ChemMedChem

Trifluoromethyl Oxetanes: Synthesis and Evaluation as a tert-Butyl Isostere ()
The synthesis of a new trifluoromethyl oxetane was developed utilizing a Corey-Chaykovsky epoxidation/ring-expansion reaction of trifluoromethyl ketones. The reaction was shown to proceed under mild conditions and displays a broad substrate scope. The trifluoromethyl oxetane was also evaluated as a tert-butyl isostere in the context of the γ-secretase modulator (GSM) program. We demonstrate that the trifluoromethyl oxetane-containing GSM has reduced lipophilicity, improved lipophilic efficiency (LipE) and metabolic stability relative to the corresponding tert-butyl GSM analog, thus highlighting several benefits of trifluoromethyl oxetane as a more polar tert-butyl isostere.
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Biophysical Screening of a Focused Library for the Discovery of CYP121 Inhibitors as Novel Antimycobacterials ()
The development of novel antimycobacterial agents against Mycobacterium tuberculosis (Mtb) is urgently required due to the appearance of multi-drug resistance (MDR) combined with a complicated long-term treatment. CYP121 was shown to be a promising novel target for inhibition of mycobacterial growth. In this study, we describe the rational discovery of new CYP121 inhibitors by a systematic screening based on biophysical and microbiological methods. Best hits originating from only one structural class gave first information about molecular motifs required for binding and activity. The initial screening procedure was followed by mode of action studies and further biological characterizations. The results demonstrate a superior antimycobacterial efficacy and a reduced toxicity profile of our frontrunner compound compared to the reference econazole. Due to its low molecular weight, promising biological profile and physicochemical properties this compound displays an excellent starting point for further rational optimization.
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Design and synthesis of terephthalic acid-based histone deacetylase inhibitors with dual stage anti-Plasmodium activity ()
In this work we aimed to develop parasite-selective HDAC inhibitors with activity against the disease-causing asexual blood stages of Plasmodium as well as causal prophylactic and/or transmission blocking properties. We report the design, synthesis and biological testing of a series of 13 terephthalic acid-based histone deacetylase inhibitors. All compounds showed low cytotoxicity against human embryonic kidney (HEK293) cells (IC50 8 - >51 µM), with 11 also having sub-micromolar in vitro activity against drug-sensitive (3D7) and multi-drug resistant (Dd2) asexual blood stage Plasmodium falciparum parasites (IC50 ~ 0.1 - 0.5 μM). A sub-set of compounds were examined for activity against early and late stage P. falciparum gametocytes and P. berghei exo-erythrocytic stage parasites. While only moderate activity was observed against gametocytes (IC50 >2 μM), the most active compound (N1-((3,5-dimethylbenzyl)oxy)-N4-hydroxyterephthal-amide, 1f) showed sub-micro molar activity against P. berghei exo-erythrocytic stages (IC50 0.18 µM) and >270-fold better activity for exo-erythrocytic forms than for HepG2 cells. This, together with asexual stage in vitro potency (IC50 ~ 0.1 μM) and selectivity of this compound versus human cells (SI >450), suggests that 1f may be a valuable starting point for the development of novel antimalarial drug leads with low host cell toxicity and multi-stage anti-plasmodial activity.
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Reducing the Flexibility of Type II Dehydroquinase Enzyme for Inhibition - A Fragment-Based Approach and Molecular Dynamics Simulation Study ()
A multidisciplinary approach was used to identify and optimize a quinazolinedione-based ligand that would reduce the flexibility of the substrate-covering loop (catalytic loop) of the type II dehydroquinase from Helicobacter pylori. This enzyme, which is essential for the survival of this bacterium, is involved in the biosynthesis of the aromatic amino acids. A computer-aided fragment-based protocol (ALTA) was first employed to identify the aromatic fragments able to block the interface pocket that separates two neighbor enzyme subunits and is located at the active site entrance. Chemical modification of its non-aromatic moiety through an olefin cross metathesis and Seebach´s self-reproduction of chirality synthetic principle allowed the development of a quinazolinedione derivative that disables the catalytic loop plasticity, which is essential for the enzyme catalytic cycle. Molecular dynamics simulation studies revealed that the ligand would force the catalytic loop into an inappropriate arrangement for catalysis by strong interactions with the catalytic tyrosine and by expelling the essential arginine out of the active site.
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Application of the Pentafluorosulfanyl Group as a Bioisosteric Replacement ()
The success of fluorinated molecules in drug design has led medicinal chemists to search for new fluorine-containing substituents. A major recently developed group is the pentafluorosulfanyl group. This group is stable under physiological conditions and displays unique physical and chemical properties. Currently, few synthetic methods exist that install the SF5 group, yet efforts to integrate this group into lead optimization continue unabated. Typically, the SF5 group has been used as a replacement for a trifluoromethyl, a tert-butyl, a halogen, or a nitro group. In this review, the use of the SF5 group as a bioisosteric replacement for each of these three functionalities will be compared and contrasted across different groups of biologically active molecules. The organization and presentation of these data are instructive to medicinal chemists considering to design synthetic strategies to access SF5 substituted molecules.
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Development of substrate-derived mechanistic sirtuin inhibitors with potential anti-cancer activity ()
RhoGDIα is a key regulator for Rho-proteins coordinating their GTP/GDP- and membrane/cytosol-cycle. Recently, it was demonstrated by quantitative mass spectrometry that RhoGDIα is heavily targeted by post-translational lysine-acetylation. For one site in its N-terminal domain, namely K52, we reported that acetylation completely switches off RhoGDIα function. Here, we show that K52-acetylated RhoGDIα is specifically deacetylated by the sirtuin deacetylase Sirt2.. We show that acetylation at K52 decelerates cervical cancer cell proliferation, suggesting RhoGDIα acetylation to be a promising therapeutic target. We demonstrate that treatment of cervical cancer cells with a RhoGDIα-derived K52-trifluoroacetylated, mechanism-based peptidic sirtuin inhibitor severely impairs cell proliferation. Finally, we conclude that the potency of mechanism-based sirtuin inhibitors depends on structural features, the substrate-derived amino acid sequence as well as the presence of the acetyl-lysine analog. These data reveal a prospective therapeutic potential of novel substrate-derived mechanistic sirtuin inhibitors.
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Silicon Phthalocyanines Axially Disubstituted with Erlotinib towards Small Molecular Target-Based Photodynamic Therapy ()
Small molecular target-based photodynamic therapy—a promising targeted anticancer strategy—has been developed by the conjugation between zinc (II) phthalocyanine and small molecular target-based anticancer drug. To inhibit the self-aggregation and avoid the isomeride problem of phthalocyanine, two silicon phthalocyanines di-substituted axially with erlotinib have been synthesized and fully characterized. These conjugates exist as a monomeric form in different solvents and the culture media. Cellular experiments showed that these conjugates localize in lysosomes and mitochondria, and keep high photodynamic activities (IC₅₀ is as low as 8 nM under 1.5 J/cm² light dose). Having erlotinib as the targeting moiety, two conjugates exhibited high specificity to the high EGFR expression cancer cells. The different PEG linker length was proven to have an effect on photophysical/photochemical properties and further in vitro phototoxicity.
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Simultaneous Multiple MS Binding Assays addressing D₁ and D₂ Dopamine receptors ()
MS Binding Assays represent a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, however, employ a nonlabelled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer - due to the selectivity of mass spectrometric detection - basically the opportunity to monitor binding of different reporter ligands towards different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays addressing selectively D₁ and D₂ Dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable to quantify both, SCH23390 selectively addressing D₁ receptors and raclopride selectively addressing D₂ receptors, was established therefor and validated. Based thereon simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both, D₁ and D₂ receptors, were performed and analyzed by LC-MS/MS within a single chromatographic cycle. Thus, the present study demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays.
