N-Acyl-3-amino-5H-furanone derivatives as new inhibitors of LuxR-dependent quorum sensing: Synthesis, biological evaluation and binding mode study

New N-acyl homoserine lactone analogues, N-acyl-3-amino-5H-furanone derivatives and some 4-halogeno counterparts, were synthesised and tested for their ability to modulate LuxR-dependent bacterial quorum sensing. Both types of analogues proved to be inhibitors, the halogenated compounds being significantly more active. Molecular modelling suggested that the conjugated enamide group induces two preferential conformations leading to specific binding modes. In addition, the presence of the halogen atom could enhance the fitting of the lactone ring through specific interactions with strictly conserved or conservatively replaceable residues in the LuxR protein family, namely Asp79, Trp94 and Ile81.     

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC₅₀ 216 μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC₅₀ values of 3.5 and 44.2 μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.     

Position—Specific contribution of interface tryptophans on membrane protein energetics

Interface tryptophans are key residues that facilitate the folding and stability of membrane proteins. Escherichia coli OmpX possesses two unique interface tryptophans, namely Trp76, which is present at the interface and is solvent-exposed, and Trp140, which is relatively more lipid solvated than Trp76 in symmetric lipid membranes. Here, we address the requirement for tryptophan and the consequences of aromatic amino acid substitutions on the folding and stability of OmpX. Using spectroscopic measurements of OmpX-Trp/Tyr/Phe mutants, we show that the specific mutation W76 → Y allows barrel assembly > 1.5-fold faster than native OmpX, and increases stability by ~ 0.4 kcal mol^− 1. In contrast, mutating W140 → F/Y lowers OmpX thermodynamic stability by ~ 0.4 kcal mol^− 1, without affecting the folding kinetics. We conclude that the stabilizing effect of tryptophan at the membrane interface can be position—and local environment—specific. We propose that the thermodynamic contributions for interface residues be interpreted with caution.     

Molecular interaction of human serum albumin with paracetamol: Spectroscopic and molecular modeling studies

The interaction between paracetamol and human serum albumin (HSA) under physiological conditions has been investigated by fluorescence, circular dichroism (CD) and docking. Fluorescence data revealed that the fluorescence quenching of HSA by paracetamol was the result of the formed complex of HSA–paracetamol, and the binding constant (K_a) and binding number obtained is 1.3 × 10⁴ at 298 K and 2, respectively for the primary binding site. Circular dichorism spectra showed the induced conformational changes in HSA by the binding of paracetamol. Moreover, protein–ligand docking study indicated that paracetamols (two paracetamols bind to HSA) bind to residues located in the subdomain IIIA.     

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2^W208F, HET-C2^W208A and HET-C2^F149Y all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2^F149A transfer activity decreased to ~ 55% but displayed ~ 120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~ 85–90%) originates from Trp109. This conclusion was supported by HET-C2^W109Y/F149Y which displayed ~ 8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λ_max by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and λ_max blue-shifts (~ 12 nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λ_max blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λ_max blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.     

New coumarin derivatives: Design,synthesis and use as inhibitors of hMAO

A series new 2H-chromene-3-carboxamides (4a–4i) and S-2H-chromene-3-carbothioates (5j–5t) were synthesized and evaluated as monoamine oxidase A and B inhibitors. Among them, compound 5k (IC₅₀ = 0.21 μM, IC_{50 iproniazid} = 7.65 μM) showed the most activity and higher MAO-B selectivity (189.2-fold vs 1-fold) with respect to the MAO-A isoform. The need to clarify at a 3D level some important molecular aspects of discussed SAR, we undertaked a number of docking simulations to better assess. The steric effect was analyzed interms of both posing and scoring by investigating the nature of the binding interactions. The docking results of active compound 5k with hMAO-B complex indicated that conserved residue ILE 199 was important for ligand binding via Sigma–Pi interaction.     

