A Surface Plasmon Resonance-based Assay for Small Molecule Inhibitors of Human Cyclophilin A

Overview

Journal Anal Biochem

Publisher Elsevier

Specialty Biochemistry

Date 2005 Aug 17

PMID 16102717

Citations 13

Authors

Martin A Wear

Alan Patterson

Kirk Malone

Colin Dunsmore

Nicholas J Turner

Malcolm D Walkinshaw

Affiliations

Soon will be listed here.

Abstract

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.

Citing Articles

High-Efficiency Trifluoromethyl-Methionine Incorporation into Cyclophilin A by Cell-Free Synthesis for F NMR Studies.

Zhu W, Monnie C, Kitoka K, Gronenborn A Angew Chem Int Ed Engl. 2024; 64(7):e202419709.

PMID: 39571097 PMC: 11813676. DOI: 10.1002/anie.202419709.

Discovery and molecular basis of subtype-selective cyclophilin inhibitors.

Peterson A, Rangwala A, Thakur M, Ward P, Hung C, Outhwaite I Nat Chem Biol. 2022; 18(11):1184-1195.

PMID: 36163383 PMC: 9596378. DOI: 10.1038/s41589-022-01116-1.

IMB-T130 targets 3-dehydroquinate synthase and inhibits Mycobacterium tuberculosis.

Zhu N, Wang X, Li D, Lin Y, You X, Jiang J Sci Rep. 2018; 8(1):17439.

PMID: 30487577 PMC: 6262012. DOI: 10.1038/s41598-018-35701-z.

Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties.

Gur M, Blackburn E, Ning J, Narayan V, Ball K, Walkinshaw M J Chem Phys. 2018; 148(14):145101.

PMID: 29655319 PMC: 5891347. DOI: 10.1063/1.5019457.

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

Wear M, Nowicki M, Blackburn E, McNae I, Walkinshaw M FEBS Open Bio. 2017; 7(4):533-549.

PMID: 28396838 PMC: 5377415. DOI: 10.1002/2211-5463.12201.

References

Pahl A, Zhang M, Torok K, Kuss H, Friedrich U, Magyar Z . Anti-inflammatory effects of a cyclosporine receptor-binding compound, D-43787. J Pharmacol Exp Ther. 2002; 301(2):738-46. DOI: 10.1124/jpet.301.2.738. View

Cannon M, Papalia G, Navratilova I, Fisher R, Roberts L, Worthy K . Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology. Anal Biochem. 2004; 330(1):98-113. DOI: 10.1016/j.ab.2004.02.027. View

Zhang Y, Erdmann F, Baumgrass R, Schutkowski M, Fischer G . Unexpected side chain effects at residue 8 of cyclosporin a derivatives allow photoswitching of immunosuppression. J Biol Chem. 2004; 280(6):4842-50. DOI: 10.1074/jbc.M409178200. View

Dornan J, Taylor P, Walkinshaw M . Structures of immunophilins and their ligand complexes. Curr Top Med Chem. 2003; 3(12):1392-409. DOI: 10.2174/1568026033451899. View

Rich R, Myszka D . A survey of the year 2002 commercial optical biosensor literature. J Mol Recognit. 2004; 16(6):351-82. DOI: 10.1002/jmr.649. View

Rovira P, Mascarell L, Truffa-Bachi P . The impact of immunosuppressive drugs on the analysis of T cell activation. Curr Med Chem. 2000; 7(7):673-92. DOI: 10.2174/0929867003374778. View

Abdiche Y, Myszka D . Probing the mechanism of drug/lipid membrane interactions using Biacore. Anal Biochem. 2004; 328(2):233-43. DOI: 10.1016/j.ab.2004.01.018. View

Towers G, Hatziioannou T, Cowan S, Goff S, Luban J, Bieniasz P . Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors. Nat Med. 2003; 9(9):1138-43. DOI: 10.1038/nm910. View

Yoo S, Myszka D, Yeh C, McMurray M, Hill C, Sundquist W . Molecular recognition in the HIV-1 capsid/cyclophilin A complex. J Mol Biol. 1997; 269(5):780-95. DOI: 10.1006/jmbi.1997.1051. View

10.

Galat A . Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity--targets--functions. Curr Top Med Chem. 2003; 3(12):1315-47. DOI: 10.2174/1568026033451862. View

11.

Handschumacher R, Harding M, Rice J, Drugge R, Speicher D . Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science. 1984; 226(4674):544-7. DOI: 10.1126/science.6238408. View

12.

Kallen J, Mikol V, Taylor P, Walkinshaw M . X-ray structures and analysis of 11 cyclosporin derivatives complexed with cyclophilin A. J Mol Biol. 1998; 283(2):435-49. DOI: 10.1006/jmbi.1998.2108. View

13.

Bua J, Ruiz A, Potenza M, Fichera L . In vitro anti-parasitic activity of Cyclosporin A analogs on Trypanosoma cruzi. Bioorg Med Chem Lett. 2004; 14(18):4633-7. DOI: 10.1016/j.bmcl.2004.07.003. View

14.

Kofron J, Kuzmic P, Kishore V, Rich D . Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry. 1991; 30(25):6127-34. DOI: 10.1021/bi00239a007. View

15.

Huber W, Perspicace S, Kohler J, Muller F, Schlatter D . SPR-based interaction studies with small molecular weight ligands using hAGT fusion proteins. Anal Biochem. 2004; 333(2):280-8. DOI: 10.1016/j.ab.2004.05.058. View

16.

Altschuh D, Braun W, Kallen J, Mikol V, Spitzfaden C, Thierry J . Conformational polymorphism of cyclosporin A. Structure. 1994; 2(10):963-72. DOI: 10.1016/s0969-2126(94)00098-0. View

17.

Zeder-Lutz G, Van Regenmortel M, Wenger R, Altschuh D . Interaction of cyclosporin A and two cyclosporin analogs with cyclophilin: relationship between structure and binding. J Chromatogr B Biomed Appl. 1994; 662(2):301-6. DOI: 10.1016/0378-4347(94)00211-8. View

18.

Gavigan C, Kiely S, Hirtzlin J, Bell A . Cyclosporin-binding proteins of Plasmodium falciparum. Int J Parasitol. 2003; 33(9):987-96. DOI: 10.1016/s0020-7519(03)00125-5. View

19.

Woodfield K, Price N, Halestrap A . cDNA cloning of rat mitochondrial cyclophilin. Biochim Biophys Acta. 1997; 1351(1-2):27-30. DOI: 10.1016/s0167-4781(97)00017-1. View

20.

Fanghanel J, Fischer G . Thermodynamic characterization of the interaction of human cyclophilin 18 with cyclosporin A. Biophys Chem. 2003; 100(1-3):351-66. DOI: 10.1016/s0301-4622(02)00292-2. View