title: Substrate-based design of reversible Pin1 inhibitors.
abstract: Human Pin1, a peptidyl-prolyl cis/trans isomerase with high specificity to -Ser/Thr(PO(3)H(2))-Pro- motifs, is required for cell cycle progression. In an effort to design reversible Pin1 inhibitors by using a substrate structure based approach, a panel of peptides were applied to systematically analyze the minimal structural requirements for Pin1 substrate recognition. Pin1 catalysis (k(cat)/K(m) < 5 mM(-1) s(-1)) for Ala-Pro, Ser-Pro, and Ser(PO(3)H(2))-Pro was detected using direct UV-visible spectrophotometric detection of prolyl isomerization, while weak competitive inhibition of Pin1 by these dipeptides was observed (K(i) > 1 mM). Substrates with chain lengths extending from either the P2 to P1' or the P1 to P2' subsite gave k(cat)/K(m) values of 100 mM(-1) s(-1) for Ala-Ser(PO(3)H(2))-Pro and 38 mM(-1) s(-1) for Ser(PO(3)H(2))-Pro-Arg. For both Pin1 and its yeast homologue Ess1, the optimal subsite recognition elements comprise five amino acid residues with the essential Ser(PO(3)H(2)) in the middle position. The resulting substrate Ac-Ala-Ala-Ser(PO(3)H(2))-Pro-Arg-NH-4-nitroanilide possesses a very low cis/trans interconversion barrier in the presence of either Pin1 or Ess1, with k(cat)/K(m) = 9300 mM(-1) s(-1) and 12000 mM(-1) s(-1), respectively. The D-Ser(PO(3)H(2)) residue preceding proline could serve as a substrate-deactivating determinant without compromising ground state affinity. Similarly, substitution of the amide bond preceding proline with a thioxo amide bond produces a potent inhibitor. Pin1 is reversibly inhibited by such substrate analogue inhibitors with IC(50) values in the low micromolar range. The D-amino acid containing inhibitor also exhibits remarkable stability against phosphatase activity in cell lysate.
authors.affiliation: Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle/Saale, Germany.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Zhang
authors.forename: Susanne
authors.initials: S
authors.lastname: Füssel
authors.forename: Ulf
authors.initials: U
authors.lastname: Reimer
authors.forename: Mike
authors.initials: M
authors.lastname: Schutkowski
authors.forename: Gunter
authors.initials: G
authors.lastname: Fischer
mesh_terms: D001667:Binding, Competitive; D004151:Dipeptides / Q000138:chemical synthesis / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D018716:Molecular Mimicry; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology; D019696:Peptidylprolyl Isomerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000502:physiology; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity / Q000187:drug effects; D013816:Thermodynamics
chemical_list: D004151:Dipeptides; D004791:Enzyme Inhibitors; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D009842:Oligopeptides; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase