Zombie cyclophilins catalyze HIV capsid isomerization

If you're going to study the role an enzyme plays in a biological pathway, it's often useful to "kill" it with a mutation. For example, the proline cis-trans isomerase cyclophilin A (CypA) needs a particular arginine residue for its chemistry, so mutations that remove or alter that functional group, like R55K and R55A, should destroy the protein's function and have effects on the related pathways that help illustrate its role. The hydrophobic pocket it uses to bind substrates is made by residues like H126, F113, and W121. Growing or shrinking those residues should alter the shape of the pocket and change binding or activity, leaving the enzyme "dead".

Using model reactions and various binding assays, researchers have previously examined a number of these mutants (4,7) and found that they diminish isomerase activity and alter inhibition. However, a detailed study of the effects of the mutations on CypA's catalytic cycle has not been performed. Former Kern lab members Daryl Bosco (now a professor at UMass Medical) and Elan Eisenmesser (now at UCHSC) examined these mutants in greater detail to see how they really behaved. I also contributed some data at the last minute, when the third reviewer requested we study an additional mutant, prompting a scene that I promise was not too much like that Downfall parody. In every case we found that these enzymes, although significantly impaired, weren't as dead as they had seemed.

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Gold dust vs. the virus

A recent episode of South Park featured a story in which two of the main characters got infected with HIV and discovered that the cure for AIDS is an injection of about $200,000. As any viewer of the series would expect, the episode is crude and vulgar, and it wobbles to and fro over the line between humor and offensiveness. Yet the episode might also turn out to be oddly prescient, if research described in an upcoming JACS article bears further fruit. As it turns out, researchers from UNC, the University of Colorado, and NC State have had some success in inhibiting HIV activity using drug-coated nanoparticles, made out of gold.

The approach Bowman et al. use is based on the idea of multivalence, which is the operating principle of Velcro. A single hook-loop interaction between two pieces of fabric usually isn't enough to keep them fastened together. However, by having a large number of relatively weak interactions a strong connection can be made. Many biological systems make use of the same principle, using many weak interactions between repeating units to produce high overall affinity. The researchers set out to apply this idea to medicine, using many copies of a low-affinity drug attached to a nanoparticle.

The drug the authors used is based on a compound designated TAK-779, which is effective at preventing HIV virions from fusing with T cells, but also has some unpleasant properties for patients. The authors lopped off the part of the molecule that causes these problems, but doing so also removes most of its ability to fight HIV. So, they linked this new compound (called SDC-1721) to the gold particles at a ratio of about 12 molecules SDC-1721 per particle. In cultured cells, the nanoparticle-linked drug had an IC50 similar to TAK-779, even though SDC-1721 by itself was totally ineffective at preventing infection. Cutting the number of SDC-1721 molecules per particle to ~1 removed the inhibitory effect, proving that the multivalent approach was critical.

This is of course no demonstration of in vivo effectiveness, and there's no telling whether the nanoparticle will have side-effects that are better or worse than TAK-779. However, if this initial success is borne out by further trials this may be a promising angle on treatments. One of the advantages to this approach is that it has some ability to counter resistance built into it because of the multivalent binding. Even if a virus evolves a lower affinity for the drug, the weak binding of many ligands, and the increased effective local concentration of those ligands, may be enough to rescue inhibitory activity. Injecting yourself with money is no way to cure anything, but it is possible that in the future we will attack viruses by injecting patients with (drug-laden) gold.

1. Bowman, M., Ballard, T.E., Ackerson, C.J., Feldheim, D.L., Margolis, D.M., Melander, C. (2008). Inhibition of HIV Fusion with Multivalent Gold Nanoparticles. Journal of the American Chemical Society DOI: 10.1021/ja710321g

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Merck HIV vaccine trial fails

Bad news today from the fight against AIDS: the Merck STEP trial has been discontinued due to evidence that the vaccine neither prevents nor attenuates HIV infection. This is a serious setback, as the trial was utilizing a new strategy and had sufficiently promising results two years ago that it was actually expanded. While this doesn't mark the death-knell of the CTL approach by any means, it is certainly a major disappointment and will probably send a number of approaches in development back to the drawing board.

The classic approaches to vaccination in humans have failed for HIV for a variety of reasons. Part of the difficulty is that HIV has such a high degree of variation. There's no guarantee that any single vaccine could protect against every strain for any length of time. Moreover, vaccination using the virus itself is incredibly risky — even a virus that is missing the essential Nef protein from its RNA can damage the immune system. Experiments in SIV show that a damaged or attenuated virus only confers protection if it reproduces at low levels in the host, but at the same time this gives it a greater opportunity to revert to pathogenic status. For this reason, chemical inactivation, which truly kills the virus, doesn't produce a viable vaccine because the virus does not stimulate sufficient antibodies to protect the patient.

The Merck vaccine used a different approach entirely, based on the idea of activating cytotoxic T lymphocytes. Rather than using inactivated HIV, the Merck team transplanted three HIV genes into an adenovirus (one of the viruses that produces the common cold). The idea was that these genes would get expressed into protein and displayed on the cell surface in the major histocompatibility complex. This would train T cells to attack any cell that the HIV virus had infected. Thus, the approach would not be to immobilize the virus with anitbodies, but rather to destroy any infected hosts cells before virus production really swung into gear. Even if it failed to prevent infection per se, it was hoped that this approach would arrest the development of an HIV infection into AIDS.

Unfortunately, this clever tack seems to have failed, at least in this application. It may take some time to understand why things did not work out, and maybe there was just an unfortunate choice of which HIV proteins were used or some other idiosyncratic issue with Merck's particular formulation of the approach. Until a detailed post-mortem is complete, however, CTL-stimulation approaches will have to be evaluated in a harsher light, with a little less hope.

NOTE: IAVI probably won't have the study status updated until Monday.

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