Drug-like Compound Libraries Research Articles

CROSSBETA STABLE Aβ OLIGOMERS: DISEASE-RELEVANT DRUG TARGETS AND VERSATILE REFERENCE STANDARDS IN BIOMARKER ASSAYS IN AD

There is accumulating evidence that soluble amyloid beta (Aβ) oligomers, rather than the amyloid fibrils, are the principal pathogenic species in Alzheimer disease (AD). Because they exhibit acute synaptic toxicity and induce neurodegenerative processes, and because the Aβ oligomer levels in cerebrospinal fluid appear to correlate with disease severity and progression, these species constitute an optimal drug target and possible useful diagnostic marker in early AD. Unfortunately, the instability and limited characterization of oligomer preparations have seriously impeded research and development in this field. Crossbeta has addressed this problem by developing a technology to reproducibly generate stable soluble Aß1–42 oligomers that are devoid of monomeric or fibrillar Aß and show biophysical properties indistinguishable from the native species. Furthermore, Crossbeta oligomers demonstrate typical functionality of pathological Aβ species such as neurotoxicity, in vivo LTP depression and induction of inflammatory response. The availability of the stable oligomers opens previously inaccessible avenues for R&D. For example, it enabled high throughput screening of drug-like compound libraries. Derived from a 100k compound screening effort, small molecule CBB68 was identified and shown to neutralize oligomer-induced neurotoxicity in rat primary neurons and to rescue the synaptic deficit induced by Aβ oligomers in vivo as well as in vitro. In particular, the in vivo LTP assay demonstrated the efficacy and brain penetration of CBB68 after intravenous administration. In another application, Crossbeta's oligomers have also been used to select shark antibodies that specifically recognize a conformational oligomeric epitope and bind to Aß1–42 oligomers with high affinity (sub-nM), but not to the monomers or fibrils. This antibody provides potential to selectively quantify Aß1–42 oligomers in patient material as part of a high sensitivity biomarker assay together with the stable Crossbeta oligomers as calibrator. Furthermore, the oligomers are excellent research tools, e.g. in disease-relevant bioassays. We show and conclude that the stable oligomers enable multiple applications and constitute optimum drug targets.

Abstract B25: “TargetDBR”—A DNA repair drug and target discovery collaboration: Exploiting synthetic lethal, high content, and functional cellular reporter assays to accelerate DNA repair targeted drug discovery

Abstract Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Inhibition of DSB repair sensitizes cells to these therapies. Mutations have been reported in nearly every DNA repair pathway and these pathways often exhibit redundancy. Inhibition of functional repair factors can induce synthetic lethality in repair-deficient tumors even in the absence of exogenous DNA damage and whilst sparing healthy tissue. We demonstrate a compound and target discovery platform comprising the integration of isogenic cell line screening, high content repair foci assays and relative DSB repair pathway reporting cells, along with chemical biology and medicinal chemistry. Two diverse drug-like compound library screens have been conducted. Screening for synthetic lethality with deficiency in FANCD2 and BRCA2 led to a hit series broadly active against a range of homologous recombination (HR) repair deficiencies, which is now undergoing medicinal chemistry optimization and target deconvolution studies. Outcome of these initial chemoproteomic target pulldown experiments will be presented. A second cellular screen, (50k compounds) for inhibition of BRCA1 and Rad51 foci formation following radiomimetic drug induced DNA damage, has led to identification of compounds that inhibit damage response after ionizing radiation and selectively inhibit HR repair. Focusing on therapeutic targeting of proliferating tumor cells, our research platform has enabled novel hit compound identification, mechanistic profiling, hit-to-lead optimization chemistry and proof of concept efficacy in in vitro tumor models. Effects on tumor cell clonogenic survival and sensitization towards chemotherapeutics and ionizing radiation will be presented, along with comparisons to known DNA repair inhibitors that demonstrate these compounds' differentiated mechanisms of action. TargetDBR has created a technology platform focused on DNA repair inhibitor discovery and has identified differentiated hits and lead series with potential for development and opportunity to meet the need for new druggable targets in oncology. Citation Format: Jonathan J. Hollick, Laura Abriola, Francoise Bono, Denise Hegan, Pamela Klingbeil, Yanfeng Liu, Ranjini Sundaram, Yulia V. Surovtseva, Mark Whittaker, Ranjit S. Bindra, Peter M. Glazer. “TargetDBR”—A DNA repair drug and target discovery collaboration: Exploiting synthetic lethal, high content, and functional cellular reporter assays to accelerate DNA repair targeted drug discovery [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B25.

