Synthesis,molecular docking and biological evaluation of N,N-disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase inhibitors

A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed, synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit AChE; the most active compounds inhibited BChE and CaE with IC₅₀ values of 0.22–2.3 μM. Pyridine-containing compounds were more selective toward BChE; compounds with the para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active compounds indicated non-competitive inhibition of CaE and varied mechanisms (competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations predicted key binding interactions of compounds with BChE and CaE and revealed that the best docked positions in BChE were at the bottom of the gorge in close proximity to the catalytic residues in the active site. In contrast, the best binding positions for CaE were clustered rather far from the active site at the top of the gorge. Thus, the docking results provided insight into differences in kinetic mechanisms and inhibitor activities of the tested compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<30 μM), none of the tested compounds significantly affected viability of human fetal mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal applications.     

Synthesis and biological activity of new salicylanilide N,N-disubstituted carbamates and thiocarbamates

The development of novel antimicrobial drugs represents a cutting edge research topic. In this study, 20 salicylanilide N,N-disubstituted carbamates and thiocarbamates were designed, synthesised and characterised by IR, ¹H NMR and ¹³C NMR. The compounds were evaluated in vitro as potential antimicrobial agents against Mycobacterium tuberculosis and nontuberculous mycobacteria (Mycobacterium avium and Mycobacterium kansasii) as well as against eight bacterial and fungal strains. Additionally, we investigated the inhibitory effect of these compounds on mycobacterial isocitrate lyase and cellular toxicity. The minimum inhibitory concentrations (MICs) against mycobacteria were from 4 μM for thiocarbamates and from 16 μM for carbamates. Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, were inhibited with MICs from 0.49 μM by thiocarbamates, whilst Gram-negative bacteria and most of the fungi did not display any significant susceptibility. All (thio)carbamates mildly inhibited isocitrate lyase (up to 22%) at a concentration of 10 μM. The (thio)carbamoylation of the parent salicylanilides led to considerably decreased cytotoxicity and thus improved the selectivity indices (up to 175). These values indicate that some derivatives are attractive candidates for future research.     

Identification of serine esterases in tissue homogenates

Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP. The nature of the enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and alpha-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity.     

GDSL family of serine esterases/lipases

GDSL esterases and lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity. They have potential for use in the hydrolysis and synthesis of important ester compounds of pharmaceutical, food, biochemical, and biological interests. This new subclass of lipolytic enzymes possesses a distinct GDSL sequence motif different from the GxSxG motif found in many lipases. Unlike the common lipases, GDSL enzymes do not have the so called nucleophile elbow. Studies show that GDSL hydrolases have a flexible active site that appears to change conformation with the presence and binding of the different substrates, much like the induced fit mechanism proposed by Koshland. Some of the GDSL enzymes have thioesterase, protease, arylesterase, and lysophospholipase activity, yet they appear to be the same protein with similar molecular weight (22–60 kDa for most esterases), although some have multiple glycosylation sites with higher apparent molecular weight. GDSL enzymes have five consensus sequence (I–V) and four invariant important catalytic residues Ser, Gly, Asn, and His in blocks I, II, III, and V, respectively. The oxyanion structure led to a new designation of these enzymes as SGNH-hydrolase superfamily or subfamily. Phylogenetic analysis revealed that block IIA which belonged to the SGNH-hydrolases was found only in clade I. Therefore, this family of hydrolases represents a new example of convergent evolution of lipolytic enzymes. These enzymes have little sequence homology to true lipases. Another important differentiating feature of GDSL subfamily of lipolytic enzymes is that the serine-containing motif is closer to the N-terminus unlike other lipases where the GxSxG motif is near the center. Since the first classification of these subclass or subfamily of lipases as GDSL(S) hydrolase, progress has been made in determining the consensus sequence, crystal structure, active site and oxyanion residues, secondary structure, mechanism of catalysis, and understanding the conformational changes. Nevertheless, much still needs to be done to gain better understanding of in vivo biological function, 3-D structure, how this group of enzymes evolved to utilize many different substrates, and the mechanism of reactions. Protein engineering is needed to improve the substrate specificity, enantioselectivity, specific activity, thermostability, and heterologous expression in other hosts (especially food grade microorganisms) leading to eventual large scale production and applications. We hope that this review will rekindle interest among researchers and the industry to study and find uses for these unique enzymes.     

