NeoR6 inhibits HIV-1-CXCR4 interaction without affecting CXCL12 chemotaxis activity.

Aminoglycoside-arginine conjugates (AACs) are multi-target HIV-1 inhibitors. The most potent AAC is neomycin hexa-arginine conjugate, NeoR6. We here demonstrate that NeoR6 interacts with CXCR4 without affecting CXCL12-CXCR4 ordinary chemotaxis activity or loss of CXCR4 cell surface expression. Importantly, NeoR6 alone does not affect cell migration, indicating that NeoR6 interacts with CXCR4 at a distinct site that is important for HIV-1 entry and mAb 12G5 binding, but not to CXCL12 binding or signaling sites. This is further supported by our modeling studies, showing that NeoR6 and CXCL12 bind to two distinct sites on CXCR4, in contrast with other CXCR4 inhibitors, e.g. T140 and AMD3100. This complementary utilization of chemical, biology, and computation analysis provides a powerful approach for designing anti-HIV-1 drugs without interfering with the natural function of CXCL12/CXCR4 binding.

Involvement of the CXCL12/CXCR4 pathway in the recovery of skin following burns.

Burn wound healing is a complex process consisting of an inflammatory phase, the formation of granulation tissue, and remodeling. The role of the CXCL12/CXCR4 pathway in the recovery of skin following burns is unknown. We found that CXCL12 is similarly expressed in human, swine, and rat skin by pericyte and endothelial cells, fibrous sheet, fibroblasts, and axons. Following burns, the levels of CXCL12 were markedly increased in human burn blister fluids. One day after injury, there was a gradual increase in the expression of CXCL12 in the hair follicles and in blood vessel endothelium surrounding the burn. Three to 11 days following burns, an increased number of fibroblasts expressing CXCL12 were observed in the recovering dermis of rat, swine, and human skin. In contrast to CXCL12, CXCR4 expression was detected in proliferating epithelial cells as well as in eosinophils and mononuclear cells infiltrating the skin. In vitro, CXCL12 was expressed by primary human skin fibroblasts, but not by keratinocytes, and was stimulated by wounding a confluent cell layer of these fibroblasts. Blocking the CXCR4/CXCL12 axis resulted in the significant reduction in eosinophil accumulation in the dermis and improved epithelialization. Thus, blocking CXCR4/CXCL12 interaction may significantly improve skin recovery after burns.

Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis.

Hormone refractory metastatic prostate cancer remains an incurable disease. We found that high expression levels of the chemokine receptor CXCR4 correlated with the presence of metastatic disease in prostate cancer patients. Positive staining for CXCL12, the ligand for CXCR4, was mainly present in the tumor-associated blood vessels and basal cell hyperplasia. Subcutaneous xenografts of PC3 and 22Rv1 prostate tumors that overexpressed CXCR4 in NOD/SCID mice were two- to threefold larger in volume and weight vs. controls. Moreover, blood vessel density, functionality, invasiveness of tumors into the surrounding tissues, and metastasis to the lymph node and lung were significantly increased in these tumors. Neutralizing the interactions of CXCL12/CXCR4 in vivo with CXCR4 specific antibodies inhibited the CXCR4-dependent tumor growth and vascularization. In vitro, CXCL12 induced the proliferation and VEGF secretion but not migration of PC3 and 22Rv1 cells overexpressing CXCR4. Similar effects of CXCR4 overexpression on tumor growth in vivo were also noted in two breast cancer lines, suggesting that the observed effect of CXCR4 is not unique to prostate tumor cells. Thus high levels of the chemokine receptor CXCR4 induce a more aggressive phenotype in prostate cancer cells and identify CXCR4 as a potential therapeutic target in advanced cases of metastatic prostate cancer.