Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment,Molecular Docking,and Enzyme Activity/Binding Assays

Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non‐HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo‐phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3‐disubstituted‐4‐amino‐pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC₅₀) in the nanomolar range with monomeric substrate diC₄PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co‐ordinates were available) and results analyzed to develop next‐generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high‐throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets.     

Synthesis,Molecular Modeling,and Biological Evaluation of Novel 1, 3‐Diphenyl‐2‐propen‐1‐one Based Pyrazolines as Anti‐inflammatory Agents

A novel series of 1,3‐diphenyl‐2‐propen‐1‐one (chalcone) derivatives was synthesized by a simple, eco‐friendly, and efficient Claisen–Schmidt condensation reaction and used as precursors for the synthesis of new pyrazoline derivatives. All the synthesized compounds were screened for anti‐inflammatory related activities such as inhibition of phospholipase A₂ (PLA₂), cyclooxygenases (COX‐1 and COX‐2), IL‐6, and TNF‐α. The results of the above studies show that the compounds synthesized are effective inhibitors of above pro‐inflammatory enzymes and cytokines. Overall, the results of the studies reveal that the pyrazolines with chlorophenyl substitution ( 1b – 6b ) seem to be important for inhibition of enzymes and cytokines. Molecular docking experiments were performed to clarify the molecular aspects of the observed COX‐inhibitory activities of the investigated compounds.     

Click chemistry‐based synthesis and cytotoxic activity evaluation of 4α‐triazole acetate podophyllotoxin derivatives

A series of novel 4α‐triazole acetate podophyllotoxin derivatives were synthesized via click chemistry. In vitro cytotoxic activity evaluation showed that most of the derivatives exhibited potent inhibitory activities against the tested cancer cell lines with low nanomolar IC₅₀ values. Further studies demonstrated that compound 31 exhibited broad‐spectrum cytotoxic activities, effectively overcame drug‐resistance, and showed relatively weak cytotoxicity on non‐cancer cells. Preliminary mechanistic studies indicated that 31 might have action on microtubule, cause cell cycle arrest at G₂/M phase, and induce apoptosis in human PC‐3 cancer cells.     

Effects of Novel Diarylpentanoid Analogues of Curcumin on Secretory Phospholipase A2, Cyclooxygenases,Lipo‐oxygenase,and Microsomal Prostaglandin E Synthase‐1

Arachidonic acid and its metabolites have generated a heightened interest due to their significant role in inflammation. Inhibiting the enzymes involved in arachidonic acid metabolism has been considered as the synergistic anti‐inflammatory effect. A series of novel curcumin diarylpentanoid analogues were synthesized and evaluated for their inhibitory effects on activity of secretory phospholipase A₂, cyclooxygenases, soybean lipo‐oxygenase as well as microsomal prostaglandin E synthase‐1. Among the curcumin analogues, compounds 3 , 6 , 9 , 12, and 17 exhibited strong inhibition of secretory phospholipase A₂ activity, with IC₅₀ values ranging from 5.89 to 11.02 μm . Seven curcumin analogues 1 , 3 , 6 , 7 , 9 , 11, and 12 showed inhibition of cyclooxygenases‐2 with IC₅₀ values in the range of 46.11 to 94.86 μm , which were lower than that of curcumin. Compounds 3 , 6 , 7 , 12, and 17 showed strong inhibition of lipo‐oxygenase enzyme activity. Preliminary screening of diarylpentanoid curcumin analogues for microsomal prostaglandin E synthase‐1 activity revealed that four diarylpentanoid curcumin analogues 5 , 6 , 7 , and 13 demonstrated higher inhibition of microsomal prostaglandin E synthase‐1 activity with IC₅₀ ranging from 2.41 to 4.48 μm , which was less than that of curcumin. The present results suggest that some of these diarylpentanoid analogues were able to inhibit the activity of these enzymes. This raises the possibility that diarylpentanoid analogues of curcumin might serve as useful starting point for the design of improved anti‐inflammatory agents.     

Synthesis,Antibacterial Activities,and 3D‐QSAR of Sulfone Derivatives Containing 1, 3, 4‐Oxadiazole Moiety

A series of sulfone derivatives containing 1, 3, 4‐oxadiazole moiety were prepared and evaluated for their antibacterial activities by the turbidimeter test. Most compounds inhibited growth of Ralstonia solanacearum (R. solanacearum) from tomato and tobacco bacterial wilt with high potency, among which compounds 5a and 5b exhibited the most potent inhibition against R. solanacearum from tomato and tobacco bacterial wilts with EC₅₀ values of 19.77 and 8.29 μg/mL, respectively. Our results also demonstrated that 5a, 5b , and a number of other compounds were more potent than commercial bactericides Kocide 3000 and Thiodiazole Copper, which inhibited R. solanacearum from tomato bacterial wilt with EC₅₀ values of 93.59 and 99.80 μg/mL and tobacco bacterial wilt with EC₅₀ values of 45.91 and 216.70 μg/mL, respectively. The structure–activity relationship (SAR) of compounds was studied using three‐dimensional quantitative structure–activity relationship (3D‐QSAR) models created by comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) based on compound bioactivities against tomato and tobacco bacterial wilts. The 3D‐QSAR models effectively predicted the correlation between inhibitory activity and steric–electrostatic properties of compounds.     

