Figure 1.
INVDOCK-predicted binding model of AGS-IV molecule (shown in ball and stick model) with cyclophilin (Cyp) and calcineurin (Cn).
CnA and CnB are the catalytic subunit and the calcium-binding regulatory subunit of calcineurin, respectively. In the protein secondary structure, red, blue and grey colors represent alpha-helices, beta-sheets and loops, respectively; for the ligand structure, red and grey colors correspond to oxygen and carbon atoms, respectively.
Table 1.
Functional classes of putative therapeutic targets of AGS-IV identified by an INVDOCK search of a candidate protein 3D structure data set.
Figure 2.
Evaluation of AGS-IV on representative protein targets.
(A) Calcineurin activity in VSMCs following a 24-h pretreatment with AGS-IV (0.10∼1.02 µM), while cyclosporin A (CsA) was used as positive control. (B) PE-induced calcineurin activation in VSMCs following a 24-h pretreatment with AGS-IV (0.10∼1.02 µM). (C) ACE activity in HUVECs following a 20-min pretreatment AGS-IV (0.10∼1.02 µM), while enalapril maleate (EM) was used as positive control. (D) AGS-IV (1.02 µM) inhibited LPS-induced activation of JNK for at least 20 min. JNK phosphorylation levels were determined by Western blot, analyzed by Quantity One™ software (BioRad Inc.) and normalized to β-actin.
Figure 3.
The protective effect of AGS-IV in adriamycin-induced injury of cardiomyocytes compared with CN inhibitor CsA.
(A) Activity of LDH in culture supernatant of the ADR-injured cardiomyocytes treated by AGS-IV (0.32∼32 µM). (B) Activity of LDH in culture supernatant of the ADR-injured cardiomyocytes treated by CsA (0.21∼4.2 µM). Data are shown as mean±SD (n = 4) (**P<0.01). (C) The effects of CsA and AS-IV on the expressions of apoptotic proteins. The expression of BCL-2 and Bax in ADR-injured cardiomyocytes treated by CsA or AGS-IV, the effects of CsA and AS-IV on the Bcl-2/Bax level. All data are mean±SD (n = 3).
Table 2.
Potency of AGS-IV against CVD-associated targets.
Figure 4.
Network analysis of the efficacy of AGS-IV on myocardial injuries.
(A) Constructed minimum protein-protein interaction network between targets of AGS-IV. Red and green nodes denote putative targets of AGS-IV. Red nodes have been reported in the literature as drug targets or potential targets for CVD therapy; green nodes are predicted by the INVDOCK but with no literature support. Grey nodes are proteins that link the targets together. The network was decomposed into topological compact modules, labeled with numbers, by a simulated annealing algorithm. (B) Drug-target network for cardiovascular drugs related to AGS-IV. Circles and triangles represent drugs and targets, respectively. AGS-IV and its putative direct targets are highlighted in red. Nodes for other drugs are colored according to their ATC codes at the 4th level (ATC codes are codes in the Anatomical Therapeutic Chemical (ATC) Classification System used for the classification of drugs, which is controlled by the WHO Collaborating Centre for Drug Statistics Methodology (WHOCC). Drugs are classified into groups at 5 different levels in this coding system, in which the 4th level of the code indicates the chemical/therapeutic/pharmacological subgroup). (C) Target-pathway network for putative targets of AGS-IV. Circles and squares represent targets and pathways, respectively. Target nodes are colored according to Drugbank drug classes. Known targets of approved CVD drugs are highlighted in red. (D) Pathway associated with myocardial ischemia injury. This pathway was constructed by integrating pathways reported to be involved in the process of myocardial ischemia injury, including the calcium signaling, MAPK, JAK-STAT and NF-KB pathway. Red and pink boxes indicate putative target proteins of AGS-IV; targets in red were experimentally validated in this study.