Cyclohexyl Ketone Inhibitors of Pin1 Dock in a Trans-Diaxial Cyclohexane Conformation

Abstract

Cyclohexyl ketone substrate analogue inhibitors (Ac–pSer-Ψ[C = OCH]-Pip–tryptamine) of Pin1, the cell cycle regulatory peptidyl-prolyl isomerase (PPIase), were designed and synthesized as potential electrophilic acceptors for the Pin1 active site Cys113 nucleophile to test a proposed nucleophilic addition-isomerization mechanism. Because they were weak inhibitors, models of all three stereoisomers were docked into the active site of Pin1. Each isomer consistently minimized to a trans-diaxial cyclohexane conformation. From this, we hypothesize that Pin1 stretches substrates into a trans-pyrrolidine conformation to lower the barrier to isomerization. Our reduced amide inhibitor of Pin1 adopted a similar trans-pyrrolidine conformation in the crystal structure. The molecular model of 1, which mimics the l-Ser-l-Pro stereochemistry, in the Pin1 active site showed a distance of 4.4 Å, and an angle of 31° between Cys113-S and the ketone carbon. The computational models suggest that the mechanism of Pin1 PPIase is not likely to proceed through nucleophilic addition.

Kinetic Isotope Effects Support the Twisted Amide Mechanism of Pin1 Peptidyl-Prolyl Isomerase

“…(22) Similarly, poor inhibition of Pin1 by cyclohexyl ketone inhibitors suggested that the nucleophilic addition mechanism with the Pin1 Cys113 thiol is unlikely. (23)…”

Structure and function of the human parvulins Pin1 and Par14/17

“…By variation of these ground state analogs, the same lab developed phosphorylated transition state inhibitors for Pin1 . Moreover, the Etzkorn group described cyclohexyl ketone Inhibitors of Pin1 that dock in a trans-diaxial conformation to the Pin1 active site in crystal structures (Xu et al, 2012). The information obtained from these inhibitors verified the twisted-amide mechanism for Pin1 catalysis.…”

“…In a series of publications, Felicia Etzkorn and her coworkers finally verified the non-covalent mechanism. They used transition state inhibitors (Xu et al, , 2012 of Pin1 to confirm a twisted amide ω-90°-syn-exo transition state conformation (Mercedes-Camacho et al, 2013). Based on these measurements a transition state was proposed that is stabilized by an intramolecular hydrogen bond from the amide Pro +1 to the prolyl nitrogen ( Figure 6).…”

“…In cells In animal model -Is able to selectively inhibit Pin1 and targets it for degradation -It binds covalently to the catalytic site of Pin1 Small molecule with unknown mechanism Cyclohexyl ketone Inhibitors -- (Xu et al, 2012) Disulfide-cyclized nona-peptide (CRYPEVEIC)…”

Polyubiquitination inhibition of estrogen receptor alpha and its implications in breast cancer

“…PIN1 is overexpressed in breast cancer and is related to mammary tumor growth, and epithelial-mesenchymal transition, and natural and synthetic inhibitors are being probed to control its activity[ 55 , 57 , 80 - 87 ]. Similarly, LMTK3 overexpression stimulates cellular proliferation and tumor formation, and correlates with shorter survival times in ERα+ breast cancer, and resistance to Tam treatment, but these events are reduced when LMTK3 expression is decreased[ 58 , 88 - 90 ].…”