The selectivity and promiscuity of brain-neuroregenerative inhibitors between ROCK1 and ROCK2 isoforms: An integration of SB-QSSR modelling, QM/MM analysis and in vitro kinase assay

The Rho-associated kinases (ROCKs) have long been recognized as an attractive therapeutic target for various neurological diseases; selective inhibition of ROCK1 and ROCK2 isoforms would result in distinct biological effects on neurogenesis, neuroplasticity and neuroregeneration after brain surgery and traumatic brain injury. However, the discovery and design of isoform-selective inhibitors remain a great challenge due to the high conservation and similarity between the kinase domains of ROCK1 and ROCK2. Here, a structure-based quantitative structure–selectivity relationship (SB-QSSR) approach was used to correlate experimentally measured selectivity with the difference in inhibitor binding to the two kinase isoforms. The resulting regression models were examined rigorously through both internal cross-validation and external blind validation; a nonlinear predictor was found to have high fitting stability and strong generalization ability, which was then employed to perform virtual screening against a structurally diverse, drug-like compound library. Consequently, five and seven hits were identified as promising candidates of 1-o-2 and 2-o-1 selective inhibitors, respectively, from which seven purchasable compounds were tested in vitro using a standard kinase assay protocol to determine their inhibitory activity against and selectivity between ROCK1 and ROCK2. The structural basis, energetic property and biological implication underlying inhibitor selectivity and promiscuity were also investigated systematically using a hybrid quantum mechanics/molecular mechanics (QM/MM) scheme.

An Automated Microwave-Assisted Synthesis Purification System for Rapid Generation of Compound Libraries.

A novel methodology for the synthesis and purification of drug-like compound libraries has been developed through the use of a microwave reactor with an integrated high-performance liquid chromatography-mass spectrometry (HPLC-MS) system. The strategy uses a fully automated synthesizer with a microwave as energy source and robotic components for weighing and dispensing of solid reagents, handling liquid reagents, capper/crimper of microwave reaction tube assemblies, and transportation. Crude reaction products were filtered through solid-phase extraction cartridges and injected directly onto a reverse-phase chromatography column via an injection valve. For multistep synthesis, crude products were passed through scavenger resins and reintroduced for subsequent reactions. All synthetic and purification steps were conducted under full automation with no handling or isolation of intermediates, to afford the desired purified products. This approach opens the way to highly efficient generation of drug-like compounds as part of a lead discovery strategy or within a lead optimization program.

Discovery of novel new Delhi metallo-β-lactamases-1 inhibitors by multistep virtual screening.

The emergence of NDM-1 containing multi-antibiotic resistant "Superbugs" necessitates the needs of developing of novel NDM-1inhibitors. In this study, we report the discovery of novel NDM-1 inhibitors by multi-step virtual screening. From a 2,800,000 virtual drug-like compound library selected from the ZINC database, we generated a focused NDM-1 inhibitor library containing 298 compounds of which 44 chemical compounds were purchased and evaluated experimentally for their ability to inhibit NDM-1 in vitro. Three novel NDM-1 inhibitors with micromolar IC50 values were validated. The most potent inhibitor, VNI-41, inhibited NDM-1 with an IC50 of 29.6 ± 1.3 μM. Molecular dynamic simulation revealed that VNI-41 interacted extensively with the active site. In particular, the sulfonamide group of VNI-41 interacts directly with the metal ion Zn1 that is critical for the catalysis. These results demonstrate the feasibility of applying virtual screening methodologies in identifying novel inhibitors for NDM-1, a metallo-β-lactamase with a malleable active site and provide a mechanism base for rational design of NDM-1 inhibitors using sulfonamide as a functional scaffold.

