Capturing the Direct Binding of CFTR Correctors to CFTR by Using Click Chemistry

Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX‐809 has been reported to facilitate the folding and trafficking of F508del‐CFTR and augment its channel function. The mechanism of action of VX‐809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX‐809: does it bind CFTR directly in order to exert its action? We synthesized two VX‐809 derivatives, ALK‐809 and SUL‐809, that possess an alkyne group and retain the rescue capacity of VX‐809. By using Cu^I‐catalyzed click chemistry, we provide evidence that the VX‐809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.     

A Systematic Investigation of Iminosugar Click Clusters as Pharmacological Chaperones for the Treatment of Gaucher Disease

A series of 18 mono‐ to 14‐valent iminosugars with different ligands, scaffolds, and alkyl spacer lengths have been synthesized and evaluated as inhibitors and pharmacological chaperones of β‐glucocerebrosidase (GCase). Small but significant multivalent effects in GCase inhibition have been observed for two iminosugar clusters. Our study provides strong confirmation that compounds that display the best affinity for GCase are not necessarily the best chaperones. The best chaperoning effect observed for a deprotected iminosugar cluster has been obtained with a tetravalent 1‐deoxynojirimycin (DNJ) analogue (3.3‐fold increase at 10 μM ). In addition, our study provides the first evidence of the high potential of prodrugs for the development of potent pharmacological chaperones. Acetylation of a trivalent DNJ derivative, to give the corresponding acetate prodrug, leads to a pharmacological chaperone that produces higher enzyme activity increases (3.0‐fold instead of 2.4‐fold) at a cellular concentration (1 μM ) reduced by one order of magnitude.     

Synthetic 1‐Deoxynojirimycin N‐Substituted Peptides Offer Prolonged Disruption to N‐Linked Glycan Processing

A panel of 1‐deoxynojirimycin (DNJ) N‐linked peptides were synthesized. Their IC₅₀ values were measured in vitro against α‐glucosidases I and II and were found to be in the micromolar range for both isozymes, and better than that of the iminosugar NB‐DNJ (miglustat, 3 ) against α‐glucosidase II. Cell‐based studies revealed that although the free iminosugar 3 is most effective at disrupting N‐linked glycan processing for short‐term incubations (one day), when the cell‐based studies were extended to three days, the DNJ N‐linked tetrapeptide KDEL, which is an endoplasmic reticulum (ER)‐retaining sequence, performed far better than 3 . In low inhibitor washout studies, NB‐DNJ inhibition was decreased to zero after 24 h, but DNJ–KDEL retained 13 % activity. This method offers a general approach for targeting drugs to the ER and prolonging their activity. Moreover, it is modular, so as new iminosugars of increased potency are discovered, they can be added to this template for targeting.     

Targeting of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Protein with a Technetium‐99m Imaging Probe

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to almost total absence of CFTR at the plasma membrane, a defect potentially corrected via drug‐based therapies. Herein, we report the first proof‐of‐principle study of a noninvasive imaging probe able to detect CFTR at the plasma membrane. We radiolabeled the CFTR inhibitor, CFTR_inh‐172a, with technetium‐99m via a pyrazolyl‐diamine chelating unit, yielding a novel ^99mTc(CO)₃ complex. A non‐radioactive surrogate showed that the structural modifications introduced in the inhibitor did not affect its activity. The radioactive complex was able to detect plasma membrane CFTR, shown by its significantly higher uptake in wild‐type versus mutated cells. Furthermore, assessment of F508del CFTR pharmacological correction in human cells using the radioactive complex revealed differences in corrector versus control uptake, recapitulating the biochemical correction observed for the protein.     

Highly Diverse Protein Library Based on the Ubiquitous (β/α)8 Enzyme Fold Yields Well‐Structured Proteins through in Vitro Folding Selection

