Thermo‐kinetic analysis space expansion for cyclophilin‐ligand interactions – identification of a new nonpeptide inhibitor using Biacore™ T200

Abstract

We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his‐tagged human cyclophilin‐A. Our orientation‐specific stabilisation approach captures his‐tagged protein under ‘physiological conditions’ (150 mm NaCl, pH 7.5) and covalently stabilises it on Ni2+‐nitrilotriacetic acid surfaces, very briefly activated for primary amine‐coupling reactions, producing very stable and active surfaces (≥ 95% specific activity) of cyclophilin‐A. Variation in protein concentration with the same contact time allows straightforward generation of variable density surfaces, with essentially no loss of activity, making the protocol easily adaptable for studying numerous interactions; from very small fragments, ~ 100 Da, to large protein ligands. This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo‐kinetic analysis space, and to determine accurate and robust thermodynamic parameters for the cyclophilin‐A–cyclosporin‐A interaction. Furthermore, the increased sensitivity of the surface allowed identification of a new nonpeptide inhibitor of cyclophilin‐A, from a screen of a fragment library. This fragment, 2,3‐diaminopyridine, bound specifically with a mean affinity of 248 ± 60 μm. The X‐ray structure of this 109‐Da fragment bound in the active site of cyclophilin‐A was solved to a resolution of 1.25 Å (PDB: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5LUD), providing new insight into the molecular details for a potential new series of nonpeptide cyclophilin‐A inhibitors.

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis–Trans Isomerization and the Chameleonic Roles of Calcium

“…In 20 mM ammonium acetate with pH = 6.8, charge states as low as +7 can be observed, consistent with the work by Barran and co-workers . The plot of CCSs versus charge state (Figure B) and comparison to theoretical CCSs of CypA X-ray structures (PDB IDs 5LUD, 1AWR, and 1AWV ) show that the protein retains its native state at z = +7 and +8. The large increase seen in charge states from z = +8 to z = +9 indicates the unfolding of the native CypA structure as extra charge states accrue, a phenomenon that is well documented for many proteins. − …”

“…(B) CCS of each ion species with its respective charge state for CypA shown in blue. The pink data points correlate to the theoretical CCS of three CypA structures (PDB IDs 5LUD, 1AWR, and 1AWV ). The inset shows the +8 and +9 charge states with their respective CCS of the CycA:CypA complex, and the pink lines show the theoretical CCSs of the X-ray crystal structure (PDB 1CWA ) and NMR structure (PDB 3CYS ) of the complex.…”

Kinetic and structural studies of Trypanosoma and Leishmania phosphofructokinases show evolutionary divergence and identify AMP as a switch regulating glycolysis versus gluconeogenesis

“…Sensor chip surfaces with active N-terminal His 6 -tagged T. brucei, T. cruzi and L. infantum PFK were generated by using a capture/stabilization method [43,44]. Surface densities corresponding to 1400-3200 resonance units (RU) were immobilized on Ni 2+ -nitrilotriacetic acid sensor chips (GE Healthcare, GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK).…”

Structural and Functional Insights into Human Nuclear Cyclophilins

“…Most drug development has focused on the connection between secreted cyclophilins and infectious disease, or the identification of PPIF as a modulator of the mitochondrial permeability transition pore complex [6,7,70]. The majority of this work continues to target the S1 pocket; highly conserved and extremely accessible to solvent, this approach is nearly guaranteed to generate pan-inhibitors with significant off-target activity [8,71,72]. In 2010, we published a study in which we identified and characterized a site contiguous to S1.…”