Proteome scale comparative modeling for conserved drug and vaccine targets identification in Corynebacterium pseudotuberculosis

Corynebacterium pseudotuberculosis (Cp) is a pathogenic bacterium that causes caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous to a broad spectrum of hosts, including ruminants, thereby threatening economic and dairy industries worldwide. Currently there is no effective drug or vaccine available against Cp. To identify new targets, we adopted a novel integrative strategy, which began with the prediction of the modelome (tridimensional protein structures for the proteome of an organism, generated through comparative modeling) for 15 previously sequenced C. pseudotuberculosis strains. This pan-modelomics approach identified a set of 331 conserved proteins having 95-100% intra-species sequence similarity. Next, we combined subtractive proteomics and modelomics to reveal a set of 10 Cp proteins, which may be essential for the bacteria. Of these, 4 proteins (tcsR, mtrA, nrdI, and ispH) were essential and non-host homologs (considering man, horse, cow and sheep as hosts) and satisfied all criteria of being putative targets. Additionally, we subjected these 4 proteins to virtual screening of a drug-like compound library. In all cases, molecules predicted to form favorable interactions and which showed high complementarity to the target were found among the top ranking compounds. The remaining 6 essential proteins (adk, gapA, glyA, fumC, gnd, and aspA) have homologs in the host proteomes. Their active site cavities were compared to the respective cavities in host proteins. We propose that some of these proteins can be selectively targeted using structure-based drug design approaches (SBDD). Our results facilitate the selection of C. pseudotuberculosis putative proteins for developing broad-spectrum novel drugs and vaccines. A few of the targets identified here have been validated in other microorganisms, suggesting that our modelome strategy is effective and can also be applicable to other pathogens.

Abstract 1635: Discovery of novel SMAC mimetics as selective IAP inhibitors

Abstract Inhibitor of apoptosis (IAP) proteins are widely considered as promising cancer drug targets, especially for drug-resistant tumors. Mimicking the IAP-binding motif of second mitochondria-derived activator of caspases (SMAC) is a rational strategy to design potential IAP inhibitors. In this report, we used the bioactive conformation of AVPI tetrapeptide in the N-terminus of SMAC as a template and performed a shape-based virtual screening against a drug-like compound library to identify novel IAP inhibitors. Top hits were subsequently docked to available IAP crystal structures as a secondary screening followed by validation using in vitro biological assays. Four novel hit compounds were identified to potently inhibit cell growth in two human melanoma (A375 and M14) and two human prostate (PC-3 and DU145) cancer cell lines. The best compound, UC-112, has IC50 values ranging from 0.7 to 3.4 µM. UC-112 also potently inhibits the growth of P-glycoprotein (P-gp) overexpressed multidrug-resistant cancer cells, strongly activates capase-3/7 and caspase-9 activities, and selectively down-regulates survivin level at a concentration as low as 1 µM. Co-incubation of UC-112 with a known proteasome inhibitor (MG132) rescued survivin inhibition, consistent with the anticipated mechanism of action for UC-112. As a single agent, UC-112 strongly inhibits tumor growth and reduces both XIAP and survivin levels in an A375 human melanoma xenograft model. Overall, our study identified novel scaffolds, especially UC-112, as new platforms on which potent and selective IAP antagonists can be developed. Citation Format: Jin Wang, Wei Li. Discovery of novel SMAC mimetics as selective IAP inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1635. doi:10.1158/1538-7445.AM2014-1635

Identification of Novel Inhibitors of Daboia russelli Phospholipase A2 Using the Combined Pharmacophore Modeling Approach

Crystal structures available for Daboia russelli venom PLA(2) confirm that it undergoes dimerization with asymmetry and hence difference in the conformation of active site of the two subunits. The active site of subunit A is open and that of subunit B is closed. Pharmacophore models were generated based on the interaction of different types of inhibitors with their preferred subsites in the active site of subunit A. Particularly, the features responsible for recognizing subsites 1-3 and those of subsites 4-6 were combined as these two are involving in inflammation and anticoagulation processes, respectively. Pharmacophore model was edited to make the geometry suitable for the active site of both the subunits A and B. Final model is validated and subjected for screening a library of druglike compounds. Eight compounds were shortlisted and subjected for molecular docking and dynamics simulation to assess their binding mode with both the subunits. Based on the hydrophobic interactions and binding free energy, four compounds were selected for further biochemical assay. The overall results suggest that two compounds can bind both the subunits of PLA(2) of Daboia russelli venom in spite of its aggregated form and other two inhibit structurally very similar Naja naja PLA(2).