Design,synthesis and biological evaluation of N,N-3-phenyl-3-benzylaminopropanamide derivatives as novel cholesteryl ester transfer protein inhibitor

A series of N,N-3-phenyl-3-benzylaminopropanamide derivatives were identified as novel CETP (cholesteryl ester transfer protein) inhibitors. In our previous study, lead compound L10 was discovered by pharmacophore-based virtual screening (Dong-Mei Zhao et al., 2014). Based on L10 (IC₅₀ 8.06 μM), compound HL6 (IC₅₀ 10.7 μM) was discovered following systematic structure variation and biological tests. Further optimization of the structure–activity relationship (SAR) resulted in N,N-3-phenyl-3-benzylaminopro panamides derivatives as novel CETP inhibitors. They were synthesized and evaluated against CETP by BODIPY-CE fluorescence assay. Among them, HL16 (IC₅₀ 0.69 μM) was a highly potent CETP inhibitor in vitro. In addition, HL16 exhibited favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. The molecular docking of HL16 into the CETP was performed. The binding mode demonstrated that HL16 occupied the CETP binding site and formed interactions with the key amino acid residues.     

Synthesis,biological evaluation,and docking analysis of a novel family of 1-methyl-1H-pyrrole-2,5-diones as highly potent and selective cyclooxygenase-2 (COX-2) inhibitors

As a continuous research for discovery of new COX-2 inhibitors, we present the simple chemical synthesis, in vitro biological screening, and molecular docking study of 1H-pyrrole-2,5-dione derivatives. New synthetic compounds were evaluated for the inhibitory activities on LPS-induced PGE₂ production in RAW 264.7 macrophage cells as well as the COX-1 and COX-2 inhibitory potency. Among them, compound 9d (MPO-0029) was identified as more potent and selective COX-2 inhibitor [PGE₂ IC₅₀ = 8.7 nM, COX-2 IC₅₀ = 6.0 nM; COX-2 selectivity index (SI) = >168] than celecoxib. Molecular docking experiments were further performed against COX-2 and COX-1 isozymes to determine their probable binding models. Results of molecular docking studies revealed that compound 9d (MPO-0029) has stronger binding interaction with COX-2 than with COX-1 isozyme, and provided successfully complementary theoretical support for the obtained experimental biological data.     

Design and synthesis of potent and selective pyridazin-4(1H)-one-based PDE10A inhibitors interacting with Tyr683 in the PDE10A selectivity pocket

Utilizing structure-based drug design techniques, we designed and synthesized phosphodiesterase 10A (PDE10A) inhibitors based on pyridazin-4(1H)-one. These compounds can interact with Tyr683 in the PDE10A selectivity pocket. Pyridazin-4(1H)-one derivative 1 was linked with a benzimidazole group through an alkyl spacer to interact with the OH of Tyr683 and fill the PDE10A selectivity pocket. After optimizing the linker length, we identified 1-(cyclopropylmethyl)-5-[3-(1-methyl-1H-benzimidazol-2-yl)propoxy]-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (16f) as having highly potent PDE10A inhibitory activity (IC₅₀ = 0.76 nM) and perfect selectivity against other PDEs (>13,000-fold, IC₅₀ = >10,000 nM). The crystal structure of 16f bound to PDE10A revealed that the benzimidazole moiety was located deep within the PDE10A selectivity pocket and interacted with Tyr683. Additionally, a bidentate interaction existed between the 5-alkoxypyridazin-4(1H)-one moiety and the conserved Gln716 present in all PDEs.     

The synthesis of 4,7-disubstituted-2H-benzo[b][1,4]-oxazin-3(4H)-ones using Smiles rearrangement and their in vitro evaluation as platelet aggregation inhibitors

A series of novel benzo[b][1,4]oxazin-3(4H)-one derivatives were synthesized as platelet aggregation inhibitors for structure–activity relationships (SAR) analysis. The synthetic pattern, involved Smiles rearrangement for the preparation of benzoxazine, was proven to be more efficient than the conventional methods. Biological evaluation demonstrated that among all the synthesized compounds, compound 9u (IC₅₀ = 9.20 μM) exhibited the most potent inhibition activity compared with aspirin, the positive control (IC₅₀ = 7.07 μM). Molecular docking revealed that these set of compounds could be the GPIIb/IIIa antagonist for that they could be situated in the binding site of GPIIb/IIIa receptor quite well.     

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity

Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF’s tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein’s flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC₅₀ values lower than 10 μM (three hits with IC₅₀ lower than 1 μM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC₅₀ = 300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF’s tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.     

