Steffen Hahn

Dual-target virtual screening by pharmacophore elucidation and molecular shape filtering.

Dual-target inhibitors gained increased attention in the past years. A novel in silico approach was employed for the discovery of dual 5-lipoxygenase/soluble epoxide hydrolase inhibitors. The ligand-based approach uses excessive pharmacophore elucidation and pharmacophore alignment in conjunction with shape-based scoring. The virtual screening results were verified in vitro, leading to nine novel inhibitors including a dual-target compound.

Synthesis and structure-activity relationship studies of novel dual inhibitors of soluble epoxide hydrolase and 5-lipoxygenase.

Current research leads to the assumption that drugs affecting more than one target could result in a more efficient treatment of diseases and fewer safety concerns. Administration of drugs inhibiting only one branch of the arachidonic acid cascade is usually accompanied by side effects. We therefore designed and synthesized a library of hybrid molecules incorporating an imidazo[1,2-a]pyridine and an urea moiety as novel soluble epoxide hydrolase (sEH)/5-lipoxygenase (5-LO) dual inhibitors. Evaluation of the compounds was accomplished by in vitro testing using recombinant enzyme assays.

Evaluation of structure-derived pharmacophore of soluble epoxide hydrolase inhibitors by virtual screening

The soluble epoxide hydrolase (sEH) is an enzyme located downstream of the CYP 450 branch of the arachidonic acid cascade and can be linked to a number of indications, including cardiovascular disorders, diabetes and inflammatory processes. Numerous inhibitors (sEHI) have been reported, mostly based on urea or amide scaffolds. The search for valid bioisosteric replacements is an ongoing challenge in the discovery of sEHI. We developed a receptor-based pharmacophore model on the basis of 13 crystal structures of the sEH and performed a virtual screening for novel compounds. The virtual screening hits were verified in vitro proving the basic applicability of the model and leading to novel non-urea sEHI.

Complementary Screening Techniques Yielded Fragments that Inhibit the Phosphatase Activity of Soluble Epoxide Hydrolase

Soluble epoxide hydrolase (sEH) is one of the key enzymes in the arachidonic acid cascade. The C-terminal domain of the protein catalyzes the transformation of epoxides to their corresponding diols. This activity has been linked with inflammation and altered vascular homeostasis. The N-terminal domain of sEH dephosphorylates fatty acid phosphates, such as farnesyl pyrophosphate, geranylgeranyl pyrophosphate and farnesylmonophosphate, 3] but the physiological consequences of this reaction are unclear. However, as the known substrates of sEH phosphatase are involved in pathophysiologically relevant processes, including proliferation and apoptosis, compounds that inhibit sEH phosphatase may have pronounced biological effects and would therefore be considered useful pharmacological tools. No potent inhibitor of the sEH lipid phosphatase activity has been reported to date, and although lipid sulfates and sulfonates can affect the activity, they lack the desirable pharmacological properties to make them suitable for further evaluation in cell-based assays and in vivo models. The X-ray structure of sEH has been resolved and offers a good starting point for structure-based virtual screening, even in the absence of a co-crystallized ligand in the phosphatase binding site. 7] Fragment screening has become an important source of chemical entities that can be used as a starting point for drug discovery. 9] Therefore, we decided to search for molecular fragments that are able to interact with the catalytic center of sEH phosphatase. Although fragments with a molecular weight (MW) less than 250 Da usually exhibit low binding affinities, they can be superior to drug-sized screening hits (300 Da< MW<500 Da) in terms of ligand efficiency, which can be expressed as a binding efficiency index (BEI = pIC50/MW). [10] Thus, a fragment hit can be rationally evolved towards a highly efficient lead structure. Typically, a screening of a library with 1000–3000 fragments is required to ensure an adequate hit rate. Due to the low potency of the hit structures, high concentrations (100 mm–10 mm) are necessary to detect an effect. This concentration range limits the variety of assay systems that can be employed. In fact, the most wide-spread techniques used for fragment screening are high-throughput X-ray crystallography and NMR-based methods, even though other types of assays have also been successfully employed for this task. 14] Here, we propose a combination of three methods— molecular docking (virtual screening), saturation transfer difference (STD)-NMR screening, and a fluorescence-based activity assay—to successfully screen a fragment library. We assume that the combination of these methods can dramatically reduce the experimental effort needed for success concerning the number of compounds to be screened and, because of the complementary nature of the techniques used, enhance the reliability of the screening results. We started with a commercially available compound library available from Specs Inc. (Delft, The Netherlands). We applied a set of criteria, often referred to as the “Astex Rule of 3” 16] to obtain a fragment-like subset of compounds from the library. These criteria include the following: MW <300 Da, total number of H-bond donors 3, total number of H-bond acceptors 3, clog P 3. Additionally, we excluded compounds with a total polar surface area (TPSA) 120 . Although it is more common to filter compounds with TPSA 60 , we loosened this criteria to get additional fragments for screening. The screening compounds were docked into the phosphatase binding site of the X-ray structure of sEH available from the Protein Data Bank (PDB code: 1ZD3) using MOE software suite 2009.11 (Chemical Computing Group, Montreal, Canada). The molecules were sorted according to the docking score, and the distribution of the docking scores is shown in Figure 1. We considered compounds with docking scores higher than the 99th percentile of the compound library as potentially interesting for further evaluation, which gave 60 fragments for further evaluation. The binding modes of these 60 compounds were visually inspected. Although we did not filter the database for metal chelating groups to ensure high structural diversity, we paid special attention to the possible interaction of the fragment with the Mg + ion located at the active site of sEH phosphatase. Figure 2 a displays the binding mode of compound 1 bound to the active site proposed by the docking software, which displays the required interactions. From the initial subset of 60 compounds, we selected 30 diverse molecular fragments for experimental validation, and then performed STD-NMR experi[a] S. Hahn, J. Achenbach, E. Buscat , F.-M. Klingler, M. Schroeder, K. Meirer, M. Hieke, Prof. Dr. M. Schubert-Zsilavecz, Prof. Dr. D. Steinhilber, Prof. Dr. E. Proschak Institute of Pharmaceutical Chemistry, LiFF/OSF/ZAFES Goethe University, Max-von-Laue Str. 9, 60438 Frankfurt/M (Germany) E-mail : proschak@pharmchem.uni-frankfurt.de [b] J. Heering, Dr. F. Loehr, Prof. Dr. V. Doetsch Institute of Biophysical Chemistry Goethe University, Max-von-Laue Str. 9, 60438 Frankfurt/M (Germany) [c] Dr. E. Barbosa-Sicard, Prof. Dr. I. Fleming Institute of Vascular Signaling, Center for Molecular Medicine Goethe University, Theodor Stern Kai 7, 60596 Frankfurt/M (Germany) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100433.

