Stereoisomeric cis and trans substrate analogues for Pint were designed and synthesized. The central phosphoSer-Pro core of the Pin1 substrate was replaced by cis and trans amide isosteres in Ac-Phe-Phe-pser-Psi[(Z and E)CH=C]-Pro-Arg-NH2, 1 and 2, peptidomimetics. They were synthesized on solid phase in 17% yield for the cis analogue 1, and 16% yield for the trans analogue 2. A second trans amide isostere with a C-terminal N-methylamide 3 was synthesized in 7% yield. The protease-coupled Pint assay showed that all three compounds inhibited the Pint peptidyl-prolyl isomerase (PPlase) enzymatic activity. The cis isostere 1 was 23 times more potent (K-i = 1.74 +/- 0.08 muM) than its trans counterpart 2 (K-i = 40 +/- 2 muM) in competitive inhibition of Pint. These results suggest that the catalytic site of Pin1 binds cis substrates more tightly in aqueous solution. Antiproliferative activity toward the A2780 human ovarian cancer cell line by the cis and trans analogues correlates with Pint inhibition results.