Herman Schreuder

Evidence for CCl/CBr⋅⋅⋅π Interactions as an Important Contribution to Protein–Ligand Binding Affinity†

Attractive chlorine: Noncovalent interactions between chlorine or bromine atoms and aromatic rings in proteins open up a new method for the manipulation of molecular recognition. Substitution at distinct positions of two factor Xa inhibitors improves the free energy of binding by interaction with a tyrosine unit. The generality of this motif was underscored by multiple crystal structures as well as high-level quantum chemical calculations (see picture).

Fragment Deconstruction of Small, Potent Factor Xa Inhibitors: Exploring the Superadditivity Energetics of Fragment Linking in Protein-Ligand Complexes.

Predictable thermodynamic additivity is one of the cornerstones of classical covalent chemistry, allowing accurate calculation of energy terms for complete processes by addition of terms for individual components. However this principle breaks down in complex noncovalent systems, such as biological systems, in which the energetics of individual components are not truly independent of each other. This complicates predicting protein structure and folding and, the focus of this work, the prediction of ligand binding to proteins. Molecular recognition in protein–ligand complexes predominantly occurs through multiple noncovalent interactions, whereas their contribution to the total free-energy of binding (DG) is often unevenly distributed over the contact interface. The identification of ligands as “molecular anchors” for high affinity regions in proteins (“hot spots”) is fundamental for fragment-based drug discovery, 3] indicating the similarity of ligandand protein-centric concepts. Often highaffinity ligands encompass more than one fragment in proximal protein sites; in a few cases, individual fragments in two neighboring sites could be linked to result in high binding affinity. Ideally, the DG of linked fragments should be significantly greater than the sum of DG increments from each fragment. This overproportional increase (“superadditivity”) is attributed to the fact that each fragment loses a significant part of its rigid body rotational and translational entropy upon complex formation. Thus, the sum of DG for two fragments includes two unfavorable rigid body entropy barrier terms, whereas the joined molecule is only affected by one of these terms. Any ligand has to overcome this barrier because of entropy loss upon association to its site. The nonadditivity for DG contributions is defined as linker coefficient E corresponding to the difference between the sums of fragment affinity and the final ligand [Eq. (1)]. DGfinal 1⁄4 DGfrag1 þ DGfrag2 þ DGlink with DGlink 1⁄4 R T ln E ð1Þ

Novel β-amino acid derivatives as inhibitors of cathepsin A.

Cathepsin A (CatA) is a serine carboxypeptidase distributed between lysosomes, cell membrane, and extracellular space. Several peptide hormones including bradykinin and angiotensin I have been described as substrates. Therefore, the inhibition of CatA has the potential for beneficial effects in cardiovascular diseases. Pharmacological inhibition of CatA by the natural product ebelactone B increased renal bradykinin levels and prevented the development of salt-induced hypertension. However, so far no small molecule inhibitors of CatA with oral bioavailability have been described to allow further pharmacological profiling. In our work we identified novel β-amino acid derivatives as inhibitors of CatA after a HTS analysis based on a project adapted fragment approach. The new inhibitors showed beneficial ADME and pharmacokinetic profiles, and their binding modes were established by X-ray crystallography. Further investigations led to the identification of a hitherto unknown pathophysiological role of CatA in cardiac hypertrophy. One of our inhibitors is currently undergoing phase I clinical trials.

Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C

Glutarylamidase is an important enzyme employed in the commercial production of 7‐aminocephalosporanic acid, a starting compound in the synthesis of cephalosporin antibiotics. 7‐aminocephalosporanic acid is obtained from cephalosporin C, a natural antibiotic, either chemically or by a two‐step enzymatic process utilizing the enzymes D‐amino acid oxidase and glutarylamidase. We have investigated possibilities for redesigning glutarylamidase for the production of 7‐aminocephalosporanic acid from cephalosporin C in a single enzymatic step. These studies are based on the structures of glutarylamidase, which we have solved with bound phosphate and ethylene glycol to 2.5 Å resolution and with bound glycerol to 2.4 Å. The phosphate binds near the catalytic serine in a way that mimics the hemiacetal that develops during catalysis, while the glycerol occupies the side‐chain binding pocket. Our structures show that the enzyme is not only structurally similar to penicillin G acylase but also employs essentially the same mechanism in which the α‐amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the Oγ through a hydrogen bond relay network involving the α‐amino group of the serine and a bound water molecule. A plausible model of the enzyme–substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7‐aminocephalosporanic acid.

Design, synthesis, and structure-activity relationship of a new class of amidinophenylurea-based factor VIIa inhibitors.

Selective inhibition of coagulation factor VIIa has recently gained attraction as interesting approach towards antithrombotic treatment. Using parallel synthesis supported by structure-based design and X-ray crystallography, we were able to identify a novel series of amidinophenylurea derivatives with remarkable affinity for factor VIIa. The most potent compound displays a K(i) value of 23 nM for factor VIIa.

A Combination of Spin Diffusion Methods for the Determination of Protein-Ligand Complex Structural Ensembles.

Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinase A and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis.

Novel Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) from Natural Product Anabaenopeptin

Anabaenopeptins isolated from cyanobacteria were identified as inhibitors of carboxypeptidase TAFIa. Cocrystal structures of these macrocyclic natural product inhibitors in a modified porcine carboxypeptidase B revealed their binding mode and provided the basis for the rational design of small molecule inhibitors with a previously unknown central urea motif. Optimization based on these design concepts allowed for a rapid evaluation of the SAR and delivered potent small molecule inhibitors of TAFIa with a promising overall profile.

Inhibition of CatA: an emerging strategy for the treatment of heart failure

The lysosomal serine carboxypeptidase CatA has a very important and well-known structural function as well as a, so far, less explored catalytic function. A complete loss of the CatA protein results in the lysosomal storage disease galactosialidosis caused by intralysosomal degradation of β-galactosidase and neuraminidase 1. However, mice with a catalytically inactive CatA enzyme show no signs of this disease. This observation establishes a clear distinction between structural and catalytic functions of the CatA enzyme. Recently, several classes of orally bioavailable synthetic inhibitors of CatA have been identified. Pharmacological studies in rodents indicate a remarkable influence of CatA inhibition on cardiovascular disease progression and identify CatA as a promising novel target for the treatment of heart failure.

Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site.

Crystal Structure of Cathepsin A, a Novel Target for the Treatment of Cardiovascular Diseases.

The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order-disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253-Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains. This network can only form if at least half of the carboxylate groups involved are protonated, which explains the acidic pH optimum of the enzyme.