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Structure-guided design of peptides as tools to probe the protein-protein interaction between Cullin-2 and Elongin BC substrate adaptor in Cullin RING E3 ubiquitin ligases ()
Cullin RING E3 ubiquitin ligases (CRLs) are large dynamic multi-subunit complexes that control the fate of many proteins in cells. CRLs constitute attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. Here we describe a structure-guided biophysical approach to probe the protein-protein interaction (PPI) between the Cullin-2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel-Lindau complex (CRL2VHL) using peptides. Two peptides were shown to bind at the targeted binding site on Elongin C, named the "EloC site", with micromolar dissociation constants, providing a starting point for future optimization. Our results suggest ligandability of the EloC binding site to short linear peptides, unveiling the opportunity and challenges to develop small molecules that have the potential to target selectively the Cul2-adaptor PPI within CRLs.
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Characterization of Small Molecule Scaffolds that Bind to the Shigella Type III Secretion System Protein IpaD ()
Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salts sterols. Here, we identified four new small molecule scaffolds that bind to IpaD based on the methylquinoline, pyrrolidin-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small molecule inhibitors of IpaD that could lead to new anti-infectives.
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What do we miss? The detergent Triton-X 100 added to avoid compound aggregation can affect assay results in an unpredictable manner ()
In this study we show that the detergent Triton X-100 being widely used in screening campaigns significantly reduces the binding affinities of some known specific inhibitors of HIV-1 protease and the well-established model protease endothiapepsin in a fluorescence-based assay. Surprisingly, other structurally related inhibitors remain entirely unaffected. In consequence, those compounds that were affected would most likely have been misclassified as unspecific binders although they actually are true positives, hence being considered excellent starting points for the further hit optimization.
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Anticancer gold N-heterocyclic carbene complexes: a comparative in vitro and ex vivo study ()
A series of organometallic Au(I) N-heterocyclic carbene (NHC) complexes was synthesized and characterized on anticancer activity in four human cancer cell lines. The compounds' toxicity in healthy tissue was determined using precision cut kidney slices (PCKS) as a tool to determine the potential selectivity of the Au complexes ex vivo. All evaluated compounds presented cytotoxic activity towards the cancer cells in the nano- or low micromolar range. The mixed Au(I) NHC complex - (ter-butylethynyl)-1,3-bis-(2,6-diisopropylphenyl)-imidazol-2-ylidene gold(I), bearing an alkynyl moiety as ancillary ligand, showed high cytotoxicity in cancer cells in vitro, while being barely toxic in healthy rat kidney tissues. The obtained results open new perspectives towards the design of mixed NHC-alkynyl gold complexes for cancer therapy.
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Targeting of a helix-loop-helix transcriptional regulator by a short helical peptide ()
The Id proteins (Id1-4) are cell cycle regulators that play a key role during development, in cancer and vascular disorders. They contain a conserved helix-loop-helix (HLH) domain that folds into a parallel four-helix bundle upon self- or heteroassociation with basic-HLH transcription factors. By using such protein-protein interactions, the Id proteins inhibit cell differentiation and promote cell cycle progression. Accordingly, their supporting role in cancer has been convincingly demonstrated, which makes these proteins interesting therapeutic targets. Here we present a short peptide containing a (i,i+4)-lactam bridge and a hydrophobic (Φ) three-residue motif Φ(i)-Φ(i+3)-Φ(i+6), which adopted a helical conformation in water, showed Id-protein binding in the low-micromolar range, penetrated into breast (MCF7 and T47D) and bladder (T24) cancer cells, accumulated in the nucleus, and reduced cell viability to about 50%. Thus, this cyclopeptide is a promising scaffold to develop Id-protein binders impairing cancer cell viability.
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Regulated Drug Release Abilities of Calcium Carbonate-Gelatin Hybrid Nanocarriers Fabricated via a Self-Organizational Process ()
In this study, we investigated the drug-releasing behavior of a calcium carbonate (CaCO3)-gelatin hybrid nanocarrier, fabricated via a single process using biomimetic mineralization. The organic scaffold (gelatin) of the fabricated nanocarrier is responsible for its ability to load anionic drugs and for controlling the morphology of the inorganic matrix (CaCO3). We studied the drug-releasing properties of the nanocarrier by investigating the response of the CaCO3 matrix to acidic conditions. We found that under neutral conditions, drug release from the nanocarrier was inhibited, while under acidic conditions, the drug was efficiently released. In conclusion, the drug release from the nanocarrier is largely dependent on the surrounding pH.
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Design and Synthesis of New 4-Alkylidene-beta-lactams: Benzyl-and Phenethyl-carbamate as Key Fragments to Switch on the Antibacterial Activity ()
The emergence of multidrug resistant bacterial strains is particularly important in some chronic pathologies such as cystic fibrosis (CF), where persistent colonization and selection of resistant strains being favoured by the frequent and repeated use of antibacterial agents. Staphylococcus aureus is a common pathogen in CF patients with an associated increased multidrug resistance. In a previous research we demonstrated that the presence of a 4-alkylidene side chain directly linked to a beta-lactam appeared to strengthen the potency against S. aureus, expecially against MRSA strains. In the present study 21 new 4-alkyliden-beta-lactams were synthesized and evaluated for antibacterial activity. We designed the new molecules to have an aryl, benzyl, or phenethyl-carbamate groups on the C-3 hydroxyethyl side chain. We found a correlation between the biological activity and the N-substituent of the carbamate group, and phenethyl-carbamate beta-lactams 3s and t resulted being valuable antibacterials against some selected strains, comprising linezolid-resistant isolates, with a MIC potency of 2-4 mg/L
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Rational Design, Chemical Development and Stability Assessment of a New Macrocyclic, Four-Hydroxamates-Bearing Bifunctional Chelating Agent for 89Zr ()
89Zr is a positron-emitting radionuclide being of high interest for medical imaging applications with Positron Emission Tomography. For the introduction of this radiometal into biologically active targeting vectors, the chelating agent desferrioxamine B (DFO) is commonly applied which is however known to form 89Zr-complexes of limited in vivo stability. We herein describe the rational design and chemical development a new, macrocyclic, four hydroxamates-bearing chelating agent (CTH36) for the stable complexation of Zr4+. For this purpose, we first performed computational studies to determine the optimal chelator geometry before we developed different synthesis pathways towards the target structures. The best results were obtained using an efficient solution phase-based synthesis strategy towards the target chelating agent. To enable an efficient and chemoselective conjugation to biomolecules, a tetrazine-modified variant of CTH36 was also developed. The excellent conjugation characteristics of the so-functionalized chelator were demonstrated on the example of the model peptide TCO-c(RGDfK). In the following, we determined the optimal 89Zr-radiolabeling parameters for CTH36 as well as its bioconjugate and found that the 89Zr-radiolabeling proceeds efficiently under very mild reaction conditions. Finally, we performed comparative complex stability tests for 89Zr-CTH36-c(RGDfK) and 89Zr-DFO-c(RGDfK), showing an improved complex stability for the newly developed chelator CTH36.
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Matched Molecular Pair Analysis on Large Melting Point Datasets: a Big Data Perspective ()
A matched molecular pair (MMP) analysis was used to examine the change in melting point (MP) between pairs of similar molecules in a set of ca. 275k compounds. We found many cases where the change of MP (ΔMP) of compounds correlates with respect to changes in functional groups. In line with a previous study of Schultes et al, correlations between ΔMP and simple molecular descriptors, such as the number of hydrogen bond donors were identified. In using a larger dataset, covering a wider chemical space and range of melting points, we observed that this method remains stable and scales well with larger datasets: This MMP based method presents the opportunity for its use as a simple privacy-preserving technique to analyse large proprietary databases and share findings between participating groups.