Proper protein folding is a prerequisite for protein stability and enzymatic activity. Although directed evolution can be a powerful tool to investigate enzymatic function and to isolate novel activities, well‐designed libraries of folded proteins are essential. In vitro selection methods are particularly capable of searching for enzymatic activities in libraries of trillions of protein variants, yet high‐quality libraries of well‐folded enzymes with such high diversity are lacking. We describe the construction and detailed characterization of a folding‐enriched protein library based on the ubiquitous (β/α)₈ barrel fold, which is found in five of the six enzyme classes. We introduced seven randomized loops on the catalytic face of the monomeric, thermostable (β/α)₈ barrel of glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima. We employed in vitro folding selection based on protease digestion to enrich intermediate libraries containing three to four randomized loops for folded variants, and then combined them to assemble the final library (10¹⁴ DNA sequences). The resulting library was analyzed by using the in vitro protease assay and an in vivo GFP‐folding assay; it contains ～10¹² soluble monomeric protein variants. We isolated six library members and demonstrated that these proteins are soluble, monomeric and show (β/α)₈‐barrel fold‐like secondary and tertiary structure. The quality of the folding‐enriched library improved up to 50‐fold compared to a control library that was assembled without the folding selection. To the best of our knowledge, this work is the first example of combining the ultra‐high throughput mRNA display method with selection for folding. The resulting (β/α)₈ barrel libraries provide a valuable starting point to study the unique catalytic capabilities of the (β/α)₈ fold, and to isolate novel enzymes.     

Chaperone Activity of Bicyclic Nojirimycin Analogues for Gaucher Mutations in Comparison with N‐(n‐nonyl)Deoxynojirimycin

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by mutations of lysosomal β‐glucosidase (acid β‐Glu, β‐glucocerebrosidase); these mutations result in protein misfolding. Some inhibitors of this enzyme, such as the iminosugar glucomimetic N‐(n‐nonyl)‐1‐deoxynojirimycin (NN‐DNJ), are known to bind to the active site and stabilize the proper folding for the catalytic form, acting as “chemical chaperones” that facilitate transport and maturation of acid β‐Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp² iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal β‐Glu. We have now evaluated the glycosidase inhibitory profile of a series of six compounds within this family, namely 5‐N,6‐O‐(N′‐octyliminomethylidene‐NJ (NOI‐NJ), the 6‐thio and 6‐amino‐6‐deoxy derivatives (6S‐NOI‐NJ and 6N‐NOI‐NJ) and the corresponding galactonojirimycin (GNJ) counterparts (NOI‐GNJ, 6S‐NOI‐GNJ and 6N‐NOI‐GNJ), against commercial as well as lysosomal glycosidases. The chaperone effects of four selected candidates (NOI‐NJ, 6S‐NOI‐NJ, 6N‐NOI‐NJ, and 6S‐NOI‐GNJ) were further evaluated in GD fibroblasts with various acid β‐Glu mutations. The compounds showed enzyme enhancement on human fibroblasts with N188S, G202R, F213I or N370S mutations. The chaperone effects of the sp² iminosugar were generally stronger than those observed for NN‐DNJ; this suggests that these compounds are promising candidates for clinical treatment of GD patients with a broad range of β‐Glu mutations, especially for neuronopathic forms of Gaucher disease.     

The Effects of Conformational Constraints in the Polyamine Moiety of Philanthotoxins on AMPAR Inhibition

Philanthotoxin‐433 (PhTX‐433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subtype by using two‐electrode voltage‐clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX‐343, with trans‐cyclopropyl‐PhTX‐343 being the most potent (～28‐fold) and cis‐cyclopropyl‐PhTX‐343 least potent (～4‐fold). Both cis‐ and trans‐cyclopropyl‐PhTX‐444 had intermediate potency (both ～12‐fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX‐444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted.     

Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment

Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta^®/Kaftrio^®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60–70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.     

Discovery and characterization of atropisomer PH-797804, a p38 MAP kinase inhibitor, as a clinical drug candidate

PH‐797804 ((aS)‐3‐{3‐bromo‐4‐[(2,4‐difluorobenzyl)oxy]‐6‐methyl‐2‐oxopyridin‐1(2H)‐yl}‐N,4‐dimethylbenzamde) is a diarylpyridinone inhibitor of p38 mitogen‐activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. Due to steric constraints imposed by the pyridinone carbonyl group and the 6‐ and 6′‐methyl substituents of PH‐797804, rotation around the connecting bond of the pyridinone and the N‐phenyl ring is restricted. Density functional theory predicts a remarkably high rotational energy barrier of >30 kcal mol^?1, corresponding to a half‐life of more than one hundred years at room temperature. This gives rise to discrete conformational spaces for the N‐phenylpyridinone group, and as a result, two atropic isomers that do not interconvert under ambient conditions. Molecular modeling studies predict that the two isomers should differ in their binding affinity for p38α kinase; whereas the atropic S (aS) isomer binds favorably, the opposite aR isomer incurs significant steric interference with p38α kinase. The two isomers were subsequently identified and separated by chiral chromatography. IC₅₀ values from p38α kinase assays confirm that one atropisomer is >100‐fold more potent than the other. It was ultimately confirmed by small‐molecule X‐ray diffraction that the more potent atropisomer, PH‐797804, is the aS isomer of the racemic pair. Extensive pharmacological characterization supports that PH‐797804 carries most activity both in vitro and in vivo, and it has a stability profile compatible with oral formulation and delivery options.     

