Metallacarboranes: Boron-Based
Compounds Inhibit Key HIV Enzyme
Parimal M.
Prajapati*, Yatri Shah, D.J. Sen and C.N.
Patel
ABSTRACT:
Human
immunodeficiency virus (HIV) is a lentivirus (a member of the retrovirus
family) that causes acquired immunodeficiency syndrome (AIDS), a condition in humans
in which the immune system begins to fail, leading to life-threatening opportunistic
infections. Infection with HIV occurs by the transfer of blood, semen, vaginal
fluid, pre-ejaculate, or breast milk. Within these bodily fluids, HIV is
present as both free virus particles and virus within infected immune cells.
The four major routes of transmission are unsafe sex, contaminated needles,
breast milk, and transmission from an infected mother to her baby at birth.
Metallacarboranes derived from the transition metals represent a large family
of aromatic borane derivatives which, when equipped with a radiometal, are
potentially useful in radioimaging and radiotherapy of tumors. The
radiometallacarborane may be localized in tumor by a tumor cell-selective
antibody molecule to which it is attached or by other means (biomolecule,
liposome). A particular advantage of radiometallacarboranes in these
applications is their extraordinarily great kinetic stability and invisibility
to enzyme systems which normally degrade organic radiometal carriers (chelates)
with release of the radiometal in an unwanted way.
INTRODUCTION
The genes of most living things, including
humans, are made of DNA. The DNA exists as a sequence of a code that can be read
like a book. In the cell the code is read to make RNA which is then used as the
code for the construction of proteins. In other words, the flow of genetic
information in the cell is usually from DNA to RNA to protein1.
FIGURE 1. HIV VIRUS
The HIV virus, on the other hand, has its
genetic material made from RNA. It has to insert its genetic code into that of
the host cell in order to replicate.
In order to achieve this it must first make a DNA copy
so that it is compatible with the DNA of the host cell. DNA is then made using
the code of the RNA. Since this is the opposite of the usual case the viruses
that do this are called retroviruses2.
What does an HIV virus look
like?
This is an artist's drawing of a single HIV virus. As
you can see, the virus has several layers, including an outer layer, an inner
layer and a core shell. The outer layer is interrupted by glycoproteins (gp)
which play an important role in the lifecycle of the virus, and are targeted by
a new type of drugs called "entry inhibitors". Within the core shell
lies the genetic material of the virus (RNA), as well as some of the enzymes
the virus needs to replicate. This includes Reverse Transcriptase, an enzyme
that catalyzes the production of DNA based on the RNA genetic code. NRTI and
NNRTI drugs inhibit the Reverse Transcriptase enzyme and thus prevent the virus
from replicating3.
FIGURE.2 ANATOMY OF AIDS VIRUS
How does HIV live in humans?
HIV uses human cells, mainly white blood cells, as host
cells to in which to replicate and thrive. The white blood cells that HIV uses
as host cells are called CD4 T-lymphocytes, which are commonly called "CD4
cells" or "T-cells" for short. The numbers of these cells are
what are referred to when someone discusses a "CD4 count," or a
"T-cell count." CD4 cells are used to fight infection and are a key
element of the immune system. In the process of replication, HIV kills CD4
cells. By replicating and killing CD4 cells, HIV degrades the immune system and
renders the patient more4.
FIGURE 3: HIV USES HUMAN CELLS
What is AIDS?
HIV is present as both free virus particles
and virus within infected immune cells. When CD4+ T cell numbers
decline below a critical level of 200 cells per µL, cell-mediated immunity is
lost, and infections with a variety of opportunistic microbes appear. The first
symptoms often include moderate and unexplained weight loss, recurring respiratory
tract infections (such as sinusitis, bronchitis, otitis media, pharyngitis),prostatitis,
skin rashes, and oral ulcerations. Common opportunistic infections and tumors,
most of which are normally controlled by robust CD4+
T cell-mediated immunity then start to affect the patient. Typically,
resistance is lost early on to oral Candida species and to Mycobacterium
tuberculosis, which leads to an increased susceptibility to oral
candidiasis(thrush) and tuberculosis. Later, reactivation of latent herpes
viruses may cause worsening recurrences of herpes simplex eruptions, shingles, Epstein-Barr
virus-induced B-cell lymphomas, or Kaposi's sarcoma. Pneumonia caused by the
fungus Pneumocystis
jirovecii is
common and often fatal. In the final stages of AIDS, infection with cytomegalovirus
(another herpes virus) or Mycobacterium avium complex is more prominent. Not
all patients with AIDS get all these infections or tumors, and there are other
tumors and infections that are less prominent but still significant5.