Stereospecific inhibition of CETP by chiral N,N-disubstituted trifluoro-3-amino-2-propanols

Chiral N,N-disubstituted trifluoro-3-amino-2-propanols represent a recently discovered class of compounds that inhibit the neutral lipid transfer activity of cholesteryl ester transfer protein (CETP). These compounds all contain a single chiral center that is essential for inhibitory activity. (R,S)SC-744, which is composed of a mixture of the two enantiomers, inhibits CETP-mediated transfer of [(3)H]cholesteryl ester ([(3)H]CE) from HDL donor particles to LDL acceptor particles with an IC(50) = 200 nM when assayed using a reconstituted system in buffer and with an IC(50) = 6 microM when assayed in plasma. Upon isolation of the enantiomers, it was found that the (R,+) enantiomer, SC-795, was about 10-fold more potent than the mixture, and that the (S,-) enantiomer, SC-794, did not have significant inhibitory activity (IC(50) > 0.8 microM). All of the activity of the (S,-)SC-794 enantiomer could be accounted for by contamination of this sample with a residual 2% of the highly potent (R,+) enantiomer, SC-795. The IC(50) of (R,+)SC-795, 20 nM, approached the concentration of CETP (8 nM) in the buffer assay. These chiral N,N-disubstituted trifluoro-3-amino-2-propanols were found to associate with both LDL and HDL, but did not disrupt overall lipoprotein structure. They did not affect the on or off rates of CETP binding to HDL disk particles. Inhibition was highly specific since the activities of phospholipid transfer protein and lecithin cholesterol acyl transferase were not affected. Competition experiments showed that the more potent enantiomer (R)SC-795 prevented cholesteryl ester binding to CETP, and direct binding experiments demonstrated that this inhibitor bound to CETP with high affinity and specificity. It is estimated, based on the relative concentrations of inhibitor and lipid in the transfer assay, that (R)SC-795 binds approximately 5000-fold more efficiently to CETP than the natural ligand, cholesteryl ester. We conclude that these chiral N,N-disubstituted trifluoro-3-amino-2-propanol compounds do not affect lipoprotein structure or CETP-lipoprotein recognition, but inhibit lipid transfer by binding to CETP reversibly and stereospecifically at a site that competes with neutral lipid binding.     

Solid-phase synthesis of benzisothiazolones as serine protease inhibitors

An efficient solid-phase synthesis of benzisothiazolone-1,1-dioxide-based serine protease inhibitors involving alkylation of carboxylic acids with N-(bromomethyl)benzisothiazolone-1,1-dioxide has been developed. An example using this procedure for preparation of a library of human mast cell tryptase inhibitors is described.     

Potential of pyrazolooxadiazinone derivatives as serine protease inhibitors

As a part of an investigation on molecular hybrids as new serine protease inhibitors, the pyrazolo [4,3-c][1,2,5]oxadiazin-3(5H)-one ring system was selected as a model of potential mechanism-based inhibitors. Due to the inherent reactivity of this system an optimal balance between susceptibility to nucleophilic attack and stability in solvents was sought prior to development as therapeutic agents. Substitutions on N5 and C7 of the supporting pyrazole ring with either aliphatic or aromatic groups (compounds 2 a-m) and the replacement of the carbonyl oxygen on the reactive oxadiazinone ring with sulfur (compounds 3a,i) were explored. Two members (2i and 2k) of this class of inhibitors displayed time-dependent inhibition of HLE suggesting mechanism-based inhibition. The observation that HLE generated a product(s) from compound 2i which displayed an identical UV-Visible spectrum to that observed during non-enzymatic hydrolysis further supports this proposal. FlexX-based docking of these compounds into a model of the human leukocyte elastase (HLE) active site produced a molecular model of the inhibitor-enzyme interaction.     

Design and synthesis of conformationally constrained N,N-disubstituted 1,4-diazepanes as potent orexin receptor antagonists

Orexins are neuropeptides that regulate wakefulness and arousal. Small molecule antagonists of orexin receptors may provide a novel therapy for the treatment of insomnia and other sleep disorders. In this Letter we describe the design and synthesis of conformationally constrained N,N-disubstituted 1,4-diazepanes as orexin receptor antagonists. The design of these constrained analogs was guided by an understanding of the preferred solution and solid state conformation of the diazepane central ring.     

