selectivity against mGPDH, it PD1-PDL1 inhibitor 2 provided insight into the structural elements essential to mGPDH inhibition and useful clues as to which chemical features should likely be targeted in future 871361-88-5 distributor optimization studies. The most selective inhibitor, iGP-1, progressively inhibited H2O2 production by mGPDH as its concentration was increased from 0.25 to 80 mM, with a half-maximal effect at about 8 mM. Only above 10 mM did iGP-1 start to inhibit H2O2 production by site IQ, demonstrating its good specificity. This effect on H2O2 production by mGPDH was mirrored over the same concentration range by significant and specific lowering of DYm driven by glycerol phosphate, and significant and specific inhibition of respiratory rates in mitochondria supplied with glycerol phosphate, suggesting that iGP-1 inhibited enzymatic activity of mGPDH. iGP-1 decreased H2O2 production by site IQ and DYm driven by low succinate but not by high succinate indicating a subtle off-target effect on succinate oxidation. It had no effect on H2O2 production, DYm, or respiration driven by oxidation of a variety of substrates including glutamate, malate, pyruvate, and palmitoylcarnitine. Similarly, iGP-5 significantly inhibited H2O2 production by mGPDH as its concentration was increased from 0.08 to 80 mM, with a half-maximal effect at about 1 mM. It also lowered DYm and inhibited respiration driven by glycerol phosphate. iGP-5 lowered DYm driven by glycerol phosphate more potently than iGP-1. However, this structural analog also displayed less selectivity in assays of H2O2 production and also significantly increased DYm in the absence but not the presence of nigericin, suggesting a negative effect on DpH. Further, iGP-5 inhibited respiration driven by pyruvate and perhaps succinate. We next determined if iGP-1 was cell-permeant and, therefore, potentially useful for inhibiting mGPDH in situ. Several iGPs exhibited fluorescence with excitation maxima between 320�C 360 nm and emission maxima between 360�C480 nm. Cells treated with 100 mM iGP-1 displayed fluorescence above background, with bright puncta in the cytosol, low levels in the nucleus, and intermediate levels distributed diffusely within
