More on LTR Inhibitors: No Drug Resistance to a Tat Inhibitor

The rapid development of drug resistance by HIV continues to be a
major obstacle in finding better treatments. Resistance to AZT and
similar drugs is well known; resistance to protease inhibitors, a new
class of experimental drugs, is now also being found. A number of
different strategies may help to deal with the problem, however.

This article looks at what can be learned from one drug (Ro 24-7429,
the Hoffmann-La Roche Tat inhibitor) to which HIV developed no
resistance at all, in two years of laboratory studies which attempted
to create such resistance.(1) We will also examine the class of drugs
to which Ro 24-7429 belongs -- the LTR inhibitors. A look at how
LTR inhibitors work suggests that they will need to be used in well-
designed combinations in order to work most effectively.

[Editor's note: Ro 24-7429 was tested alone, and rejected by
Hoffmann-La Roche for further development, after it failed to reduce
the level of p24 antigen in people in a small clinical trial; this data
was presented at the Ninth International Conference on AIDS, in
Berlin, in June 1993. Since that time, laboratory tests have shown
that the drug works many times better when combined with
pentoxifylline, a readily available prescription drug. Unfortunately
the company had been trying to kill the project for several years, for
business reasons, and had only kept it alive because of pressure from
scientists, who understood the importance of this unique drug; AIDS
TREATMENT NEWS has covered these events over the years. The
negative result presented in Berlin provided the necessary excuse to
stop development; and as a result of this drug's political history,
there is virtually no chance that the company will run combination
tests, which it could have done long ago. Ro 24-7429 remains
important, not only in itself, but also for the doors it opens for
developing better treatments. These possibilities urgently need more
attention. JSJ]

Note: This article is the second in a series, which began with "LTR
Inhibitors, a New Kind of Potential AIDS Treatment: Interview with
Arthur B. Pardee, Ph.D.," published in AIDS TREATMENT NEWS #192,
February 4, 1994. Parts of this article are more technical than what
usually appears in AIDS TREATMENT NEWS; for those who do not
have a background in this area, we suggest reading the previous
article first. If you do not already have a copy, you can obtain one by
sending a self-addressed stamped envelope to: AIDS TREATMENT
NEWS, P.O. Box 411256, San Francisco, CA 94141; be sure to mention
"LTR" (or "issue #192") so that we will know which issue to send.

HIV and Drug Resistance

Nucleoside analog drugs (such as AZT or ddI) and protease inhibitors
invite drug resistance because they are directed against viral
proteins, which are highly prone to mutations (genetic changes)
when HIV reproduces. Frequent mutations occur because reverse
transcriptase (RT), the viral enzyme that produces copies of viral
genes, is a thousand times more error-prone than the human enzyme
that cells use when copying their own genes. Altered genes result in
altered proteins. Therefore, while most essential human proteins are
fairly accurately and consistently produced by human cells, viral
proteins mutate at a rate hundreds of times faster. When mutations
accumulate, the altered viral proteins are less able to bind to the
drugs, so the drugs become less effective.

Background: The HIV Life Cycle

HIV is a retrovirus; and in retroviruses, the genetic information is in
the form of RNA, not DNA as with most living things. After HIV
infects a cell, the information in RNA is copied to DNA by reverse
transcriptase (which is the enzyme inhibited by AZT). This DNA
becomes incorporated into the DNA of the infected cell, whereupon it
may either lie dormant or become active. If this "integrated" DNA is
activated, more viral RNA is produced; some of it is "translated" into
viral proteins, and some is encapsulated directly into new viral
particles.

While some viral proteins are used in constructing the viral coat,
others regulate the production and processing of viral RNA from its
(integrated) DNA "template". The most important of these regulatory
proteins is Tat. Tat activates high levels of viral RNA production --
the first step in viral replication in an HIV-infected cell.

