Theoretical Investigation on the Mechanism of Alzheimer’s Disease Detection through Firefly Bioluminescence

Abstract Development of precise, non-invasive, and dynamic methods for detecting amyloid-β (Aβ) aggregates is of vital importance for the early diagnosis and therapeutic evaluation of Alzheimer’s disease (AD). Recently, the Akalumine (AkaL)-based firefly bioluminescent probe has emerged as a promising tool for deep-tissue Aβ imaging. However, despite the successful experimental application of this probe, an understanding that links its microscopic mechanisms to the observed bioluminescence (BL) intensity, kinetics, and imaging performance is still lacking. In this work, a multiscale theoretical investigation combining classical molecular dynamics (cMD) simulations, absolute binding free energy (ABFE) calculations, quantum mechanics/molecular mechanics (QM/MM), and QM/MM metadynamics simulations was performed to comprehensively elucidate the mechanism of this bioluminescent system. Our cMD and ABFE results reveal that Aβ acts as a reservoir capable of storing AkaL via hydrophobic interactions, thereby dynamically modulating the concentration of free AkaL and the evolution of BL intensity. Changes in BL intensity can serve as an indicator of the concentration and distribution of Aβ. Furthermore, the complete BL process of AkaL catalyzed by firefly luciferase was systematically studied. The initial adenylation follows the SN2 mechanism. The water molecule was found to play a crucial catalytic role in the subsequent deprotonation by mediating proton transfer, significantly lowering the energy barrier. Then, oxygenation occurs via a triplet-to-singlet intersystem crossing (ISC) process to generate the dioxetanone intermediate. The decomposition of the dioxetanone intermediate follows the gradually reversible charge-transfer initiated luminescence (GRCTIL) mechanism that generates the light emitter Oxy-AkaL in the S1 state. The calculated emission wavelength is in reasonable agreement with the experimental BL wavelength. Overall, this work provides theoretical insights into the mechanism of firefly BL-based AD detection and offers guidance for the rational design of future bioluminescent probes for AD.