A high-throughput screening strategy identifies adenosine 5-monophosphate as a novel enhancing molecule of the cardio-protective enzyme 5-oxoprolinase (OPLAH)

Abstract Background/Introduction Oxidative stress plays an essential role in the pathophysiology of heart failure (HF), and reducing it might improve outcomes in patients suffering from it. We recently identified 5-oxoproline to be elevated in patients with HF and that the increased levels are associated with a poor prognosis. 5-oxoprolinase (OPLAH) is an enzyme that converts toxic 5-oxoproline to harmless glutamate. We previously showed that the overexpression of OPLAH prevented cardiac damage in experimental models. Purpose To identify small molecules that hold the potential to increase the activity of the antioxidant enzyme OPLAH. Methods and results OPLAH expression vectors were established containing a 6xHis-tag in the N-terminal region and were used to efficiently overexpress enzymatically active recombinant OPLAH protein (rOPLAH) in human embryonic kidney cells (HEK293A) and Spodoptera frugiperda cells (SF9). rOPLAH expressing His-tag was purified under native conditions using an affinity chromatography matrix, and enzymatic activity was proven by measuring the conversion of 5-oxoproline to glutamate using LC-MS/MS. After miniaturization and optimization of a luminescence-based technique that measures OPLAH's ATPase activity, a high-throughput screening campaign of 1280 FDA-approved compounds was performed. Adenosine 5′-monophosphate (5'-AMP) was identified to enhance the ATPase activity of OPLAH by >50%. In addition, an LC-MS/MS dose-response curve exhibited that increasing 5'-AMP concentration resulted in a higher glutamate production using isolated rOPLAH. A thermal shift assay confirmed the interaction of 5'-AMP and OPLAH. Since 5’-AMP is unable to cross the cellular plasma membrane, acetalated dextran spermine-modified nanoparticles loaded with 5'-AMP were produced (AcDXSp@AMP NPs) to target cells. NPs were prepared by the water-in-oil-in-water double emulsion technique. Particle size, polydispersity index and z–potential of the NPs were characterized by dynamic and electrophoretic light scattering. HPLC tested the quantification of encapsulated 5'-AMP and its release over time. The efficacy of OPLAH activation by 5'-AMP-loaded NPs was tested in HeLa cells cultured in the presence of 10 mM 13C-5-oxoproline. Employing AcDXSp@AMP NPs for 12h to cells resulted in about 50% elevation of 13C-glutamate production, showing successful activation of OPLAH by AMP in cells. Conclusion Here, we developed a high-throughput screening for 5-oxprolinase and identified a non-cell permeable enhancer compound, adenosine 5′-monophosphate. Orthogonal studies confirmed 5'-AMP affinity to OPLAH. Finally, we successfully utilized nanoparticles as a drug-delivery system for 5'-AMP and proved its intracellular effect. Our efforts are a blueprint for bringing target discovery to treatment identification. As such, nanoparticle-based delivery systems hold great potential for future trials utilizing impermeable modulators to target heart failure and other diseases.Illustrative workflow