About five percent of the viral DNA does not code for proteins at all,
but instead acts as a binding site for a class of proteins known as
activators. This portion of viral DNA is called the Long Terminal
Repeat (or LTR); it functions as the "on" switch for the production of
viral RNA. A number of proteins bind onto the LTR to activate it; the
two most important are Tat (produced by HIV) and NF-kB (NF-kappa
B, which is a normal and necessary human protein, but one which
often becomes overactive in HIV disease and certain other chronic
illnesses). Drugs which reduce activation of the virus by the LTR are
called LTR inhibitors. Three potential treatments (topotecan, beta
lapachone, and curcumin) have been found through a test to screen
for LTR inhibitors (see AIDS TREATMENT NEWS issue #174, May 7,
1993).

Antioxidants -- such as vitamins C and E, or curcumin (from the spice
turmeric), or the amino acid N-acetyl cysteine.(2,3) (NAC), or the
drug pentoxifylline -- are examples of one type of LTR inhibitor.
These compounds work by preventing the binding of NF-kB to its
DNA binding site, and those which have been tested have been
observed in the laboratory to partially inhibit the production of HIV.
Normal immune responses, inflammation, opportunistic infections
and co-infection with other viruses all increase NF-kB binding and
subsequent production of viral RNA. Individuals with HIV have
impaired antioxidant defenses -- for example, they have abnormally
low levels of cysteine. Conversely, a high cysteine level inside the
cells (which provides for greater DNA-protective, antioxidant
activity) is required for the DNA synthesis preceding T-cell
division.(4) Therefore, not only does "oxidative stress" activate the
LTR, it also inhibits the functioning of T-cells.(2)

No Resistance with Tat Inhibitor Ro 24-7429

Unlike other activators, Tat binds onto recently formed RNA instead
of onto DNA. Its binding site is a small region of the LTR known as
TAR. There are only about 15 proteins coded for by the virus;
therefore, HIV uses certain cellular (i.e., human) proteins to assist in
its own replication. The Tat inhibitor, Ro 24-7429, however, prevents
LTR activation by binding onto a cellular protein instead of onto Tat
itself. Because this target is a human protein, not a viral one,
resistance is unlikely to occur. Furthermore, when researchers
flooded cells with the Tat protein in a 25-fold excess over that
required for maximal activation, there was no decrease in the
effectiveness of Ro 24-7429. This suggests that it does not bind to
Tat, since if it did, the large excess of Tat would have overwhelmed
the drug.(1)

Recent work by Alan Kingsman and colleagues at Oxford has now
formally proven that Ro 24-7429 does not bind onto Tat at all, but
rather to a cellular "loop binding protein." This protein, in turn, binds
onto a small loop adjacent to the Tat binding site. Kingsman and
colleagues suggest that the loop binding protein cooperates with Tat,
and that Ro 24-7429 inhibits this interaction.(5) The hope from this
research is that once the loop binding protein is physically isolated,
its 3-dimensional structure will be determined. Knowing the
structure could lead to the development of second-generation drugs
with greater binding activity, smaller effective doses, and fewer side
effects.

In recently infected cell cultures exposed to very low doses of Ro
24-7429 alone, viral production is suppressed for a while, then it
returns to higher levels. The lower the dose, the less the inhibition
and the shorter the interval before viral production rebounds to
higher levels. Low-dose monotherapy may result in only a partial
suppression. This effect is probably not due to HIV resistance, but to
increasing numbers of infected cells and accumulating amounts of
viral proteins. Infected cells respond to these proteins by secreting
inflammatory substances that increase NF-kB activity, thereby
driving viral production higher. This result also suggests the
importance of combination therapy, to prevent viral activation by
Tat, while also preventing excessive activation by NF-kB.

Synergy in LTR Activation and Inhibition

The interrelationship of NF-kB and Tat in LTR activation (and hence
viral production) is far more than additive. A modified LTR, in which
the Tat activation pathway has been disabled, is activated by NF-kB
alone to 15-fold above unstimulated (or basal) levels. An LTR
without NF-kB (but with an intact Tat activation pathway) can be
activated by Tat alone to 80-fold above basal levels. But HIV LTR
with both NF-kB and Tat pathways intact can be activated 388- to
760-fold above basal levels by the combination of Tat and NF-kB
(depending on the amount of Tat added).(6) And when both
pathways are disabled, the virus cannot replicate at all. Such
experiments indicate that NF-kB and Tat are required for replication
and that they function synergistically. [Definition: "synergism" is
defined by Steadman's Medical Dictionary as "coordinated or
correlated action of two or more structures, agents, or physiologic
processes so that the combined action is greater than that of each
acting separately."]

