1. IMPDH2 Is an Intracellular Target of the Cyclophilin A and Sanglifehrin A Complex 
Khian Hong Pua, Dylan T. Stiles, Mathew E. Sowa, Gregory L. Verdine 
Cell Reports 
Volume 18, Issue 2, Pages (January 2017)
DOI: /j.celrep
Copyright © 2017 The Authors Terms and Conditions

2. Cell Reports 2017 18, 432-442DOI: (10.1016/j.celrep.2016.12.030)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1 Interaction of IMPDH2 with PPIA Is SFA-Dependent
(A) Structure of sanglifehrin A.
(B) Silver staining of GST-PPIA pull-down with Jurkat cell lysate (lanes in between ladder and DMSO control were cropped).
(C) Fold change of the total spectral counts for the top 25 interacting proteins from HA immunoprecipitation with PPIA as the bait with HeLa cell lysates treated with SFA or DMSO.
(D) IP from HeLa cell lysates stably expressing HA-PPIA are performed in the presence or absence of SFA and western blot probed with HA and IMPDH2 antibody.
(E) IP from HeLa cell lysates stably expressing HA-IMPDH1 or HA-IMPDH2 are performed in the presence or absence of SFA and western blot probed with HA and PPIA antibody.
See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 2
Macrocyclic Analog SFM Does Not Bind IMPDH2 in the Presence of PPIA and Is Biologically Inactive
(A) Synthetic degradation of SFA to SFM via the oxidative cleavage at olefinic C26-C27.
(B) Representative SPR sensorgram of SFA binding to immobilized PPIA. 1:1 Langmuir model fits to the kinetic data are shown in black.
(C) Representative SPR sensorgram of SFM binding to immobilized PPIA. 1:1 Langmuir model fits to the kinetic data are shown in black.
(D) GST-PPIA pull-down assay with IMPDH2 in the presence or absence of SFA, SFM, and CSA.
(E) TR-FRET assay of IMPDH2 ternary complex formation with SFA, SFM, and CSA. No ternary complex is observed with CSA. Representative data showing mean ± SD for experiments performed in triplicate (n = 3).
(F) TR-FRET assay showing no ternary complex formation with IMPDH1 in the presence of SFA, SFM, and CSA. Representative data showing mean ± SD for experiments performed in triplicate (n = 3).
(G) SFM has a marked reduction in potency in K562 cells. Representative data showing mean ± SD for experiments performed in triplicate (n = 3 biological replicates).
See also Figure S2.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 3
PPIA-SFA Complex Does Not Block Dehydrogenase Function of IMPDH2
(A) IMPDH inhibition assay with SFA, PPIA-SFA, and MPA. Enzymatic activity of IMPDH2 is performed by incubating IMPDH2 with IMP and NAD (in the absence or presence of inhibitors), and absorbance at 340 nM is measured every 1 min for 10 min.
(B) Viability of K562 cells overexpressing GFP or IMPDH2 after treatment with SFA. Representative data showing mean ± SD for experiments performed in triplicate (n = 3 biological replicates).
(C) Viability of K562 cells overexpressing GFP or IMPDH2 after treatment with MPA. Representative data showing mean ± SD for experiments performed in triplicate (n = 3 biological replicates).
(D) K562 cells supplemented with 10 μM nucleosides, treated with SFA and assayed for viability. Representative data showing mean ± SD for experiments performed in triplicate (n = 3 biological replicates).
(E) TR-FRET assay showing that the catalytic dead IMPDH2_C331A mutant can form a ternary complex with SFA, and to a lesser extent with SFM. No ternary complex is observed with CSA. Representative data showing mean ± SD for experiments performed in triplicate (n = 3).
See also Figure S3.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 4 CBS Domains of IMPDH2 Are Necessary for PPIA-SFA Binding
(A) Structure of human IMPDHs. The canonical hIMPDH1 and hIMPDH2 are 514-amino-acids long and share 84% sequence homology at the amino acid level. In human IMPDHs, the catalytic cysteine residue is located at position 331 (green).
(B) Monomeric IMPDH2 with its CBS domains shown in red. The CBS-domain-deleted IMPDH2 construct (IMPDH2_ΔCBS) retains catalytic activity (PDB: 1B3O) (Colby et al., 1999).
(C) GST-PPIA pull-down assay with IMPDH2_ΔCBS.
(D) IMPDH chimeric constructs with IMPDH2 core bearing the CBS domains of IMPDH1 and vice versa are designed (IMPDH2_CBS1 and IMPDH1_CBS2). GST-PPIA pull-down assay showing chimeric IMPDH1 bearing the CBS domain of IMPDH2 binds to PPIA-SFA, whereas the other chimera loses its ability to bind to PPIA-SFA.
(E) Identifying the sequence determinants for the binding of PPIA-SFA to IMPDH2 via site-directed mutagenesis of IMPDH2 at I192, E214, and D215 to that corresponding to IMPDH1. Residues I192 and E214 are identified as critical for protein-drug interaction.
(F) TR-FRET assay of double mutant IMPDH1_V192I/D214E ternary complex formation with various cyclophilin binding ligands. Representative data showing mean ± SD for experiments performed in triplicate (n = 3).
(G) TR-FRET assay of double mutant IMPDH2_I192V/E214D ternary complex formation with various cyclophilin binding ligands. Representative data showing mean ± SD for experiments performed in triplicate (n = 3).
See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 5 CBS Domains of IMPDH2 Are Involved in Cellular Proliferation
(A) CRISPR/Cas9 knockout of IMPDH2 in K562 cells.
(B) Proliferation of IMPDH2 knockout clonal parental line A3 and A3 with overexpression of various rescue constructs.
(C) Rescue cell lines and their sensitivity to SFA. Cells are treated with 100 nM SFA for 72 hr. Treatments are performed in triplicate, and each data point shown is an average of replicates ± SD.
See also Figure S5.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Authors Terms and Conditions