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Dissertation
Mechanism of action studies defining the activity of the biguanide-based, human immunodeficiency virus type 1 co-receptor inhibitor NB325
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2008
DOI:
https://doi.org/10.17918/00008856
Abstract
Our preclinical microbicide development efforts have focused on NB325, which was produced through studies of the biguanide-based compound polyethylene hexamethylene biguanide (PEHMB). Preliminary experiments designed to determine the mechanism by which NB325 inhibits HIV-1 infection suggested the involvement of the viral co-receptors, CXCR4 and CCR5. A number of in vitro assays were used to document the impact of NB325 exposure on CXCR4 and CCR5 detection and function. Flow cytometric assays using antibodies with distinct epitope specificities were used to probe changes in CXCR4 and CCR5 detection induced by NB325 exposure. Competitive binding assays with the sole CXCR4 ligand, CXCL12, and peptides derived from extracellular domains of CXCR4 were used to examine interaction specificity. Inhibition of CXCR4-induced chemotaxis was also assessed. These mechanistic studies demonstrated that NB325 altered the detection of CXCR4 on primary human memory CD4+ T lymphocytes in an epitope-specific manner. Differential detection of CXCR4 epitopes after NB325 exposure was consistent with inhibition of CXCL12-induced chemotaxis, no effect on CXCL12 binding, and a lack of CXCR4 internalization induced by NB325. Peptide competition experiments supported the hypothesis that NB325 interacts with CXCR4 extracellular loop 2 (ECL2). Further analyses demonstrated that NB325 persistently inhibited X4 HIV-1 infection and that mechanisms that provide immediate protection from infection also operate to provide antiviral memory activity and extended inhibition of CXCR4-induced chemotaxis. Similar studies involving CCR5 indicated that CCR5 detection was increased in an epitope-independent manner despite effective inhibition of R5 HIV-1 infection. Interestingly, NB325-induced changes in CCR5 detection were dependent on cellular stimulation. NB325 also conferred antiviral memory activity against R5 HIV-1 infection using a mechanism that also resulted in persistent elevation of CCR5 detection. Collectively, these findings indicate that NB325 provides immediate and persistent protection from X4 and R5 HIV-1 infection through interactions with CXCR4 and CCR5 that perturb their functions as HIV-1 co-receptors during virus attachment and entry into host cells. In conclusion, NB325 functions as a dual co-receptor inhibitor with immediate and memory antiviral activity against HIV-1.
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Details
- Title
- Mechanism of action studies defining the activity of the biguanide-based, human immunodeficiency virus type 1 co-receptor inhibitor NB325
- Creators
- Nina Thakkar Rivera
- Contributors
- Fred C. Krebs (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xix, 305 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991021888961504721