Biological Evaluation of 2‐(4‐Amino‐Phenyl)‐3‐(3,5‐Dihydroxylphenyl) Propenoic Acid

Abstract: 2‐(4‐Aminophenyl)‐3‐(3,5‐dihydroxylphenyl) propenoic acid (CSN‐07001) is a new compound based on the combination of resveratrol and propenoic acid derivatives. In vitro cyclooxygenase (COX)/5‐lipoxygenase (5‐LOX) inhibition assays showed that the test compound exhibited a dual inhibitory activity against the COX (COX‐1 IC₅₀ = 2.20 μM, COX‐2 IC₅₀ = 1.76 μM) and 5‐LOX (IC₅₀ = 0.28 μM) enzymes. Further, the enhanced COX‐1/COX‐2/5‐LOX expression in lipopolysaccaride‐induced lung inflammation in mice was also suppressed by CSN‐07001 in a concentration‐dependent manner. In vivo studies showed that CSN‐07001 exhibited potent anti‐inflammatory and antinociceptive effects in different experimental models. We further examined the risk of gastric damage induced by CSN‐07001. The test compound was gastric‐sparing in that it elicited markedly fewer stomach lesions than indomethacin in rats. Taken together, our data indicate that CSN‐07001 exhibits a novel class of dual inhibitors of COX and 5‐LOX having therapeutic potential as non‐steroidal anti‐inflammatory agents with an enhanced gastric safety profile.     

Synthesis and biological evaluation of phloroglucinol derivatives possessing α‐glycosidase,acetylcholinesterase, butyrylcholinesterase,carbonic anhydrase inhibitory activity

A series of novel phloroglucinol derivatives were designed, synthesized, characterized spectroscopically and tested for their inhibitory activity against selected metabolic enzymes, including α‐glycosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human carbonic anhydrase I and II (hCA I and II). These compounds displayed nanomolar inhibition levels and showed K_i values of 1.14–3.92 nM against AChE, 0.24–1.64 nM against BChE, 6.73–51.10 nM against α‐glycosidase, 1.80–5.10 nM against hCA I, and 1.14–5.45 nM against hCA II.

     

A novel Ca<Superscript>2+</Superscript>-dependent phospholipase D from <Emphasis Type="Italic">Streptomyces tendae</Emphasis>, possessing only hydrolytic activity

An extracellular phospholipase D (PLD _St ) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLD _St was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60°C, and the enzyme was stable at or below 60°C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca²⁺, and the maximum activity was at 2 mM CaCl₂. The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 μmol min⁻¹ mg⁻¹, respectively. More importantly, PLD _St could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLD _St does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLD _St may therefore be a novel type of PLD enzyme.     

Identification of 1, 4‐Dihydrothieno[3′, 2′:5, 6]thiopyrano[4, 3‐c]pyrazole Derivatives as Human 5‐Lipo‐oxygenase Inhibitors

A series of novel 1,4‐dihydrothieno[3′,2′:5,6]thiopyrano[4,3‐c]‐pyrazole‐3‐carboxamide derivatives were synthesized and evaluated for their inhibitory activity to human 5‐lipo‐oxygenase (5‐LOX). Compound 7c was found to exhibit significant inhibition to human 5‐LOX with IC₅₀ value of 5.7 ± 0.9 μm . Compound 7c was further studied using molecular docking in order to delineate its structure–activity relationship and to gain insight into the design of effective 5‐LOX inhibitors.     

Design and synthesis of leucine‐linked quinazoline‐4(3H)‐one‐sulphonamide molecules distorting malarial reductase activity in the folate pathway

Optimization of a modified Grimmel's method for N‐heterocyclization of a leucine‐linked sulfonamide side‐arm at position 2 leading to 2,3‐disustituted‐4‐quinazolin‐(3H)‐ones was accomplished. Further, 22 hybrid quinazolinone motifs ( 4a‐v ) were synthesized by N‐heterocyclization reaction under microwave irradiation using the ionic liquid [Bmim][BF₄]‐H₂O as green solvent as well as the catalyst. The in vitro screening of the hybrid entities against the malarial species Plasmodium falciparum yielded five potent molecules 4l, 4n, 4o, 4t , and 4u owning antimalarial activity comparable to those of the reference drugs. In continuation, an in silico study was carried out to obtain a pharmacophoric model and quantitative structure–activity relationship. We also built a 3D‐QSAR model to procure more information that could be applied to design new molecules with more potent Pf‐DHFR inhibitory activity. The designed pharmacophore was recognized to be more potent for the selected molecules, exhibiting five pharmacophoric features. The active scaffolds were further evaluated for enzyme inhibition efficacy against alleged receptor Pf‐DHFR computationally and in vitro, proving their candidature as lead dihydrofolate reductase inhibitors, and the selectivity of the test candidates was ascertained by toxicity study against Vero cells. Good oral bioavailability was also proved by studying pharmacokinetic properties.     

