Mechanism Of Action Of HIV-1Fusion Inhibitor HR212and Anti-HIV-1Activities Of The Novel Gossypol Derivatives

Acquired immunodeficiency syndrome (AIDS) was recognized in1981, and the first human immunodeficiency virus (HIV) was isolated2years later, heralding a new era in the fight against pathogenic viruses. Since then, HIV infection has become a major public health problem worldwide. The introduction of highly active antiretroviral therapy (HAART) has markedly decreased mortality and morbidity. However, drug resistance and high toxicity are serious limitations to current treatments that justify the continuation of research efforts for new strategies and interventions. Viral entry currently represents one of the most attractive targets in the search for new drugs to treat HIV infection. HIV entry inhibitors appear to be a rational step forward in ARV therapy, because they prevent the virus from infecting new host cells, and may potentially stop or significantly limit HIV transmission.HR212, a recombinant protein composed of the heptad repeat, is a rationally designed human immunodeficiency virus type1(HIV-1) fusion inhibitor. This protein can be easily produced by E. coli at a low cost. Previously, studies indicated that HR212can efficiently inhibit the entry and replication of both laboratory and clinical HIV-1strains, and this protein is more stable and less sensitive to proteinases than the T20. However, the potential mechanism of action of HR212is still not clear.The procedure of HIV-1entry into the host cells can be divided into three main steps:(Ⅰ) attachment of the viral gp120to the CD4T cell receptor,(Ⅱ) binding of the gp120to CCR5or CXCR4co-receptors and (Ⅲ) gp41changes its conformation by forming N-helix trimer between N-heptad repeats (NHRs) and then six-helix bundle between the N-trimer and the C-heptad repeats (CHRs), the viral and cellular membranes fusion. In the present study, we demonstrated that HR212does not block gp120-CD4binding or interfere with the HIV-1coreceptor CXCR4. However, HR212inhibited the fusion-active gp41core formation mimicked by peptides derived from the viral gp41N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), as determined by ELISA, native polyacrylamide gel electrophoresis, and circular dichroism analysis. Moreover, the fluorescence native polyacrylamide gel electrophoresis (FN-PAGE) indicated that HR212could form a complex with peptide N36to block the gp41fusogenic core formation. These results suggest that HR212inhibits HIV-1entry by targeting the NHR region of gp41. Therefore, HR212can be potentially developed as a novel, high-efficiency HIV-1entry inhibitor. Gossypol is a polyphenolic aldehyde extracted from cotton plants. In order to explore the antiviral effect of gossypol, from the aspects of improving hydrophilicity and lower toxicity, a series of amino acids, amino sugars, and oligopeptides have been used to modify gossypol’s structure and a number of novel gossypol derivatives have been synthesized and screened in vitro for their anti-HIV-1activity. We found that replacing the aldehyde groups of gossypol with some amino acids not only reduced the cytotoxicity but also enhanced the activities against HIV-1. Further study indicated that the neutral amino acids with aliphatic group derivatives of (-)-gossypol showed the strongest inhibitory activity and the lowest cytotoxicity in vitro among all the derivatives tested. The selectivity index of the novel (-)-gossypol-neutral amino acid conjugates is increased100fold when compared with gossypol alone. Additionally, the inhibitory activities of the (-)-gossypol bis L-alanine sodium scruff’s base (50#) on infection of PBMC by primary HIV-1strains were also determined. This compound had potent inhibitory activity on infection by primary HIV-1strains with distinct genotypes and biotypes. This result suggest that the novel (-)-gossypol derivative has potent antiviral activity against a broad spectrum of HIV-1strains.It is widely accepted that gossypol and gossypol derivatives inhibit HIV-1replication by targeting reverse transcriptase. However, from the results of our time-of-addition assay, HIV-1-mediated cell fusion assay and pseudotyped virus assay, we demonstrate that the (-)-gossypol bis L-alanine sodium schiff’s base (50#) is an effective HIV-1entry inhibitor. Thus it may be used as lead compound of a new type of anti-HIV entry inhibitors for further study.