Acidification of the cellular lysosome is an important factor in
infection of mammalian cells by SARS-CoV-2. Therefore, raising the pH of the lysosome would theoretically be beneficial in prevention or treatment of
SARS-CoV-2 infection.
Sodium bicarbonate,
carbicarb, and THAM are
buffers that can be used clinically to provide base to patients. To examine whether these bases could raise lysosomal pH and therefore be a primary or adjunctive treatment of
SARS-CoV-2 infection, we measured lysosomal and intracellular pH of mammalian cells after exposure to each of these bases. Mammalian HEK293 cells expressing RpH-LAMP1-3xFLAG, a ratiometric sensor of lysosomal
luminal pH, were first exposed to
Hepes which was then switched to
sodium bicarbonate,
carbicarb, or THAM and lysosomal pH measured. In
bicarbonate buffer the mean lysosomal pH was 4.3 ± 0.1 (n = 20); p = NS versus
Hepes (n = 20). The mean lysosomal pH in
bicarbonate/
carbonate was 4.3 ± 0.1 (n = 21) versus
Hepes (n = 21), p = NS. In THAM
buffer the mean lysosomal pH was 4.7 ± 0.07 (n = 20) versus
Hepes (4.6 ± 0.1, n = 20), p = NS. In addition, there was no statistical difference between pHi in
bicarbonate,
carbicarb or THAM solutions. Using the membrane permeable base NH4Cl (5 mM), lysosomal pH increased significantly to 5.9 ± 0.1 (n = 21) compared to
Hepes (4.5 ± 0.07, n = 21); p < 0.0001. Similarly, exposure to 1 mM
hydroxychloroquine significantly increased the lysosomal pH to (5.9 ± 0.06, n = 20) versus
Hepes (4.3 ± 0.1, n = 20), p < 0.0001. Separately steady-state pHi was measured in HEK293 cells bathed in various
buffers. In
bicarbonate pHi was 7.29 ± 0.02 (n = 12) versus
Hepes (7.45 ± 0.03, [n = 12]), p < 0.001. In cells bathed in
carbicarb pHi was 7.27 ± 0.02 (n = 5) versus
Hepes (7.43 ± 0.04, [n = 5]), p < 0.01. Cells bathed in THAM had a pHi of 7.25 ± 0.03 (n = 12) versus
Hepes (7.44 ± 0.03 [n = 12]), p < 0.001. In addition, there was no statistical difference in pHi in
bicarbonate,
carbicarb or THAM solutions. The results of these studies indicate that none of the
buffers designed to provide base to patients alters lysosomal pH at the concentrations used in this study and therefore would be predicted to be of no value in the treatment of
SARS-CoV-2 infection. If the goal is to raise lysosomal pH to decrease the infectivity of SARS-CoV-2, utilizing lysosomal permeable
buffers at the appropriate dose that is non-toxic appears to be a useful approach to explore.