Life Cycle of AIDS:
The HIV virus’s life cycle begins
as it is born in a host cell. The virus exits the cell through its cell
membrane, and searches for a new T-cell to infect. The virus can identify the
cell it wishes to find, the CD4+ T-cell, because they have CD4 molecules on
their surface. Once the virus reaches a victim, it attaches itself to the CD4
molecules that make up its surface, and the virus’s outer envelope merges with
the cells and becomes one.
FIGURE. 4
LIFE CYCLE OF AIDS
After getting inside the cell, HIV
uses a chemical called reverse transcriptase to convert RNA into DNA. This is
the form of nucleic acid which can be used to produce copies of the virus.
When the DNA enters the cells
nucleus, the next stage of the process is complete. This is done with the help
of a chemical known as integrase. Next, the HIV DNA is turned into RNA that can
use the cells machinery. After this is complete, this “messenger” RNA moves out
of the nucleus and into the cytoplasm of the cell to take control of it. It
creates proteins needed for more viruses using the host cells parts. The last
stage occurs when these proteins join together and create young viruses. These
are not yet ready to infect other cells. A chemical called protease cleans up
the proteins and enzymes in these immature HIV strains, and then they gather up
in the cells membrane and burst out. It is this eruption of new viruses that
destroys the host cell 6-7
What is the treatment for HIV or AIDS?
Every person is different and your doctor
will design a specific medical plan for you. It is essential that you follow
this plan exactly, taking your medications exactly as prescribed. Some of the
medications you will receive are intended to fight opportunistic infections.
Other medications target the HIV virus in order to block replication and
protect the body's immune system. These drugs are called "antiretroviral
drugs." Often several antiretroviral drugs are prescribed for maximum
effect. This is sometimes called "cocktail" therapy or highly active
antiretroviral therapy (HAART). HIV protease (PR)
represents a prime target for rational drug design, and protease inhibitors
(PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide
compounds with limited bioavailability and stability, and their use is
compromised by high costs, side effects, and development of resistant strains.
In our search for novel PI structures, we have identified a group of inorganic
compounds, icosahedral metallacarboranes, as candidates for a novel class of
nonpeptidic PIs. Here, we report the potent, specific, and selective
competitive inhibition of HIV PR by substituted metallacarboranes. The most
active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt
bis(1,2-dicarbollide)]di-ate, exhibited a K i value of 2.2
nM and a submicromolar EC50 in antiviral tests, showed no toxicity
in tissue culture, weakly inhibited human cathepsin D and pepsin, and was
inactive against trypsin, papain, and amylase. The structure of the parent
cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Ĺ
resolution by protein crystallography and represents the first
carborane-protein complex structure determined. It shows the following mode of
PR inhibition: two molecules of the parent compound bind to the hydrophobic
pockets in the flap-proximal region of the S3 and S3′ subsites of PR. We
suggest, therefore, that these compounds block flap closure in addition to
filling the corresponding binding pockets as conventional PIs. This type of
binding and inhibition, chemical and biological stability, low toxicity, and
the possibility to introduce various modifications make boron clusters
attractive pharmacophores for potent and specific enzyme inhibition 8-9.
FIGURE.5 A metallacarborane-based molecular motor devised lab rotates by
charge transfer and is stabilized by a hydrogen bond.
Metallacarboranes and Related Species as Radionuclide Carriers for
Antibody-Mediated Diagnosis and Therapy
Metallacarboranes derived from the
transition metals represent a large family of aromatic borane derivatives
which, when equipped with a radiometal, are potentially useful in radioimaging
and radiotherapy of tumors. The radiometallacarborane may be localized in tumor
by a tumor cell-selective antibody molecule to which it is attached or by other
means (biomolecule, liposome). A particular advantage of radiometallacarboranes
in these applications is their extraordinarily great kinetic stability and
invisibility to enzyme systems which normally degrade organic radiometal
carriers (chelates) with release of the radiometal in an unwanted way. The
space-filling model shown below is a representation of a radiometallacarborane
utilizing a generalized Co3+ nucleus and the Venus Flytrap ligand
system which is, in turn, connected to a tumor-selective antibody. Use of 55Co,
a positron emitter, is under investigation for application as a positron
emission tomography (PET) agent. PET is extraordinarily useful in diagnosis.
Other radioimaging systems based upon the invincibility of aromatic boranes to
enzyme attack involve simple radioiodination of the ubiquitous nido-7,8-C2B9H12-
ion and its derivatives. This is a facile process and the iodine remains fixed
to the boron atom to which it is bound. These and other useful systems are
described in M. Frederick Hawthorne and Andreas Maderna, "Applications of
Radiolabeled Boron Clusters to the Diagnosis and Boron Neutron Capture Therapy
of Cancer," Chem. Rev., 99, 3421 (1999). The use of
radionuclides which emit alpha or beta particles would be useful as therapeutic
agents against cancer and work of this type is also underway. Enzymes are
protein molecules which catalyze important chemical reactions in our body. To
perform their action, enzymes bind to other molecules called ligands or
substrates. Sometimes, enzymes must be blocked to prevent them to cause
diseases, like in the case of HIV-1 protease which is involved in AIDS. A small
ligand (drug) is therefore designed which binds to the enzyme blocking it.10-12.