An exhaustive yet simple virtual screening campaign against Sortase A from multiple drug resistant Staphylococcus aureus.

Methicillin resistant Staphylococcus aureus (MRSA) is one of the challenging bacterial pathogen due to its acquired resistance to the β lactam antibiotics. The Sortase A is an enzyme of Gram-positive bacteria including S. aureus to anchor surface proteins to the cell wall. Sortase A is well studied enzyme and considered as the drug target against MRSA. Sortase A plays active role in anchoring the virulence proteins on the cell wall of the Gram-positive bacteria. The inhibition of Sortase A activity results in the separation of S. aureus from the host cells and ultimately alleviation of the infection. Here, we adapted a structure-based virtual screening protocol which helped in identification of novel potential inhibitors of Sortase A. The protocol involved the docking of a chemical library of druglike compounds with the Sortase A binding site represented by multiple crystal structures. The compounds were ranked by multiple scoring functions and shortlisted for future experimental screening. The method resulted in shortlisting of three compounds as potential novel inhibitors of Sortase A out of a large chemical library. The high rankings of shortlisted compounds estimated by multiple scoring functions showed their binding potential with Sortase A. The results are proved to be a simple yet efficient choice of structure-based virtual screening. The identified compounds are druglike and show high rankings among all set protocols of the virtual screening. We hope that the study would eventually help to expedite the discovery of novel drug candidates against MRSA.

Discovery of Novel Second Mitochondria-Derived Activator of Caspase Mimetics as Selective Inhibitor of Apoptosis Protein Inhibitors

Inhibitor of apoptosis (IAP) proteins are widely considered as promising cancer drug targets, especially for drug-resistant tumors. Mimicking the IAP-binding motif of second mitochondria-derived activator of caspases (SMAC) is a rational strategy to design potential IAP inhibitors. In this report, we used the bioactive conformation of AVPI tetrapeptide in the N terminus of SMAC as a template and performed a shape-based virtual screening against a drug-like compound library to identify novel IAP inhibitors. Top hits were subsequently docked to available IAP crystal structures as a secondary screening followed by validation using in vitro biologic assays. Four novel hit compounds were identified to potently inhibit cell growth in two human melanoma (A375 and M14) and two human prostate (PC-3 and DU145) cancer cell lines. The best compound, UC-112 [5-((benzyloxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol], has IC50 values ranging from 0.7 to 3.4 µM. UC-112 also potently inhibits the growth of P-glycoprotein (P-gp)-overexpressed multidrug-resistant cancer cells, strongly activates caspase-3/7 and caspase-9 activities, and selectively downregulates survivin level at a concentration as low as 1 µM. Coincubation of UC-112 with a known proteasome inhibitor Z-Leu-Leu-Leu-CHO (MG-132) rescued survivin inhibition, consistent with the anticipated mechanism of action for UC-112. As a single agent, UC-112 strongly inhibits tumor growth and reduces both X chromosome-linked IAP and survivin levels in an A375 human melanoma xenograft model in vivo. Overall, our study identified novel scaffolds, especially UC-112, as new platforms on which potent and selective IAP antagonists can be developed.

Targeting protein–protein interactions to rescue Δf508‐cftr: a novel corrector approach to treat cystic fibrosis

