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design such molecules on the basis of the trypanosomal TIM structure by a linked-fragment approach
[57]. In that strategy small building blocks are designed to be complementary to the targeted surface of a
protein. Such fragments can then be synthesized or purchased, tested for their effect on enzyme kinetics
and for their binding mode by crystallography. Promising fragments are then linked together into larger
molecules. The idea behind this stepwise approach is to obtain early experimental feedback in the drugdesign
cycle.
The crystallographic follow-up of our linked-fragment approach design for trypanosomal TIM was
disappointing. Two designed fragments were soaked into a crystal of trypanosomal TIM, namely 4hydroxy-2-butanone
and D-asparagine. Despite high concentrations of these molecules in the mother
liquor, 220mM and 30 mM respectively, no convincing electron density could be seen in difference
Fourier maps calculated between 10.0-2.8 Å [72]. Common to both molecules is that they are fairly
polar, rather flexible, and were expected to displace crystallographically observed water molecules.
Apparently, de novo design of tightly binding small ligands is far from trivial.
We also tried to find new leads by a completely experimental approach. For that purpose the
crystallographic cocktail soak (CCS) approach was developed. In this method cocktails of fine
chemicals are soaked into a crystal in the hope of finding crystallographic evidence of binding for one of
the molecules from the cocktail. The identification of such a molecule might not be clear immediately
because several molecules in the cocktail might be compatible
with the shape of the electron density, especially if the resolution is not very high. An outcome would be
provided by a dichotomic approach (Figure 9), in which the crystallographic soaking experiment is
repeated with ever smaller subcocktails of the original one. For example, if a ligand shows up from a
cocktail soak of 32 compounds, a second experiment should be done with only half of the compounds. If
the ligand fails to show up, one knows that it is one of the alternative 16 compounds. After at most six
experiments the identity of the ligand is known. One might like to think of the CCS approach as the
experimental analog of the computational methods in programs like GRID [73] or MCSS [74], but with
thirty-two compounds at a time.
Figure 9
Dichotomic search for unknown ligand from
cocktail 1. The number of compounds in the sub
cocktail is indicated by n, and the
interpretation about the presence of compound
X in the subcocktail is given as Y/?/N.
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