The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over 6,000,000 cases and nearly 400,000 deaths reported worldwide by the end of May 2020. A rush to find a cure prompted re-evaluation of a range of existing therapeutics vis-à-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the noncovalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease in its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-p I ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19.

The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over six million cases and nearly four hundred thousand deaths reported worldwide by the end of May 2020. A rush to find the cures prompted re-evaluation of a range of existing therapeutics vis-à-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the non-covalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease of its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-pI ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19.

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Purpose: The present study was aimed to investigate whether combination treatment with progesterone and rehabilitation training would be more effective than monotherapy after cerebral ischemia. Methods: C57BL/6 mice were subjected to focal ischemia by photothrombosis and were treated with progesterone (2 mg/kg), rehabilitation training or their combination. 2,3,5-triphenyltetrazolium chloride (TTC) staining and Nissl staining were used to measure infarct size at day 3 and day 7 after surgery, and rotarod test and grip strength test were conducted to evaluate behavioral outcomes. Results: TTC staining indicated that progesterone, rehabilitation training and their combination produced a different degree of reduction in infarct volume compared with vehicle control at day 3 after ischemia (progesterone: 16.70 ± 0.93 mm 3 , p < 0.01, rehabilitation training: 22.19 ± 0.93 mm 3 , p < 0.05, progesterone + rehabilitation training: 14.76 ± 0.92 mm 3 , p < 0.01, vehicle control: 28.73 ± 1.05 mm 3 ). Nissl staining revealed that prolonged treatment of progesterone, rehabilitation training and their combination led to a significant decrease in infarct volume at day 7 after ischemia (progesterone: 18.64 ± 1.83 mm 3 , p < 0.01, rehabilitation training: 25.07 ± 1.70 mm 3 , p < 0.05, progesterone + rehabilitation training: 17.09 ± 0.92 mm 3 , p < 0.01, vehicle control: 30.31 ± 1.36 mm 3 ). No accumulative effect in the reduction of infarct volume was observed in combination therapy at both day 3 and day 7 after ischemia. However, combination therapy significantly improved behavioral performances in the first week after photothrombosis. Combination treatment significantly enhanced rotarod performance and forelimb grip strength at all time points within 7 days after ischemia compared with rehabilitation alone, and significantly improved rotarod performance and forelimb grip strength from day 2 after ischemia compared with progesterone alone. Conclusion: Our results suggested that combination treatment with progesterone and rehabilitation training had no additive effect in reducing infarct volume, but combination therapy exhibited enhanced efficacy in promoting functional recovery after ischemic stroke, suggesting progesterone and rehabilitation training may exert their effects via different mechanisms.