Conjugation of Cholesterol to HIV-1 Fusion Peptide CP32M Improves its Antiviral Potency and Retainment at the Host Cell Membrane

Research Article

Ann Transl Med Epidemiol. 2016; 3(1): 1009.

Conjugation of Cholesterol to HIV-1 Fusion Peptide CP32M Improves its Antiviral Potency and Retainment at the Host Cell Membrane

Xiaoqian Zhang¹, Jian Hu1,2, Jie Li1,3 and Ying-Jie Wang1*

¹State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China

²Chengkou Agriculture Commission of Chongqing City, Chongqing 405900, China

3Department of Infectious Diseases, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China

*Corresponding author: Ying-Jie Wang, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qing Chun Road, Hangzhou 310003, China

Received: June 10, 2016; Accepted: July 11, 2016; Published: July 14, 2016


Peptides derived from the C-terminal heptad repeat (CHR) of the HIV-1 gp41 can effectively block viral entry by binding to the trimeric viral N-terminal heptad repeat (NHR) coiled-coil core. CP32M, a peptide modified from the CHR peptide CP621-652, has high antiviral potency, broad antiviral spectrum, and superior antiviral profile against CHR-resistant HIV-1 variants. However, peptide inhibitors including CP32M are expected to reach their target sites by simple diffusion in vivo which is rather inefficient, and the majority of the administered peptides will be metabolized and nullified. To overcome the problem, in this study, we designed a peptide called “CP32M-12aa-chol” in which the carboxylterminus of CP32M is extended with a flexible 12 amino acid (aa) linker followed by a cysteine residue where a cholesterol group is directly conjugated. Compared with its parental peptides that contain no cholesterol conjugation, the CP32M-12aa-cholpeptide had significantly enhanced antiviral potency but no appreciable cytotoxicity. Consistent with its anticipated mechanism of action, the antiviral potency of CP32M-12aa-chol was only reduced by 28-fold when it was pre-incubated with the host cells followed by thorough washes while under the same conditions all other tested peptides with no cholesterol conjugation lost their antiviral potencies by 159- to 3816-fold. Thus, adding a cholesterol group to the carboxyl-terminus of CP32M is likely to facilitate its targeting to and retention at the host cell membrane where the cell membrane/viral membrane fusion-mediated HIV-1 entry occurs, and such a strategy holds promise for further developing highly efficacious yet safe drugs against HIV-1.

Keywords: HIV-1; Fusion peptide; CP32M; Cholesterol; Cell membrane


Numerous studies over the past two decades have provided definitive evidence that the gp41 “prehairpin intermediate” is an effective target for peptide- and neutralizing antibody-based fusion inhibitors to block HIV-1 viral entry [1]. Peptide-based fusion inhibitors mainly include the Class 1 inhibitors that target the NHR trimeric coiled-coil core and the Class 2 inhibitors that bind to the CHR region [2,3]. The best-characterized example of the Class 1 inhibitors is the CHR peptide T20 (residues 638-673 of gp41), also known as DP178 or enfuvirtide [4]. Unfortunately, the clinical application of T20 is limited because of its relatively low potency, low genetic barrier to drug resistance and short half-life. Subsequently developed CHR peptide C34 (residues 628-661 of gp41) or CP621- 652 (residues 621-652 of gp41) which included the pocket-binding domain (PBD) only or PBD plus the “QIWNNMT” motif (Figure 1A), respectively, exhibited more potent HIV-1 fusion inhibitory activity than T20 [2,3]. The “QIWNNMT” motif and in particular the M-T hook-like structure could stabilize the interaction between NHR and CHR in the 6-HB core or between the CHR peptide and the deep pocket on the NHR trimer and thus increasing the anti-HIV-1 activity of CHR peptides [5]. However, CHR peptides with the wild type HIV-1 gp41 sequences all tend to develop viral drug-resistance in a relatively short time.

To further improve the drug-resistant profiles and the pharmacokinetics of the CHR peptides, He et al. took the CP621–652 as a template and mutated 11 of 32 residues that are not essential for the activity or stability of the peptide, resulting in a peptide termed CP32M (Figure 1B) [6]. CP32M is highly effective against a panel of primary HIV-1 strains with distinct genotypes (group M, subtypes A–G) and phenotypes (R5 and R5X4), and is exceptionally potent against HIV-1 variants resistant to T20 and other CHR peptides including C34. Although its parental peptide CP621–652 is also potent against the drug-resistant HIV-1 viruses, CP32M exhibited much improved antiviral potencies against some T20-resistant HIV- 1 variants, with IC50s even in the picomolar range.