Jörg Czech

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

Glucuronide prodrugs of hydroxy compounds for antibody directed enzyme prodrug therapy (ADEPT) : A phenol nitrogen mustard carbamate

Abstract A prodrug consisting of a β-D-glucuronic acid linked to a self-immolative spacer (a N-( ortho -hydroxyphenyl)-N-methylcarbamate) and a phenolic nitrogen mustard was synthesised. As this prodrug was easily cleaved by a β-glucuronidase enzyme and displayed low cytotoxicity, it must be considered as appropriate for an ADEPT approach.

Identification of High-Affinity P2Y12 Antagonists Based on a Phenylpyrazole Glutamic Acid Piperazine Backbone

A series of novel, highly potent P2Y₁₂ antagonists as inhibitors of platelet aggregation based on a phenylpyrazole glutamic acid piperazine backbone is described. Exploration of the structural requirements of the substituents by probing the structure-activity relationship along this backbone led to the discovery of the N-acetyl-(S)-proline cyclobutyl amide moiety as a highly privileged motif. Combining the most favorable substituents led to remarkably potent P2Y₁₂ antagonists displaying not only low nanomolar binding affinity to the P2Y₁₂ receptor but also a low nanomolar inhibition of platelet aggregation in the human platelet rich plasma assay with IC₅₀ values below 50 nM. Using a homology and a three-dimensional quantitative structure-activity relationship model, a binding hypothesis elucidating the impact of several structural features was developed.

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors

From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure.

Pharmacological characterization of a new 4-amidinophenyl-alanine thrombin-inhibitor (CRC 220)

The new thrombin inhibitor CRC 220 was characterized in vivo for its antithrombotic effects. CRC 220 led to a dose-dependent prolongation of clotting parameters as determined in rats, rabbits, dogs, sheeps, pigs and monkeys. We evaluated the efficacy of CRC 220 to prevent thrombus formation in arteries and in the microcirculation in different animal models. In a rabbit model of tissue factor-induced coagulation activation, infusion of 0.5 mg/kg x h CRC 220 (3 hours) led to a significant prevention of fibrinogen decrease. In a rat model of lethal LPS-induced DIC CRC 220 significantly prevented the mortality rate after a 4h-infusion of 0.75 mg/kg x h. Thrombin-induced platelet aggregation in rat lungs could be prevented by the i.v. bolus injection of CRC 220. A dose of 0.3 mg/kg leads to a reduction of more than 80% of platelet deposition in the lung, significant inhibition was still observed 90 minutes after CRC 220 administration; at this time the inhibitor had already been cleared from plasma. Arterial thrombosis was induced in rabbits by squeezing and stenosis of the A. carotis. The i.v. bolus administration of CRC 220 dose-dependently prevented thrombus formation, an ED50 of 0.03 mg/kg was calculated. This dose was associated with only a minor prolongation of aPTT.

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.

Synthesis and cytotoxic activity of a glucuronylated prodrug of nornitrogen mustard

A new glucuronylated prodrug of nornitrogen mustard, incorporating the same spacer group as the doxorubicin prodrug HMR 1826, has been prepared. Upon exposure to E. coli beta-glucuronidase, fast hydrolysis occurs but a lower cytotoxicity against LoVo cancer cells is observed compared to the nornitrogen mustard alone. This is explained by cyclization of the intermediate carbamic acid to the inactive chloroethyl oxazolidinone.

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.

Influence of Recombinant Hirudin on Tissue-Factor-Induced Activation of Coagulation in Rabbits

Uncontrolled activation of the tissue-factor (TF)-dependent extrinsic pathway of coagulation can lead to severe impairment of the hemostatic balance. AS thrombin plays the central role in the initiation of clotting, we used the highly specific thrombin inhibitor recombinant hirudin to prevent TF-induced coagulation activation in a rabbit model. Infusion of 0.5 micrograms.kg-1.h-1 TF in rabbits for 7 h led to a decrease in fibrinogen and platelets, to an increase in fibrin monomers and to a prolongation of TT, aPTT and PT. Recombinant hirudin was administered in doses of 0.5, 1 and 2 mg.kg-1 body weight (intravenous bolus), the protocol included a pre-TF (recombinant hirudin given at t = 0) and a post-TF study group (recombinant hirudin given at t = 2 h after the start of the TF infusion). Fibrinogen plasma levels, platelet counts and recombinant hirudin plasma levels were measured at baseline (t = 0) at 0.5, 1, 2, 3, 4, 5, 6, and 7 h; the deceleration rate of fibrinogen and platelets per hour was calculated for the control and the recombinant-hirudin-treated groups. The deceleration rate for fibrinogen in the TF group was -0.227 g.l-1.h-1 and was reduced by recombinant hirudin to -0.119, -0.116 and -0.095 g.1-1.h-1 for 0.5, 1 or 2 mg.kg-1, respectively (significant differences to control group, Jonckheere-Terpstra test). The inhibitor similarly prevented the decrease of platelets dose-dependently. Recombinant hirudin was cleared from plasma with a terminal half-life of about 100 min; however, even after its clearance from plasma, recombinant hirudin significantly prevented the fibrinogen and platelet drop. Recombinant hirudin was effective when given in the pre-TF as well as in the post-TF phase.