Biophysical studies of membrane proteins/peptides. Interaction with ...

Chapter III ‐ Binding of Inhibitors to a Putative Binding
Domain of V‐ATPase.
The studies conducted allowed us to know in detail the properties of the indole type
of inhibitors in lipid membranes. The molecules studied, which correspond to the initial
(low efficiency) and final stage (high efficiency) in the development of these inhibitors
as potential drugs in osteoporosis treatment, present almost identical properties in the
liposomes environment. The preference of the lipid phase over the aqueous phase, the
position in the bilayer and the orientation of the molecules were all almost identical for
both molecules studied.
According to the results of Gagliardi et al. (1998a), the change in inhibition
efficiency of the V-ATPase inhibitors within the indole class presented some extent of
correlation to the hydrophobicity of the molecules, i.e. the more hydrophobic indole
inhibitor molecules had also a tendency to be more efficient inhibitors of V-ATPase. It
was possible that the interaction of the molecules with the lipid milieu was significantly
different within this class of inhibitors, and this could correspond to dramatically
different efficiencies of inhibition, explaining the 10 3 difference in IC 50 values of INH-1
and SB242784. In this way, the process of inhibitor binding to the lipid bilayer would
be crucial to the efficiency of the inhibition event. However, the results obtained point
to a non-determinant role of inhibitor interaction with the lipid bilayer, and the specific
molecular recognition processes with the enzyme’s inhibition site are very likely to be
responsible for dictating the large differences of inhibition efficiencies within the V-
ATPase indole inhibitors class.
The binding studies of bafilomycin and SB242784 to the peptide corresponding to
the 4 th TM helix of subunit c of V-ATPase revealed no affinity at all for SB242784 and
very low affinities for bafilomycin. These results can be seen as an indicator that the
binding pocket for the inhibitors is not solely composed of the lipid exposed face of the
4 th TM helix of subunit c, but also involves contributions of amino acids from other
domains of the protein. This proposal is in agreement with the hypothesis of the
inhibitors operating as a stone in a gear, blocking movements of the TM domains of
subunit c and interrupting proton transport.
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