Synthetic Amine Linkers for Efficient Sortagging.

Enzymatic site-specific bioconjugation techniques, in particular sortase-mediated ligation, are increasingly used to generate conjugated proteins for a wide array of applications. Extension of the utility and practicality of sortagging for diverse purposes is critically dependent on further improvement of the efficiency of sortagging reactions with a wider structural variety of substrates. We present a comprehensive comparative mass spectrometry screening study of synthetic nonpeptidic incoming amine nucleophile substrates of Staphylococcus aureus Sortase A enzyme. We have identified the optimal structural motifs among the chemically diverse set of 452 model primary and secondary amine-containing sortagging substrates, and we demonstrate the utility of representative amine linkers for efficient C-terminal biotinylation of nanobodies.

Synthesis of methotrexate-betulonic acid hybrids and evaluation of their effect on artificial and Caco-2 cell membranes.

An efficient protocol for the synthesis of novel methotrexate-betulonic acid hybrids with a (tert-butoxycarbonylamino)-3,6-dioxa-8-octanamine (Boc-DOOA) linkage has been developed. Reaction of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-betulonamide with methotrexate resulted in a mixture of isomeric conjugates which were separated by column chromatography. Their structures and composition have been fully established by 1H NMR, 13C spectra, FAB mass spectrometry and elemental analysis. The identity of conjugates was confirmed by LC-MS data. Membranotropic properties of the new hybrids were assessed on the basis of their interactions with artificial lipid membranes by differential scanning calorimetry (DSC) method. The ability of the conjugates to penetrate Caco-2 cells is inferior to methotrexate. Probably, this is due to the increasing lipophilicity, the affinity of these hybrid molecules for the lipid bilayer increases, which is confirmed by experiments with artificial membranes.

Impact of ß-perfluoroalkyl substitution of proline on the proteolytic stability of its peptide derivatives.

A series of all stereoisomers of ß-CF3 or ß-C2F5 substituted prolines and their dipeptide derivatives were synthesized. Mouse plasma stability assay was carried out to study the impact of fluoroalkyl substituents on the proteolytic stability of proline-derived peptides. The effect of the (R)-/(S)-configuration at the C-2 atom in combination with electronic and steric effects imposed by fluoroalkyl groups was addressed to rationalize the difference in the half-life stability of diastereomeric ß-CF3-Pro-Gly and ß-C2F5-Pro-Gly derivatives and compared to those of parent (S)-Pro-Gly and (R)-Pro-Gly dipeptides. The steric effect was predominant when the ß-CF3 or ß-C2F5 group was placed properly to create a spatial interference within the pockets of proteases, thereby protecting the substances from degradation (e.g., for cis-isomeric derivatives). Otherwise, a smaller electronic effect accelerating proteolysis was in charge (i.e., for the (2S,3S) isomers).

Strained contacts with the cell membrane may influence ligand affinity to G protein coupled receptors: a case of free fatty acid receptor 1 agonists.

A set of 1,3,4-thiadiazole-2-carboxamides bearing a substituted biphenyl in the amide portion was synthesised and tested for agonistic activity towards free fatty acid receptor 1 (FFA1). The observed activity trends were impossible to rationalised based solely on the docking energy scores of Glide SP. On the contrary, when the phospholipid cell membrane bilayer was reconstructed around FFA1, it became apparent that inactive compounds displayed significant strained contacts with the membrane while for active compounds the strain was noticeably lower. These findings justify using the improved docking protocol for modelling GPCR-ligand interactions which uses the crystal structure of the receptor and a reconstructed portion of a cell membrane.

Exploration of the nitrogen heterocyclic periphery around the core of the advanced FFA1 agonist fasiglifam (TAK-875).

Three types of heterocyclic moieties-piperidines fused to a heteroaromatic moiety-were explored as potential periphery motifs for the pharmacophoric core of fasiglifam (TAK-875), with fasiglifam being the most advanced agonist of free fatty acid receptor 1, a promising target for therapeutic intervention in type 2 diabetes. Several observed structure-activity relationship trends were corroborated by in silico docking results. Balanced selection based on potency and Caco-2 permeability advanced six compounds to cellular efficacy tests (glucose-stimulated insulin secretion in rat insulinoma INS1E cells). This led to the nomination of compound 16a (LK1408, 3-[4-({4-[(3-{[(2-fluorobenzyl)oxy]methyl}-1-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl]benzyl}oxy)phenyl]propanoic acid hydrochloride) as the lead for further development.

Small-Molecule Chemical Knockdown of MuRF1 in Melanoma Bearing Mice Attenuates Tumor Cachexia Associated Myopathy.