Cystic fibrosis (CF) is one of the most prevalent life‐threatening autosomal recessive disorders in the Western World, with an estimated incidence of 1 in 2000–3000 Caucasian newborns and a median age of survival of 40 years. The first report of CF as a distinct pathology was published in 1938 by Dorothy Andersen, a physician at the Babies & Children's Hospital of Columbia University, who differentiated its diagnosis from celiac disease in the pancreas (Andersen, 1938). This seminal description was followed by other contributions that linked CF to altered Cl− permeability in epithelial cells in the gastrointestinal and hepato‐billiary systems, respiratory tract, reproductive system and sweat glands (Kreindler, 2010); and culminated with the discovery of the gene responsible of the disease, referred to as the cystic fibrosis transmembrane regulator (CFTR) (Riordan et al, 1989). The CFTR is a cAMP‐regulated chloride and bicarbonate channel that controls anion conductance in epithelial cells located in mucosal surfaces. Functional alteration of CFTR leads to the accumulation of mucous secretions that cannot be cleared favouring inflammation and infection. Cumulative evidence shows a strong correlation between the severity of the CF phenotype and the degree of CFTR dysfunction, being stronger for those patients where the channel function is absent (Kreindler, 2010). The CF phenotype is caused by more than 1000 mutations of the CFTR gene including missense, such as R117H or G551D that significantly reduce channel activity, and nonsense like G542X, R553X or W1282X, which abrogate protein expression (Kreindler, 2010). However, the most prevalent mutation in CF patients (70%) is the in‐frame deletion of Phenylalanine at position 508 (ΔF508‐CFTR) (Kreindler, 2010; Rowe & Verkman, 2013). This deletion produces a partially unfolded CFTR protein that is retained in the endoplasmic reticulum and diverted to degradation by the proteosome, resulting in a virtually absent CFTR activity …

Inhibition of Histo-blood Group Antigen Binding as a Novel Strategy to Block Norovirus Infections

Noroviruses (NoVs) are the most important viral pathogens that cause epidemic acute gastroenteritis. NoVs recognize human histo-blood group antigens (HBGAs) as receptors or attachment factors. The elucidation of crystal structures of the HBGA-binding interfaces of a number of human NoVs representing different HBGA binding patterns opens a new strategy for the development of antiviral compounds against NoVs through rational drug design and computer-aided virtual screening methods. In this study, docking simulations and virtual screening were used to identify hit compounds targeting the A and B antigens binding sites on the surface of the capsid P protein of a GII.4 NoV (VA387). Following validation by re-docking of the A and B ligands, these structural models and AutoDock suite of programs were used to screen a large drug-like compound library (derived from ZINC library) for inhibitors blocking GII.4 binding to HBGAs. After screening >2 million compounds using multistage protocol, 160 hit compounds with best predicted binding affinities and representing a number of distinct chemical classes have been selected for subsequent experimental validation. Twenty of the 160 compounds were found to be able to block the VA387 P dimers binding to the A and/or B HBGAs at an IC50<40.0 µM, with top 5 compounds blocking the HBGA binding at an IC50<10.0 µM in both oligosaccharide- and saliva-based blocking assays. Interestingly, 4 of the top-5 compounds shared the basic structure of cyclopenta [a] dimethyl phenanthren, indicating a promising structural template for further improvement by rational design.

SVM Based Descriptor Selection and Classification of Neurodegenerative Disease Drugs for Pharmacological Modeling.

Systems pharmacological modeling of drug mode of action for the next generation of multitarget drugs may open new routes for drug design and discovery. Computational methods are widely used in this context amongst which support vector machines (SVM) have proven successful in addressing the challenge of classifying drugs with similar features. We have applied a variety of such SVM-based approaches, namely SVM-based recursive feature elimination (SVM-RFE). We use the approach to predict the pharmacological properties of drugs widely used against complex neurodegenerative disorders (NDD) and to build an in-silico computational model for the binary classification of NDD drugs from other drugs. Application of an SVM-RFE model to a set of drugs successfully classified NDD drugs from non-NDD drugs and resulted in overall accuracy of ∼80 % with 10 fold cross validation using 40 top ranked molecular descriptors selected out of total 314 descriptors. Moreover, SVM-RFE method outperformed linear discriminant analysis (LDA) based feature selection and classification. The model reduced the multidimensional descriptors space of drugs dramatically and predicted NDD drugs with high accuracy, while avoiding over fitting. Based on these results, NDD-specific focused libraries of drug-like compounds can be designed and existing NDD-specific drugs can be characterized by a well-characterized set of molecular descriptors.

Development of Chemical Compound Libraries for In Silico Drug Screening

Chemical compound libraries are the basic database for virtual (in silico) drug screening, and the number of entries has reached 20 million. Many drug-like compound libraries for virtual drug screening have been developed and released. In this review, the process of constructing a database for virtual screening is reviewed, and several popular databases are introduced. Several kinds of focused libraries have been developed. The author has developed databases for metalloproteases, and the details of the libraries are described. The library for metalloproteases was developed by improving the generation of the dominant-ion forms. For instance, the SH group is treated as S- in this library while all SH groups are protonated in the conventional libraries. In addition, metal complexes were examined as new candidates of drug-like compounds. Finally, a method for generating chemical space is introduced, and the diversity of compound libraries is discussed.

