What are PPIases?
The principal function of peptidyl-prolyl cis-trans isomerases is to participate in protein folding by catalysing the cis-trans isomerisation of the X-Pro peptide bond in polypeptide chains (where X is any amino acid). PPIases comprise the subfamilies of cyclophilins, FK506-binding proteins (FKBP) and parvulins (including Pin1). They are abundant and highly conserved across species.
Inhibitors of PPIases
Some PPIases bind known immunosuppressive drugs: Cyclophilins bind cyclosporin A (CsA) and FKBPs bind FK506 and rapamycin. PPIases are important regulators of the activity of their client proteins. Inhibitors of these enzymes have important therapeutic potential in the area of viral infection, inflammation, cancer and neuroprotection.
Functional PPIase Assays: Selcia has established a panel of PPIase assays that can be used to profile customer compounds and determine enzyme specificity. We have developed functional assays for a number of PPIases (see table below). The functional assays, which measure the enzyme catalysed cis-trans isomerisation rate of a peptide substrate, are technically challenging due to the background isomerisation rate of the substrate in the absence of the enzyme.
TR-FRET: 384 well, competitive ligand displacement assays using either fluorescently labelled cyclosporin A or FK506 have been developed for cyclophlins A, B, C and D and for FKBP12, 51 and 52, respectively. These assays are high throughput and can be used to confirm that inhibitors are competitive with the known ligands. Small molecules, peptides and/or complex natural products can be screened.
Drug Screening for Novel Inhibitors of PPIases:
Natural Product Screening: The majority of pharmacologically successful PPIase inhibitors are natural product derived inhibitors eg cyclosporins and sanglifehrin, inhibitors of cyclophilins; FK506 and rapamycin, inhibitors of FKBPs. It is likely that there are other, as yet undiscovered, potent natural product inhibitors of cyclophilins and other PPIases. To identify these, Selcia is able to offer screening of microbial extracts using the TR-FRET assays. Selcia has access to libraries of pure natural products as well as extracts.
Table: Selcia’s PPIase Profiling Portfolio
|Functional PPIase Assay1||Competitive TR-FRET Assay2|
|Cyclophilin A||Cyclophilin A|
|Cyclophilin B||Cyclophilin B|
|Cyclophilin C||Cyclophilin C|
|Cyclophilin D*||Cyclophilin D*|
|Pin 1||In progress|
- Jankowski B. et. el., Anal. Biochem., 1997, 252, 299 – 307.
- 384 well competitive binding time-resolved fluorescence resonance energy transfer assay.
To see how the two assays differ
If your enzyme of interest is not in this list, please contact us to discuss further.