Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs
Divya Prakash Gnanadhas, Prasanta K. Dash, Brady Sillman, Aditya N. Bade, Zhiyi Lin, Diana L. Palandri, Nagsen Gautam, Yazen Alnouti, Harris A. Gelbard, JoEllyn McMillan, R. Lee Mosley, Benson Edagwa, Howard E. Gendelman, Santhi Gorantla
Divya Prakash Gnanadhas, Prasanta K. Dash, Brady Sillman, Aditya N. Bade, Zhiyi Lin, Diana L. Palandri, Nagsen Gautam, Yazen Alnouti, Harris A. Gelbard, JoEllyn McMillan, R. Lee Mosley, Benson Edagwa, Howard E. Gendelman, Santhi Gorantla
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Research Article AIDS/HIV

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

Abstract

Long-acting anti-HIV products can substantively change the standard of care for patients with HIV/AIDS. To this end, hydrophobic antiretroviral drugs (ARVs) were recently developed for parenteral administration at monthly or longer intervals. While shorter-acting hydrophilic drugs can be made into nanocarrier-encased prodrugs, the nanocarrier encasement must be boosted to establish long-acting ARV depots. The mixed-lineage kinase 3 (MLK-3) inhibitor URMC-099 provides this function by affecting autophagy. Here, we have shown that URMC-099 facilitates ARV sequestration and its antiretroviral responses by promoting the nuclear translocation of the transcription factor EB (TFEB). In monocyte-derived macrophages, URMC-099 induction of autophagy led to retention of nanoparticles containing the antiretroviral protease inhibitor atazanavir. These nanoparticles were localized within macrophage autophagosomes, leading to a 4-fold enhancement of mitochondrial and cell vitality. In rodents, URMC-099 activation of autophagy led to 50-fold increases in the plasma drug concentration of the viral integrase inhibitor dolutegravir. These data paralleled URMC-099–mediated induction of autophagy and the previously reported antiretroviral responses in HIV-1–infected humanized mice. We conclude that pharmacologic induction of autophagy provides a means to extend the action of a long-acting, slow, effective release of antiretroviral therapy.

Authors

Divya Prakash Gnanadhas, Prasanta K. Dash, Brady Sillman, Aditya N. Bade, Zhiyi Lin, Diana L. Palandri, Nagsen Gautam, Yazen Alnouti, Harris A. Gelbard, JoEllyn McMillan, R. Lee Mosley, Benson Edagwa, Howard E. Gendelman, Santhi Gorantla

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Figure 5

URMC-099–induced autophagy affects HIV-1 clearance.

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URMC-099–induced autophagy affects HIV-1 clearance. HIV-1ADA–infected hu...
HIV-1ADA–infected human MDMs were treated with 1 μM nanoATV on day 1 or day 3 after infection and incubated with or without 400 ng/ml URMC-099 and in the presence of the autophagy inhibitors (A) 3-MA (100 μM) or (B) chloroquine (10 μM). Supernatants were collected on different days after infection and analyzed for HIV-1 RT activity (n = 5). The same HIV-1 infection control plot is presented in A and B. (A and B) The mean values of RT activity were assessed by 2-way factorial ANOVA, which showed a significant time-dependent treatment effect (P < 0.02). Pairwise comparisons using Bonferroni’s post-hoc test were assessed for URMC-099–treated cultures, with P < 0.05 compared with HIV-infected controls in the absence (acontrol) or presence (bHIV) of an autophagy inhibitor. (C–E) MDMs were treated in the presence or absence of 400 ng/ml URMC-099 for 14 days. Twenty-four hours or twelve hours before harvesting, cells were treated with 10 μM cycloheximide (CHX) to inhibit translation. Total cell lysates were analyzed by Western blotting. (D) Values represent the mean ± SEM of LC3BII/LC3BI ratios and were compared by Student’s t test and adjusted for multiple comparisons using the Benjamini-Hochberg method. *P ≤ 0.05 and **P ≤ 0.01 (n = 3). (E) Differences in mean fold changes were assessed by 2-way ANOVA and pairwise comparison with the respective proteins was done using Bonferroni’s post-hoc test. P ≤ 0.05 for ano URMC-099/no CHX control and bURMC-099/no CHX control (n = 3). (F) Human MDMs treated with 400 ng/ml URMC-099 were transfected with either TFEB siRNA or ATG13 siRNA on days 3 and 7. On day 14, cell lysates were analyzed for by Western blotting. (G) URMC-099–treated (400 ng/ml) and untreated (control) MDMs were transfected with LC3B-GFP on day 12, and 48 hours later were stained and imaged with a confocal microscope. Scale bars: 20 μm. Data are representative of 3 independent experiments.