HALO384: A Halo-Based Potency Prediction Algorithm for High-Throughput Detection of Antimicrobial Agents

A high-throughput (HT) agar-based halo assay is described, which allows for rapid screening of chemical libraries for bioactivity in microorganisms such as yeast and bacteria. A pattern recognition algorithm was developed to identify halo-like shapes in plate reader optical density (OD) measurements. The authors find that the total growth inhibition within a detected halo provides an accurate estimate of a compound's potency measured in terms of its EC(50). The new halo recognition method performs significantly better than an earlier method based on single-point OD readings. An assay based on the halo algorithm was used to screen a 21,120-member library of drug-like compounds in Saccharomyces cerevisiae, leading to the identification of novel bioactive scaffolds containing derivatives of varying potencies. The authors also show that the HT halo assay can be performed with the pathogenic bacterium Vibrio cholerae and that liquid culture EC(50) values and halo scores show a good correlation in this organism. These results suggest that the HT halo assay provides a rapid and inexpensive way to screen for bioactivity in multiple microorganisms.

Parallel Synthesis of Bis-heterocyclic Isoxazolylmethyl- and Isoxazolinylmethylpyrazoles

The solution-phase parallel synthesis of a 136-member library of isoxazol(in)e-CH(2)-pyrazoles is described. X-ray crystallographic structure determination verified the regioselectivities of the N-alkylation and nitrile oxide 1,3-dipolar cycloaddition steps. The construction of these pharmaceutically relevant heterocycles on solid support under microwave irradiation is also demonstrated. The resulting library of drug-like compounds has been added to the National Institutes of Health repository (approximately 10 mg of each with >or=90% purity) for pilot-scale biomedical studies with bioassay data available at the National Center for Biotechnology Information PubChem database. A subset of these compounds has been broadly screened by Dow AgroSciences for herbicidal, fungicidal, and insecticidal activity.

A High-Throughput Approach for Identification of Novel General Anesthetics

Anesthetic development has been a largely empirical process. Recently, we described a GABAergic mimetic model system for anesthetic binding, based on apoferritin and an environment-sensitive fluorescent probe. Here, a competition assay based on 1-aminoanthracene and apoferritin has been taken to a high throughput screening level, and validated using the LOPAC1280 library of drug-like compounds. A raw hit rate of ∼15% was reduced through the use of computational filters to yield an overall hit rate of ∼1%. These hits were validated using isothermal titration calorimetry. The success of this initial screen and computational triage provides feasibility to undergo a large scale campaign to discover novel general anesthetics.

Aerobic oxidation of indolomorphinan without the 4,5-epoxy bridge and subsequent rearrangement of the oxidation product to spiroindolinonyl- C-normorphinan derivative

Aerobic oxidation of indolomorphinan 1 without a 4,5-epoxy bridge proceeded in the presence of platinum catalyst to give indoleninomorphinan 2 or quinolono- C-normorphinan 5. The 4-hydroxy group would play an important role in deciding the course of the reaction. Treatment of 2a with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave spiroindolinonyl- C-normorphinan 3a whose structure resembles that of δ opioid receptor agonist spiroindanyloxymorphone (SIOM). Boron trichloride was effective for the reverse reaction from 3a to 2a without side reaction. This practical interconversion method between hydroxyindolenine and spiroindolinone would be useful for the design and construction of drug-like compound libraries. Although the compound 3b was expected to show the selectivity for δ opioid receptor because of the structural resemblance to SIOM, it was rather selective for μ opioid receptor (μ: K i = 0.75 nM; δ: K i = 2.90 nM; κ: K i = 13.4 nM). The result suggests that the slight difference of the spatial location of the benzene rings in these compounds may definitively affect the binding affinity for δ opioid receptor.

