Herein, we report the effect of nine FDA approved protease inhibitor drugs against a new HIV-1 subtype C mutant protease, E35D↑G↑S. The mutant has five mutations, E35D, two insertions, position 36 (G and S), and D60E. Kinetics, inhibition constants, vitality, Gibbs free binding energies are reported. The variant showed a decreased affinity for substrate and low catalytic efficiency compared to the wild type. There was a significant decrease in the binding of seven FDA approved protease inhibitors against the mutant ( p < .0001). Amprenavir and ritonavir showed the least decrease, but still significant reduced activity in comparison to the wildtype (4 and 5 folds, respectively, p = .0021 and .003, respectively). Nelfinavir and atazanavir were the worst inhibitors against the variant as seen from the IC 50 , with values of 1401 ± 3.0 and 685 ± 3.0 nM, respectively. Thermodynamics data showed less favourable Gibbs free binding energies for the protease inhibitors to the mutant.

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HIV-1 protease (HIV PR) is considered as one of the most attractive targets for the treatment of HIV and the impact of flap dynamics of HIV PR on the binding affinities of protease inhibitors (PIs) is a crucial ongoing research field. Recently, our research group evaluated the binding affinities of different FDA approved PIs against the South African HIV-1 subtype C (C-SA) protease (PR). The CSA-HIV PR displayed weaker binding affinity for most of the clinical PIs compared to HIV-1 B subtype for West and Central Europe, the Americas. In the current work, the flap dynamics of four different systems of HIV-1 C-SA PR complexed to FDA approved second generation PIs and its impact on binding was explored over the molecular dynamic trajectories. It was observed that the interactions of the selected drugs with the binding site residues of the protease may not be the major contributor for affinity towards PIs. Various post-MD analyses were performed, also entropic contributions, solvation free energies and hydrophobic core formation interactions were studied to assess how the flap dynamics of C-SA PR which is affected by such factors. From these contributions, large van der Waals interactions and low solvation free energies were found to be major factors for the higher activity of ATV against C-SA HIV PR. Furthermore, a comparatively stable hydrophobic core may be responsible for higher stability of the PR flaps of the ATV complex. The outcome of this study provides significant guidance to how the flap dynamics of C-SA PR is affected by various factors as a result of the binding affinity of various protease inhibitors. It will also assist with the design of potent inhibitors against C-SA HIV PR that apart from binding in the active site of PR can interacts with the flaps to prevent opening of the flaps resulting in inactivation of the protease.

Source of materialAsolution of the cbz (cbz =benzyloxycarbon)protected (S)-N-((S)-1-phenylethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (1.0 g, 2.41 mmol) in THF (20 ml) was added to a suspension of activated 10 wt %Pd/C (0.5 g) in MeOH (20 ml). The mixture was connected to ahydrogen source at 1atmosphere and stirred at room temperature for 2hours. Completion of the reaction wasm onitored through TLC in hexane/ethyla cetate (50/50, R f =0.3). ThePd/C was filtered through acelite pad and washed with methanol (20 ml). The filtrate was evaporated under reduced pressure affording the crude amino ester, which was purified by columnc hromatography using ethyl acetate/hexane (50:50) as the eluent to yield the precusor (0.60 g, 89%) as awhite solid. M.p. =414-416 K. This compound was then dissolved in acetone and by slow evaporation, crystals suitable for x-ray diffraction were obtained. Experimental detailsAll hydrogen atomswere positioned geometrically with C-H distances ranging from 0.95 Åto1.00 Åand refined as riding on their parent atoms, with U iso (H) =1.2 -1.5 U eq (C). Crystals were very fragile and readily crumble when cut, so needles with length beinglargerthanthe x-raybeam size were used. The structure containssolvent accessible voidsof180.0 Å 3 andtwo residualswith largeelectrondensities of 1.53 and1.26e/Å -3 were observed. Attempts to model the solvent molecule failed despite data having been collectedtwice on twodifferent crystals. The solvent molecule waslocated on thefourfoldscrew axis whichrunning along [001]d irection and was severely disordered. Therefore the solvent was omitted from the final structure model. With unmerged reflections the Flack xp arameter equals to -0.7836 with esd 2.5357. So the final structure was refined with averaging Friedel pairs and the Rfactor is 0.05. Structure Contains Solvent Accessible VOIDS of 178 A 3 .SQUEEZE estimated atotal count of 40 electrons per unit cell contributed by the disordered solvents, which could not be modelled as discrete atomic sites. DiscussionAs part of our ongoing study into novel tetrahydroisquinoline (TIQ)based catalysts, the precusor to the title compound was synthesised [1][2][3]. In attempt to grow as ingle crystal of this compound, arange of different solvents were used by the method of slow evaporation. To ours urprise, the titlec ompound had recrystallised when acetone was used as the solvent. From literature tetrahydroindole based compounds undergo asimilar cyclisation in the presence of acetone [4]. These compounds are of interest due to its biological activity for its dual-acting ability to reverseresistanceand anti-cancer activity [5]. It is the first report of this tetrahydroisquinoline class with cyclisation between the secondary amine in the 6-membered ringa nd the amide nitrogen. Theabsolute stereochemistry of the crystal was confirmed to be S,S at C9 andC11 positions, respectively by proton NMRspectroscopy in keepingwiththe chiral starting aminoacidand amine. From theplain formed by theatoms C8-C7-C2-C1-N1-C9the di...