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Direct and Topoisomerase II Mediated DNA Damage by Bis-3-chloropiperidines: The Importance of Being an Earnest G ()
Bis-3-chloropiperidines are a new class of DNA-active compounds capable of alkylating nucleobases and inducing strand cleavage. In this study, we investigated the reactivity of these mustard-based agents with both single- and double-stranded DNA constructs. Polyacrylamide gel electrophoresis (PAGE) and electrospray ionization mass spectrometry (ESI-MS) were used to obtain valuable insight into their mechanism at the molecular level and to investigate their time- and concentration-dependent activity. The results revealed the preferential formation of mono- and bifunctional adducts at nucleophilic guanine sites. In a stepwise fashion, alkylation was followed by depurination and subsequent strand scission at the ensuing apurinic site. We demonstrated that the covalent modifications introduced by this new class of compounds can inhibit the activity of essential DNA-processing proteins, such as topoisomerase IIα, thereby suggesting that bis-3-chloropiperidines may have excellent anticancer potential. The assault on guanines! In exploring the molecular details of the reactivity of bis-3-chloropiperidines with DNA, our results demonstrate that the initial attack is directed toward the N7 position of guanine, generating both mono- and bi-functional adducts. The DNA damage inflicted by bis-3-chloropiperidines has meaningful properties on the activity of human topoisomerases IIα to develop this class of DNA-active compounds as anticancer agents.
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Heme Oxygenase Database (HemeOxDB) and QSAR Analysis of Isoform 1 Inhibitors ()
Due to increasing interest in the field of heme oxygenases (HOs), we built a ligand database called HemeOxDB that includes the entire set of known HO-1 and HO-2 inhibitors, resulting in more than 400 compounds. The HemeOxDB is available online at http://www.researchdsf.unict.it/hemeoxdb/, and having a robust search engine allows end users to build complex queries, sort tabulated results, and generate color-coded two- and three-dimensional graphs. This database will grow to be a tool for the design of potent and selective HO-1 or HO-2 inhibitors. We were also interested in virtually searching for alternative inhibitors, and, for the first time in the field of HOs, a quantitative structure–activity relationship (QSAR) model was built using half-maximal inhibitory concentration (IC50) values of the whole set of known HO-1 inhibitors, taken from the HemeOxDB and employing the Monte Carlo technique. The statistical quality suggested that the model is robust and possesses desirable predictive potential. The screening of US Food and Drug Administration (FDA)-approved drugs, external to our dataset, suggested new predicted inhibitors, opening the way for replacing imidazole groups. The HemeOxDB and the QSAR model reported herein may help in prospectively identifying or repurposing new drugs with optimal structural attributes for HO enzyme inhibition. Heme oxygenase inhibitors: A collection of the whole set of heme oxygenase (HO) inhibitors is reported, and inhibitors of heme oxygenase-1 (HO-1) were used in creating a Monte Carlo based QSAR model. The screening of a number of FDA-approved drugs with the developed model revealed that top-scored compounds share common sulfonylurea or sulfonyl acetamide functions, which may be novel replacements for the imidazole used in the vast majority of HO-1 inhibitors.
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Optimized Solid-Phase-Assisted Synthesis of Oleic Acid Containing siRNA Nanocarriers ()
Cationic lipo-oligomers containing unsaturated oleic acid are potent siRNA carriers based on electrostatic and hydrophobic lipo-polyplex formation and endosomal membrane destabilization. Lipo-oligomers can be produced by solid-phase-supported synthesis in sequence-defined form. However, the trifluoroacetic acid (TFA)-mediated removal of acid-labile protecting groups and cleavage from the resin can be accompanied by side products caused by the addition of TFA to the double bonds of oleic acid. Under aqueous conditions, these TFA adducts of oleic acid are converted into hydroxystearic acid derivatives. The cleavage protocol was optimized to decrease TFA adducts. The pure oleic acid (C18:1) containing lipo-oligomer was compared with analogous structures containing saturated or modified hydrophobic moieties (stearic acid (C18:0), hydroxystearic acid, and 8-nonanamidooctanoic acid). The structure containing intact oleic acid shows favorable pH dependency of lytic activity, efficient gene silencing, and excellent cell tolerability relative to its counterparts. Better carriers for RNAi: Cationic lipo-oligomers containing cis-unsaturated fatty acids such as oleic acid are potent siRNA carriers which can be assembled by solid-phase-assisted synthesis. Trifluoroacetic acid (TFA)-mediated deprotection and cleavage of carriers from the resin may lead to side products initiated by the addition of TFA to the double bonds. The cleavage protocol was optimized to minimize TFA adducts, resulting (in comparison with the modified analogues) in beneficial siRNA delivery at low cytotoxicity.
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Immunization with a Synthetic Human MUC1 Glycopeptide Vaccine against Tumor-Associated MUC1 Breaks Tolerance in Human MUC1 Transgenic Mice ()
Breaking tolerance is crucial for effective tumor immunotherapy. We showed that vaccines containing tumor-associated human MUC1 glycopeptides induce strong humoral antitumor responses in mice. The question remained whether such vaccines work in humans, in systems where huMUC1 is a self-antigen. To clarify the question, mice transgenic in expressing huMUC1, mimicking the self-tolerant environment, and wild-type mice were vaccinated with a synthetic vaccine. This vaccine comprised STn and Tn antigens bound to a MUC1 tandem repeat peptide coupled to tetanus toxoid. The vaccine induced strong immune responses in wild-type and huMUC1-transgenic mice without auto-aggressive side effects. All antisera exhibited almost equivalent binding to human breast tumor cells. Similar increases of activated B-, CD4+ T-, and dendritic cells was found in the lymph nodes. The results demonstrate that tumor-associated huMUC1 glycopeptides coupled to tetanus toxoid are promising antitumor vaccines. Booster shot for cancer immunotherapy: A synthetic human tumor-associated MUC1 glycopeptide vaccine that contains tetanus toxoid as a carrier protein is able to induce a strong humoral immune response within human MUC1 transgenic mice. These results suggest that the induction of a selective immune response to an altered cancer-related glycopeptide antigen is possible without autoimmune side effects against the self-antigen.
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A Ligand-Based NMR Screening Approach for the Identification and Characterization of Inhibitors and Promoters of Amyloid Peptide Aggregation ()
Over the years a significant amount of effort has been put into the development of rapid and reliable methods to monitor the aggregation dynamics of the β1–42 amyloid peptide in real time. We present an alternative approach based on a suitable reporter or spy molecule and three different NMR experiments: WaterLOGSY, 1H selective T1 filter, and 19F T2 filter, for monitoring the initial self-aggregation process kinetics of the β1–42 amyloid peptide and identifying molecules that retard or accelerate the self-aggregation process. Although the proposed method is not a high-throughput assay, it avoids problems associated with interference events that are sometimes observed in fluorescence-based assays. Real-time surveillance: A ligand-based NMR screening approach with a spy molecule and using WaterLOGSY, 1H selective T1 filter, and 19F T2 filter experiments is proposed for monitoring the initial self-aggregation process kinetics of the β1–42 amyloid peptide. This methodology can be used in the screening of large chemical mixtures for the identification of molecules that delay or accelerate the initial aggregation process.
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Sulfonylureas as Concomitant Insulin Secretagogues and NLRP3 Inflammasome Inhibitors ()
Insulin-secretory sulfonylureas are widely used, cost-effective treatments for type 2 diabetes (T2D). However, pancreatic β-cells are continually depleted as T2D progresses, thereby rendering the sulfonylurea drug class ineffective in controlling glycaemia. Dysregulation of the innate immune system via activation of the NLRP3 inflammasome, and the consequent production of interleukin-1β, has been linked to pancreatic β-cell death and multiple inflammatory complications of T2D disease. One proposed strategy for treating T2D is the use of sulfonylurea insulin secretagogues that are also NLRP3 inhibitors. We report the synthesis and biological evaluation of nine sulfonylureas that inhibit NLRP3 activation in murine bone-marrow- derived macrophages in a potent, dose-dependent manner. Six of these compounds inhibited NLRP3 at nanomolar concentrations and can also stimulate insulin secretion from a murine pancreatic cell line (MIN6). These novel compounds possess unprecedented dual modes of action, paving the way for a new generation of sulfonylureas that may be useful as therapeutic candidates and/or tool compounds in T2D and its associated inflammatory complications. Pattern recognition: Hybridisation of the potent NLRP3 inhibitor MCC950 with antidiabetic sulfonylureas has created dual mode of action compounds which concomitantly inhibit NLRP3 and stimulate insulin secretion. These hybrids are interesting tool compounds for studying the role of inflammation in type 2 diabetes, promising therapeutics and highlight the ability to functionalise MCC950 with other bioactive motifs to create small-molecule chimeras.
