#98 from R&D Innovator Volume 3, Number 5          May 1994

Buckyballs and HIV—You’ve Got to be Kidding!
by Simon H. Friedman

Mr. Friedman is a graduate student at the department of pharmaceutical chemistry, University of California, San Francisco.

You may have heard about the seemingly unrelated topics that came together in my Ph.D. work--the AIDS virus (HIV) and buckyballs, buckminsterfulleres, those interesting carbon molecules that have increasingly caught the attention of chemists in the past few years.

I doubt I would even have started thinking about buckyballs—in relation to HIV—at all except for a friend's chance comment during a late-night lab bull session.  Diana Roe and I were members of research groups that were looking for an inhibitor of the HIV protease, one which might become the basis for an AIDS drug.  The HIV protease plays a critical role for virus proliferation; therefore, and inhibitor may eventually protect people from the disease.  Talking about work that had been done in the field, we agreed that some truly interesting and strange molecules had been assayed as potential HIV protease inhibitors.

As we mulled over this list, Diana said, “Yeah, what are they going to try next, buckyballs?”

We both chuckled, I thought about it for a minute and responded, “You know, that might actually work."  I was thinking, the protease active site—the physical target for any inhibitor—is hydrophobic (in water it prefers to associate with other hydrophobic surfaces instead of water), and it is rather round.  Likewise, buckyballs are hydrophobic, and they are round too.  I went to the chalk board and drew a picture of a Pacman (remember the voracious creature in that primordial video game?) devouring the energizer dot, it's target on the screen.  The pacman represented the protease, and its jaws stood for the flaps on the active site.  The dot, of course, was the buckyball. I drew two negatively charged aspartates (a type of amino acid) which were known to be a part of the active site, in the mouth of the pacman/protease.  Then I drew two positively charged amino groups on the surface of the buckyball, forming salt bridges with the negatively charged aspartates.

Over the next couple of weeks the idea became a bit of a joke in our group; people suggested that I publish it in the Journal of Irreproducible Results.  I thought this juxtaposition of two unrelated fields was pretty weird, but I also felt there were reasons that the right fullerene derivative could be an effective inhibitor.

I'd spent many hours staring at images of the HIV protease's active site, since our department focused on a program of structurally based drug design to block this essential enzyme.  I therefore knew that most of the active site was lined with hydrophobic amino acids.  In water, hydrophobic surfaces tend to associate with each other, much in the same way that drops of oil on the surface of water coalesce into larger drops.  Fullerenes are also hydrophobic. 

Furthermore, the active site is large, open and tube-like (a circular cross section), with two flaps that wrap around its natural substrates, an arrangement that struck me as ideal for interacting with the spherical fullerene.  The key question was:  Would the active site be wide enough to accomodate the fullerence derivatives, and allow the two hydrophobic surfaces to contact each other?

Enough Jokes

Thinking about these issues was definitely a sideline to the main project I was working on, but since that project was going slowly, it was easy to allow my attention to wander over to the fullerenes.

One night, finding myself in front of the graphics workstation, I decided to finally check if the active site would accommodate a fullerene.  The well-known 60-carbon fullerene (C60) has the same arrangement of pentagons and hexagons as a soccer ball, and it forms a nearly spherical molecule.  So I began constructing the C60 structure on my computer using a figure from a paper as a guide.

It was slow going, trying to keep all the pentagons and hexagons aligned with each other, but I finally finished and viewed the familiar spherical shape on the screen.  Then I opened up a protease structure on the same screen and began moving the fullerene towards the active site.

It was looking good—the diameter of the fullerene was in the right ballpark—so I continued nudging the molecules to optimize the interaction.  Finally, I scrutinized the complex I had created by “hand docking,” examining thin cross sections for close surface contacts (which are good) and surface overlaps (which are not).  The outcome was undeniable:  C60 fit almost perfectly into the cylindrical protease active site.

This was quite promising.  The clincher, though, came from a “back of the envelope” calculation estimating of the binding affinity between the fullerene and the active site.  I estimated a binding constant in the nanomolar range, which would be a very respectable affinity. 

The key now was to find a suitable compound to test. Talk of this project continued among my friends.  Once, when I was leaving a cafe, I heard some semi-playful jabs about my “fullerene/protease” project.”  I took aside a bright and arrogant friend and asked him, “Seriously, doesn’t it make sense that fullerenes could be inhibitors?  They're the right shape, and they're hydrophobic?”

He predicted that I would never get the fullerenes solubilized.  I pointed out that if the fullerene surface had some amino groups, it should be water soluble.  But he retorted in the voice of the wise old master from Kung Fu, “Ah, Grasshopper, you can't disturb the molecule's surface without affecting the binding!”  That comment, and his oh-so-certain tone, bothered me even though I believed that he was wrong. 

To jump from theory to experiment, I needed a fullerene with positively charged amino groups attached.  Not only would they solubilize the molecule, but they could also bind to the negatively charged aspartates in the protease active site.  I needed to talk to Fred Wudl at the University of California at Santa Barbara, who was the only person I knew of who had pursued adding amino groups to fullerenes.

Time to Tell the Boss

I had been working on this side project in my “spare” time and thought it was time to tell George Kenyon, my thesis advisor.  I went to his office and described the idea, the modeling, the calculations, and showed him images of the modeled complexes.  He could easily have squelched the project and directed me to get on with my main project, but instead he said, “Go for it!”

The next day I described the project in detail to Professor Wudl via e-mail, and the following day he responded, eager to collaborate.  Although he didn’t have the exact compound I'd envisioned, he did come up with a soluble buckyball with enough of the desired features that I knew we should try it out. 

Diane DeCamp, a postdoc in the department who was running the HIV assay, ran the sample.  A couple of weeks later, she came back with the result:  The compound had indeed inhibited HIV protease.  While it wasn’t quite as effective as my back-of-the-envelope calculation had estimated it would be, it was still a respectable value for a first compound.

About a year later, after I had done some more detailed molecular modeling and enzymology on the compound, the paper on this discovery was published.  It has attracted quite a bit of interest, being the first biochemical application of a fullerene.  We are continuing to apply computer-based design techniques to find tighter binding, more effective compounds. 

In the end, I feel the story illustrates the importance of allowing freedom in the discovery process:  a freedom to follow a direction others didn’t take seriously, and a freedom to pursue a project I wasn’t assigned.

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