Characterization of three serine esterases isolated from human IL-2 activated killer cells

Human peripheral blood mononuclear cells, activated for 14 to 20 days with 1000 U/ml rIL-2, develop strong cytotoxicity for NK sensitive and resistant targets. This process is accompanied by the acquisition of cytoplasmic granules in approximately 60% of the cells and by the expression of esterase activity cleaving the synthetic substrate BLT. The esterase activity, localized in the cytoplasmic granules, was purified and characterized. Three proteins with 3H-DFP binding activity were isolated and had the following properties. Following the proposed nomenclature by Masson et al., the esterases were named human granzymes 1, 2, and 3. Human granzyme 1 on SDS-PAGE has an unreduced relative m.w. of 43,000 and can form disulfide-linked oligomers of relative higher m.w. All forms of granzyme 1 bind 3H-DFP. Upon reduction, granzyme 1 migrates with Mr 30,000 on SDS-PAGE. Additional proteolytic fragments of Mr 24,000 and Mr 28,000 are observed in some reduced preparations. Granzyme 1 cleaves the substrate BLT and appears homologous with murine granzyme A. Human granzyme 2 has an unreduced relative m.w. of 30,000; after reduction, it migrates at Mr 32,000. Even though granzyme 2 binds 3H-DFT, it does not cleave BLT. Human granzyme 2 has properties similar to those of murine granzymes B-H. Human granzyme 3 has unreduced and reduced relative m.w. of 25,000 and 28,000, respectively. It is active in cleaving the substrate BLT. A murine analog for human granzyme 3 has not been described previously. N-terminal sequencing of the purified human granzymes revealed that human granzyme 1 is the gene product of human Hanuka factor cDNA clone and that it represents the human homolog to murine granzyme A. Similarly, human granzyme 2 revealed absolute identity with cDNA-derived N-terminal sequence of a putative human lymphocyte protease cDNA clone.     

Reactions of serine palmitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors

Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A to 3-ketodihydrosphingosine. We have succeeded in the overproduction of a water-soluble homodimeric SPT from Sphingomonas paucimobilis EY2395(T) in Escherichia coli. The recombinant SPT showed the characteristic absorption and circular dichroism spectra derived from its coenzyme pyridoxal 5'-phosphate. On the basis of the spectral changes of SPT, we have analyzed the reactions of SPT with compounds related to L-serine and product, and showed the following new aspects: First, we analyzed the binding of L-serine and 3-hydroxypropionate and found that the spectral change in SPT by the substrate is caused by the formation of an external aldimine intermediate and not by the formation of the Michaelis complex. Second, various serine analogues were also examined; the data indicated that the alpha-carboxyl group of L-serine was quite important for substrate recognition by SPT. Third, we focused on a series of SPT inhibitors, which have been used as convenient tools to study the cell responses caused by sphingolipid depletion. The interaction of SPT with myriocin suggested that such product-related compounds would strongly and competitively inhibit enzyme activity by forming an external aldimine in the active site of the enzyme. Beta-chloro-L-alanine and L-cycloserine were found to generate characteristic PLP-adducts that produced inactivation of SPT in an irreversible manner. The detailed mechanisms for the SPT inactivation were discussed. This is the first analysis of the inhibition mechanisms of SPT by these compounds, which will provide an enzymological basis for the interpretation of the results from cell biological experiments.     

Granules of cytolytic T-lymphocytes contain two serine esterases.

Cytoplasmic granules from cytolytic T-lymphocytes (CTL) contain two proteins which react with the serine esterase-specific affinity label diisopropylfluorophosphate (DFP). One of these is a trypsin-like esterase which consists of two disulfide-linked 35-kd subunits. The other consists of a single 29-kd chain. Both molecules are induced concomitantly with cytolytic activity and perforin activity in a CTL-derived T-cell hybrid.     

Substituted isocoumarins as inhibitors of complement serine proteases.

Inhibition of complement proteins D, B, C2, C1s, C1r, I, and the catalytic fragments Bb and C2a by substituted isocoumarins was investigated. 3,4-Dichloroisocoumarin, a general serine protease inhibitor, inhibited factor D, C1r, and C1s moderately with second-order inhibition constants (kobs/[I]) of 40 to 190 M-1 s-1, but it did not inhibit C2, factor B, C2a, or Bb. The best inhibitor for factors D and B was 4-chloro-7-guanidino-3-methoxyisocoumarin with kobs/[I] values of 250 and 290 M-1 s-1, respectively. Most isocoumarins did not inhibit C2 or C2a; only 4-chloro-3-isothiureidoalkoxyisocoumarins were slightly inhibitory. 3-Alkoxy-4-chloro-7-guanidinoisocoumarins inhibited C1r and C1s moderately. The best inhibitor for C1r and C1s was 4-chloro-3-(3-isothiureidopropoxy)isocoumarin with kobs/[I] values of 6,600 and 130,000 M-1 s-1, respectively. Fifty amino acid or peptide thioesters containing Arg or other amino acids at the P1 site were tested as substrates of factor I, however none was hydrolyzed. Isocoumarins substituted with chloro and basic groups such as guanidino and isothiureidoalkoxy inhibited factor I activity with its natural substrate C3b, but kobs/[I] values were low. 4-Chloro-3-ethoxy-7-guanidinoisocoumarin inhibited activation of the alternative pathway and, to a lesser extent, of the classical pathway in serum. Several other substituted isocoumarins also inhibited cobra venom factor-initiated activation of the alternative pathway in serum.     