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Bridging Pharmaceutical Chemistry with Drug and Nanoparticle Targeting to Investigate the Role of the 18-kDa Translocator Protein TSPO ()
An interesting mitochondrial biomarker is the 18-kDa mitochondrial translocator protein (TSPO). Decades of study have shown that this protein plays an important role in a wide range of cellular functions, including opening of the mitochondrial permeability transition pore as well as programmed cell death and proliferation. Variations in TSPO expression have been correlated to different diseases, from tumors to endocrine and neurological disorders. TSPO has therefore become an appealing target for both early diagnosis and selective mitochondrial drug delivery. The number of structurally different TSPO ligands examined has increased over time, highlighting the scientific community′s growing understanding of the roles of TSPO in normal and pathological conditions. However, only few TSPO ligands are characterized by the presence of groups that are potentially derivatizable; therefore only few such ligands are well suited for the preparation of targeted prodrugs or nanocarriers able to deliver therapeutics and/or diagnostic agents to mitochondria. This review provides an overview of the very few examples of drug delivery systems characterized by moieties that target TSPO. Ligand logistics: Compounds with high affinity and selectivity for the 18-kDa mitochondrial translocator protein (TSPO) are useful for intracellular drug and nanoparticle targeting. The number of identified structurally distinct TSPO ligands has increased in recent years, highlighting our growing understanding of the roles of TSPO in normal and pathological conditions. This review summarizes some examples of drug delivery systems characterized by TSPO-targeting compounds.
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Investigating Structural Requirements for the Antiproliferative Activity of Biphenyl Nicotinamides ()
A number of trimethoxybenzoic acid anilides, previously studied as permeability glycoprotein (P-gp) modulators, were screened with the aim of identifying new anticancer agents. One of these compounds, which showed antiproliferative activity against resistant MCF-7 cell line, was selected as the hit structure. Replacement of the trimethoxybenzoyl moiety with a nicotinoyl group, in order to overcome solubility issues, led to a new series of N-biphenyl nicotinoyl anilides, among which a nitro derivative, N-(3′,5′-difluoro-3-nitro-[1,1′-biphenyl]-4-yl)nicotinamide (3), displayed antiproliferative activity against MCF-7 and MDA-MB-231 cells in the nanomolar range. The search for a bioisostere of the nitro group led to nitrile analogue N-(3-cyano-4′-fluoro-[1,1′-biphenyl]-4-yl)nicotinamide (36), which shows a strong increase in activity against MCF-7 and MDA-MB-231 cells. Compound 36 induced a dose-dependent accumulation of G2- and M-phase MCF-7 cell populations, and a decrease in S-phase cells. Relative to vinblastine, a well-known potent antimitotic agent, compound 36 also induced G1-phase arrest at low doses (20–40 nm), but did not inhibit in vitro tubulin polymerization. Stopping the cell cycle: The screening of a small library of trimethoxybenzoic and nicotinic acid anilides, previously studied as P-gp modulators, allowed us to identify some N-biphenyl nicotinoyl anilides that show very interesting antiproliferative activities. The next hit refinement by a bioisosteric approach led us to observe potent antiproliferative activity of the cyano derivative 36 against MCF-7 and MDA-MB-231 cell lines.
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A Small-Molecule Inhibitor of Prion Replication and Mutant Prion Protein Toxicity ()
Into the fold: Prion diseases are neurodegenerative disorders characterized by the accumulation in the brain of a self-replicating, misfolded isoform (PrPSc) of the cellular prion protein (PrPC). No therapies are available for these pathologies. We capitalized on previously described cell-based assays to screen a library of small molecules, and identified 55, a compound capable of counteracting both prion replication and toxicity. Compound 55 may represent the starting point for the development of a completely new class of therapeutics for prion diseases.
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Discovery of wtRET and V804MRET Inhibitors: From Hit to Lead ()
Oncogenic activation of RET kinase has been found in several neoplastic diseases, like medullary thyroid carcinoma, multiple endocrine neoplasia, papillary thyroid carcinoma, and non-small-cell lung cancer. Currently approved RET inhibitors were not originally designed to be RET inhibitors, and their potency against RET kinase has not been optimized. Hence, novel compounds able to inhibit both wild-type RET (wtRET) and its mutants (e.g., V804MRET) are needed. Herein we present the development and the preliminary evaluation of a new sub-micromolar wtRET/V804MRET inhibitor, N-(2-fluoro-5-trifluoromethylphenyl)-N′-{4′-[(2′′-benzamido)pyridin-4′′-ylamino]phenyl}urea (69), endowed with a 4-anilinopyridine structure, starting from our previously identified 4-anilinopyrimidine hit compound. Profiling against a panel of kinases indicated 69 as a multi cKIT/wtRET/V804MRET inhibitor. Intentional inhibition: RET kinases are involved in the onset and development of various types of cancer. Currently approved RET inhibitors were not originally designed to be RET inhibitors, and their potency against RET kinase has not been optimized. Starting from our previously identified dual cKIT/PDGFRβ inhibitor, we report the development of a novel multi-cKIT/wtRET/V804MRET inhibitor.
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Development of a New Structural Class of Broadly Acting HCV Non-Nucleoside Inhibitors Leading to the Discovery of MK-8876 ()
Studies directed at developing a broadly acting non-nucleoside inhibitor of HCV NS5B led to the discovery of a novel structural class of 5-aryl benzofurans that simultaneously interact with both the palm I and palm II binding regions. An initial candidate was potent in vitro against HCV GT1a and GT1b replicons, and induced multi-log reductions in HCV viral load when orally dosed to chronic GT1 infected chimpanzees. However, in vitro potency losses against clinically relevant GT1a variants prompted a further effort to develop compounds with sustained potency across a broader array of HCV genotypes and mutants. Ultimately, a biology and medicinal chemistry collaboration led to the discovery of the development candidate MK-8876. MK-8876 demonstrated a pan-genotypic potency profile and maintained potency against clinically relevant mutants. It demonstrated moderate bioavailability in rats and dogs, but showed low plasma clearance characteristics consistent with once-daily dosing. Herein we describe the efforts which led to the discovery of MK-8876, which advanced into Phase 1 monotherapy studies for evaluation and characterization as a component of an all-oral direct-acting drug regimen for the treatment of chronic HCV infection. Both palms open: Studies directed at developing a broadly acting non-nucleoside inhibitor of HCV NS5B led to the discovery of a novel structural class of 5-aryl benzofurans that simultaneously interact with both the palm I and palm II binding regions. Herein we describe the efforts that led to the discovery of MK-8876, which advanced into Phase 1 monotherapy studies for evaluation and characterization as a component of an all-oral direct-acting drug regimen for the treatment of chronic HCV infection.
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Searching for Novel Inhibitors of the S. aureus NorA Efflux Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-benzothiazine Analogues ()
Bacterial resistance to antimicrobial agents has become an increasingly serious health problem in recent years. Among the strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sub-lethal concentration of the antibacterial agent at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei. None of the new synthesized compounds showed any appreciable intrinsic antibacterial activity, and, in particular, 2-(3,4-dimethoxyphenyl)quinoline (6 c) was able to decrease, in a concentration-dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S. aureus SA-K2378 (norA++) and SA-1199B (norA+/A116E GrlA). Contrasting bacterial resistance to antimicrobial agents by the inhibition of efflux pumps is a promising strategy aimed at restoring antibacterial MIC in resistant strains. In an attempt to improve the benzothiazine class of Staphylococcus aureus NorA inhibitors in terms of efflux inhibitory activity and chemical stability, the synthesis of six different sets of derivatives was performed. The 2-phenylquinoline series provided the best derivatives, suggesting this scaffold as the best suited for further optimization.