It is exactly this type of synergism that Debajit K. Biswas and
colleagues (at Harvard University's Dana-Farber Cancer Institute)
built upon when they analyzed the effects of an NF-kB inhibitor
(pentoxifylline) combined with a Tat-inhibitor (Ro 24-7429). They
showed that when pentoxifylline and Ro 24-7429 were used
together, instead of separately, only one-tenth as much of each
compound was needed to produce the same LTR inhibition.(7)

These results are important for two reasons. First, the synergism of
Ro 24-7429 with other LTR inhibitors would allow the use of lower
doses, and might therefore eliminate the side effects of the drugs.
Second, lower doses of pentoxifylline would lead to less marked
inhibition of NF-kB and leave unhindered certain important immune
functions still dependent on NF-kB.

More laboratory work is needed to determine the most effective and
least toxic combination of LTR inhibitors. These would
simultaneously reduce viral production and oxidative stress. Ro 24-
7429 should certainly be tested as a candidate for such a
combination, as should the other recently developed Tat inhibitor
from Allelix, Alx40-4C, which is presently in early clinical trials. LTR
inhibitors with good toxicity profiles might sometimes show low
efficacy as a monotherapy, but combinations would be expected to
work much better.

LTR combination therapy is definitely a here-and-now possibility
that should go into clinical trials immediately. This new approach to
treatment might become a valuable therapy in itself. And also, by
reducing viral replication, it might help to delay the development of
resistance to other classes of drugs, such as nucleoside analogs or
protease inhibitors.

References

1. Hsu MC, Dhingra U, Earley JV, and others. Inhibition of type 1
human immunodeficiency virus replication by a Tat antagonist to
which the virus remains sensitive after prolonged exposure in vitro.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA. July
1993; volume 90, pages 6395-6399.

2. Roederer M, Staal FJT, Ela SW, Herzenberg LA, and Herzenberg LA.
N-Acetylcysteine: Potential for AIDS Therapy. PHARMACOLOGY.
1993; volume 46, pages 121-129.

3. Mihm S, Ennen J, Pessara U, Kurth R, and Droge W. Inhibition of
HIV-1 replication and NF-kB activity by cysteine and cysteine
derivatives. AIDS. 1991; volume 5, number 5, pages 497-503.

4. Gmnder H, Roth S, Eck H-P, Gallas H, Mihm S, Droge W.
Interleukin-2 mRNA Expression, Lymphokine Production and DNA
Synthesis in Glutathione-Depleted T Cells. CELLULAR IMMUNOLOGY.
1990; volume 24, pages 520-528.

5. Braddock M, Cannon P, Muckenthaler M, Kingsman AJ, and
Kingsman SM. Inhibition of human immunodeficiency virus type 1
Tat-dependent activation of translation of Xenopus oocytes by the
benzodiazepine Ro 24-7429 requires trans-activation response
element loop sequences. JOURNAL OF VIROLOGY. January 1994; pages
25-33.

6. Liu J, Perkins ND, Schmid RM, and Nabel GJ. Specific NF-kB
subunits act in concert with Tat to stimulate human
immunodeficiency virus type 1 transcription. JOURNAL OF VIROLOGY.
June 1992; volume 66, number 6, pages 3883-3887.

7. Biswas DK, Ahlers CM, Dezube BJ, and Pardee AB. Cooperative
inhibition of NF-kB and Tat-induced superactivation of human
immunodeficiency virus type 1 long terminal repeat. PROCEEDINGS
OF THE NATIONAL ACADEMY OF SCIENCES, USA. December 1993;
volume 90, pages 11044-11048.