Terazosin activates Pgk1 and Hsp90 to promote stress resistance

Abstract

Drugs that can protect against organ damage are urgently needed, especially for diseases such as sepsis and brain stroke. We discovered that terazosin (TZ), a widely marketed α1-adrenergic receptor antagonist, alleviated organ damage and improved survival in rodent models of stroke and sepsis. Through combined studies of enzymology and X-ray crystallography, we discovered that TZ binds a new target, phosphoglycerate kinase 1 (Pgk1), and activates its enzymatic activity, probably through 2,4-diamino-6,7-dimethoxyisoquinoline's ability to promote ATP release from Pgk1. Mechanistically, the ATP generated from Pgk1 may enhance the chaperone activity of Hsp90, an ATPase known to associate with Pgk1. Upon activation, Hsp90 promotes multistress resistance. Our studies demonstrate that TZ has a new protein target, Pgk1, and reveal its corresponding biological effect. As a clinical drug, TZ may be quickly translated into treatments for diseases including stroke and sepsis.

Figures

Figure 1. TZ blocked apoptosis in cultured mammalian cells

(a) Examples of bright field and fluorescent images of the RAW 264.7 cells stained with Annexin V after LPS and IFN-γ. The bar represents 25 μm. 3 independent experiments were performed. (b) The LDH assay of cell death induced by hydrogen peroxide. Th e R A W 264.7 cells treated with 0.1 % DMSO (control), H2O2 (1 mM), H2O2 plus Z-vad (100 μM) or H2O2 plus TZ (10 μM) in 0.1 % DMSO. The percentage of cell death (%) = LDHmedium/(LDHmedium + LDHcell) × 100%; Trial n = 3. All data are presented as the mean + standard error, unless otherwise indicated. The P values of a two-tailed t-test for the comparison of the two data sets comparison, the one-way ANOVA with post-hoc multiple comparison Sidak for three or more data sets comparisons, and the Kaplan-Meier survival analysis with a log rank algorithm are represented as *** for P<0.001, ** for P<0.01, and * for P<0.05, respectively, throughout all figures. (c) The statistical result of the Western blot to detect the active form of caspase 3. The same condition as (b). The relative ratio of caspase 3/β-actin from the H2O2 treatment was set as 1, and the relative ratio of other treatments were shown. Trial n = 3. (d) The effect of TZ on the activity of caspase 3/7 measured by a DEVDase activity assay. The data were normalized to the control conditions (without treatment). Trial n = 3.

Figure 2. Pgk1 as the target of TZ

(a) The chemical structures of TZ, modified TZ (TZ-md) and the bait compound (TZ-TA). (b) The in vitro pull-down assay to identify the TZ target. Naked Affi-Gel beads andAffi-Gel-TZ-TA beads with saturated soluble competitor of TZ were used as controls. The full gel was shown on Supplementary Fig. 15. (c) The in vitro pull down of the recombinant mouse His-tagged Pgk1 protein by Affi-Gel-TZ-TA. The full gel was shown on Supplementary Fig. 15. (d) TZ effect on purified mouse Pgk1 enzymatic activity. The dosage TZ was indicated. Trial n = 4. (e) The effect of TZ on the ATP and pyruvate production in the cell lysate of RAW 264.7 cells. For the ATP level, the transient 1st minute was measured. Trial n = 5. For the pyruvate production, the steady state (the 10th minute) was measured. Trial n = 3. (f) The effect of stably expressed shRNA for Pgk1 (scrambled shRNA as the control) on cell death mediated by H2O2. The percentage of cell death was determined by the LDH assay; Trial n = 5. (g) The effect of stably expressing Pgk1 or EGFP (control) on the PARP1 cleavage upon H2O2 treatment. The full gel was shown on Supplementary Fig. 15. Trial n = 3.

Figure 3. Crystal structure of Pgk1 and TZ binding

(a) The relative positions of TZ, ADP and ATP were shown. The red, white, and blue colors indicated acidic, neutral, and alkalic distributions, respectively. (b) The structure of mPgk1-3PG-TZ. The enlarged view showed a hydrophobic cleft at the C-terminal domain of Pgk1. The dashed lines indicated predicted hydrogen bonds of TZ with the amino acids from Pgk1. Interactions of potential water molecules with TZ were also shown (in pink). (c) Overlaid of the mPgk1-3PG-TZ structure onto the ADP-bound open form structure of human Pgk1. “2XE7” indicated the open form of hPgk1. (d) Overlaid of the mPgk1-3PG-TZ structure onto the ATP-bound closed form structure of human Pgk1. “2X15” indicated the closed form of hPgk1.

Figure 4. The anti-apoptosis effect of TZ was dependent on Hsp90

(a) The effect of TZ and GA on the ATP levels in the cell lysate of RAW 264.7 cells. The steady state (after the 10th minute) ATP level in the cell lysate was measured after 0.1% DMSO (control), TZ (10 μM), GA alone (100 μM) or TZ plus GA (100 μM) were added. Tail n = 3. (b) The co-immunoprecipitation of Pgk1-HA and Hsp90. Pgk1-HA was transfected into RAW 264.7 cells. An anti-HA antibody was used for a protein pull-down from the cell lysate. The full gel was shown on Supplementary Fig. 15. (c) Effect of TZ against H2O2-induced cell death in the presence of GA (10 nM). The LDH release assay was shown. Trial n = 3.

Figure 5. TZ showed multiple stress resistance in flies and rodents

(a) The TZ effect on adult flies under oxidative stress. Survival rates of different genotypes of flies feeding on 20 mM paraquat were shown with or without feeding 10 μM TZ. Trial n = 3, each trial tested 60 flies. (b) The effect of Pgk1 overexpression on apoptosis in Drosophila. GMR-rpr represents a transgene with the GMR promoter directly upstream of the reaper cDNA, which causes eye-specific apoptosis in flies. The bars represent a length of 50 μm. (c) The CLP model of sepsis. TZ (0.08 mg/kg was injected s.c. at 1.5 and 24 hours after CLP), or saline (vehicle) was injected as a control. The Kaplan-Merier survival analysis was performed, and the number of mice tested was shown. (d) Effect of TZ on MCAO in rats. The micrograph showed the representative TTC staining from a sectioned whole brain. The quantification data was shown on the bar graph.