Arginase from Bacillus thuringiensis SK 20.001: Purification,characteristics, and implications for l-ornithine biosynthesis

An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2 units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000 Da as evaluated by SDS-PAGE and 191,000 Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40 °C. It was stable from pH 8.0–12.0 and <50 °C without Mn²⁺. The presence of Mn²⁺ and Ni²⁺ had strong effects on the enzyme activity, and Mn²⁺ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca²⁺, Fe²⁺ and Zn²⁺. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The K_m and V_max for l-arginine were estimated to be 15.6 mM and 538.9 μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7 g/L with the enzyme.     

Active-site mutations improved the transglycosylation activity of Stenotrophomonas maltophilia chitinase A

Transglycosylation (TG) by family 18 chitinases is of special interest due to the many biological applications of long-chain chitooligosaccharides (CHOS). In the current study, the TG activity of chitinase A from Stenotrophomonas maltophilia (StmChiA) was improved through structure-guided mutations within and around the active site. Three independent mutants were created, targeting Trp residues from the − 3 and − 1 subsites and the central catalytic Asp from the DxDxE motif of StmChiA. The former was replaced with Ala and the latter with Asn. Changes in the hydrolytic and TG activities of the enzymes were assessed by monitoring the product profile of each mutant by high-performance liquid chromatography. All three mutants showed increased TG activity. Increased in the higher TG activity of mutant W306A was accompanied by increased hydrolysis. However, this mutant also accumulated substantial amounts of TG products during the first 15–30 min of the reaction. In contrast, mutants D464N and W679A showed reduced hydrolysis, which was accompanied by the gradual accumulation of TG products up to 12 h. Molecular docking studies with chitohexaose showed that the side chains of Trp residues mediate stacking interactions with sugar residues from the − 3 and − 1 subsites, indicating the importance of these residues in the enzymatic activity of StmChiA. Overall, mutants of the glycon-binding site (W306A and W679A) appear to produce long-chain CHOS more efficiently than the catalytic mutant D464N.     

Microwave-assisted synthesis,molecular docking and antitubercular activity of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives

Based on bioisosteric similarities with isoniazid, a series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives has been designed. The target compounds have been synthesized by multicomponent reaction which involves one-pot organic reactions using ethylcyanoacetate, urea/thiourea and arylaldehydes in presence of ethanolic K₂CO₃. Two methodologies, conventional and microwave-assisted, have been adopted for the synthesis. The later strategy gave high yields in less than 10 min as compared to long hours using the former approach. Molecular docking of the target compounds into the enzyme Mycobacterium tuberculosis enoyl reductase (InhA) revealed important structural information on the plausible binding interactions. Major binding interactions were found to be of dispersion type (residues Tyr158, Ile215, Met103 and Met199) and a hydrogen bond with Tyr158. Binding poses of the all the compounds were energetically favorable and showed good interactions with the active site residues. Few selected compounds were also evaluated for antitubercular activity in vitro against drug-sensitive M. tuberculosis H37Rv strain and clinically isolated S, H, R and E resistant M. tuberculosis by luciferase reporter phage (LRP) assay method. Some compounds displayed promising antimycobacterial activity comparable or less than the standard drugs isoniazid and rifampicin.     

Rational design and synthesis of dihydropyrimidine based dual binding site acetylcholinesterase inhibitors

Based on the pharmacological importance of dihydropyrimidine (DHPM) scaffold, substituted DHPMs linked with acetamide linker to substituted aromatic anilines were synthesized and evaluated for their potency as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The good AChE inhibitory activity of 4-dihydropyrimidine-2-thione (4a–h) and 2-amino-1,4-dihyropyrimidines (5a–h) series was observed with compound 4a and 4d identified as the most potent compounds with IC₅₀ values of 0.17 ± 0.01 and 0.39 ± 0.04 μM respectively. The inhibition of BChE was found in a broader range of concentrations (2.37–56.32 μM). To explore the binding insights into the enzyme, molecular docking study was carried out using GOLD software. The binding mode analysis indicated that all of these inhibitors are well accommodated in the active site and interact with the key amino acid residues of Catalytic anionic site (CAS) and peripheral anionic site (PAS). Furthermore, in silico ADMET predictions suggest that these compounds are non-AMES toxic with good blood brain barrier (BBB) penetration, human intestinal absorption.     

