Background: Antiretroviral therapy (ART) has improved life quality and longevity for infected people. However, despite ART viral replication continues, albeit at low levels. Eradication of infection remains the major goal of HIV scientists and clinicians alike. Recently, a novel mixed-lineage kinase-3 (MLK3) inhibitor (URMC-099) with promising immune and neural modulatory activities was found. Surprisingly, the drug was found to potentiate the antiretroviral activities of our nanoformulated long-acting ART Methodology: Isobaric tag for relative and absolute quantitation (iTRAQ) was performed in URMC-099 treated monocyte-derived macrophages (MDM) seeking potential antiretroviral actions. Nanoformulated atazanavir (nanoATV) with/out dose escalated URMC-099 was tested for antiretroviral activities in HIV-1ADA exposed MDM. Viral DNA and RNA, Gag proteins and reverse transcriptase (RT) activities were assessed. Subcellular endosomal trafficking (Rab 5, 7, 8 and 14) of the ART nanoparticles were investigated by Western blot, confocal microscopy and HPLC. Humanized NOD/scid-IL-2Rγcnull (NSG) mice were infected with HIV-1ADA followed by administration of URMC-099 daily and nanoformulated atazanavir and ritonavir (nanoART/r) for three weeks beginning at 10 weeks post-infection. Peripheral viral load (VL), human CD4+ and CD8+ T cells, and lymphoid and brain tissues were investigated for virus and immune profiling. Results: URMC-099 increased expression of the RAS-related GTP-binding proteins Rab 7, 8 and 14 in HIV-1 infected MDM. NanoATV antiretroviral activities were potentiated by URMC-099 with reductions in viral DNA and RNA, HIV-1gag and RT activity. URMC-099 increased ATV concentrations in Rab14 endosomal compartments. Highest levels of CD4+ T cells numbers were seen in infected humanized mice treated with URMC-099 and nanoATV with VL in plasma below the limit of assay detection in the mice (<500 copies/ml). Such URMC-099-ART synergy was not seen with native drug. Histopathological tests of spleen and lymph nodes showed decreased numbers of HIV-1p24 cells with both drugs as compared to nanoATV alone. Preservation of synaptic structures was also seen. iTRAQ and tandem mass spectrometry tests were confirmed by Western blot tests showing significant Rab7 and 8 changes in HIV-1 infected MDM. Conclusions: URMC-099 significantly potentiates nanoATV responses to restrict HIV-1 and protect CD4+ T lymphocytes. This appears to occur, in part, by sustaining ART levels in specific endosomal compartments of the cells. Such new effects of URMC-099 may represent a novel means for viral clearance.
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