David W. Banner

Crystal structure of the soluble human 55 kd TNF receptor-human TNFβ complex: Implications for TNF receptor activation

The X-ray crystal structure of the complex of the extracellular domain of the human 55 kd tumor necrosis factor (TNF) receptor with human TNF beta has been determined at 2.85 A resolution. The complex has three receptor molecules bound symmetrically to one TNF beta trimer. The receptor fragment, a very elongated end to end assembly of four similar folding domains, binds in the groove between two adjacent TNF beta subunits. The structure of the complex defines the orientation of the ligand with respect to the cell membrane and provides a model for TNF receptor activation. The novel fold of the TNF receptor structure is likely to be representative of the nerve growth factor (NGF)/TNF receptor family as a whole.

Fluorine in Medicinal Chemistry

Fluorinated compounds are synthesized in pharmaceutical research on a routine basis and many marketed compounds contain fluorine. The present review summarizes some of the most frequently employed strategies for using fluorine substituents in medicinal chemistry. Quite often, fluorine is introduced to improve the metabolic stability by blocking metabolically labile sites. However, fluorine can also be used to modulate the physicochemical properties, such as lipophilicity or basicity. It may exert a substantial effect on the conformation of a molecule. Increasingly, fluorine is used to enhance the binding affinity to the target protein. Recent 3D‐structure determinations of protein complexes with bound fluorinated ligands have led to an improved understanding of the nonbonding protein–ligand interactions that involve fluorine.

Systematic Investigation of Halogen Bonding in Protein-Ligand Interactions.

Halogen bonding (XB) refers to the noncovalent interaction of general structure DX···A between halogen-bearing compounds (DX: XB donor, where X=Cl, Br, I) and nucleophiles (A: XB acceptor). Since the first observation in cocrystal structures of 1,4-dioxane and Br2 by Hassel and Hvoslef in 1954, XB has been widely used in crystal engineering and solid-state supramolecular chemistry. The nature of the interaction and the underlying electronic prerequisite, the s hole in the XB donor, have been the subject of extensive theoretical studies. 7–9] Most recently, the attractive nature of XB between 1-iodoperfluoroalkanes and various donors has also been demonstrated and quantified in solution studies. Novel inhibitors of human Cathepsin L (hCatL) were discovered which bind covalently to the side chain of the catalytic Cys25 residue in the S1 pocket under formation of thioimidates, which are stabilized by the oxyanion hole of the protease. These ligands form hydrogen bonds to the backbone NH and C=O groups of Gly68 and Asp162, respectively, and fill the S2 and S3 pockets, thereby interacting with the enzyme through multiple lipophilic contacts. During the course of this research, we obtained an indication of an XB contact between a 4-chlorophenyl moiety of a ligand, whose binding affinity was enhanced by a factor of 13 compared to the unsubstituted phenyl derivative, and the backbone C=O group of Gly61 in the S3 pocket (Figure 1). This finding stimulated the prepa-

Fluorine Interactions at the Thrombin Active Site: Protein Backbone Fragments HCαCO Comprise a Favorable CF Environment and Interactions of CF with Electrophiles

In a systematic fluorine scan of a rigid inhibitor to map the fluorophilicity/fluorophobicity of the active site in thrombin, one or more F substituents were introduced into the benzyl ring reaching into the D pocket. The 4‐fluorobenzyl inhibitor showed a five to tenfold higher affinity than ligands with other fluorination patterns. X‐ray crystal‐structure analysis of the protein–ligand complex revealed favorable CF⋅⋅⋅HCαCO and CF⋅⋅⋅CO interactions of the 4‐F substituent of the inhibitor with the backbone HCαCO unit of Asn98. The importance of these interactions was further corroborated by the analysis of small‐molecule X‐ray crystal‐structure searches in the Protein Data Base (PDB) and the Cambridge Structural Database (CSD). In the CF⋅⋅⋅CO interactions that are observed for both aromatic and aliphatic CF units and a variety of carbonyl and carboxyl derivatives, the F atom approaches the CO C atom preferentially along the pseudotrigonal axis of the carbonyl system. Similar orientational preferences are also seen in the dipolar interactions CF⋅⋅⋅CN, CF⋅⋅⋅CF, and CF⋅⋅⋅NO2, in which the F atoms interact at sub‐van der Waals distances with the electrophilic centers.