: Patients with malignant tumors frequently suffer during disease progression from a syndrome referred to as cancer cachexia (CaCax): CaCax includes skeletal muscle atrophy and weakness, loss of bodyweight, and fat tissues. Currently, there are no FDA (Food and Drug Administration) approved treatments available for CaCax. Here, we studied skeletal muscle atrophy and dysfunction in a murine CaCax model by injecting B16F10 melanoma cells into mouse thighs and followed mice during melanoma outgrowth. Skeletal muscles developed progressive weakness as detected by wire hang tests (WHTs) during days 13-23. Individual muscles analyzed at day 24 had atrophy, mitochondrial dysfunction, augmented metabolic reactive oxygen species (ROS) stress, and a catabolically activated ubiquitin proteasome system (UPS), including upregulated MuRF1. Accordingly, we tested as an experimental intervention of recently identified small molecules, Myomed-205 and -946, that inhibit MuRF1 activity and MuRF1/MuRF2 expression. Results indicate that MuRF1 inhibitor fed attenuated induction of MuRF1 in tumor stressed muscles. In addition, the compounds augmented muscle performance in WHTs and attenuated muscle weight loss. Myomed-205 and -946 also rescued citrate synthase and complex-1 activities in tumor-stressed muscles, possibly suggesting that mitochondrial-metabolic and muscle wasting effects in this CaCax model are mechanistically connected. Inhibition of MuRF1 during tumor cachexia may represent a suitable strategy to attenuate skeletal muscle atrophy and dysfunction.

Synthesis, biological evaluation, and modeling studies of 1,3-disubstituted cyclobutane-containing analogs of combretastatin A4.

With the aim of circumventing the adverse cis/trans-isomerization of combretastatin A4 (CA4), a naturally occurring tumor-vascular disrupting agent, we designed novel CA4 analogs bearing 1,3-cyclobutane moiety instead of the cis-stilbene unit of the parent compound. The corresponding cis and trans cyclobutane-containing derivatives were prepared as pure diastereomers. The structure of the target compounds was confirmed by X-ray diffraction study. The title compounds were evaluated for their cytotoxic properties in human cancer cell lines HepG2 (hepatocarcinoma) and SK-N-DZ (neuroblastoma), and the overall activity was found in micromolar range. Molecular docking studies and molecular dynamics simulation within the colchicine binding site of tubulin were in good agreement with the obtained cytotoxicity data.

Exploring bulky natural and natural-like periphery in the design of p-(benzyloxy)phenylpropionic acid agonists of free fatty acid receptor 1 (GPR40).

Six derivatives of 3-phenylpropionic acid bearing various natural and natural-like, spatially defined peripheral motifs have been synthesized and evaluated in vitro for free fatty acid receptor 1 (FFA1) activation. Two frontrunner compounds (bearing a bornyl and cytosine groups) were evaluated in an oral glucose tolerance test in mice where both demonstrated the ability to sustain blood glucose levels following a glucose challenge. The bornyl compound displayed a somewhat superior, dose-dependent efficacy and, therefore, can be regarded as a lead compounds for further development as a therapeutic agent for type 2 diabetes mellitus. Its high affinity to FFA1 was rationalized by docking experiments.

Screening of benzenesulfonamide in combination with chemically diverse fragments against carbonic anhydrase by differential scanning fluorimetry.

The differential scanning fluorimetry (DSF) screening of 5.692 fragments in combination with benzenesulfonamide (BSA) against bovine carbonic anhydrase (bCA) delivered >100 hits that either caused, on their own, a significant thermal shift (ΔTm, °C) in the protein melting temperature or significantly influenced the thermal shift observed for BSA alone. Three hits based on 1,2,3-triazole moiety represent the periphery of the recently reported potent inhibitors of hCA II, IX and XII which were efficacious in vivo. Such a re-discovery of suitable BSA periphery essentially validates the new fragment-based approach to the discovery of future CAIs. Structures of other validated fragment hits are reported.

Further validation of strecker-type α-aminonitriles as a new class of potent human carbonic anhydrase II inhibitors: hit expansion within the public domain using differential scanning fluorimetry leads to chemotype refinement.

Testing of an expanded, 800-compound set of analogues of the earlier described Strecker-type α-aminonitriles (selected from publicly available Enamine Ltd. Screening Collection) in thermal shift assay against bovine carbonic anhydrase (bCA) led to further validation of this new class of inhibitors and identification a new, refined chemotype represented by inhibitors with 10-improved potency. [Formula: see text].

A prospective compound screening contest identified broader inhibitors for Sirtuin 1.

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

Difluoro-Substituted Bicyclo[1.1.1]pentanes for Medicinal Chemistry: Design, Synthesis, and Characterization.

A practical synthetic approach to the difluoro-substituted bicyclo[1.1.1]pentanes was developed. The key step was an addition of difluorocarbene (:CF2) to electron-rich bicyclo[1.1.0]butanes by the CF3TMS/NaI system. The obtained difluoro-bicyclo[1.1.1]pentanes are suggested to be used as saturated bioisosteres of benzene rings for the purpose of drug discovery projects.

Photochemical In-Flow Synthesis of 2,4-Methanopyrrolidines: Pyrrolidine Analogues with Improved Water Solubility and Reduced Lipophilicity.

