AIDS Treatment News Issue #192, February 4, 1994
Charles Davidson and John S. James

To help the AIDS community understand LTR inhibitors as an approach to drug development, AIDS TREATMENT NEWS interviewed Arthur B. Pardee, Ph.D., who is head of the group which initiated the research cited above. Dr. Pardee is Professor of Biological Chemistry and Molecular Pharmacology at Dana-Farber Cancer Institute and Harvard Medical School, and Chief of the Division of Cell Growth and Regulation at Dana-Farber. He is a member of the National Academy of Sciences, and has co-authored 400 technical papers and other publications. He is a molecular biologist; two other researchers on the project, Clyde S. Crumpacker, M.D., and Bruce J. Dezube, M.D., have run AIDS clinical trials for years.

The laboratory test for LTR inhibitors is well established in basic research. (The particular version of the test used by Pardee's group was donated by the Herzenberg's laboratory at Stanford University -- which has studied NAC, an experimental HIV treatment which may reduce LTR activation indirectly.) Dr. Pardee's team has used this test to study anti-HIV properties of available, known, nontoxic compounds -- to look for a new class of potential drugs which target the on-off switch of the virus. And they have used the same test to show that these kinds of HIV inhibitors may work far better together, in certain combinations which block different paths involved in LTR activation.(2) (This recent paper, on combining a Tat inhibitor with pentoxifylline in laboratory tests, was described in AIDS TREATMENT NEWS #188, December 3, 1993).

Clearly this line of work should be pursued rapidly -- especially since other approaches have had only limited success. But much of this research is not funded; only the fact that the laboratory tests are inexpensive enabled it to be done at all. The problem seems to be that searching for AIDS treatments by screening for LTR inhibitors has not yet become an influential part of the mainstream thinking, which has focused on other kinds of drugs -- mostly AZT, etc., or high-tech approaches which may not be ready for years.

One great barrier stopping new AIDS drugs today is the lack of funding and other support for preclinical work needed to move promising ideas from the laboratory into early human tests. This barrier is now impeding the practical development of LTR inhibitors, slowing or preventing the fairly obvious research steps that are needed next.

Background for Non-Technical Readers

After HIV infects a cell, its genetic information becomes part of the DNA (the genetic inheritance) of the cell. Each cell contains a complete copy of all the person's DNA -- the information needed to construct all the organs of the body. Of course not all of this DNA is active in any particular cell. Instead, complex regulatory mechanisms determine which genes are expressed, and when. (Each gene is a part of the DNA which codes the information for creating one protein.)

When a cell has been infected and contains the HIV DNA, that DNA is often inactive, not making new virus or doing any other harm. Only when the HIV is activated is it harmful. Various different substances can increase or decrease this activation, and many of them have their effect through the LTR (the long terminal repeat of the AIDS virus).

An LTR inhibitor is a compound which prevents or reduces this activation, and thereby blocks viral replication and other viral functions.

There is a rapid, inexpensive test which can be used to screen substances to identify the ones which show promise as LTR inhibitors. This test, created by genetic engineering, uses living cells which have additional, artificially added DNA. This additional DNA includes the LTR of HIV (or of another virus being studied), together with a "reporter" gene. A reporter gene is a gene which produces a protein (usually an enzyme) that can easily be detected in the laboratory (for example by causing a color change in a test tube). In the test cells, the reporter gene is controlled by the HIV LTR, so that it produces its protein only when the LTR is activated.

To run the test, the cells are treated with a chemical (such as tumor necrosis factor) which is known to activate the LTR, and the resulting activity of the reporter gene is observed. A substance being tested can be added; if it inhibits the LTR, it will turn off or reduce the activity of the reporter Gene. This test is easy to do, and it does not require live HIV, so it does not require the elaborate safety precautions needed to work with HIV itself. It can be done in most laboratories, and could quickly screen many substances to look for potential LTR inhibitors. Positive results must be confirmed using laboratory tests with the live virus, but this only needs to be done for the much smaller number of substances which show good results from the initial screening. (These screening programs often test hundreds, or even thousands, of random chemicals to find one which may have value. Pardee's group had better success looking for LTR inhibitors -- three found out of a dozen chemicals tested -- because they were not funded for mass screening, and instead used intelligent guesses about what was likely to work.)