Halogen Bonding at the Active Sites of Human Cathepsin L and MEK1 Kinase: Efficient Interactions in Different Environments

In two series of small‐molecule ligands, one inhibiting human cathepsin L (hcatL) and the other MEK1 kinase, biological affinities were found to strongly increase when an aryl ring of the inhibitors is substituted with the larger halogens Cl, Br, and I, but to decrease upon F substitution. X‐ray co‐crystal structure analyses revealed that the higher halides engage in halogen bonding (XB) with a backbone CO in the S3 pocket of hcatL and in a back pocket of MEK1. While the S3 pocket is located at the surface of the enzyme, which provides a polar environment, the back pocket in MEK1 is deeply buried in the protein and is of pronounced apolar character. This study analyzes environmental effects on XB in protein–ligand complexes. It is hypothesized that energetic gains by XB are predominantly not due to water replacements but originate from direct interactions between the XB donor (CarylX) and the XB acceptor (CO) in the correct geometry. New X‐ray co‐crystal structures in the same crystal form (space group P212121) were obtained for aryl chloride, bromide, and iodide ligands bound to hcatL. These high‐resolution structures reveal that the backbone CO group of Gly61 in most hcatL co‐crystal structures maintains water solvation while engaging in XB. An arylCF3‐substituted ligand of hcatL with an unexpectedly high affinity was found to adopt the same binding geometry as the aryl halides, with the CF3 group pointing to the CO group of Gly61 in the S3 pocket. In this case, a repulsive F2CF⋅⋅⋅OC contact apparently is energetically overcompensated by other favorable protein–ligand contacts established by the CF3 group.

Molecular recognition at the thrombin active site: structure-based design and synthesis of potent and selective thrombin inhibitors and the X-ray crystal structures of two thrombin-inhibitor complexes

BACKGROUND The serine protease thrombin is central in the processes of hemostasis and thrombosis. To be useful, thrombin inhibitors should combine potency towards thrombin with selectivity towards other related enzymes such as trypsin. We previously reported the structure-based design of thrombin inhibitors with rigid, bicyclic core structures. These compounds were highly active towards thrombin, but showed only modest selectivity. RESULTS Here, we describe the rational design of selective thrombin inhibitors starting from the X-ray crystal structure of the complex between the previously generated lead molecule and thrombin. The lead molecule bound with a Ki value of 90nM and a selectivity of 7.8 for thrombin over trypsin. Our design led to inhibitors with improved activity and greatly enhanced selectivity. The binding mode for two of the new inhibitors was determined by X-ray crystallography of their complexes with thrombin. The results confirmed the structures predicted by molecular modeling and, together with the binding assays, provided profound insight into molecular recognition phenomena at the thrombin active site. CONCLUSIONS A novel class of nonpeptidic, selective thrombin inhibitors has resulted from structure-based design and subsequent improvement of the initial lead molecule. These compounds, which are preorganized for binding to thrombin through a rigid, bicyclic or tricyclic central core, could aid in the development of new antithrombotic drugs. Correlative binding and X-ray structural studies within a series of related, highly preorganized inhibitors, which all prefer similar modes of association to thrombin, generate detailed information on the strength of individual intermolecular bonding interactions and their contribution to the overall free energy of complexation.

Crystal structures of uninhibited factor VIIa link its cofactor and substrate-assisted activation to specific interactions.