In silico structural characterization and molecular docking studies of first glucuronoxylan-xylanohydrolase (Xyn30A) from family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum

CtXynGH30 is a carbohydrate active modular enzyme and component of cellulosome of Clostridium thermocellum. The full length CtXynGH30 contains an N-terminal catalytic module named as Xyn30A and a family 6 carbohydrate binding module (CBM6) at C-terminus. Xyn30A was modeled by computer program Modeller9v8 taking crystal structure of XynC from B. subtilis as a template to generate the molecular model. Model refinement was done using energy minimization by implementing steepest descent algorithm with GROMOS96 43a1 force field. Quality assessment by Ramachandran plot showed that 91% amino acids lie in most favourable region and 9% in additional allowed region. Structural analysis depicted that Xyn30A has a (β/α)₈ barrel fold. Additionally, it had a β-strand rich structure called ‘side β-structure’ attached with main catalytic core. Structural superimposition reflected that Glu136 act as a catalytic acid/base while Glu225 act as a catalytic nucleophile. Multiple sequence alignment showed that these catalytic residues are conserved within the family. The docking results showed that these residues display polar interaction with linear and substituted xylo-oligosaccharides. The binding interaction of ligands depicted that aromatic amino acids Trp81, Tyr139, Trp143, Phe172, His198, Tyr200, Tyr227, Trp264 and Tyr265 create binding site pocket around the active site. We report overall structural feature, conserved active site residues and enzyme-ligand docking of first glucuronoxylan-xylanohydrolase (Xyn30A) of family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum.     

Synthesis,biological evaluation,and docking studies of novel heterocyclic diaryl compounds as selective COX-2 inhibitors

Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1 IC₅₀ = 0.061 μM and COX-2 IC₅₀ = 0.325 μM; selectivity index (SI) = 0.19). Among the 1H-pyrazole derivatives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2 IC₅₀ = 0.011 μM and 0.398 μM, respectively), but showed no selectivity for COX-2 isoform. Compound 11c demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC₅₀ = 1 μM, COX-2 IC₅₀ = 0.011 μM; SI = ～92). Molecular docking studies of compounds 6b and 11b–d into the binding sites of COX-1 and COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.     

In vitro characterization of CYP102G4 from Streptomyces cattleya: A self-sufficient P450 naturally producing indigo

Self-sufficient CYP102As possess outstanding hydroxylating activity to fatty acids such as myristic acid. Other CYP102 subfamily members share substrate specificity of CYP102As, but, occasionally, unusual characteristics of its own subfamily have been found. In this study, only one self-sufficient cytochrome P450 from Streptomyces cattleya was renamed from CYP102A_scat to CYP102G4, purified and characterized. UV–Vis spectrometry pattern, FAD/FMN analysis, and protein sequence comparison among CYP102s have shown that CYP102 from Streptomyces cattleya belongs to CYP102G subfamily. It showed hydroxylation activity toward fatty acids generating ω-1, ω-2, and ω-3-hydroxyfatty acids, which is similar to the general substrate specificity of CYP102 family. Unexpectedly, however, expression of CYP102G4 showed indigo production in LB medium batch flask culture, and high catalytic activity (k_cat/K_m) for indole was measured as 6.14 ± 0.10 min^− 1 mM^− 1. Besides indole, CYP102G4 was able to hydroxylate aromatic compounds such as flavone, benzophenone, and chloroindoles. Homology model has shown such ability to accept aromatic compounds is due to its bigger active site cavity. Unlike other CYP102s, CYP102G4 did not have biased cofactor dependency, which was possibly determined by difference in NAD(P)H binding residues (Ala984, Val990, and Tyr1064) compared to CYP102A1 (Arg966, Lys972 and Trp1046). Overall, a self-sufficient CYP within CYP102G subfamily was characterized using purified enzymes, which appears to possess unique properties such as an only prokaryotic CYP naturally producing indigo.     

Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38

Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme. Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes. We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity. Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency (k_cat/K_m, NADPH) by more than three orders of magnitude. Similarly, substitution of Asp for Ala-37 decreased k_cat/K_m, NADPH 1000-fold but had little effect on k_cat/K_m, NADH. The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.