Of various factors which increase HIV activity by activating the LTR, the most powerful is the protein produced by the Tat gene of HIV. Another important cause of viral activation is excessive levels of a different protein, NF-kB (NF-kappa B) -- which (unlike Tat) is normally present in the body. Each of these binds to the LTR (at different sites), activating HIV. NF-kB can be induced by a variety of stimulants, including T-cell activation, inflammation, bacterial products, and oxidative stress.

Other research groups had found that HIV is much more active when both the Tat protein, and also high levels of NF-kB, are present.(3) It appears that the two together act in concert in a way that is far more than additive; in one experiment, NF-kB alone upregulated the virus by 16 times, Tat alone by 80 times, and both together 388 to 760 times, depending on the concentration of Tat used.

Dr. Pardee and his colleagues have built on basic research to find potential LTR inhibitors, and especially synergistic combinations of these compounds. Recently Debajit K. Biswas, Ph.D., and others showed that a combination of drugs that inhibit both NF-kB and Tat worked much better than either one alone -- so much better that the concentrations of both drugs (pentoxifylline, and the Roche Tat inhibitor Ro 24-7429) could be reduced ten times, and still give the same inhibition as the larger concentration of either one alone.(2)

This paper gives some insights as to why the Hoffmann-La Roche trials of its Tat inhibitor failed to reduce viral activity in humans. Since NF-kB activity is likely to be excessive in people with HIV, due to oxidative stress caused by infections, it may be necessary to combine a Tat inhibitor with another treatment (such as pentoxifylline) to reduce NF-kB activity. Further improvements may be made by adding other LTR inhibitors with different mechanisms of action.

The scientific search for LTR inhibitors also led Dr. Pardee's lab to investigate topotecan, which inhibits a DNA-unwinding enzyme which upregulates many genes (in other words, makes them more active), including those governed by the HIV LTR. This inhibitor, now in use in cancer trials but never tested in persons with HIV, can be tolerated in patients at a concentration that is 200 times that which inhibits 80 percent of viral production in laboratory tests.(1)

Dr. Pardee's lab is also considering a number of substances to modify levels of pro-inflammatory cytokines, secreted by certain immune cells, which upregulate the LTR. (In addition to pentoxifylline to decrease TNF, another example is tenidap, to inhibit IL-6 production). Also, as certain enzyme systems become upregulated in people with AIDS, inhibitors of these pathways might also be targets for inhibiting the LTR.

The synergistic inhibition of viral production, using novel combinations of drugs at tolerable doses, is a here-and-now approach. In view of the urgent need for better treatments for AIDS, we must not overlook a potentially effective antiviral strategy that is possibly within reach today.

Interview

Dr. Pardee was interviewed by John S. James of AIDS TREATMENT NEWS, and by Charles Davidson, a graduate student with a strong interest in Tat and LTR inhibitors, who brought much of this information to our attention.

James: Could you summarize the rationale for your work?

Pardee: Our approach is a simple one. We look at the LTR of HIV-1, and see what substances respond. It's a quick, easy test, and you don't need special safety precautions [which would be required for working with the live virus]. We're pretty much limited to that test [because of lack of funding to do the live-virus tests]. But we've come up with pentoxifylline, and the other three compounds we reported a year ago. We've done other work on tenidap, which looks interesting, and we have more potential drugs in the pipeline. This approach has been productive. We have shown an effect on the LTR system; now it's pretty much up to someone else to carry it forward. Bruce [Dr. Bruce Dezube] can carry it into the clinic if it looks good, but you need somebody in between [to do the preclinical development], and we do not have funds for that.

