Franca-Maria Klingler

Approved Drugs Containing Thiols as Inhibitors of Metallo-β-lactamases: Strategy To Combat Multidrug-Resistant Bacteria.

Resistance to β-lactam antibiotics can be mediated by metallo-β-lactamase enzymes (MBLs). An MBL inhibitor could restore the effectiveness of β-lactams. We report on the evaluation of approved thiol-containing drugs as inhibitors of NDM-1, VIM-1, and IMP-7. Drugs were assessed by a novel assay using a purchasable fluorescent substrate and thermal shift. Best compounds were tested in antimicrobial susceptibility assay. Using these orthogonal screening methods, we identified drugs that restored the activity of imipenem.

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.

Exploring the Chemical Space of Multitarget Ligands Using Aligned Self-Organizing Maps

Design of multitarget drugs and polypharmacological compounds has become popular during the past decade. However, the main approach to design such compounds is to link two selective ligands via a flexible linker. Although such chimeric ligands often have reasonable potency in vitro, the in vivo efficacy is low due to high molecular weight, low ligand efficiency, and poor pharmacokinetic profile. We developed an unprecedented in silico approach for fragment-based design of multitarget ligands. It relies on superposition of the chemical spaces related to the affinity on single targets represented by self-organizing maps. We used this approach for screening of molecular fragments, which bind to the enzymes 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). Using STD-NMR and activity-based assays, we were able to identify fragments binding to both targets. Furthermore, we were able to expand one of the fragments to a potent dual inhibitor bearing a reasonable molecular weight (MW = 446) and high affinity to both targets (IC50 of 0.03 μM toward 5-LO and 0.17 μM toward sEH).

From a multipotent stilbene to soluble epoxide hydrolase inhibitors with antiproliferative properties.

Inspired by nature: Natural product isopropylstilbene was identified as an inhibitor of soluble epoxide hydrolase exhibiting antiproliferative properties. Following the natural product inspired design approach, a library of (E)-styryl-1H-benzo[d]imidazoles was synthesized and evaluated with recombinant enzyme and on several cancer cell lines.

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.

NSAIDs Ibuprofen, Indometacin, and Diclofenac do not interact with Farnesoid X Receptor.

The nuclear farnesoid X receptor (FXR) is a ligand activated transcription factor and acts as cellular sensor for bile acids. In this role, FXR is a highly important liver protector and FXR inhibition by antagonists or knockout has shown several deleterious effects. A recent report characterized non-steroidal anti-rheumatic drugs (NSAIDs) such as ibuprofen or diclofenac as FXR antagonists and linked hepatotoxic effects of these drugs with antagonistic activity on FXR. Since this would guide a way to develop safer anti-inflammatory agents by sparing FXR, we intended to further characterize the reported antagonistic activity and intensively investigated ibuprofen, indometacin and diclofenac. However, we conclude that these agents do not interact with FXR and that the reported reduced FXR signaling induced by CDCA in presence of NSAIDs is merely a consequence than a cause of hepatotoxicity.

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.

Probing metallo-β-lactamases with molecular fragments identified by consensus docking

Bacterial resistance mediated by metallo-β-lactamases (MBLs) is a major problem for the treatment of infections. An MBL inhibitor could restore the potency of β-lactam antibiotics. Fragment-based design might deliver valuable starting points for the discovery of novel MBL inhibitors. In this study, we chose an in silico approach to search for fragments able to bind and inhibit NDM-1, VIM-1, and IMP-7. We used consensus docking to identify low molecular weight compounds from a commercially available library. Most promising compounds were evaluated in a sensitive fluorescence-based activity assay and by the orthogonal biophysical technique saturation transfer difference (STD)-NMR. (1)H-(15)N chemical shift perturbation NMR was used to confirm the reversible binding and measure the dissociation constant of the most promising compound qualifying it as a high-quality starting point for further optimization.

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.