X-ray Structures and Analysis of 11 Cyclosporin Derivatives ...

436 Cyclosporin Derivatives Complexed with CypA
Table 1. Formulae of CsA analogues and their biological
activities
Figure 1. Residue labelling scheme for CsA-analogues
(see also Table 1). Atom labelling is according to the
IUPAC convention. CN is the methyl carbon atom of
the N-methylated amino acids. MLE for MeLeu; MVA
for MeVal; BMT for MeBmt; ABU for Abu; SAR for Sar;
VAL for Val; ALA for Ala; DAL for D-Ala. All amino
acid residues with the exception of D-Ala8 (and Sar3)
are in the L-con®guration.
activity (Sigal et al., 1991). Even small chemical
changes to residues of the effector loop can destroy
the immunsuppressive effect without reducing the
ability to bind cyclophilin (Papageorgiou et al.,
1994a).
This paper describes the structures of eight
chemically distinct CsA-analogues complexed with
CypA which show modi®cations in both the cyclophilin-binding
residues and the effector loop.
A comparison of each complex with the native
CypA/CsA structure shows differences in conformation,
molecular rigidity and water binding. This
small library of closely related ligand structures
also provides a picture of the frequently unpredictable
effects of small chemical changes on 3D structure
and biological activity.
Results
Side-chains for the amino acids at position 1, 2, 3 and 4 are
shown for the 11 different CsA derivative complexes. The horizontal
line represents the Ca-C b bond for the side-chain. The
CypA value in column 6 gives a measure of the strength of
binding of the derivative relative to CsA value in column 6
gives a measure of the strength of binding of the derivative
relative to CsA as determined using an ELISA assay
(Quesniaux et al., 1988). The measure of the effect of suppressing
the production of interleukin-2 relative to CsA in a whole
cell assay is given in the column labeled IL2 (Fliri et al., 1993;
Bollinger et al., 1990).
CsA ˆ Abu2-CS; 116450 ˆ MeBm 2 t-CS; 33804 ˆ Val2-CS;
27402 ˆ Thr2-CS; 224698 ˆ (5-hydroxy)Nva2-CS; 209313 ˆ
D-MeSer3-CS; 209650 ˆ Val2-D-MeAla3-CS; 209217 ˆ Val2-D-(2-
S-methyl) Sar3-CS; 209825 ˆ (6,7-dihydro)MeBmt-1-Val2-D-(2-
S-methyl)Sar3-CS; 211810 ˆ (4-hydroxy)MeLeu4-CS; 211811 ˆ
MeIle4-CS.
a IC 50 (derivative)/IC 50 CsA.
b 26 times less binding than CsA.
c 4.2 times less immunosuppressive than CsA.
d Three times better binding than CsA.
X-ray results
X-ray structures of a total of 11 cyclosporines cocrystallised
with cyclophilin are presented. The labelling
scheme is shown in Figure 1 and chemical
structures of the cyclosporines are described in
Table 1. For completeness, these include three previously
published structures: native CsA (Mikol
et al., 1993), 116450 ((4-methyl)MeBmt1-CS) (Mikol
et al., 1994) and 224698 ((5-hydroxy)Nva2-CS)
(Mikol et al., 1995). Most of the CypA/CsA-analogue
complexes were grown using a cross-seeding
technique (Mikol & Duc, 1994). These crystals
grew isomorphously with space group P2 1 2 1 2 1
and cell dimensions a ˆ 36.4 AÊ , b ˆ 60.7 AÊ ,
c ˆ 72.2 AÊ . The re®ned cell dimensions for the
different isomorphous complexes containing CsAanalogues
varied by a maximum of 0.6 AÊ in the a-
dimension, 1.6 AÊ in the b-dimension and 1.4 AÊ in
the c-dimension (see Table 2). The unliganded
CypA structure showed a shrinkage of over 2 AÊ
in the b and c-dimensions with a ˆ 36.46 AÊ ,
b ˆ 57.32 AÊ , c ˆ 70.73 AÊ . The complex CypA/
209313 was crystallised in space group P2 1 2 1 2
with unit cell dimensions a ˆ 62.9 AÊ , b ˆ 65.3 AÊ ,
c ˆ 40.8 AÊ .
The 11 structures are re®ned to a resolution of
between 2.2 AÊ and 1.76 AÊ . and give R-factors