Factor VIIa initiates the extrinsic coagulation cascade; this event requires a delicately balanced regulation that is implemented on different levels, including a sophisticated multi-step activation mechanism of factor VII. Its central role in hemostasis and thrombosis makes factor VIIa a key target of pharmaceutical research. We succeeded, for the first time, in recombinantly producing N-terminally truncated factor VII (rf7) in an Escherichia coli expression system by employing an oxidative, in vitro, folding protocol, which depends critically on the presence of ethylene glycol. Activated recombinant factor VIIa (rf7a) was crystallised in the presence of the reversible S1-site inhibitor benzamidine. Comparison of this 1.69A crystal structure with that of an inhibitor-free and sulphate-free, but isomorphous crystal form identified structural details of factor VIIa stimulation. The stabilisation of Asp189-Ser190 by benzamidine and the capping of the intermediate helix by a sulphate ion appear to be sufficient to mimic the disorder-order transition conferred by the cofactor tissue factor (TF) and the substrate factor X. Factor VIIa shares with the homologous factor IXa, but not factor Xa, a bell-shaped activity modulation dependent on ethylene glycol. The ethylene glycol-binding site of rf7a was identified in the vicinity of the 60 loop. Ethylene glycol binding induces a significant conformational rearrangement of the 60 loop. This region serves as a recognition site of the physiologic substrate, factor X, which is common to both factor VIIa and factor IXa. These results provide a mechanistic framework of substrate-assisted catalysis of both factor VIIa and factor IXa.

A fluorine scan of the phenylamidinium needle of tricyclic thrombin inhibitors: effects of fluorine substitution on pKa and binding affinity and evidence for intermolecular C–F⋯CN interactions

The H-atoms of the phenylamidinium needle of tricyclic thrombin inhibitors, which interacts with Asp189 at the bottom of the selectivity pocket S1 of the enzyme, were systematically exchanged with F-atoms in an attempt to improve the pharmacokinetic properties by lowering the pK(a) value. Both the pK(a) values and the inhibitory constants K(i) against thrombin and trypsin were decreased upon F-substitution. Interestingly, linear free energy relationships (LFERs) revealed that binding affinity against thrombin is much more affected by a decrease in pK(a) than the affinity against trypsin. Surprising effects of F-substitutions in the phenylamidinium needle on the pK(a) value of the tertiary amine centre in the tricyclic scaffold of the inhibitors were observed and subsequently rationalised by X-ray crystallographic analysis and ab initio calculations. Evidence for highly directional intermolecular C-F...CN interactions was obtained by analysis of small-molecule X-ray crystal structures and investigations in the Cambridge Structural Database (CSD).

Crystal Structures of Candida albicans N-Myristoyltransferase with Two Distinct Inhibitors

Myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a monomeric enzyme that catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine residue of a variety of eukaryotic and viral proteins. Genetic and biochemical studies have established that Nmt is an attractive target for antifungal drugs. We present here crystal structures of C. albicans Nmt complexed with two classes of inhibitor competitive for peptide substrates. One is a peptidic inhibitor designed from the peptide substrate; the other is a nonpeptidic inhibitor having a benzofuran core. Both inhibitors are bound into the same binding groove, generated by some structural rearrangements of the enzyme, with the peptidic inhibitor showing a substrate-like binding mode and the nonpeptidic inhibitor binding differently. Further, site-directed mutagenesis for C. albicans Nmt has been utilized in order to define explicitly which amino acids are critical for inhibitor binding. The results suggest that the enzyme has some degree of flexibility for substrate binding and provide valuable information for inhibitor design.

Cation–π Interactions at the Active Site of Factor Xa: Dramatic Enhancement upon Stepwise N‐Alkylation of Ammonium Ions

A new class of potent inhibitors of factor Xa features a quaternary ammonium ion to fill the aromatic box in the S4 pocket and a 2-chlorothiophenyl group to occupy the S1 pocket (see picture; red O, blue N, yellow S, green Cl). Changing from a primary to a quaternary ammonium ion increases the binding affinity by a factor of 1000. The poor affinity in the former case suggests negligible cation- interactions between Lys and Trp.