1) Compounds of the type shown in FIG. 1
were prepared by the reaction of the tetrathallium salt of “Z compound” shown
in FIG. 8C with two equivalents of (Ph 3 P) 2 HCl 2 (where
M=Pt, Pd, Co, Ni) in dry THF at room temperature under dry nitrogen conditions
for 1 hour (reaction appeared to be complete after about 10 minutes). The TlCl
precipitated, leaving the desired product in solution. This air-stable product
was filtered and pumped to dryness in vacuo giving yields of 40-75%. Compounds
were characterised by IR, NMR, elemental analysis, mass spectrometry and
magnetic measurements. By way of example, Tl 4 [1,4-(1-C 6 H
5 —C 2 B 9 H 9 ) 2 C 6
H 4 ] (328 mg, 0.25 mmol) was suspended in stirred thf (40 ml)
under ambient conditions, and brown (PPh 3 ) 2 Col 2
(420 mg, 0.50 mmol) was added. This immediately caused the formation of
an off-white precipitate (Thallium (I) iodide), which turned grey on standing
in air. After 30 minutes stirring, the bright green solution was isolated by
filtration and the thf removed in vacuo, yielding Bis-Co (PPh 3 ) 2
-1,4-(1-C 5 H 5 —C 2 B 9 H 9
) 2 C 6 . Appearance: Bright green powder Yield:
311 mg, 75% boron-containing compound can inhibit HIV protease, a key
enzyme involved in replicating the virus that causes Aids. The finding is
potentially signficant because the compounds - metallacarboranes - attack the
enzyme in a different way to most existing drugs and could help overcome
problems of resistance. Carboranes are polyhedral cages composed of carbon and
boron atoms. The cages can be paired through a metal ion - in this case cobalt
- to form metallacarboranes. linked two of these pairs together using a short
chain with a quaternary amine in the centre. A range of these compounds with
different substituents attached to the quaternary amine, and tested their
effect on HIV protease in the test tube 13-15.
The team found that
significant inhibition of the enzyme could be achieved - with varying degrees
of potency depending on the nature of the substituent groups on the amine. Importantly
the compounds also inhibited variants of the enzyme that had developed
resistance to existing treatments16.
FIGURE.6 TWO METABLLOBORANES ARE JOINED BY A LINKER CHAIN WITH A CENTRAL
QUATERNARY AMINE
X-ray structure of
how the compounds bind to HIV protease
X-ray
crystallography of the inhibitor binding to the enzyme showed that the while
the molecules were occupying the enzyme's active site, the same as existing
drugs and the natural peptide, the metallacarborane cages additionally interfered
with other key parts of the enzyme's structure. Based on these findings
that metallacarboranes are worth pursuing as HIV protease inhibitors. 'Their
chemical and biological stability, low toxicity, and the possibility to
introduce heteroatoms into the cage or polar group modifications to the side
chains make boron clusters very attractive pharmacophores for development of
potent HIV protease inhibitors 17-19.
FIGURE.7 X-RAY
STRACTURE ANALYSIS OF THE BINDING OF COMPOUND TO HIV-PR.
(A) Overall structure of
the HIV PR-compound complex. The PR dimer is in ribbon representation with the
two catalytic aspartates shown in sticks. Two compound molecules are
represented by their van der Waals surfaces and gray stick model, with cobalt
ions shown as magenta spheres. Autoproteolytic peptide product is represented
as stick model. (B) Superposition of PR-compound complex with
PR-lopinavir complex and with the free PR structure. Protease complex with
lopinavir is represented in yellow
ribbons, lopinavir is shown as a stick model, free PR structure (PDB ID code
1HHP) is shown in green ribbons, and
color coding for PR-compound complex is
the same in A 20-21 .
CONCLUSION:
Metallacarboranes are worth pursuing as
HIV protease inhibitors. 'Their chemical and biological stability, low
toxicity, and the possibility to introduce heteroatoms into the cage or polar
group modifications to the side chains make boron clusters very attractive
pharmacophores for development of potent HIV protease inhibitors. A particular
advantage of radiometallacarboranes in these applications is their
extraordinarily great kinetic stability and invisibility to enzyme systems
which normally degrade organic radiometal carriers (chelates) with release of
the radiometal in an unwanted way.
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Received on 27.01.2010
Accepted on 20.03.2010
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Research J. Pharmacology and
Pharmacodynamics. 2(3): May-June 2010, 205-210