Search

Results from a novel approach which uses protein crystallography for the screening of a low affinity inhibitor fragment library are analyzed by comparing the X-ray structures with bound fragments to the structures with the corresponding full length inhibitors. The screen for new phospho-tyrosine mimics binding to the SH2 domain of ^pp60src was initiated because of the limited cell penetration of phosphates. Fragments in our library typically had between 6 and 30 atoms and included compounds which had either millimolar activity in a Biacore assay or were suggested by the ab initio design program LUDI but had no measurable affinity. All identified fragments were located in the phospho-tyrosine pocket. The most promising fragments were successfully used to replace the phospho-tyrosine and resulted in novel nonpeptidic high affinity inhibitors. The significant diversity of successful fragments is reflected in the high flexibility of the phospho-tyrosine pocket. Comparison of the X-ray structures shows that the presence of the H-bond acceptors and not their relative position within the pharmacophore are essential for fragment binding and/or high affinity binding of full length inhibitors. The X-ray data show that the fragments are recognized by forming a complex H-bond network within the phospho-tyrosine pocket of SH2. No fragment structure was found in which this H-bond network was incomplete, and any uncompensated H-bond within the H-bond network leads to a significant decrease in the affinity of full length inhibitors. No correlation between affinity and fragment binding was found for these polar fragments and hence affinity-based screening would have overlooked some interesting starting points for inhibitor design. In contrast, we were unable to identify electron density for hydrophobic fragments, confirming that hydrophobic interactions are important for inhibitor affinity but of minor importance for ligand recognition. Our results suggest that a screening approach using protein crystallography is particularly useful to identify universal fragments for the conserved hydrophilic recognition sites found in target families such as SH2 domains, phosphatases, kinases, proteases, and esterases.

A total of 11 structures of the ^pp60src SH2 domain with non-peptidic inhibitors based on the same two closely related inhibitor scaffolds were determined using X-ray crystallography. Surprisingly, the inhibitors that have an IC50 value between 4 and 2700 nM bind in three different binding modes. Structure comparisons show that the inhibitors aim to maximize the interaction between the hydrophobic substituent and the hydrophobic pY+3 pocket. This is achieved either by an alternative binding mode of the phenyl phosphate or by including water molecules that mediate the interaction between the inhibitor scaffold and a rigid surface of the SH2 domain. The combination of the rigid pY+3 pocket and the rigid protein surface to which the scaffolds bind results in severe distance and angular restraints for putative scaffolds and their substituents. The X-ray data suggest that these restraints seem to be compensated in our system by including water molecules, thereby increasing the flexibility of the system.

^pp60Src is a protein involved in signal transduction and is mainly expressed in neurones, platelets, and osteoclasts. Its precise biological role was recently discovered with the KO experiments by Soriano that gave rise to no other apparent phenotype than osteopetrosis, a disease resulting in excedent bone formation. The SH2 domain of the Src family specifically recognizes a sequence of tetrapeptide featuring a phosphotyrosine and a lipophilic aminoacid at the +1 and +3 positions. Recently we engaged in the search for SH2 ligands via modular peptidomimicry of this tetrapetide. This gave rise to several families of nanomolar inhibitors; the best one incorporated a caprolactam scaffold, a biphenyl moiety, and a phosphotyrosine. However, these inhibitors still incorporated the phosphate group that confers good binding affinity to the protein. Phosphates have undesirable features for drug candidates, namely, high rate of hydrolysis of the phosphate group by phosphatases and high charge content precluding cell penetration. Therefore, while searching for optimal non-peptide ligands for Src SH2, we looked for phosphate replacements. For this, we have designed an SAR by fragment crystallography approach. The start of this work resulted from two experimental observations. First, the fact that phenyl phosphate itself displayed detectable binding affinity for Src SH2 permitted us to perform a screening of small aromatic compounds as phenyl phosphate surrogates. Second, the obtention of large Src SH2 crystals displaying a channel large enough for soaking purposes allowed structure determination of over 40 of these small aromatic compounds bound in the phosphotyrosine binding pocket. This search and the way it gave rise to low nanomolar range Src SH2 inhibitors devoid of phosphate groups will be the subject of the present paper.

During the course of a study aimed at the search for new potent aromatase inhibitors, several new androstenedione analogs were synthesized and evaluated. This study led to the discovery of 19-[(methylthio)methyl]androsta-4,9(11)-diene-3,17-dione (7; RU54115) already described by our laboratory. The object of the present series of papers is to disclose the result of the structure−activity relationship studies that gave rise to this compound. This first part deals mainly with the substitution in the 19-position of the steroid nucleus. Several parameters were varied, the length of the chain and its rigidity and branching, as well as the nature of the heteroatom itself and its substitution. The interaction of these new compounds with human placental aromatase in competition with the substrate androstenedione was studied by difference visible spectroscopy. The in vivo aromatase-inhibiting activities were evaluated by measuring the estradiol lowering after oral administration of the compounds to PMSG-primed female rats.

Several extracts from Epilobium parviflorum, a plant used in Central Europe for the treatment of prostate disorders, were evaluated in a biochemical assay with 5-α-reductase. The aqueous extract displaying inhibition of the enzyme was analyzed, the fraction responsible for this activity was purified, and the active compound identified as a macrocyclic tannin, oenothein B (1).