Design and synthesis of dual modulators of soluble epoxide hydrolase and peroxisome proliferator-activated receptors.

Metabolic syndrome is a complex condition which often requires the use of multiple medications as a treatment. The resulting problems of polypharmacy are increase in side effects, drug-drug interactions, and its high economic cost. Development of multitarget compounds is a promising strategy to avoid the complications arising from administration of multiple drugs. Modulators of peroxisome proliferator-activated receptors (PPARs) are established agents in the treatment of dyslipidaemia, hyperglycaemia, and insulin resistance. Inhibitors of soluble epoxide hydrolase (sEH) are under evaluation for their use in cardiovascular diseases. In the present study, a series of dual sEH/PPAR modulators containing a pyrrole acidic headgroup and a urea pharmacophore were designed, synthesized, and evaluated in vitro using recombinant enzyme and cell-based assays. Compounds with different activity profiles were obtained which could be used in the treatment of metabolic syndrome.

Reprint of “Cell-free production of G protein-coupled receptors for functional and structural studies” [J. Struct. Biol. 158 (2007) 482–493]

G-protein coupled receptors (GPCRs) are key elements in signal transduction pathways of eukaryotic cells and they play central roles in many human diseases. So far, most structural and functional approaches have been limited by the immense difficulties in the production of sufficient amounts of protein samples in conventional expression systems based on living cells. We report the high level production of six different GPCRs in an individual cell-free expression system based on Escherichia coli extracts. The open nature of cell-free systems allows the addition of detergents in order to provide an artificial hydrophobic environment for the reaction. This strategy defines a completely new technique for the production of membrane proteins that can directly associate with detergent micelles upon translation. We demonstrate the efficient overproduction of the human melatonin 1B receptor, the human endothelin B receptor, the human and porcine vasopressin type 2 receptors, the human neuropeptide Y4 receptor and the rat corticotropin releasing factor receptor by cell-free expression. In all cases, the long chain polyoxyethylene detergent Brij78 was found to be highly effective for solubilization and milligram amounts of soluble protein could be generated in less than 24h. Single particle analysis indicated a homogenous distribution of predominantly protein dimers of the cell-free expressed GPCR samples, with dimensions similar to the related rhodopsin. Ligand interaction studies with the endothelin B receptor and a derivative of its peptide ligand ET-1 gave further evidence of a functional folding of the cell-free produced protein.

