HIV-1
Protease and Inhibitor Complex
Kaleb Keyserling '09 and Ken Noguchi '10
Contents:
I. Introduction
The AIDS
epidemic currently affects 40 million
people
worldwide and has caused over 22 million deaths. AIDS is caused by the
retrovirus HIV which attacks the human immune system leaving the victim
susceptible to disease. Due to the severity of the HIV virus, much
research has been devoted to developing antiviral therapy. In recent
years many reverse transcriptase and protease inhibitors have been
synthesized to fight HIV (Brik and Wong, 2002). Although
these inhibitors do not cure the
disease, they are able to improve the quality and prolong the life of
those
infected. Here
is a diagram of the HIV life cycle and targets where inhibition occurs.
(Pomerantz
and Horn, 2003)
This Jmol tutorial focuses on the protease inhibitor.
HIV-1 protease
is
important in HIV replication and infectious capacity because it cleaves
polypeptide chains to
create mature enzymes and structural components of the virus (including
itself). HIV protein
synthesis occurs in the host cell. Once translation of the viral mRNA
occurs, the
components
of the HIV virus assemble in the golgi apparatus and the virion
begins to bud from the host cell. As the budding occurs or shortly
after the virion emerges, it matures and
infects other host cells. It is during this maturation
stage when the HIV-1 protease cleaves gag and pol polyprotein
precursors into
mature
functional proteins. Research
has shown that HIV viruses containing inactive protease cannot
replicate and infect further cells (Wlodawer et al., 1989).
There are
currently 9 FDA approved
protease inhibitors and several more in late stages of clinical
development (Wu et al., 2008). Here
are
the structures of six of the FDA
approved HIV-1 inhibitors. The AIDS virus is constantly evolving to
resist
inhibitors; therefore, there is a constant need for the development of
new inhibitors. Many of the current HIV protease inhibitors have poor
pharmacokinetic properties such as low aqueous solubility, poor
membrane permeability, high protein binding, and insufficient metabolic
stability. The inhibitor/protease complex shown in this tutorial was
crystallized by Wu et al. in 2008. It is a novel inhibitor
because it has a two
carbon elongation with a tertiary-alcohol-containing transition state
mimic. This
inhibitor has been shown to bind HIV-1 protease in vitro, but has yet
to be
tested
in vivo.
II. HIV-1 Protease Structure
HIV-1 protease
is a homodimer containing 99
amino acids
.
It
is an aspartyl protease with one active
site which
is C2-symmetric when ligand free
.
The ligand inhibitor
binds to the active
site preventing polyprotein
cleavage
.
The
protease contains two β-sheet
flaps
which
are connected by glycine
rich loops
.
These
flaps are part of the active site and play an
important part in ligand binding.
More
than 140 structures of
protease (wild type and mutant) have been complexed with various
inhibitors. Here is an example of HIV-1 protease complexed with an
inhibitor lacking the two carbon extension (Duskova et al., 2006)
.
III.
Active Site
HIV-1
protease belongs to the class of aspartic proteases. The
active site of HIV-1
protease is made up of two loops with the sequence
Asp-Thr-Gly, the active site triad, which is conserved among
proteases of this family
.
These
active site loops are held together by
a network of
hydrogen bonds
between
the active site amino acids and the surrounding residues.
The
rigid structure of the active site is due to the
“fireman’s grip” which is formed when
each of the Thr 26
residues accept a
hydrogen bond from the amino group of the Thr 26 in the
opposing chain and donate a hydrogen
bond
to the carbonyl oxygen of the Leu 24 residue located next to each
catalytic
aspartate
.
Each Asp
25
residue is also bound to the backbone NH
group
of the opposing Gly 27.
Researches
have found through mutational analysis that Asp
25
is
important for proteolytic activity
.
When
these aspartic acids
are replaced by other amino acids the protein becomes inactive. All
aspartyl proteases have a similar mechanism for proteolytic cleavage.
One of the aspartic residues, Asp
25,
is always deprotonated in the
physiological pH
range and can act as a hydrogen acceptor. Asp
25
can activate a
nearby water molecule
which attacks a carbonyl carbon on the peptide
chain forming a oxyanion tetrahedral intermediate
.
The
intermediate is then protonated and broken down
into its hydrolysis products. Click here
to see a more detail mechanism of this aspartic protease cleavage.
Here is an example of an HIV-1 protease complexed with its cleaved
protein product (Das et al., 2006)
.
IV.
Inhibitor Structure
A commonality in all HIV-1 protease inhibitors is that they
mimic the
tetrahedral transition state of the proteolytic reaction. A
nonhydrolyzable hydroxyethylene or hydroxyethylamine moiety is the
basic
core of almost all inhibitors as well as other non-cleavable protein
transition states. More than 2000 inhibitors have been synthesized, but
only 9 have been FDA approved. The inhibitor in this complex (Wu et
al., 2008) has the
basic structure of the FDA approved Indinavir inhibitor
(a
hydroxyethylene inhibitor) with two main differences
.