Synthesis and erythropoietin receptor binding affinities of N,N-disubstituted amino acids

N,N-Dicinnamyl, N-benzyl-N-cinnamyl, and N,N-dibenzyl amino acids were prepared and evaluated in an EPO binding assay. Several derivatives of aspartic acid, glutamic acid, and lysine exhibited moderate (10-50 microM) affinity for EBP; 'dimerization' of the most potent analogues by coupling with linear diamines led to EPO competitors having 1-2 microM binding affinities.     

N,N-disubstituted aminomethyl benzofuran derivatives: synthesis and preliminary binding evaluation

A series of new N-substituted 2,3-dihydro-2-aminomethyl-2H-1-benzofuran derivatives was prepared and evaluated for affinity at 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, D2, and D3 receptors. Compound 9, 8-[4-[N-propyl-N-(7-hydroxy-2,3-dihydro -2H-1-benzofuran-2-yl)methyl]aminobutyl]-8-azaspiro[4,5]decane-7,9 -dione, bound at 5-HT1A sites with nanomolar affinity (IC50= 1.5 nM) and high selectivity over 5-HT2A, 5-HT2C, 5-HT3, D2, and D3 receptors.     

Pseudo-peptides derived from isomannide as potential inhibitors of serine proteases

Summary. Hepatitis C, Dengue and West Nile virus are among of the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes, and are a primary target in the drug development field. In this paper, we describe the synthesis and preliminary molecular modeling studies of a novel class of N-t-Boc amino acid amides derived of isomannide as potential serine proteases inhibitors.     

Isosorbide-based peptidomimetics as inhibitors of hepatitis C virus serine protease

Hepatitis C infection is a cause of chronic liver diseases such as cirrhosis and carcinoma. The current therapy for hepatitis C has limited efficacy and low tolerance. The HCV encodes a serine protease which is critical for viral replication, and few protease inhibitors are currently on the market. In this paper, we describe the synthesis and screening of novel isosorbide-based peptidomimetic inhibitors, in which the compounds 1d, 1e, and 1i showed significant inhibition of the protease activity in vitro at 100 µM. The compound 1e also showed dose-response (IC₅₀ = 36 ± 3 µM) and inhibited the protease mutants D168A and V170A at 100 µM, indicating it as a promising inhibitor of the HCV NS3/4A protease. Our molecular modeling studies suggest that the activity of 1e is associated with a change in the interactions of S2 and S4 subsites, since that the increased flexibility favors a decrease in activity against D168A, whereas the appearance of a hydrophobic cavity in the S4 subsite increase the inhibition against V170A strain.     

Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents

We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.     

Salicylaldehyde derivatives as new protein kinase CK2 inhibitors

Protein kinase CK2 is a Ser/Thr kinase, with a constitutive activity, that is considered as a promising target for cancer therapy. The currently available CK2 inhibitors lack the potency and the pharmacological properties necessary to be suitable and successful in clinical settings. We report the development of new potent CK2 inhibitors from salicylaldehyde derivatives identified by automated screening of a proprietary small-molecule library. Docking simulations and analysis of the structure-activity relationship for the hits allowed to determine their binding modes on CK2, and to carry out the optimization of their structures. This strategy led to the discovery of potent CK2 inhibitors with novel structures, one of which was able to inhibit CK2 activity in living cells and promote tumor cell death. The essential features required for potent CK2 inhibitory activity of this class of compounds are discussed.     

The interaction of alkynyl carboxylates with serine enzymes. A potent new class of serine enzyme inhibitors

The recently reported alkynyl esters, propynyl benzoate and propynyl p-methoxybenzoate, were found to interact with a variety of serine enzymes. alpha-Chymotrypsin was inhibited very rapidly by an equivalent amount of the esters. Trypsin, elastase and pronase were also inhibited by the esters. On the other hand, liver esterase started to hydrolyze the alkynyl esters rapidly, but the enzyme became inhibited during the course of reaction. The inhibited enzymes exhibited slow reactivation which could be considerably enhanced by hydroxylamine.     

Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors

Serine hydrolases are a diverse enzyme class representing ～1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.