Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV‑1 by Peptide Triazole Thiols

We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine–ferrocenyltriazole-proline–tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)­phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol–disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection

I_to reduction in cardiomyocytes isolated from the left atrial poisterior wall (LAPW).

<p>(a) Example voltage-sensitive, Ca²⁺ independent, macroscopic K⁺ currents evoked in cardiomyocytes isolated from the left atrial appendage (LAA, left) and LAPW (right). Voltage protocol is shown inset. (b-d) LAA and LAPW I/V relationships for the peak outward K⁺ current, I_to and steady state K⁺ current. Data presented as mean ± SEM. * denotes P<0.05 LAA (N = 16 cells) v LAPW (N = 12 cells), two way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis.</p

Action potential (AP) prolongation and heterogeneity in the left atrial posterior wall (LAPW).

<p>(a) Examples of left atrial (LA) isochronal action potential duration (APD) distribution maps at 30 and 70% repolarisation. (b) A raw fluorescence image of an LA loaded with Di-4-ANEPPS, along with the 9 region grid used for quantitative regional analysis. (c) Example optical action potentials (OAPs) recorded from the 9 different LA regions during 10Hz pacing. The green dotted line indicates APD70. (d) Box and whisker plot of APD70 values measured in each LA region. * denotes P<0.05 vs regions 7,8,9 inclusive, + P<0.05 vs region 7 only, one way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis, N = 18 LA. Inset: Heat map depicting mean APD70 values of the 9 LA regions of the LA. (e) Example isochronal APD70 distribution maps of the same LA at 10 and 1Hz (same scale). (f) Mean APD70 at 10 and 1Hz for the left atrial appendage (LAA) and left atrial posterior wall (LAPW). * denotes P<0.05 LAA v LAPW, one way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis, N = 5 LA. (g) LA gradients at 10 and 1Hz. * denotes P<0.05 LAA v LAPW, paired t-test, N = 5 LA.</p

I_KACh is depleted in left atrial posterior wall (LAPW) cardiomyocytes.

<p>(a) Current traces demonstrating isolation of BaCl₂ sensitive (I_K1) and CCh induced (I_KACh) currents in a single left atrial cardiomyocyte. Voltage protocol is shown inset. (b & c) Comparison of LAA and LAPW I/V relationships for I_K1 and I_KACh. The dashed lines indicate mean best fit I_K1 and I_KACh I/V curves with liquid junction potential correction, for both LAA and LAPW. Data presented as mean ± SEM. * denotes P<0.05 LAA (N = 25 cells) v LAPW (N = 17 cells), two way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis.</p

Ion channel expression differences between the left atrial posterior wall (LAPW) and left atrial appendage (LAA).

<p>(a-c) Comparisons of K⁺, Na⁺ and background/leak channel gene expression, between the LAPW and LAA, measured using Taqman Low Density Array (TDLA). Control sample was the LAA. ** and *** denote P<0.01 and P<0.001 respectively, LAA v LAPW, paired t-test, N = 9 LA.</p

Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1 Envelope gp120 and the Host-Cell CCR5 Coreceptor at the Virus–Cell Interface

To address the urgent need for new agents to reduce the global occurrence and spread of AIDS, we investigated the underlying hypothesis that antagonists of the HIV-1 envelope (Env) gp120 protein and the host-cell coreceptor (CoR) protein can be covalently joined into bifunctional synergistic combinations with improved antiviral capabilities. A synthetic protocol was established to covalently combine a CCR5 small-molecule antagonist and a gp120 peptide triazole antagonist to form the bifunctional chimera. Importantly, the chimeric inhibitor preserved the specific targeting properties of the two separate chimera components and, at the same time, exhibited low to subnanomolar potencies in inhibiting cell infection by different pseudoviruses, which were substantially greater than those of a noncovalent mixture of the individual components. The results demonstrate that targeting the virus–cell interface with a single molecule can result in improved potencies and also the introduction of new phenotypes to the chimeric inhibitor, such as the irreversible inactivation of HIV-1

Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1 Envelope gp120 and the Host-Cell CCR5 Coreceptor at the Virus–Cell Interface

To address the urgent need for new agents to reduce the global occurrence and spread of AIDS, we investigated the underlying hypothesis that antagonists of the HIV-1 envelope (Env) gp120 protein and the host-cell coreceptor (CoR) protein can be covalently joined into bifunctional synergistic combinations with improved antiviral capabilities. A synthetic protocol was established to covalently combine a CCR5 small-molecule antagonist and a gp120 peptide triazole antagonist to form the bifunctional chimera. Importantly, the chimeric inhibitor preserved the specific targeting properties of the two separate chimera components and, at the same time, exhibited low to subnanomolar potencies in inhibiting cell infection by different pseudoviruses, which were substantially greater than those of a noncovalent mixture of the individual components. The results demonstrate that targeting the virus–cell interface with a single molecule can result in improved potencies and also the introduction of new phenotypes to the chimeric inhibitor, such as the irreversible inactivation of HIV-1

Action potential differences between cardiomyocytes in the left atrial posterior wall (LAPW) and left atrial appendage (LAA).

<p>(a) Example intracellular recording trace demonstrating the stimulation protocol used to achieve sufficient action potential rate adaptation. (b) Example transmembrane action potentials (TAPs) taken from the LAA and LAPW of the same left atrium. TAPs are aligned at the resting membrane potential (RMP). The green vertical line indicates action potential duration at 90% repolarisation (APD90). (c-f) Box and whisker plots and individual values comparing the RMP, APD50-90, action potential amplitude (APA) and dV/dt (Vmax), of the LAA and LAPW, at 10Hz pacing frequency. **, *** denotes P<0.01 and P<0.001, LAA v LAPW, one way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis, or paired t-test; N = 20 LA.</p