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2,6-Difluorobenzamide Inhibitors of Bacterial Cell Division Protein FtsZ: Design, Synthesis, and Structure–Activity Relationships ()
A wide variety of drug-resistant microorganisms are continuously emerging, restricting the therapeutic options for common bacterial infections. Antimicrobial agents that were originally potent are now no longer helpful, due to their weak or null activity toward these antibiotic-resistant bacteria. In addition, none of the recently approved antibiotics affect innovative targets, resulting in a need for novel drugs with innovative antibacterial mechanisms of action. The essential cell division protein filamentous temperature-sensitive Z (FtsZ) has emerged as a possible target, thanks to its ubiquitous expression and its homology to eukaryotic β-tubulin. In the latest years, several compounds were shown to interact with this prokaryotic protein and selectively inhibit bacterial cell division. Recently, our research group developed interesting derivatives displaying good antibacterial activities against methicillin-resistant Staphylococcus aureus, as well as vancomycin-resistant Enterococcus faecalis and Mycobacterium tuberculosis. The aim of the present study was to summarize the structure–activity relationships of differently substituted heterocycles, linked by a methylenoxy bridge to the 2,6-difluorobenzamide, and to validate FtsZ as the real target of this class of antimicrobials. Targeting the Z! The search for innovative targets and potent antibiotics is an important endeavor against a serious health problem. The present study is aimed at broadening structure–activity relationship knowledge for a novel class of antimicrobial agents and demonstrating that these compounds interact with filamentous temperature-sensitive Z (FtsZ), an essential prokaryotic cell division protein.
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Repurposing of Human Kinase Inhibitors in Neglected Protozoan Diseases ()
Human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis belong to a group of infectious diseases known as neglected tropical diseases and are induced by infection with protozoan parasites named trypanosomatids. Drugs in current use have several limitations, and therefore new candidate drugs are required. The majority of current therapeutic trypanosomatid targets are enzymes or cell-surface receptors. Among these, eukaryotic protein kinases are a major group of protein targets whose modulation may be beneficial for the treatment of neglected tropical protozoan diseases. This review summarizes the finding of new hit compounds for neglected tropical protozoan diseases, by repurposing known human kinase inhibitors on trypanosomatids. Kinase inhibitors are grouped by human kinase family and discussed according to the screening (target-based or phenotypic) reported for these compounds on trypanosomatids. This collection aims to provide insight into repurposed human kinase inhibitors and their importance in the development of new chemical entities with potential beneficial effects on the diseases caused by trypanosomatids. Drugs redirected: Neglected tropical protozoan diseases are induced by parasites called trypanosomatids. Given the limitations of drugs in current use, the development of new candidates is needed. Eukaryotic protein kinases are a major group of protein targets whose modulation may be beneficial for the treatment of these diseases. This review focuses on recent efforts in finding new hit compounds by repurposing the major known human kinase inhibitors to fight trypanosomatids.
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Comparison of the Human A2A Adenosine Receptor Recognition by Adenosine and Inosine: New Insight from Supervised Molecular Dynamics Simulations ()
Adenosine deaminase converts adenosine into inosine. In contrast to adenosine, relatively little attention has been paid to the physiological roles of inosine. Nevertheless, recent studies have demonstrated that inosine has neuroprotective, cardioprotective, immunomodulatory, and antidepressive effects. Inosine was recently shown to be a less potent agonist than adenosine at the A2A adenosine receptor. To better depict the differences in the mechanisms of receptor recognition between adenosine and inosine, we carried out supervised molecular dynamics (SuMD) simulations, and the results are analyzed herein. Close supervision: Recent studies have shown that inosine has neuroprotective, cardioprotective, immunomodulatory, and antidepressive effects. Inosine was recently found to be a less potent agonist than adenosine at the A2A adenosine receptor (A2A AR). The recognition pathways of adenosine and inosine against the human A2A AR were investigated by supervised molecular dynamics (SuMD) simulations.
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Structure–Activity Relationship Studies on 6,7-Dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline Derivatives as Multidrug Resistance Reversers ()
A series of derivatives were synthesized and studied with the aim to investigate the structure–activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar. Then, different aryl-substituted amides were inserted, and to explore the effects of varying the amide function, the corresponding isosteric ester derivatives and some alkylamine analogues were synthesized. The new compounds were studied to evaluate their P-gp interaction profile and selectivity toward the two other ABC transporters, multidrug-resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP). Investigation of the chemical stability of the amide and ester derivatives toward spontaneous or enzymatic hydrolysis showed that these compounds were stable in phosphate-buffered saline and human plasma. This study allowed us to evaluate the selectivity of the three series on the three efflux pumps and to propose the structural requirements that define the P-gp interaction profile. We identified two P-gp substrates, a P-gp inhibitor, and three ester derivatives that were active on BCRP, which opens a new scenario in the development of ligands active toward this pump. Resistance is futile: A set of 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline derivatives were designed and synthesized to develop potent and selective multidrug resistance (MDR) reversing agents. Their behavior on the three ABC transporters, P-gp, MRP1, and BCRP, was investigated. The results allow us to propose the structural requirements for defining P-gp selectivity and mechanism of interaction.
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Discovery of Potent Dual Binding Site Acetylcholinesterase Inhibitors via Homo- and Heterodimerization of Coumarin-Based Moieties ()
Acetylcholinesterase (AChE) inhibitors still comprise the majority of the marketed drugs for Alzheimer's disease (AD). The structural arrangement of the enzyme, which features a narrow gorge that separates the catalytic and peripheral anionic subsites (CAS and PAS, respectively), inspired the development of bivalent ligands that are able to bind and block the catalytic activity of the CAS as well as the role of the PAS in beta amyloid (Aβ) fibrillogenesis. With the aim of discovering novel AChE dual binders with improved drug-likeness, homo- and heterodimers containing 2H-chromen-2-one building blocks were developed. By exploring diverse linkages of neutral and protonatable amino moieties through aliphatic spacers of different length, a nanomolar bivalent AChE inhibitor was identified (3-[2-({4-[(dimethylamino)methyl]-2-oxo-2H-chromen-7-yl}oxy)ethoxy]-6,7-dimethoxy-2H-chromen-2-one (6 d), IC50=59 nm) from originally weakly active fragments. To assess the potential against AD, the disease-related biological properties of 6 d were investigated. It performed mixed-type AChE enzyme kinetics (inhibition constant Ki=68 nm) and inhibited Aβ self-aggregation. Moreover, it displayed an outstanding ability to protect SH-SY5Y cells from Aβ1-42 damage. Power in pairing: Acetylcholinesterase inhibition can be strongly improved by connecting weakly active smaller fragments through a suitable spacer. A dimerization strategy is described that leads to a coumarin-based inhibitor endowed with nanomolar affinity. The inhibitor can bind both catalytic and peripheral anionic subsites, decrease amyloid aggregation, and protect SH-SY5Y cells from Aβ1–42 insults.
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Kinome-Wide Profiling Prediction of Small Molecules ()
Extensive kinase profiling data, covering more than half of the human kinome, are available nowadays and allow the construction of activity prediction models of high practical utility. Proteochemometric (PCM) approaches use compound and protein descriptors, which enables the extrapolation of bioactivity values to thus far unexplored kinases. In this study, the potential of PCM to make large-scale predictions on the entire kinome is explored, considering the applicability on novel compounds and kinases, including clinically relevant mutants. A rigorous validation indicates high predictive power on left-out kinases and superiority over individual kinase QSAR models for new compounds. Furthermore, external validation on clinically relevant mutant kinases reveals an excellent predictive power for mutations spread across the ATP binding site. Kinome-wide prediction of kinase inhibitors: The capabilities of proteochemometric (PCM) models to make large-scale predictions on the entire kinome was explored. The combination of a compound fingerprint with a protein fingerprint 1) improves the activity prediction for each kinase relative to individually trained models and 2) enables prediction of the activity of compounds for the entire kinome, including cancer-related resistance mutations.