Camptothecin and its analog SN-38, the active metabolite of irinotecan, inhibit binding of the transcriptional regulator and oncoprotein FUBP1 to its DNA target sequence FUSE

Graphical abstract Figure. No Caption available. ABSTRACT The transcriptional regulator FUSE Binding Protein 1 (FUBP1) is overexpressed in more than 80% of all human hepatocellular carcinomas (HCCs) and other solid tumor entities including prostate and colorectal carcinoma. FUBP1 expression is required for HCC tumor cell expansion, and it functions as an important pro‐proliferative and anti‐apoptotic oncoprotein that binds to the single‐stranded DNA sequence FUSE to regulate the transcription of a variety of target genes. In this study, we screened an FDA‐approved drug library and discovered that the Topoisomerase I (TOP1) inhibitor camptothecin (CPT) and its derivative 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), the active irinotecan metabolite that is used in the clinics in combination with other chemotherapeutics to treat carcinoma, inhibit FUBP1 activity. Both molecules prevent in vitro the binding of FUBP1 to its single‐stranded target DNA FUSE, and they induce deregulation of FUBP1 target genes in HCC cells. Our results suggest the interference with the FUBP1/FUSE interaction as a further molecular mechanism that, in addition to the inactivation of TOP1, may contribute to the therapeutic potential of CPT/SN‐38. Targeting of FUBP1 in HCC therapy with SN‐38/irinotecan could be a particularly interesting option because of the high FUBP1 levels in HCC cells and their dependency on FUBP1 expression.

Design of dual ligands using excessive pharmacophore query alignment

Dual- or multi-target ligands have gained increased attention in the past years due to several advantages, including more simple pharmacokinetic and phamarcodynamic properties compared to a combined application of several drugs. Furthermore multi-target ligands often possess improved efficacy [1]. We present a new approach for the discovery of dual-target ligands using aligned pharmacophore models combined with a shape-based scoring. Starting with two sets of known active compounds for each target, a number of different pharmacophore models is generated and subjected to pairwise graph-based alignment using the Kabsch-Algorithm [2,3]. Since a compound may be able to bind to different targets in different conformations, the algorithm aligns pairs of pharmacophore models sharing the same features which are not necessarily at the exactly same spatial distance. Using the aligned models, a pharmacophore search on a multi-conformation-database is performed to find compounds matching both models. The potentially “dual” ligands are scored by a shape-based comparison with the known active molecules using ShaEP [4]. Using this approach, we performed a prospective fragment-based virtual screening for dual 5-LO/sEH inhibitors. Both enzymes play an important role in the arachidonic acid cascade and are involved in inflammatory processes, pain, cardiovascular diseases and allergic reactions [5,6]. Beside several new selective inhibitors we were able to find a compound inhibiting both enzymes in low micromolar concentrations. The results indicate that the idea of aligned pharmacophore models can be successfully employed for the discovery of dual-target ligands.

Fragment-based identification of multi-target ligands by self-organizing map alignment

In the recent years the prevalent paradigm in drug discovery of „one drug – one target – one disease“, following the assumption that highly selective ligands would avoid unwandted side effects caused by binding to seconday non-theratpeutic targets, got reconsidered. The results of post-genomic and network biology showed that proteins rarely act in isolated systems but rather as a part of a highly connected network [1]. It was further shown that the efficacy of several approved drugs is traced back to the fact that they act on multiple targets [2]. Therefore inhibiting a single target of such a network might not lead to the desired therapeutic effect. These findings lead to a shift towards polypharmacology [3] and the rational design of selective multi-target drugs, which have often improved efficacy [4]. But the design of multi target drugs is still a great challenge in regard of a sufficient activity on each target as well as an adequate pharmacokinetic profile [5]. Early design strategies tried to link the pharmacophors of known inhibitors, however these methods often lead to high molecular weight and low ligand efficiency. We present a new approach based on self-organizing maps [3,6] (SOM) for the identification of multi-target fragments. We describe a workflow that initially identifies multi-target relevant substructures with a combination of maximum common substructure search and the alignment of multiple SOMs. Furthermore, these substructures are trained together with a fragment library on additional SOMs to find new multi-target fragments, validated by saturation transfer difference (STD)-NMR and biochemical assay systems. We used our approach for the identification of new dual-acting inhibitors of 5Lipoxygenase (5-LO) and soluble Epoxide Hydrolase (sEH), both enzymes located in the arachidonic acid cascade and involved in inflammatory processes, pain and cadiovascular diseases.