Correctors Rescue CFTR Mutations in Nucleotide‐Binding Domain 1 (NBD1) by Modulating Proteostasis

We evaluated whether small molecule correctors could rescue four nucleotide‐binding domain 1 (NBD1) mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (A455E, S492F, ΔI507, and R560T). We first transfected Cos‐7 cells (green monkey kidney cells) with A455E, S492F, ΔI507, or R560T and created HEK‐293 (human embryonic kidney cells) cell lines stably expressing these CFTR mutations. The mutants showed lowered protein expression, instability at physiological temperature, and rapid degradation. After treatment with correctors CFFT‐002, CFFT‐003, C3, C4, and/or C18, the combination of C18+C4 showed the most correction and resulted in increased CFTR residing in the plasma membrane. We found a profound decrease in binding of CFTR to histone deacetylases (HDAC) 6 and 7 and heat shock proteins (Hsps) 27 and 40. Silencing Hsp27 or 40 rescued the mutants, but no additional amount of CFTR was rescued when both proteins were knocked down simultaneously. Thus, CFTR mutations in NBD1 can be rescued by a combination of correctors, and the treatment alters the interaction between mutated CFTR and the endoplasmic reticulum machinery.     

New Insights into the Pharmacological Chaperone Activity of C2‐Substituted Glucoimidazoles for the Treatment of Gaucher Disease

Mutations in acid β‐glucocerebrosidase (GCase) lead to the accumulation of the sphingolipid glucosylceramide, thereby resulting in Gaucher disease (GD). Active‐site‐specific competitive GCase inhibitors are effective pharmacological chaperones (PCs) that act as folding agents for mutant GCase folding in the endoplasmic reticulum. In this study, we prepared a series of glucoimidazole C2‐substituent derivatives, and evaluated their inhibition and PC properties with GCase. A cell‐based assay with patient‐derived lymphoblasts (N370S or L444P mutations) demonstrated that administration of these compounds can significantly increase GCase activity. Interestingly, the 3,3‐dimethyl‐N‐phenyl‐4‐amide‐1‐butyl‐substituted moderate inhibitor 11 had the greatest effect on activity: 2.1‐fold increase in N370S lymphoblasts at 2.5 μM and 1.2‐fold increase in L444P at 0.5 μM following a three‐day incubation. Computer docking studies and a protease protection assay were used to elucidate the ligand–enzyme interactions responsible for the chaperone activity of 11 . Western blot and immuno‐fluorescence assays verified restoration of GCase trafficking to the lysosome. Together, these results indicate that 11 is a promising PC for GD treatment and provide direct evidence of the mechanism of GCase chaperoning.     

An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3‐N‐Acetyl‐D‐glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose‐Binding Protein