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Residence Time, a New parameter to Predict Neurosteroidogenic Efficacy of Translocator Protein (TSPO) Ligands: the Case Study of N,N-Dialkyl-2-arylindol-3-ylglyoxylamides ()
Targeting the biosynthetic pathway of neuroactive steroids with specific 18 kDa translocator protein (TSPO) ligands may be a viable therapeutic approach for a variety of neurodegenerative and neuropsychiatric diseases. However, the lack of correlation between binding affinity and in vitro steroidogenic efficacy has limited the identification of lead compounds by traditional affinity-based drug discovery strategies. Our recent research indicates that the key factor for robust steroidogenic TSPO ligand efficacy is not the binding affinity per se, but rather the time the compound spends in the target, namely its residence time (RT). The assessment of this kinetic parameter during the in vitro characterization of compounds appears mandatory in order to obtain structure–efficacy relationships suitable for the future development of novel molecules with promising pharmacological properties. How long can you stay? The neurosteroidogenic efficacy of translocator protein (TSPO) ligands can be predicted by evaluating the amount of time—the residence time—that a given ligand spends in the target rather than the binding affinity. This aids in the development of novel compounds with promising pharmacological properties and therapeutic potential.
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Identification of Breast Cancer Inhibitors Specific for G Protein-Coupled Estrogen Receptor (GPER)-Expressing Cells ()
Together with estrogen receptors ERα and ERβ, the G protein-coupled estrogen receptor (GPER) mediates important pathophysiological signaling pathways induced by estrogens and is currently regarded as a promising target for ER-negative (ER−) and triple-negative (TN) breast cancer. Only a few selective GPER modulators have been reported to date, and their use in cancer cell lines has often led to contradictory results. Herein we report the application of virtual screening and cell-based studies for the identification of new chemical scaffolds with a specific antiproliferative effect against GPER-expressing breast cancer cell lines. Out of the four different scaffolds identified, 8-chloro-4-(4-chlorophenyl)pyrrolo[1,2-a]quinoxaline 14 c was found to be the most promising compound able to induce: 1) antiproliferative activity in GPER-expressing cell lines (MCF7 and SKBR3), similarly to G15; 2) no effect on cells that do not express GPER (HEK293); 3) a decrease in cyclin D1 expression; and 4) a sustained induction of cell-cycle negative regulators p53 and p21. Positive news for ER-negative: We show how the combination of virtual screening on a GPER model and cell-based assays quickly leads to the identification of new chemical entities that induce a specific antiproliferative effect on GPER-expressing cells (MCF7 and SKBR3), but that have no effect on cells that do not express GPER. Further in vitro studies allowed identification of pyrrolo[1,2-a]quinoxaline 14 c as the most interesting compound for hit-to-lead optimization aimed at developing new drugs to treat breast cancer.
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Identification of α7 Nicotinic Acetylcholine Receptor Silent Agonists Based on the Spirocyclic Quinuclidine-Δ2-Isoxazoline Scaffold: Synthesis and Electrophysiological Evaluation ()
Compound 11 (3-(benzyloxy)-1′-methyl-1′-azonia-4H-1′-azaspiro[isoxazole-5,3′-bicyclo[2.2.2]octane] iodide) was selected from a previous set of nicotinic ligands as a suitable model compound for the design of new silent agonists of α7 nicotinic acetylcholine receptors (nAChRs). Silent agonists evoke little or no channel activation but can induce the α7 desensitized Ds state, which is sensitive to a type II positive allosteric modulator, such as PNU-120596. Introduction of meta substituents into the benzyloxy moiety of 11 led to two sets of tertiary amines and quaternary ammonium salts based on the spirocyclic quinuclidinyl-Δ2-isoxazoline scaffold. Electrophysiological assays performed on Xenopus laevis oocytes expressing human α7 nAChRs highlighted four compounds that are endowed with a significant silent-agonism profile. Structure–activity relationships of this group of analogues provided evidence of the crucial role of the positive charge at the quaternary quinuclidine nitrogen atom. Moreover, the present study indicates that meta substituents, in particular halogens, on the benzyloxy substructure direct specific interactions that stabilize a desensitized conformational state of the receptor and induce silent activity. Shh, it's a “silent” activation: Designed quinuclidine spirocyclic derivatives were characterized as silent agonists of α7 nicotinic acetylcholine receptors (nAChRs) with electrophysiological assays. The 3-halo-substituted tertiary amine 17 a and quaternary ammonium salt 18 b exhibited the best silent activity in the set of analogues. Thus, both a protonatable and a permanently charged nitrogen atom may promote the silent activation of α7 nAChRs.
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Identification of Transthyretin Fibril Formation Inhibitors Using Structure-Based Virtual Screening ()
Transthyretin (TTR) is the primary carrier for thyroxine (T4) in cerebrospinal fluid and a secondary carrier in blood. TTR is a stable homotetramer, but certain factors, genetic or environmental, could promote its degradation to form amyloid fibrils. A docking study using crystal structures of wild-type TTR was planned; our aim was to design new ligands that are able to inhibit TTR fibril formation. The computational protocol was thought to overcome the multiple binding modes of the ligands induced by the peculiarity of the TTR binding site and by the pseudosymmetry of the site pockets, which generally weaken such structure-based studies. Two docking steps, one that is very fast and a subsequent step that is more accurate, were used to screen the Aldrich Market Select database. Five compounds were selected, and their activity toward inhibiting TTR fibril formation was assessed. Three compounds were observed to be actives, two of which have the same potency as the positive control, and the other was found to be a promising lead compound. These results validate a computational protocol that is able to archive information on the key interactions between database compounds and TTR, which is valuable for supporting further studies. Rapid and accurate! Inhibitors of transthyretin (TTR) fibril deposition were identified thanks to a structure-based virtual screening of the entire Aldrich Market Select database. The first results of this protocol, despite the unique peculiarity of the TTR site with its symmetry and multi-binding mode, allowed the detection of two very active inhibitors and one scaffold for optimization. Much information, however, is still stored in the outputs.
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Novel, Selective, and Developable Dopamine D3 Antagonists with a Modified “Amino” Region ()
This Minireview describes a presentation made at the XXIV National Meeting in Medicinal Chemistry (NMMC) held in Perugia (Italy), September 11–14, 2016. It relates to the discovery of novel templates of the so-called “amino” region of dopamine D3 receptor antagonists. Moving from the early scaffolds, which were modified in the amine portion, this review discusses the variations that led to the discovery of new systems published in 2016, which allowed the identification of compounds endowed with great selectivity over the dopamine D2 receptor and the human ether-à-go-go-related gene (hERG) ion channel. The main efforts in characterizing these compounds were devoted not only to determining their potency and selectivity relative to closely associated targets (e.g., the dopamine D2 receptor), but to ensure a large therapeutic window versus liability points such as hERG. In particular, we present examples of derivatives with selectivities greater than 2000-fold. Furthermore, much focus is devoted to the overall developability of the scaffolds, ensuring that appropriate physicochemical and pharmacokinetic parameters are present in all compounds progressing through the screening cascade. New scaffolds! This Minireview describes the discovery of novel templates of the so-called “amino” region of dopamine D3 receptor antagonists. Moving from the early scaffolds, which were modified in the amine portion, this review discusses the variations that led to the discovery of new systems published in 2016, which allowed the identification of compounds endowed with great selectivity over the dopamine D2 receptor and hERG channels.
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Cannabinoid Type 1 Receptor (CB1) Ligands with Therapeutic Potential for Withdrawal Syndrome in Chemical Dependents of Cannabis sativa ()
Cannabis sativa withdrawal syndrome is characterized mainly by psychological symptoms. By using computational tools, the aim of this study was to propose drug candidates for treating withdrawal syndrome based on the natural ligands of the cannabinoid type 1 receptor (CB1). One compound in particular, 2-n-butyl-5-n-pentylbenzene-1,3-diol (ZINC1730183, also known as stemphol), showed positive predictions as a human CB1 ligand and for facile synthetic accessibility. Therefore, ZINC1730183 is a favorable candidate scaffold for further research into pharmacotherapeutic alternatives to treat C. sativa withdrawal syndrome. Off the hook: Most drugs used to treat chemical dependence treat the symptoms but not the underlying biological target. This study was designed to identify potential CB1 agonists to treat C. sativa withdrawal syndrome. Pharmacophore-based ligand screening and classification by structural similarity revealed a group of seven compounds for which predictions of pharmacokinetics, toxicology, biological activity, and synthetic accessibility were carried out. One compound stood out as particularly well suited for further development, with the ultimate goal of decreasing the damage caused by drug dependency.
