Fw. Okumu et al., EFFECT OF RESTRICTED CONFORMATIONAL FLEXIBILITY ON THE PERMEATION OF MODEL HEXAPEPTIDES ACROSS CACO-2 CELL MONOLAYERS, Pharmaceutical research, 14(2), 1997, pp. 169-175
Purpose, To determine how restricted conformational flexibility of hex
apeptides influences their cellular permeation characteristics. Method
s, Linear (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2; X = Asp, Asn, Lys) and cyclic
(cyclo[Trp-Ala-Gly-Gly-X Ala]; X = Asp, Asn, Lys) hexapeptides were s
ynthesized, and their transport characteristics were assessed using th
e Caco-2 cell culture model. The lipophilicities of the hexapeptides w
ere determined using an immobilized artificial membrane. Diffusion coe
fficients used to calculate molecular radii were determined by NMR. Tw
o-dimensional NMR spectroscopy, circular dichroism, and molecular dyna
mic simulations were used to elucidate the most favorable solution str
ucture of the cyclic Asp-containing peptide. Results, The cyclic hexap
eptides used in this study were 2-3 times more able to permeate (e.g.,
P-app = 9.3 +/- 0.3 x 10(-8) cm/sec, X = Asp) the Caco-2 cell monolay
er than were their linear analogs (e.g., P-app = 3.2 + 0.3 X 10(-s) cm
/sec, X = Asp). In contrast to the linear hexapeptides, the flux of th
e cyclic hexapeptides was independent of charge. The cyclic hexapeptid
es were shown to be more lipophilic than the linear hexapeptides as de
termined by their retention times on an immobilized phospholipid colum
n. Determination of molecular radii by two different techniques sugges
ts little or no difference in size between the linear and cyclic hexap
eptides. Spectroscopic data indicate that the Asp-containing linear he
xapeptide exists in a dynamic equilibrium between random coil and beta
-turn structures while the cyclic Asp-containing hexapeptide exists in
a well-defined compact amphophilic structure containing two beta-turn
s. Conclusions. Cyclization of the linear hexapeptides increased their
lipophiticities. The increased permeation characteristics of the cycl
ic hexapeptides as compared to their linear analogs appears to be due
to an increase in their flux via the transcellular route because of th
ese increased lipophilicities. Structural analyses of the cyclic Asp-c
ontaining hexapeptide suggest that its well-defined solution structure
and, specifically the existence of two beta-turns, explain its greate
r lipophilicity.