First,
it has a two
carbon
elongation
between the central
alcohol and the hydrazide part of the molecule
.
This
inhibitor also has a tertiary
alcohol in the
transition-state mimicking scaffold in contrast to the secondary
alcohol of other inhibitors
.
The tertiary
alcohol
is shielded by a benzene ring
.
There
is also a
bromobenzene
group in the P'1 substitute
.
This
complex shows one inhibitor of many that
Wu et al. (2008) synthesized. A list of the other inhibitor compounds
along
with their Ki (binding affinity) and EC50(median
effective
concentration) can be found
here. The authors made several changes to the P'1 substitute
(shown as the R group in the figure).
V.
Inhibitor Complex
Here
is the active site of the HIV-1 protease in complex with the
two-carbon-elongated HIV-1 protease inhibitor
.
The
inhibitor interacts
with the active site of the protease by direct
hydrogen bonds
and
indirect
hydrogen bonds
through two water
molecules
.
The
elongated inhibitor only accepts hydrogen bonds from one of the Asp 25 in the active
site
compared
to shorter inhibitors which bind
weakly to both.
The
P'1 bromobenzene extension
of the inhibitor does not make any direct contacts with the active site
of the protease and is extended towards the solvent. However,
the P'1 extension
of many other inhibitors does interact directly with the active site.
Wu
et al. (2008)
crystallized complexes for three
protease inhibitors. Compound 12d is the inhibitor featured
in
this tutorial, compound B lacks the carbon
elongation and binds weakly to both Asp 25, and compound 15 has an
extra
central carbonyl oxygen. All three of these
inhibitors have shielded tertiary alcohols.
The
HIV proteases in the Wu et al. (2008) paper were cloned and inserted
into
E. coli cells where binding affinity was measured. The anti HIV
activity (EC50) was measured in vitro in Mt40
cells. While the in vitro
experiments show promise, the drugs must be tested in vivo before it
can
be considered as a possible drug candidate.
VI.
Mutations
While
there are currently several FDA approved protease
inhibitors,
the HIV-1 virus is able to mutate and develop resistance to these
inhibitors.
1929 isolates from patients have been shown to contain drug induced
mutation of the protease. Point mutations
are often found in the region of the active site (Gly
48-Ile 50)
, which
interacts with
the P'1 group of the inhibitor.
There
are
also 6 common point mutations found in
the HIV-1 protease that are also found in FIV
(Feline immunodeficiency virus protease): K20I,
V32I, I50V, N88D,
L90M,
Q92K
.
These similarities allows for
FIV to be used as a model for HIV-1 resistant proteases (Brik and
Wong, 2002). Due to the frequency of mutations in HIV-1 protease, it is
important to continually develop new inhibitors.
VII.
References
Brik, A., Wong, C.H., 2002. HIV-1 protease:
mechanism and drug discovery. Organic Biomolecular Chemistry
2003.
1:5-14.
Das, A., Prashar, V., Mahale, S., Serre, L., Ferrer, J.-L/, Hosur,
M.V., 2006.
Crystal structure of HIV-1 protease in situ product complex and
observation of a low-barrier hydrogen bond between caralytic
aspartates. Proc Natl.
Acad. Sci. USA 103: 18464-18469
Dun, B.M., Goodenow, M.M., Gustchina A., Wlodawer
A., 2002. Retroviral proteases. Genome Biology 2002
3(4):reviews3006.1–3006.7 253:
1001-1007.
Duskova, J., Dohnalek, J., Skaova, T., Petrokova,
H., Vondrackova, E., Hradilek, M., Konvalinka, J., Soucek, M., Brynda,
J., Faby, M., Sedlacek. J., Hasek J., 2006. On the role of the R
configuration of the reaction-intermediate isoster 1 protease-inhibitor
binding: X-ray structure as 2.0 A resolution. Acta
Crystallogr Sect.
D, 62:489-497.
Pomerantz R.j., Horn, D.L., 2003. Twenty years of
therapy for the HIV-1 infection. Nature Medicine
9(7):867-873.
Wlodawer, A., Miller M., Mariusz, J.,
Sathyanarayana, B.K., Baldwin E., Weber, I.T., Selk, L.M., Clawson, L.,
Shneider, J., Kent S.B.H., 1989. Conserved folding in retroviral
proteases: Crystal Structure of a Synthetic HIV-1 Protease. Science
245(4918):616-621.
Wu, X., Ohrngren, P., Ekegren, J.K., Unge, J.,
Unge, T., Wallberg, H., Samuelsson, B., Hallburg, A., Larhed, M., 2008.
Two-Carbon-Elongated HIV-1 Protease Inhibitors with a
Tertiary-Alcohol-Containing Transition-State Mimic. Journal
of
Medicinal Chemisty 51(4):1053-1057.
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