A fusion protein composed of β1,3‐N‐acetyl‐D ‐glucosaminyltransferase (β1,3‐GlcNAcT) from Streptococcus agalactiae type Ia and maltose‐binding protein (MBP) was produced in Escherichia coli as a soluble and highly active form. Although this fusion protein (MBP‐β1,3‐GlcNAcT) did not show any sugar‐elongation activity to some simple low‐molecular weight acceptor substrates such as galactose, Galβ(1→4)Glc (lactose), Galβ(1→4)GlcNAc (N‐acetyllactosamine), Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glc (lacto‐N‐tetraose), and Galβ(1→4)GlcβCer (lactosylceramide, LacCer), the multivalent glycopolymer having LacCer‐mimic branches (LacCer mimic polymer, LacCer primer) was found to be an excellent acceptor substrate for the introduction of a β‐GlcNAc residue at the O‐3 position of the non‐reducing galactose moiety by this engineered enzyme. Subsequently, the polymer having GlcNAcβ(1→3)Galβ(1→4)Glc was subjected to further enzymatic modifications by using recombinant β1,4‐D ‐galactosyltransferase (β1,4‐GalT), α2,3‐sialyltransferase (α2,3‐SiaT), α1,3‐L ‐fucosyltransferase (α1,3‐FucT), and ceramide glycanase (CGase) to afford a biologically important ganglioside; Neu5Aα(2→3)Galβ(1→4)[Fucα(1→3)]GlcNAcβ(1→3)Galβ(1→4)GlcCerα(IV3Neu5Acα,III3Fucα‐nLc4Cer) in 40% yield (4 steps). Interestingly, it was suggested that MBP‐β1,3‐GlcNAcT could also catalyze a glycosylation reaction of the LacCer mimic polymer with N‐acetyl‐D ‐galactosamine served from UDP‐GalNAc to afford a polymer carrying trisaccharide branches, GalNAcβ(1→3)Galβ(1→4)Glc. The versatility of the MBP‐β1,3‐GlcNAcT in the practical synthesis was preliminarily demonstrated by applying this fusion protein as an immobilized biocatalyst displayed on the amylose resin which is known as a solid support showing potent binding‐affinity with MBP.     

Kinetics of Bu‐NENA Evaporation from Bu‐NENA/NC Propellant Determined by Isothermal Thermogravimetry

Bu‐NENA (N‐butyl‐N‐nitratoethyl nitramine) base propellants have versatile qualities, such as, higher energy, reduced sensitivity, and enhanced mechanical properties. The evaporation of Bu‐NENA, which takes place in the propellant grains in the course of time, can reduce the physical properties of the propellants, weaken the propellant grains, cause the propellants to crack at stress‐concentrated points, and finally result in unfavorable increases or fluctuations of the burning rate and poor performance of the rocket motor. In this study, the evaporation of Bu‐NENA from a double base propellant was investigated using isothermal thermogravimetry. The results showed that the entire process of Bu‐NENA evaporation complied with the power law of evaporation rate with time. The values of kinetic parameters of Bu‐NENA evaporation were calculated: E _vap=67.68 kJ mol⁻¹ and A _vap=1.57×10⁵ s⁻¹. In comparison, the values of NG (nitroglycerin) evaporation were determined: E _vap=69.68 kJ mol⁻¹ and A _vap=1.33×10⁶ s⁻¹. The value of the activation energy of Bu‐NENA evaporation was close to that of NG, but the pre‐exponential factors differed by an order of magnitude. The evaporation of Bu‐NENA followed zero‐order kinetics at the early stage, and the enthalpy of Bu‐NENA evaporation was calculated to be 69.75 kJ mol⁻¹ according to Langmuir and Clausius‐Clapeyron equations.     

Identification of Potential Leukocyte Biomarkers Related to Drug Recovery of CFTR: Clinical Applications in Cystic Fibrosis

The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC–MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl⁻ transport in CF.     

Developing an Irreversible Inhibitor of Human DDAH‐1, an Enzyme Upregulated in Melanoma

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase‐1 (DDAH‐1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. In this study, N⁵‐(1‐imino‐2‐chloroethyl)‐L ‐ornithine (Cl‐NIO) is shown to be a potent time‐ and concentration‐dependent inhibitor of purified human DDAH‐1 (K_I=1.3±0.6 μM ; k_inact=0.34±0.07 min^?1), with >500‐fold selectivity against two arginine‐handling enzymes in the same pathway. An activity probe is used to measure the “in cell” IC₅₀ value (6.6±0.2 μM ) for Cl‐NIO inhibition of DDAH‐1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50/64 (78 %) of melanoma lines tested showed increased levels of DDAH‐1 relative to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl‐NIO shows a subsequent decrease in cellular nitric oxide production. Cl‐NIO is a promising tool for the study of methylarginine‐mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production, such as septic shock and idiopathic pulmonary fibrosis.     

