A Cell-penetrating Helical Peptide as a Potential HIV-1 Inhibitor

Abstract

The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer alpha-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 A. Using this structural information, we have utilized a structure-based rational design approach to stabilize the alpha-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced alpha-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein.

Stapled peptides in the p53 pathway: Computer simulations reveal novel interactions of the staples with the target protein

“…For such cases, the stabilization of peptides as pre-formed helices through stapling with hydrocarbon chains reduces the entropic penalty that a random coil peptide has to pay as it adopts a helical fold for binding; this method has so far been very successful. [47][48][49][50][51] MD simulations were carried out for a set of such peptides that were designed to bind to MDM2. 53 We find that the good binders assume helicity in solution as has also been demonstrated in other simulation studies 53,54 and now we also show that they assume extended helicity when bound to MDM2.…”

“…However, clearly the physico-chemical parameters that contribute to the outcome are far more complex as has been demonstrated for the NMR structure of the monomeric C-terminal domain of HIV-1 capsid bound to inhibiting staples; in this case, the staples are far from the surface that is being targeted. 50,51 Clearly much more needs to be discovered to understand the structure-energy-functions of these exciting molecules. Our simulations have provided insights …”

“…45 Indeed the technique has been shown to be successful in the p53 pathway, 43 NOTCH pathway, 47 BCL pathway 48,49 and in targeting HIV. 50,51 In addition, a successful…”

Value of MR contrast media in image-guided body interventions

“…MR contrast media are useful in evaluating the efficacy of therapy by allowing the determination of tissue perfusion, blood volume, vascular permeability, angiogenesis and cellular integrity [21,22] . For example, after effective chemotherapy there is a decrease in the size of the lesion and number of microvessels, which can be detected on contrast enhanced MR images.…”

“…Saeed et al [21] demonstrated the advantageous effects of vascular endothelial growth factor and hepatocyte growth factor genes, delivered transendocardially under MR-guidance, in creating new blood vessels and reducing the size of infarcted myocardium using gadolinium-based MR contrast media [22] . Others used contrast enhanced MRI for determining the effects of intracoronary stem cell therapy on left ventricular function [88] .…”