HIV protease inhibitors
Molecular dynamics simulations of the HIV protease complexed with
fullerene-based inhibitors
Ever since the complementary spatial relationship between fullerene
C60 and the cavity of the HIV protease (HIVP) was first reported,
there has been a growing interest in the possibility of designing
HIVP inhibitors based on C60 derivatives.
Using the PINYMD package developed in my group in collaboration
with Prof. Glenn J. Martyna at Indiana University, we have carried out
classical molecular dynamics (MD) simulations to investigate the
ability of such inhibitors to localize in the active site region of the
HIV protease. These simulations utilized multiple time scale
integration methodology (r-RESPA) and the CHARMM22 force field.
Using a hierarchy of intramolecular, short range and long range
force separations, the reversible multiple time scale methodology
allowed all simulations to be carried out with a time step
of 8 fs. Simulations were carried out in the gas phase, using the
recently introduced cluster Ewald method,
to reflect the type
of calculation often performed in conjunction with drug design.
Simulations including explicit solvent are currently under way.
The following depicts the structure of the HIV protease (coordinates obtained
from the Brookhaven protein data bank).
It is a dimer
composed of two noncovalently bonded 99-residue strands. The cavity
region is characterized by two flexible flaps at the top and the
active site region at the bottom.
Simulations of the HIV protease complexed with C60 alone,
two C60-based inhibitors, and a commercially available
inhibitor, Saquinavir, were carried out, each of 2 ns in length at 300 K
using canonical MD methods.
When complexed with C60 alone, a flap-opening event
was observed to occur after about 1 ns. The figure below shows the
progression of the flap distance (defined as the distance between
alpha carbons of two isoleucine residues in the flap region.
and the snapshot shows the C60 moving toward the open flaps,
away from the active site.
This suggests that the average flap distance could be an important
geometrical parameter for characterizing the efficacy of a given
inhibitor.
When HIVP is complexed with the two fullerene-based inhibitors, a correlation
is found between the average flap separation, the average distance
between the catalytic aspartic acid residues (Rasp-asp),
and the activity of the inhibitor. In particular, the
Rasp-asp values for HIVP complexed with C60
alone, with the less active and with the more active fullerene-based
inhibitors are, 9.03, 7.26 and 7.19 angstroms, respectively, while
the flap separations are 9.95, 8.95 and 5.80 angstroms, respectively.
Thus, we see that both of these parameters decrease with increasing
activity. For comparison, the values of the flap separation and
Rasp-asp for HIVP complexed with Saquinavir are
6.2 and 5.9 angstroms, respectively. Thus, the more active fullerene-based
inhibitor leads to average cavity structural features that are comparable
with an active inhibitor, Saquinavir. The figures below shows snapshots of HIVP
complexed with the more active fullerene-based inhibitor and HIVP complexed
with Saquinavir, respectively:
It should be noted that both Saquinavir and the more active fullerene-based
inhibitor share an important design feature in common, which is
an OH group that is capable of hydrogen bonding to oxygen atoms in the
catalytic aspartic acid residues.
Recently, we have performed multiple time scale molecular dynamics
and free energy calculations of the flap-opening event
in solution of the HIV protease complexed with the aforementioned
fullerene-based inhibitor. In solution, it is not known what the
protonation state of the active site residues is. In order to gain
some insight into this question and to investigate the binding
mechanism, the flap-opening free energy profile as a function of
the Asp-Asp distance defined above was computed and the water
content of the active site determined. The free energy profiles,
computed using both the bluemoon ensemble approach and the
recently introduced adiabatic free energy dynamics method, are
shown below for different protonation states of the HIV protease
alone and the HIV protease complexed with the fullerene-based
inhibitor in solution:
The figure shows a flat free energy profile for the protease
alone in solution. However, in complexes with the fullerene-based
inhibitor, a significant barrier to flap-opening develops
which increases as the protonation state of the active
site increases. Below, we show typical snapshots from the MD
simulation of water content of the active site in the absence
and presence of the inhibitor:
These show that water content is significantly reduced when
the protease is complexed with the inhibitor. What is observed
is that the cavity changes its shape to fit the inhibitor,
thereby ``squeezing out'' the water and creating a strong
hydrophobic interaction between the cavity and the
C60 moiety. The following tables
give average cavity structure and
the computed water density in the cavity region
with and without the inhibitor in different protonation states:
Note that the effective volume occupied by the drug is excluded
(Vexcl)
These water densities are to be compared with the value
0.033 for bulk water. These show a significant reduction
water density in the cavity region upon complexation
with the inhibitor and help explain the flap-opening
free energy profiles above. The results suggest that binding
is most favorable in a diprotonated state, in which an oxygen
on each Asp residue is protonated.
References
Molecular dynamics study of the connection between flap closing
and binding of fullerene based inhibitors of the HIV-1 protease.
Z. Zhu, D. I. Schuster and M.E. Tuckerman,
Biochem. (in press).
A molecular dynamics study of HIV-1 protease complexes with
C60 and fullerene-based
anti-viral agents
.
H. Mi, M.E. Tuckerman, D.I. Schuster and S.R. Wilson,
Proc. Electrochem. Soc. (1999).