Protein target identification of nitazenes and their metabolites using structural and chemoproteomics

The Department of Health and Human Services National Institutes of Health Office of the Director awarded Battelle Memorial Institute's Pacific Northwest National Laboratory Division $505,790 on August 15, 2026, under the Trans-NIH Research Support program (CFDA 93.310) to identify protein targets of nitazenes and their metabolites using structural and chemoproteomics approaches.

The recipient will design and synthesize nitazene chemical probes for affinity-based protein profiling to identify nitazene-binding proteins in human brain, heart, and lung cells, and apply limited proteolysis to map protein structural changes induced by nitazene exposure. These combined proteomics techniques will elucidate direct protein-nitazene binding interactions and downstream signaling pathways perturbed by nitazenes, generating molecular-level data on altered signaling cascades and mechanisms responsible for nitazene-induced toxicity to inform future medical countermeasure strategies for nitazene overdose treatment. The work addresses the emerging threat of novel nitazene analogs—benzimidazole opioids displaying potency equal to or exceeding fentanyl—for which limited molecular pharmacology data exists beyond opioid receptor interactions.

Performance occurs in Richland, Washington. The period of performance extends from August 15, 2026, through July 31, 2028. This is a Project Grant assistance award.

Abstract

Nitazenes are benzimidazole opioids that display similar or higher potency than other ultrapotent synthetic (UPS) opioids such as fentanyl. Although nitazenes were first synthesized and studied in the 1950S, they were never approved for medical use, and therefore we have limited data on their molecular pharmacology beyond interactions with opioid receptors. Given that the numbers of novel nitazene analogs have been increasing on an annual basis and involvement of nitazenes in drug overdoses worldwide, we need a better understanding of the mechanisms that contribute to the adverse effects of this emerging UPS opioid class. We propose to design, synthesize, and apply nitazene chemical probes for affinity-based protein profiling (afbpp) to identify nitazene-binding proteins in human brain, heart, and lung cells. In parallel, we will use limited proteolysis (LIP), a structural proteomics technique, to map protein structural changes induced by nitazene exposures to further validate and characterize the biological consequences of nitazene binding on cellular functions. The combination of these two proteomics techniques will provide rich molecular level data on both direct protein-nitazene binding interactions and the downstream signaling pathways that are perturbed by these UPS opioids. By identifying the specific biological targets that interact with nitazenes in key cell types, we will gain critical insights into the altered signaling cascades and molecular mechanisms responsible for nitazene- induced toxicity, which can inform future medical countermeasure strategies to treat nitazene overdose. The specific aims of this proposal are: aim 1: identify nitazene-binding proteins using affinity-based protein profiling (afbpp). Aim 2: elucidate protein structural and functional changes induced by nitazene exposure