Janosch Achenbach

Computational tools for polypharmacology and repurposing

Most drugs act on a multitude of targets rather than on one single target. Polypharmacology, an upcoming branch of pharmaceutical science, deals with the recognition of these off-target activities of small chemical compounds. Due to the high amount of data to be processed, application of computational methods is indispensable in this area. This review summarizes the most important in silico approaches for polypharmacology. The described methods comprise network pharmacology, machine learning techniques and chemogenomic approaches. The use of these methods for drug repurposing as a branch of drug discovery and development is discussed. Furthermore, a broad range of prospective applications is summarized to give the reader an overview of possibilities and limitations of the described techniques.

VAMMPIRE: A Matched Molecular Pairs Database for Structure-Based Drug Design and Optimization

Structure-based optimization to improve the affinity of a lead compound is an established approach in drug discovery. Knowledge-based databases holding molecular replacements can be supportive in the optimization process. We introduce a strategy to relate the substitution effect within matched molecular pairs (MMPs) to the atom environment within the cocrystallized protein-ligand complex. Virtually Aligned Matched Molecular Pairs Including Receptor Environment (VAMMPIRE) database and the supplementary web interface ( http://vammpire.pharmchem.uni-frankfurt.de ) provide valuable information for structure-based lead optimization.

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.

Molecular characterization of EP6—A novel imidazo[1,2-a]pyridine based direct 5-lipoxygenase inhibitor

5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade and catalyzes the formation of bioactive leukotrienes (LTs) which are involved in inflammatory diseases and allergic reactions. The pathophysiological effects of LTs are considered to be prevented by 5-LO inhibitors. In this study we present cyclohexyl-[6-methyl-2-(4-morpholin-4-yl-phenyl)-imidazo[1,2-a]pyridin-3-yl]-amine (EP6), a novel imidazo[1,2-a]pyridine based compound and its characterization in several in vitro assays. EP6 suppresses 5-LO activity in intact polymorphonuclear leukocytes with an IC(50) value of 0.16μM and exhibits full inhibitory potency in cell free assays (IC(50) value of 0.05μM for purified 5-LO). The efficacy of EP6 was not affected by the redox tone or the concentration of exogenous AA, characteristic drawbacks known for the class of nonredox-type 5-LO inhibitors. Furthermore, EP6 suppressed 5-LO activity independently of the cell stimulus or the activation pathway of 5-LO contrary to what is known for some nonredox-type inhibitors. Using molecular modeling and site-directed mutagenesis studies, we were able to derive a feasible binding region within the C2-like domain of 5-LO that can serve as a new starting point for optimization and development of new 5-LO inhibitors targeting this site. EP6 has promising effects on cell viability of tumor cells without mutagenic activity. Hence the drug may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer including leukemia.

Investigation of imatinib and other approved drugs as starting points for antidiabetic drug discovery with FXR modulating activity.

A self-organizing map (SOM) is a virtual screening method used for correlation of molecular structure and potential biological activity on a certain target and offers a way to represent multi-dimensional data of large databases in a two-dimensional space. Large databases, for example the DrugBank database, provide information about biological activity and chemical structure of small molecules and are widely used in drug development for identification of new lead structures. The farnesoid X receptor (FXR) is a ligand activated transcription factor involved in key regulation mechanisms within glucose and lipid homeostasis. Although FXR became an established target in drug development for diseases associated with lipid, glucose or hepatic disorders during the last decade, none of the developed compounds have reached later phases of clinical development so far. We used a SOM trained with known FXR ligands to screen the DrugBank database for potential ligands for FXR. In this article, we report the successful identification of six approved drugs out of the Drugbank as FXR modulators (ketoconazole, pentamidine, dobutamine, imatinib, papaverine and montelukast) by using a SOM for screening of the DrugBank database. We show FXR modulation by selected compounds in a full length FXR transactivation assay and modulation of a FXR target gene by imatinib.

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).

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.

Multi-dimensional target profiling of N,4-diaryl-1,3-thiazole-2-amines as potent inhibitors of eicosanoid metabolism.

Eicosanoids like leukotrienes and prostaglandins play a considerable role in inflammation. Produced within the arachidonic acid (AA) cascade, these lipid mediators are involved in the pathogenesis of pain as well as acute and chronic inflammatory diseases like rheumatoid arthritis and asthma. With regard to the lipid cross-talk within the AA pathway, a promising approach for an effective anti-inflammatory therapy is the development of inhibitors targeting more than one enzyme of this cascade. Within this study, thirty N-4-diaryl-1,3-thiazole-2-amine based compounds with different substitution patterns were synthesized and tested in various cell-based assays to investigate their activity and selectivity profile concerning five key enzymes involved in eicosanoid metabolism (5-, 12-, 15-lipoxygenase (LO), cyclooxygenase-1 and -2 (COX-1/-2)). With compound 7, 2-(4-phenyl)thiazol-2-ylamino)phenol (ST-1355), a multi-target ligand targeting all tested enzymes is presented, whereas compound 9, 2-(4-(4-chlorophenyl)thiazol-2-ylamino)phenol (ST-1705), represents a potent and selective 5-LO and COX-2 inhibitor with an IC50 value of 0.9 ± 0.2 μM (5-LO) and a residual activity of 9.1 ± 1.1% at 10 μM (COX-2 product formation). The promising characteristics and the additional non-cytotoxic profile of both compounds reveal new lead structures for the treatment of eicosanoid-mediated diseases.

Identification of novel farnesoid X receptor modulators using a combined ligand- and structure-based virtual screening

A combined ligand- and structure-based virtual screening has been applied to retrieve novel modulators of the farnesoid X receptor (FXR). Four distinct chemotypes exhibiting partial activation of FXR in a reporter gene assay could be identified. The analysis of the preliminary structure–activity relationships yielded a 3-amino-imidazo[1,2-a]pyridine derivative which showed a maximum relative FXR activation of about 14% with an EC50 = 480 nM.

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.