Davidson: You found the topoisomerase inhibitor was quite effective?

Pardee: Topotecan, the one we used in that work of a year ago, was by far the most potent drug. We think it has some promise. It was tested with the virus system very extensively, as we reported in our paper.(1) For reasons that aren't too clear to me, Smith-Kline Beecham does not seem very interested in pursuing it further. It's the company's decision at this point; it's pretty much out of our hands.

We have largely shifted to some other camptothecin analogs; they look pretty effective, too. We are working mostly with camptothecin itself; there are analogs that are clinically preferable, but there are no patents on camptothecin; you run into problems when you work with companies.

There are several ways a drug can inhibit the LTR, which is quite complicated. There are NF-kB motifs, and Tat-TAR motifs, and some others, SP-1 motifs, etc. The questions are what is the target for a particular compound, and how can we set up better assays for finding new compounds, by taking advantage of what we've learned with the first generation.

And of course curcumin [first discovered to have anti-HIV activity by Pardee's group, using the test for LTR inhibitors] is another one which needs to be investigated. I understand there will be a clinical trial in the Boston area, also [to be conducted by the Community Research Initiative of New England -- in addition to the trial by SEARCH Alliance, in Los Angeles]. I have no further information on it. We are starting further work on curcumin. We hear stories about India, where they eat a lot of turmeric; some people say there's lots of AIDS in India. I understand there is a lot of HIV infection, but not very much AIDS, so it may slow down the disease. Dr. Biswas, my Indian colleague, is currently in India; he is in close contact with top leaders in their infectious-disease work, and they are interested. There are also some other Indian plant products which may be quite interesting; we're hoping to look at them. Concerning curcumin, I have been told by another Indian scientist that there are other compounds in that plant which are more active than curcumin. There is much we can do if we get the time and resources.

James: Two callers told me that they didn't know where to get the more concentrated curcumin, so they used turmeric from a grocery store. Both said they had substantial improvement in blood work. This should be studied.

Davidson: Curcumin is classified as an antioxidant?

Pardee: Yes, but we don't know what its real mechanism of action is against HIV. Somebody published an abstract at a recent meeting saying it may inhibit the HIV protease. They did a computer study of what the structure should be to block the specific HIV protease, and came up with a curcumin structure.

Davidson: If you have several different mechanisms, and there is synergism, one should be able to use smaller doses of the drugs?

Pardee: Right, you should be able to get more effectiveness with less side effects, we hope. But people are very complicated organisms; just because it works on some simple system in the laboratory does not mean there will be no toxicity or other problem.

Pardee: I wish that what might be called the AIDS establishment was a little more open to innovative approaches. I have not been able to get a penny from the NIH to do any of our work, and we have come up with several new compounds [with potential use for AIDS], and we have several more in the pipeline. We will soon report on another class of compounds which is effective against the HIV LTR. But we can't go on working without funds. We have to pay researchers to eat and pay rent.

James: What study groups at NIH review your proposals?

Pardee: It goes into some AIDS study groups. I must say that one of the reviews, on attempts to follow up on the topoisomerase inhibitors, was erroneous. It claimed that we did the work before we submitted the grant, which is absolutely untrue. We did all this work in the two years of the grant; we published two good papers, and they said we didn't accomplish anything. And they also said we should be working on HIV-2 as well as HIV-1. Well, we are not a factory. If we discover something active against the HIV-1 LTR, it could be pursued with HIV-2 by someone else. It's pretty discouraging.

James: Over the years I have seen that this is not a new problem.

Pardee: The AIDS establishment has to produce something with all the money they are spending. They give us no encouragement or help at all [for innovative approaches]; and we get the same story from colleagues. The whole AIDS granting process has been dominated by the AZT types, and a few other interest areas. It's like molasses to get funded. This new panel [the National Task Force on Drug Development] is likely to have people who don't rock the boat.

