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Roald Hoffmann

Three Short Stories of the Real World

By way of example, here are three tales of chemical discovery:

Insects are the greatest chemists. In 1966 R. S. Berger identified the main sex pheromone of the cabbage looper moth, Trichoplusia ni (Noctuidae) as (Z)-7-dodecenyl acetate. Those were the halcyon days of early pheromone chemistry; everyone was happy with one molecule (as they were with one gene for each trait). Thirteen years later L. B. Bjostad et al. identified a second component, important especially in close-range courtship behavior—simple dodecyl acetate. Bjostad, C. E. Linn, W. L. Roelofs and their coworkers, at the New York State Agricultural Experiment Station in Geneva, New York, then began to think through the biosynthetic relations between these two components and other molecules observed in the pheromone gland. Obviously, enzymes that shorten molecules, reduce or acylate them, remove hydrogens—all the wondrous machinery of the living—are at work. Figure 2 is a complex graph from one of their papers, showing the biochemical relations between the various kinds of fatty acids present. A blend of six components, suggested by their analysis, elicited complete courting flights against a stiff breeze in a wind tunnel. Clearly one needs six for sex. And would a human master perfumer be surprised?

Figure 2. Proposed biosynthetic pathwayClick to Enlarge Image

The story is told with sufficient verve in the Bjostad, Linn, Du and Roelofs paper that even I, an outsider to the field, am pulled in by it. More than just an analysis of pheromone glands, the biochemical relations are clever. I am intrigued by their tale and begin to think of its sequel—how do the females evolve that blend? How do the males evolve the receptors to it? Thomas C. Baker and his coworkers at Iowa State University have actually located separate compartments for the six components (and one antagonist) near where the male antenna input is first processed.

Blood Red: Hemoglobin is the stumbling block to the simpleminded; there is a story in every turn in this best known of proteins. I will pick one of the oldest, that of the cooperativity of oxygen uptake by this molecule. Hemoglobin has four subunits, two consisting of 146 amino acids, two of 141, each cradling a porphyrin where an oxygen molecule binds to an iron atom. Oxygenation of one subunit makes the subsequent oxygenation of another easier. An important early phenomenological theory accounted for the kinetics. But how does it happen on the molecular level, over a distance of 25–30 angstroms between iron atoms?

Max Perutz, whose perseverance and talent first gave us the structure of hemoglobin, also built a bold theory of the cooperativity. It begins with an iron atom on one subunit moving into the heme plane upon oxygenation, pulling a proximal histidine with it. Movement of the histidine shifts a helix, it is suggested; eventually a geometry change at the subunit interface ensues, where a salt bridge between subunits is broken. A net conformational change in the protein occurs, influencing the binding of the next oxygen. Is this a Rube Goldberg (Heath Robinson in England) machine? Rube Goldberg studied some chemistry at Berkeley—maybe he learned something about reaction mechanisms. It is a mechanism, a story well told, remarkably convincing. And permitting elaboration, as work by Martin Karplus and his collaborators shows.

The Road to Cariporide: Once upon a time (1986), in a pharmaceutical company (Hoechst, now Aventis), the chemists Hans-Jochen Lang and Heinrich Englert became interested in the sodium (Na+)-hydrogen (H+) exchange (therefore called NHE) system, a fine biochemical machine for moving about protons and sodium ions, and thereby regulating cellular acidity. NHE had been first described in 1976 by Swiss physiologist Heini Murer as an ion-transport system in the proximal tubule of the kidney. There were many speculations about the role of this device, present in virtually every type of mammalian cell. For instance, might NHE affect the pathophysiology of brain edema caused by stroke?

Pharmacologists and chemists started looking for NHE inhibitors. As is often the case in drug development, the problem is not so much the chemical compounds to test, for chemists have certainly learned the lesson of Genesis, that we have been put on this earth to create. No, the problem is so often the assay. In the case at hand, a promising one, using renal membrane vesicles, proved insensitive.

At the same time Hoechst pharmacologist Wolfgang Scholz was working on a completely different system, the use of ion transport in red blood cells as an assay for the identification of diuretics. One day he was asked by a cardiovascular-research group to test the red blood cells of rabbits on a high-cholesterol diet for possible changes in their ion-transport mechanisms. Remarkably, whereas NHE activity is quite low in red blood cells under normal conditions, there was an approximately tenfold increase caused by the special cholesterol-rich diet.

Whatever the reason for the original experiment (rabbits emulating American junk-food consumers?), the Hoechst scientists saw an opportunity—these red blood cells provided an exquisitely sensitive NHE assay, 1,000 times as sensitive as the kidney membrane vesicles.

There was now momentum for synthetic chemists to ply their art. New classes of compounds were tried. One pharmacologist was reading a paper in the Russian literature, on a totally different subject, when he came across the statement that a sodium ion was roughly of the same size as a guanidinium group. Now that turns out to be somewhat farfetched, but no matter, it gave impetus to the synthesis (and testing with the new assay) of a variety of guanidine derivatives. Some of these compounds, the benzoyl guanidines, turned out to be potent and specific enough to test them for reduction of brain edema. The results were quite disappointing.

Thus, in late 1988, Lang, Englert and Scholz had in hand a new class of ion-transport inhibitors. The only problem was that there were no known clinical indications for them! It was then decided to test one of the best compounds in a broader range of pharmacological models. One of them was the isolated working rat heart in the lab of the pharmacologist Wolfgang Linz. When a benzoyl guanidine code-named HOE 694 was tested in this model, Linz was amazed to find it was about the most protective compound in cases of cardiac ischemia/reperfusion (blood vessel constriction and blood resupply) that he had ever seen.

At this point molecular biology kicks in. In 1989 the group of Jacques Pouyssegure in Nice cloned the NHE gene. In the following years, several subtypes of NHE were identified. A collaboration between the Hoechst team and Pouyssegure's group soon determined that NHE subtype 1 is not only ubiquitous but also the predominant subtype in the heart and blood cells. It was found that HOE 694 and most of the related benzoyl guanidine compounds were quite selective inhibitors of NHE subtype 1. The predominant subtype in the proximal tubule of the kidney was NHE-3. Compounds like HOE 694 were about 1,000 times more effective on NHE-1 than on NHE-3. So, finally, it was understood why the red-blood-cell assay had worked and the renal membranes had not!

All the laws that characterize the infinity of failures facing human beings apply to pharmaceutical research as well. Within weeks it was revealed that HOE 694 formed a metabolite that concentrated in the rat kidneys and precipitated in the tubular system, where it caused obstruction and inflammation. A strategy to construct compounds metabolized in a different way led to a new compound, HOE 642, synthesized by chemist Andreas Weichert. Now, HOE 642 (generic cariporide) has reached a late stage of clinical development and will it is hoped come to the market in three years. I have recounted three tales of discovery. They start simple, yet in each, the ornery complexity of the real is parlayed by the protaganist chemists into a delightful, deeper story.

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