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Structure–Activity Relationships on Cinnamoyl Derivatives as Inhibitors of p300 Histone Acetyltransferase ()
Human p300 is a polyhedric transcriptional coactivator that plays a crucial role in acetylating histones on specific lysine residues. A great deal of evidence shows that p300 is involved in several diseases, including leukemia, tumors, and viral infection. Its involvement in pleiotropic biological roles and connections to diseases provide the rationale to determine how its modulation could represent an amenable drug target. Several p300 inhibitors (i.e., histone acetyltransferase inhibitors, HATis) have been described so far, but they all suffer from low potency, lack of specificity, or low cell permeability, which thus highlights the need to find more effective inhibitors. Our cinnamoyl derivative, 2,6-bis(3-bromo-4-hydroxybenzylidene)cyclohexanone (RC56), was identified as an active and selective p300 inhibitor and was proven to be a good hit candidate to investigate the structure–activity relationship toward p300. Herein, we describe the design, synthesis, and biological evaluation of new HATis structurally related to our hit; moreover, we investigate the interactions between p300 and the best-emerged hits by means of induced-fit docking and molecular-dynamics simulations, which provided insight into the peculiar chemical features that influence their activity toward the targeted enzyme. HAT trick: Histone acetyltransferase (HAT) is an attractive anticancer target. Several HAT inhibitors have been identified, but they all exhibit low potency or pharmacodynamics limits. Herein we report the design and synthesis along with biological evaluations and theoretical investigations of potent and selective cinnamoyl compounds, highlighting the peculiar features required to develop an effective HAT inhibitor.
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A Molecular Dynamics–Shared Pharmacophore Approach to Boost Early-Enrichment Virtual Screening: A Case Study on Peroxisome Proliferator-Activated Receptor α ()
Molecular dynamics (MD) simulations can be used, prior to virtual screening, to add flexibility to proteins and study them in a dynamic way. Furthermore, the use of multiple crystal structures of the same protein containing different co-crystallized ligands can help elucidate the role of the ligand on a protein′s active conformation, and then explore the most common interactions between small molecules and the receptor. In this work, we evaluated the contribution of the combined use of MD on crystal structures containing the same protein but different ligands to examine the crucial ligand–protein interactions within the complexes. The study was carried out on peroxisome proliferator-activated receptor α (PPARα). Findings derived from the dynamic analysis of interactions were then used as features for pharmacophore generation and constraints for generating the docking grid for use in virtual screening. We found that information derived from short multiple MD simulations using different molecules within the binding pocket of the target can improve the early enrichment of active ligands in the virtual screening process for this receptor. In the end we adopted a consensus scoring based on docking score and pharmacophore alignment to rank our dataset. Our results showed an improvement in virtual screening performance in early recognition when screening was performed with the Molecular dYnamics SHAred PharmacophorE (MYSHAPE) approach. The MYSHAPE approach is a new way of studying ligand–receptor interactions. In this method, a pharmacophore model is created that exploits information derived from multiple short MD simulations, using different molecules within the binding pocket of the target. Conformational information is retrieved from the full trajectory and not only from clustered frames. This approach can help improve virtual screening performance, especially for the early enrichment of active ligands in the virtual screening process.
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Cover Picture: Discovery of Novel Potent Muscarinic M3 Receptor Antagonists with Proper Plasma Stability by Structural Recombination of Marketed M3 Antagonists (ChemMedChem 15/2017) ()
The front cover picture shows a novel potent muscarinic M3 receptor antagonist (R)-3-[2-hydroxy-2,2-di(thiophen-2-yl)acetoxy]-1,1-dimethylpyrrolidinium bromide (1a: Ki=0.16 nm, IC50=0.38 nm) inhaled to dilate bronchus of patients with chronic obstructive pulmonary disease (COPD). 1a and its enantiomer 1b have proper stability in human plasma (t1/2=9.34, 19.2 min), while the marketed M3 antagonists aclidinium bromide (t1/2=2.52 min) and glycopyrronium bromide (t1/2>60 min) are either too unstable or overstable in plasma. The newly discovered M3 antagonists 1a and 1b may have great potential to overcome the clinical drawbacks caused by the extreme instability of aclidinium bromide and the overstablility of glycopyrronium bromide in plasma. More information can be found in the Full Paper by Hongbin Sun, Xiaoan Wen et al. on page 1173 in Issue 15, 2017 (DOI: 10.1002/cmdc.201700189).
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Recent Research Advances in Selective Matrix Metalloproteinase-13 Inhibitors as Anti-Osteoarthritis Agents ()
Matrix metalloproteinase-13 (MMP-13) plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). The subtle differences between the S1′ loop of MMP-13 and that of other MMPs offer a structural base for the design of selective MMP-13 inhibitors to mitigate the unperceived risk associated with inhibiting other MMP isoforms. In this review, we summarize zinc-binding and non-zinc-binding selective MMP-13 inhibitors. The zinc-binding MMP-13 inhibitors contain a small set of zinc-binding groups (ZBGs), including hydroxamic acid, pyrimidinetrione, reversed hydroxamic acid and hydantoin, carboxylic acid, 1,2,4,-triazole, and 1,2,4,-triazolone. The non-zinc-binding MMP-13 inhibitors have different structural scaffolds, including diphenyl ethers, biaryls (aryltetrazoliums, arylfurans, pyrazole-indoles), pyrimidines, and aryl/cycloalkyl-fused pyrimidines. This review provides a systematic overview of recent developments in MMP-13 inhibitors for the treatment of OA, with emphasis on their enzyme inhibitory potency, selectivity, and biological activities, and highlights the various binding modes of typical inhibitors with MMP-13. Lucky 13: Matrix metalloproteinase-13 (MMP-13) plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). This review covers recent advances in the area of MMP-13-specific inhibitors, which lack the risks associated with the inhibition of other MMP isoforms. With greater insight into the mechanism of action of MMP-13 and the pathogenic process of OA, research into selective MMP-13 inhibitors will make significant breakthroughs for patients suffering from OA in the near future.
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Rational Design of a Highly Potent and Selective Peptide Inhibitor of PACE4 by Salt Bridge Interaction with D160 at Position P3 ()
PACE4, a member of the proprotein convertases (PCs) family of serine proteases, is a validated target for prostate cancer. Our group has developed a potent and selective PACE4 inhibitor: Ac-LLLLRVKR-NH2. In seeking for modifications to increase the selectivity of this ligand toward PACE4, we replaced one of its P3 Val methyl groups with a basic group capable of forming a salt bridge with D160 of PACE4. The resulting inhibitor is eight times more potent than the P3 Val parent inhibitor and two times more selective over furin, because the equivalent salt bridge with furin E257 is not optimal. Moreover, the β-branched nature of the new P3 residue favors the extended β-sheet conformation usually associated with substrates of proteases. This work provides new insight for better understanding of β-sheet backbone–backbone interactions between serine proteases and their peptidic ligands. Setting the PACE with concurrent control of conformation and binding: Most substrates of proteases form a β-sheet with the active site upon binding. New β-branched residues, known to favor β-sheet conformation in linear peptides, were introduced at the P3 Val position of inhibitors of the serine protease PACE4. Through this P3 strategic position, it was possible to obtain potent and selective inhibitors, by additive β-sheet conformation control and salt bridge formation with PACE4 Asp160.