How to Efficiently Include Receptor Flexibility During Computational Docking

Target-based drug design uses available 3D structural information of receptor molecules to either dock putative ligand molecules to receptor binding sites or to de-novo design new ligands. In many cases accurate prediction of putative binding geometries requires the appropriate inclusion of conformational flexibility of both the ligand as well as the receptor structure. The problem of appropriate treatment of conformational flexibility during docking is also tightly connected to the improvement of scoring a docked ligand-receptor complex. Highly accurate scoring of a ligand placement is only possible if the complex geometry has been predicted with high precision. Considerable progress has already been achieved in modeling the conformational flexibility of small organic ligands during docking. Although of similar importance, receptor flexibility has not been tackled satisfactorily despite the steady increase in computational power. Especially during virtual screening of large drug-like compound libraries the target structure often is still kept rigid. However, many protein structures undergo local structural changes (side chain or loop motions) as well as global changes in the backbone geometry upon complex formation. In recent years, several promising approaches to efficiently tackle receptor flexibility have been introduced ranging from conformational ensemble methods to explicit inclusion of the most relevant receptor degrees of freedom. Possible applications and future directions on improving flexible docking approaches will be discussed.

Solid-Phase Synthesis of 2-Amino-2-thiazolines by the Cyclization of N-(2-Hydroxyethyl)thioureas Using TFA

The 2-amono-2-thiazolines have recently gained significant interest as a scaffold that is applicable to the development of bioactive compounds such as pronounced antidepressant agents, potent human nitric oxide synthase inhibitors, octopaminergic-agonists, anthelmintics, and anti-inflammatory agents. The solid phase synthesis of small heterocycles have been considerably studied because it can be applied to the rapid generation of diverse libraries of drug-like compounds. Although there are many wellestablished reports on the solution phase synthesis of 2amino-2-thiazolines scaffolds because of their valuable pharmaceutical properties, very recently we first reported a solid phase synthetic method of 2-amino-2-thiazolines from the cyclization of N-(2-hydroxyethyl)thioureas using dicyclohexylcarbodiimide (DCC). The ring closure of N-(2hydroxyethyl)thioureas can furnish different products depending on the reaction conditions and substrates such as S-cyclized, N-cyclized, or O-cyclized products. Resinbound substrates 6 were designed as precursors to generate 2-amino-2-thiazolines by the S-cyclization, which were conveniently prepared from various commercially available aminoalcohols and isothiocyanates (Scheme 1). We wish to report an alternative and more effective way into the solid phase synthesis of 2-amino-2-thiazolines, using the cyclization of N-(2-hydroxyethyl)thioureas. Scheme 2 shows the synthetic route of the 2-amino-2thiazoline scaffold. The first step in solid phase reactions was the coupling of amino alcohol onto an ArgoGel-MBCHO resin via reductive amination, followed by the protection of the free alcohol 3 with tert-butyldimethylsilyl chloride (TBSCl) according to the previous procedures. Treatment of this intermediate with isothiocyanates afforded the thioureas resin 5, and subsequent deprotection of the silylated hydroxy group with tetrabutyl ammonium fluoride in THF yielded resin 6. The intramolecular cyclization of resin 6 using dicyclohexylcarbodiimide (DCC) gave mainly the required S-cyclized 2-amino-2-thiazolines resin 7 as reported. Then we turned to release the N-(2-hydroxyethyl)thioureas from the resin 6, key intermediate in Scheme 2, by the trifluoroacetic acid (TFA) cleavage to determine the loading capacity of the both amino alcohols and isothiocyanates. The desired thiourea was not obtained but the final product 2-amino-2-thiazoline was released in high yield and purity. This is that the cyclization and cleavage reaction in TFA simultaneously occurred at the same step, thereby saving one step procedure. The results using TFA for the cyclization and cleavage are summarized in Table 1. Resin 6 derived from either aliphatic (entry 7a-b) or aryl isothiocyanates (entry 7c-7k) furnished the required Salkylation products, but aminoalcohol was limited to the primary alcohol. In summary, a solid phase synthetic method was developed for the parallel synthesis of 2-amino-2-thiazolines, using the cyclization of N-(2-hydroxyethyl)thioureas and cleavage in TFA. This synthetic methodology is ideally suited for the automated applications, because all the reactions were carried out under ambient conditions.