the targeted protein [18]. The unique chemical characteristics of halogens are beneficial to the design of protein inhibitors and drugs [19]. The ALLINIs showed relatively higher binding affinity to the variant IN as compared to wild-type. The IN polymorphisms T66I/A, V72I, F121Y, T125K, G140C, S147G, Q148H, V151I, S153Y, M154I, and S230R mediate resistance to EVG [20]. The IN variant in this study contained M154I; however, according to the docking analysis, EVG bound more strongly to the IN variant as compared to wild-type. The binding affinity between EVG and variant IN was −8.1 kcal/mol, but −7.2 kcal/mol with the wild-type IN (Table 2). Residues Glu170 and Thr174 of both wild-type and variant form hydrogen bonds with EVG. However residues Ala124 and Ala125 of the IN variant, but not wild-type, formed pi-alkyl interactions with EVG. The polymorphisms Q148H/K/R, N155H, and Y143H of IN were associated with the resistance of HIV-1 to RAL [21]. These polymorphisms were not found in this study; however the binding affinity between RAL and the variant IN was decreased as compared to the wild-type IN, which was due to the loss of hydrogen bonding between RAL with Thr125 in variant IN and loss of several other interactions between RAL and Leu102, Ala128, Ala129, and Trp132.
    Declarations
    Introduction Human immunodeficiency virus (HIV), the etiological agent of acquired immune deficiency syndrome (AIDS), encodes three essential replication enzymes in its pol gene: reverse transcriptase (RT), protease (PR) and integrase (IN) [1]. These enzymes are the most attractive HIV-drug targets with up to approximately 40 drugs thus far approved by US Acarbose Food and Drug Administration (FDA) as major components of the highly active antiretroviral therapy (HAART) [2]. The majority of these drugs target RT and PR. Presently, only three drugs of the β-diketo acid class (DKA) Raltegravir, Elvitegravir and Dolutegravir target IN; this small number of active inhibitors is attributed to the limited structural and experimental information pertaining to the catalytic mechanism of the enzyme [3], [4], [5]. The therapeutic potential of IN remains largely unexploited, even though it has a central role in the life-cycle of HIV irreversibly inserting pro-viral cDNA into infected host cells’ chromosomes via covalent linkage, and despite the lack of functional and structural similarity to human enzymes [6]. IN is a 32 kDa protein with 288 residues having three distinct domains: N-terminal domain (NTD; residues 1–49); the catalytic core domain (CCD; residues 50–212); the C-terminal domain (CTD; residues 213–288) [7]. The CCD mainly functions as the DNA substrate recognition and catalytic domain, while NTD and CTD are involved in the enzyme oligomerization and as a stabilization platform of the IN-DNA complex respectively. CCD belongs to the polynucleotidyl transferase superfamily of enzymes that share a structural fold similar to bacterial RNase H [8]. It contains a highly conserved residue triad D-D-E that coordinates a divalent cation (Mg2+) essential for its catalytic activity. CCD viral-human DNA integration occurs in a clearly defined two-step mechanism involving both the 3′- end processing step and the strand transfer reaction [9], [10]. The available DKA drugs function by inhibiting the strand transfer reaction, and are thus called integrase strand transfer inhibitors (INSTIs). However, the constant evolution of viral strains resistant to these first-generation INSTIs remains a threat to HAART [11]. Thus, a continued search of novel INSTIs effective against recorded mutants and with minimal associated toxicities remains a top priority.