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Discovery of Novel Potent Muscarinic M3 Receptor Antagonists with Proper Plasma Stability by Structural Recombination of Marketed M3 Antagonists ()
The marketed long-acting M3 antagonists for treatment of chronic obstructive pulmonary disease have inappropriate plasma stability (either overstable or excessively unstable), which causes substantial systemic exposure or poor patient compliance. To discover novel M3 antagonists with proper plasma stability, we synthesized and biologically evaluated a series of chiral quaternary ammonium salts of pyrrolidinol esters, which were designed by structural recombination of the marketed M3 antagonists. As a result, two novel potent M3 antagonists, (R/S)-3-[2-hydroxy-2,2-di(thiophen-2-yl)acetoxy]-1,1-dimethylpyrrolidinium bromides (1 a: Ki=0.16 nm, IC50=0.38 nm, t1/2=9.34 min; 1 b: Ki=0.32 nm, IC50=1.01 nm, t1/2=19.2 min) with proper plasma stability were identified, which (particularly 1 a) hold great promise as clinical drug candidates to overcome the drawbacks caused by the inappropriate stability of the currently marketed M3 antagonists. In addition, structure–activity relationship studies revealed that the R configuration of the pyrrolidinyl C3 atom was clearly better than the S configuration. Steady as she goes: Structural recombination of the marketed muscarinic M3 receptor antagonists aclidinium bromide and glycopyrronium bromide, which have inappropriate plasma stability, led to the discovery of novel potent M3 receptor antagonists 1 a and 1 b, which have proper plasma stability. These antagonists hold great promise as clinical drug candidates to overcome the drawbacks caused by the unsuitable stability of currently marketed M3 antagonists.
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Effects of Pimozide Derivatives on pSTAT5 in K562 Cells ()
STAT5 is a transcription factor, a member of the STAT family of signaling proteins. STAT5 is involved in many types of cancer, including chronic myelogenous leukemia (CML), in which this protein is found constitutively activated as a consequence of BCR-ABL expression. The neuroleptic drug pimozide was recently reported to act as an inhibitor of STAT5 phosphorylation and is capable of inducing apoptosis in CML cells in vitro. Our research group has synthesized simple derivatives of pimozide with cytotoxic activity and that are able to decrease the levels of phosphorylated STAT5. In this work we continued the search for novel STAT5 inhibitors, synthesizing compounds in which the benzoimidazolinone ring of pimozide is either maintained or modified, in order to obtain further structure–activity relationship information for this class of STAT5 inhibitors. Two compounds of the series showed potent cytotoxic activity against BCR-ABL-positive and pSTAT5-overexpressing K562 cells and were able to markedly decrease the levels of phosphorylated STAT5. Neuroleptic goes anticancer: New derivatives of pimozide were obtained with potent growth inhibitory and apoptotic activity in K562 BCR-ABL-expressing leukemia cell lines. The detection of overexpressed phosphorylated STAT5 with a fluorophore-conjugated monoclonal antibody (anti-pSTAT5) resulted in strong inhibition with derivatives at a concentration of 10–15 μm, in contrast to pimozide, active at 30 μm under the same conditions.
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A Hollow NaGdF4/AFn Nanosystem Based on “Relay Race” Release for Therapy ()
To develop a multifunctional nanomaterial for dual-mode imaging and synergetic chemotherapy, curcumin (CUR) was physically entrapped into hollow upconversion NaGdF4 nanomaterial, then apoferritin (AFn) loaded with doxorubicin (DOX) was attached to the NaGdF4 surface. Subsequent modification with the targeting reagent folic acid (FA) led to generation of the CUR/NaGdF4–DOX/AFn–FA conjugate for cancer treatment. X-ray diffraction, scanning (SEM) and transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy demonstrated the successful preparation of hexagonal-phase NaGdF4 and NaGdF4–AFn–FA. Moreover, no toxicity was observed for NaGdF4–AFn–FA. In vitro and in vivo experiments demonstrated that the two drugs are sequentially released from the nanocomposites. This two-drug system showed strong growth inhibitory effects on MCF-7 cells. Upconversion luminescence imaging and magnetic resonance (MR) imaging of NaGdF4–AFn–FA were carried out. The results of this study show that NaGdF4–AFn–FA can be used for targeted anticancer drug delivery as well as imaging, a novel multi-pronged theranostic system for tumor treatment. Pass the baton! We synthesized hollow mesoporous structured NaGdF4 nanoparticles with both excellent upconversion luminescent and magnetic resonance capacities for in vivo imaging applications. The dual-drug system CUR/NaGdF4–DOX/AFn–FA enabled the temporal release of two drugs: curcumin (CUR) is released rapidly to inhibit P-glycoprotein (P-gp) activity and to restore apoptosis signaling pathways, while doxorubicin (DOX) undergoes sustained release and thus high accumulation in drug-resistant cells to exert its therapeutic effect thanks to CUR-mediated inactivation of P-gp.
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Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment-Based Screening ()
Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention in recent years. So far, only a few studies have discussed mass spectrometry as a screening technology for fragments. Herein, we report the application of native electrospray ionization mass spectrometry (MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screening conducted with a thermal shift assay (TSA) and represented different binding categories. Our data indicated that, beside specific complex formation, many fragments show extensive multiple binding and also charge-state shifts. Both of these factors complicate automated data analysis and decrease the attractiveness of native MS as a primary screening tool for fragments. A comparison of the hits identified by native MS and TSA showed good agreement for two of the proteins. Furthermore, we discuss general challenges, including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS to be a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening. Does mass matter? Defined sets of fragments were screened against four target proteins by using native mass spectrometry (MS). Beside specific complex formation, many fragments show multiple binding and charge-state shifts. These factors complicate automated data analysis and diminish the attractiveness of native MS as a primary fragment screening tool. However, a comparison of the hits identified by native MS and thermal shift assays shows good agreement for two of the target proteins.
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Design, Synthesis, Pharmacological Evaluation and Docking Studies of GluN2B-Selective NMDA Receptor Antagonists with a Benzo[7]annulen-7-amine Scaffold ()
Antagonists that selectively target GluN2B-subunit-containing N-methyl-d-aspartate (NMDA) receptors are of major interest for the treatment of various neurological disorders. In this study, relationships between variously substituted benzo[7]annulen-7-amines and their GluN2B affinity were investigated. 2-Nitro-5,6,8,9-tetrahydrobenzo[7]annulen-7-one (8) represents the central building block for the introduction of various substituents at the 2-position and various 7-amino moieties. N-(3-Phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amines with a 2-NO2 (7 c), 2-Cl (15 c), or 2-OBn group (22 c) show very high GluN2B affinity (Ki=1.6–3.6 nm). Docking studies revealed the same binding poses for benzo[7]annulen-7-amines and ifenprodil at the interface of GluN1b and GluN2B subunits. The large 2-OBn moiety of 22 c occupies a previously unrecognized subpocket, which explains its high GluN2B affinity (Ki=3.6 nm). In two-electrode voltage clamp experiments and cytoprotection assays, the high-affinity GluN2B ligands 7 c, 15 c, and 22 c could not inhibit the glutamate-/glycine-evoked current and cytotoxic effects. However, the analogous phenols 16 c ((3-phenylpropyl)amino moiety) and 16 d ((4-phenylbutyl)amino moiety) with 10-fold lower GluN2B affinity (Ki=28 and 21 nm, respectively) showed promising inhibition of glutamate-/glycine-evoked effects in both assays. The presence of a phenolic hydroxy group seems to be essential for inducing conformational changes of the receptor protein, which finally results in closure of the ion conduction pathway. A new subpocket: 6,7,8,9-Tetrahydro-5H-benzo[7]annulen-7-amines with a nitro group, chlorine atom, or benzyloxy moiety at the 2-position show very high affinity toward GluN2B-subunit-containing NMDA receptors, but no antagonistic effects. The large 2-benzyloxy moiety occupies a previously unrecognized subpocket at the interface between the GluN1b and GluN2B subunits. The corresponding phenols reveal 10-fold lower GluN2B affinity, but promising inhibition of glutamate-/glycine-evoked effects in two-electrode voltage clamp experiments and cytoprotection assays.
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Comprehensive Analysis of Parasite Biology: From Metabolism to Drug Discovery. Edited by Sylke Müller, Rachel Cerdan and Ovidiu Radulescu; Series Editor: Paul M. Selzer ()

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