Influence of Cleavage Site on Global Folding of an RNA‐Cleaving DNAzyme

8–17 is a DNAzyme with metal‐dependent endoribonuclease activity. Recently, a variant termed 8–17NG was reported as the first nucleic acid enzyme capable of cleaving all 16 dinucleotide junctions of RNA with rate enhancements ranging from 1000‐ to 1 000 000 000‐fold over background activity. We attributed this broad‐ranging cleavage efficiency to global folding of the DNAzyme. We sought to examine the influence of dinucleotides at the cleavage site of 8–17NG on global folding by using three‐color (3c) FRET. By comparing the folding of 8–17NG with all 16 possible dinucleotide junctions, we found all examined DNAzyme–substrate constructs adopted a two‐step folding process in the presence of Mn²⁺, which was consistent with previous metal‐induced folding studies of 8–17. Interestingly, Mn²⁺ titration experiments also suggest that the second folding step is dependent on dinucleotide identity: purine–purine junctions allowed 8–17NG to fold at lower concentrations than pyrimidine–pyrimidine linkages. This finding was corroborated by RNA cleavage assays, in which the largest improvement in cleavage yield was observed in pyrimidine–pyrimidine junctions when [Mn²⁺] was increased. Taken together, these results support the previously observed hierarchy of 8–17 activity for different cleavage sites. Complemented by earlier sequence and structure–function studies, this investigation allowed for the first detailed examination of crucial relationships between the structural influence and junction preferences of nucleic acid‐catalyzed RNA cleavage reactions.     

Ligand‐Biased and Probe‐Dependent Modulation of Chemokine Receptor CXCR3 Signaling by Negative Allosteric Modulators

Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein‐coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8‐azaquinazolinone derivative (N‐{1‐[3‐(4‐ethoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidin‐2‐yl]ethyl}‐4‐(4‐fluorobutoxy)‐N‐[(1‐methylpiperidin‐4‐yl)methyl]butanamide, 1 b ) that can inhibit CXC chemokine 11 (CXCL11)‐dependent G protein activation over β‐arrestin recruitment with 187‐fold selectivity. This compound also demonstrates probe‐dependent activity, that is, it inhibits CXCL11‐ over CXCL10‐mediated G protein activation with 12‐fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.     

Design,Synthesis, and Antibacterial Activity of Demethylvancomycin Analogues against Drug‐Resistant Bacteria

Five novel N‐substituted demethylvancomycin derivatives were rationally designed and synthesized by using a structure‐based approach. The in vitro antibacterial activities against methicillin‐resistant Staphylococcus aureus (MRSA), gentamicin‐resistant Enterococcus faecalis (GRE), methicillin‐resistant Streptococcus pneumoniae (MRS), and vancomycin‐resistant Enterococcus faecalis (VRE) were evaluated. One of the compounds, N‐(6‐phenylheptyl)demethylvancomycin ( 12 a ), was found to exhibit more potent antibacterial activity than vancomycin and demethylvancomycin. Compound 12 a was also found to be ～18‐fold more efficacious than vancomycin against MRSA; however, the two compounds were found to have similar efficacy against MRS. Furthermore, compound 12 a exhibited a favorable pharmacokinetic profile with a half‐life of 5.11±0.52 h, which is longer than that of vancomycin (4.3±1.9 h). These results suggest that 12 a is a promising antibacterial drug candidate for further preclinical evaluation.     

1‐Arylsulfonyl‐5‐(N‐hydroxyacrylamide)indolines Histone Deacetylase Inhibitors Are Potent Cytokine Release Suppressors

A series of 1‐arylsulfonyl‐5‐(N‐hydroxyacrylamide)indolines ( 7 – 15 ) has been developed; the compounds exhibited potent histone deacetylase (HDAC) inhibitory activities. Notably, almost all of this series exhibited better HDAC‐inhibitory and antiproliferative activities than 3‐(1‐benzenesulfonyl‐1H‐indol‐5‐yl)‐N‐hydroxyacrylamide ( 6 ), as reported in a previous study. Among these compounds, 3‐[1‐(4‐methoxybenzenesulfonyl)‐2,3‐dihydro‐1H‐indol‐5‐yl]‐N‐hydroxyacrylamide ( 9 ) showed a two‐ to tenfold increase in activity compared to SAHA ( 1 ) in the suppression of lipopolysaccharide‐induced cytokine production. Compound 9 also caused a marked reduction in carrageenan‐induced acute inflammation in a rat model. Taken together, these data indicated that 1‐arylsulfonyl‐5‐(N‐hydroxyacrylamide)indolines HDAC inhibitors exhibit potent anti‐inflammatory activity.