A practical synthesis of 2,4-methanopyrrolidines was elaborated. The key synthetic step was an intramolecular photochemical [2 + 2]-cycloaddition of an acrylic acid derivative in flow. In spite of a higher molecular weight, 2,4-methanopyrrolidines were shown to have higher solubility in water and lower lipophilicity than pyrrolidines, important characteristics of bioactive molecules in drug design.

(Chlorosulfonyl)benzenesulfonyl Fluorides-Versatile Building Blocks for Combinatorial Chemistry: Design, Synthesis and Evaluation of a Covalent Inhibitor Library.

Multigram synthesis of (chlorosulfonyl)benzenesulfonyl fluorides is described. Selective modification of these building blocks at the sulfonyl chloride function under parallel synthesis conditions is achieved. It is shown that the reaction scope includes the use of (hetero)aromatic and electron-poor aliphatic amines (e.g., amino nitriles). Utility of the method is demonstrated by preparation of the sulfonyl fluoride library for potential use as covalent fragments, which is demonstrated by a combination of in silico and in vitro screening against trypsin as a model enzyme. As a result, several inhibitors were identified with activity on par with that of the known inhibitor.

Straightforward hit identification approach in fragment-based discovery of bromodomain-containing protein 4 (BRD4) inhibitors.

A combination approach of a fragment screening and "SAR by catalog" was used for the discovery of bromodomain-containing protein 4 (BRD4) inhibitors. Initial screening of 3695-fragment library against bromodomain 1 of BRD4 using thermal shift assay (TSA), followed by initial hit validation, resulted in 73 fragment hits, which were used to construct a follow-up library selected from available screening collection. Additionally, analogs of inactive fragments, as well as a set of randomly selected compounds were also prepared (3 × 3200 compounds in total). Screening of the resulting sets using TSA, followed by re-testing at several concentrations, counter-screen, and TR-FRET assay resulted in 18 confirmed hits. Compounds derived from the initial fragment set showed better hit rate as compared to the other two sets. Finally, building dose-response curves revealed three compounds with IC50 = 1.9-7.4 µM. For these compounds, binding sites and conformations in the BRD4 (4UYD) have been determined by docking.

Synthesis of Multifunctional Spirocyclic Azetidines and Their Application in Drug Discovery.

The synthesis of multifunctional spirocycles was achieved from common cyclic carboxylic acids (cyclobutane carboxylate, cyclopentane carboxylate, l-proline, etc.). The whole sequence included only two chemical steps-synthesis of azetidinones, and reduction into azetidines. The obtained spirocyclic amino acids were incorporated into a structure of the known anesthetic drug Bupivacaine. The obtained analogues were more active and less toxic than the original drug. We believe that this discovery will lead to a wide use of spirocyclic building blocks in drug discovery in the near future.

Photochemical Synthesis of 2-Azabicyclo[3.2.0]heptanes: Advanced Building Blocks for Drug Discovery. Synthesis of 2,3-Ethanoproline.

Intramolecular photochemical [2 + 2]-cyclization of acetophenone enamides gave 2-azabicyclo[3.2.0]heptanes, advanced building blocks for drug discovery. Synthesis of a conformationally restricted analogue of proline, 2,3-ethanoproline, was performed.

An iterative compound screening contest method for identifying target protein inhibitors using the tyrosine-protein kinase Yes.

We propose a new iterative screening contest method to identify target protein inhibitors. After conducting a compound screening contest in 2014, we report results acquired from a contest held in 2015 in this study. Our aims were to identify target enzyme inhibitors and to benchmark a variety of computer-aided drug discovery methods under identical experimental conditions. In both contests, we employed the tyrosine-protein kinase Yes as an example target protein. Participating groups virtually screened possible inhibitors from a library containing 2.4 million compounds. Compounds were ranked based on functional scores obtained using their respective methods, and the top 181 compounds from each group were selected. Our results from the 2015 contest show an improved hit rate when compared to results from the 2014 contest. In addition, we have successfully identified a statistically-warranted method for identifying target inhibitors. Quantitative analysis of the most successful method gave additional insights into important characteristics of the method used.

Continued SAR exploration of 1,2,4-thiadiazole-containing scaffolds in the design of free fatty acid receptor 1 (GPR40) agonists.

An earlier reported series of 1,2,4-thiadiazole-based agonists of FFA1 (GPR40) was evolved into two structurally distinct series of compounds. One of the series (structurally related to known FFA1 agonist GW9508) displayed low micromolar potency while the other (representing a truncated version of the earlier reported potent FFA1 agonists) was, surprisingly, found to be devoid of agonist potency. In silico docking of representative compounds into the crystal structure of FFA1 revealed possible structural grounds for the observed SAR.

Photochemical Synthesis of 3-Azabicyclo[3.2.0]heptanes: Advanced Building Blocks for Drug Discovery.

We have developed a rapid two-step synthesis of substituted 3-azabicyclo[3.2.0]heptanes which are attractive building blocks for drug discovery. This new method utilizes very common chemicals, benzaldehyde, allylamine, and cinnamic acid, via intramolectular [2+2]-photochemical cyclization.