Anti-HIV NRTIs and the lysosomal toxicity

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PROJECT SUMMARY The nucleoside reverse transcriptase inhibitors (NRTIs) have potent activities against HIV, but their therapeutic benefit in patients undergoing NRTI therapies is limited by significant adverse drug reactions (ADR), resulting in poor patient compliance and compromised drug efficacy. Our group has recently described an indispensable role of a lysosomal nucleoside transporter ENT3 in lysosomal homeostasis via deletion of ENT3 in mice. Intriguingly, ENT3 KO mice manifest clinical phenotypes closely resembling NRTI ADR. The overall objective of this application is to evaluate ENT3-loss driven lysosomal toxicity as a putative mechanism involved in the chronic adverse sequelae of NRTIs. The central hypothesis of this proposal is that NRTIs that do not interfere with ENT3-supported lysosomal homeostasis or the inclusion of lysosomal signaling agents will minimize the occurrence of NRTI ADR. Aim 1 will evaluate the strategies to avoid NRTI toxicity without compromising drug efficacy. Our working hypothesis for this aim is that disruption of interaction between ENT3 and NRTIs or the inclusion of pharmacological agonists of lysosomal-autophagy pathway will mitigate the onset and severity of NRTI ADR. The preliminary studies that demonstrate the involvement of the cell surface NRTI transporters (e.g., ENT1, CNT) and not the lysosomal ENT3 for NRTI efficacy, the misregulation of the AMPK and mTOR signaling axis in the Ent3-/- mice, and the functional rescue of multi-organ dysfunction in Ent3-/- mice using a pharmacological AMPK activator AICAR; all support this aim. Aim 2 will elucidate the mechanism(s) of occurrence of NRTI-specific ADR signs. Our working hypothesis for this aim is that NRTIs, when present at clinically relevant blood concentrations, will inhibit the ENT3-regulated adult stem cell functions resulting in disruption of tissue repair and regeneration. In addition, we hypothesize that NRTIs will differentially impact the ENT3 function in adult tissues to bring distinct inflammatory, metabolic and degenerative changes that coupled with stem cell alterations, will explain the clinically observed NRTI ADR signs. The preliminary studies that demonstrate the transport of many ADR-producing NRTIs by ENT3, the inhibition of lysosomal adenosine transport by NRTIs and the perturbation of lysosomal recycling of adenosine in Ent3-/- mice leading to adult stem cell exhaustion, tissue inflammation and degeneration, and breaches of mesodermal tissue integrity, which taken together supports this aim. The project will utilize biochemical and molecular approaches, novel ENT3 probes, newly generated ENT3 mouse models, metabolomics, tissue engineering, pharmacophore modeling, synthetic and screening procedures and PKPD to accomplish the goals. The successful completion of the project will provide new insights into the mechanisms of occurrence of NRTI ADR and may have translational benefit for optimizing treatments (such as long-term efficacy, adherence, tolerability, etc.) in patients undergoing NRTI therapies.