A carefully worded article about how we need innovative approaches against AIDS, and need to support these innovative approaches, would hopefully do some good if it gets to the right people.

We need to find out more about the mechanism of action, why these work. We are likely to find some new targets. When we know what the target is, and we get an assay for that target, we can test for other substances that work better. We published on several potential drugs already. Nobody has clinically tried topoisomerase I inhibitors on AIDS, and yet these compounds are pretty powerful, as we reported.

We think we have a fairly clear understanding of pentoxifylline, how it blocks the LTR. It's through NF-kB, but there are additional details. It's part of another very interesting class of inhibitors of the LTR. It's not too hard to come up with possibilities. Hopefully some of them will work.

Our approach is to use the LTR as a target, just like others use the protease as a target. I think it's a very promising target.

Davidson: How much do you think has been spent so far on LTR inhibitors?

Pardee: I don't know how much companies such as Roche spend. We get a small amount to do our work on pentoxifylline, from Hoechst-Roussel Pharmaceuticals, Inc. We luckily got an NIH grant to work on topoisomerase inhibitors, which I used to work on the AIDS problem; but that is terminating and won't be renewed, so this work will stop rather fast. We have never been funded to work on this project specifically; we haven't gotten one cent. And very few people elsewhere are working on this approach. Virtually nothing has been spent on it. We'll eke out what little we can.

Davidson: Do you have any idea how the Roche Tat drug [Ro 24-7429] works?

Pardee: There are accessory proteins involved in the Tat-TAR interaction. We're trying to take these apart as fast as we can.

[Editor's note: Obstacles to the research on the Ro 24-7429, the Hoffmann-La Roche Tat inhibitor, have included not only lack of funding, but difficulty getting the drug to test, and lack of access to results from other researchers. Even though this substance failed to reduce viral activity in humans in a single-drug trial, it may be important because of the possibility that it might work in combination -- for example, with pentoxifylline, since that combination works very well in the laboratory. Also, it is worth investigating why Ro 24-7429 did not work by itself in people; there are many possible reasons that a particular drug will not work in the body, and the failure of one compound does not invalidate the whole approach. (A recent paper suggests that Ro 24-7429 alone does not inhibit the Tat protein itself, but another protein which works with Tat.(4) This might help to explain the disappointing results of human tests.)

While there are a number of ways to inhibit the LTR through the NF-kB mechanism, Tat inhibitors are still scarce. A very different Tat drug is being developed in Canada, by Allelix Biopharmaceuticals, but it is just beginning human tests (see AIDS TREATMENT NEWS #187, November 19, 1993). A prescription drug, d-penicillamine, was tested as a Tat inhibitor in Europe several years ago; it did not help patients in trials, but perhaps should be re-examined as a possible part of a combination.

We believe that the most important work at this time is human trials of topotecan, curcumin, some other LTR inhibitors being tested by Pardee's group, and the combination of Ro 24-7429 with pentoxifylline. Equally important is the laboratory search for new LTR inhibitors, and especially for better combinations of LTR inhibitors, and combinations of these with other classes of AIDS treatments. This area could move quite rapidly, if it had support instead of roadblocks from corporations and from government research agencies.]

References

1. Li CJ, Zhang LJ, Dezube BJ, Crumpacker CS, and Pardee AB. Three inhibitors of type 1 human immunodeficiency virus long terminal repeat-directed gene expression and virus replication. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA. March 1993; volume 90, pages 1839-1842.

2. 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 1, 1993; volume 90, pages 11044-11048.

3. 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.

4. Braddock M, Cannon P, Muckenthaler M, Kingsman AJ, and Kingsman SM. Inhibition of human immunodeficiency virus type 1 Tat-dependent activation of translation in Xenopus oocytes by the benzodiazepine Ro 24-7429 requires trans-activation response element loop sequences. JOURNAL OF VIROLOGY. January 1994; volume 68, number 1, pages 25-33.

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