A novel class of human 15‐LOX‐1 inhibitors based on 3‐hydroxycoumarin

Inflammations, sensitivities, and some cancers in mammals are intimately linked to the activity of lipo‐oxygenase enzymes. Owing to the importance of these enzymes, mechanistic studies, product analysis, and synthesis of inhibitors have expanded. In this study, a series of hydroxycoumarins, methoxy‐3‐hydroxy coumarins, and 7‐alkoxy‐3‐hydroxy coumarins were synthesized and evaluated as potential inhibitors of human 15‐LOX‐1. Among the synthetic coumarins, 7‐methoxy‐3‐hydroxycoumarin derivative demonstrated potent inhibitory activity and the compound, 5f , showed the best result. Radical scavenging assessment, IC₅₀, HNMR, and DPPH bleaching results indicate that the electronic properties are the major factors for the lipo‐oxygenase inhibition potency of the synthetic coumarins. Based on the theoretical studies, it was suggested that the mesomeric effect of the substituent at the seventh position of the benzene ring is one of the major factors in the stability of the oxy‐radical intermediate.     

Synthesis and in vitro Evaluation of 1,2,4‐Triazolo[1,5‐a][1,3,5]triazine Derivatives as Thymidine Phosphorylase Inhibitors

In our lead finding program, a series of 1,2,4‐triazolo[1,5‐a][1,3,5]triazine derivatives were synthesized, and their in vitro thymidine phosphorylase inhibitory potential was explored. Among the different derivatives, compounds having keto group (C = O) at C7 and thioketo group (C = S) at C5 positions showed varying degrees of TP inhibitory activity comparable with positive control, 7‐deazaxanthine ( 7‐DX , 2 ) (IC₅₀ value = 42.63 μm ). Enzyme inhibition kinetics study suggested that compound IVn behaved as a mixed‐type inhibitor of the enzyme with respect to thymidine (dThd) as a variable substrate. Compound IVn was also found to inhibit PMA‐induced MMP‐9 expression in MDA‐MB‐231 cells at sublethal concentrations. Computational docking study was performed to illustrate the enzyme inhibition kinetics and to explore the ligand–enzyme interactions.     

Antifibrotic effects of D‐limonene (5(1‐methyl‐4‐[1‐methylethenyl]) cyclohexane) in CCl4 induced liver toxicity in Wistar rats

This study was designed to assess the potential antifibrotic effect of D‐Limonene—a component of volatile oils extracted from citrus plants. D‐limonene is reported to have numerous therapeutic properties. CCl₄‐intduced model of liver fibrosis in Wistar rats is most widely used model to study chemopreventive studies. CCl₄‐intoxication significantly increased serum aminotransferases and total cholesterol these effects were prevented by cotreatment with D‐Limonene. Also, CCl₄‐intoxication caused depletion of glutathione and other antioxidant enzymes while D‐Limonene preserved them within normal values. Hydroxyproline and malondialdehyde content was increased markedly by CCl₄ treatment while D‐Limonene prevented these alterations. Levels of TNF‐α, TGF‐β, and α‐SMA were also assessed; CCl₄ increased the expression of α‐SMA, NF‐κB and other downstream inflammatory cascade while D‐Limonene co‐treatment inhibited them. Collectively these findings indicate that D‐Limonene possesses potent antifibrotic effect which may be attributed to its antioxidant and anti‐inflammatory properties.     

Structure‐based drug design and in vitro testing reveal new inhibitors of enoyl‐acyl carrier protein reductases

The need for new antibacterial agents is increasingly becoming of great importance as bacterial resistance to current drugs is quickly spreading. Enoyl‐acyl carrier protein reductases (FabI) are important enzymes for fatty acid biosynthesis in bacteria and other micro‐organisms. In this project, we conducted structure‐based virtual screening against the FabI enzyme, and accordingly, 37 compounds were selected for experimental testing. Interestingly, five compounds were able to demonstrate antimicrobial effect with variable inhibition activity against various strains of bacteria and fungi. Minimum inhibitory concentrations of the active compounds were determined and showed to be in low to medium micromolar range. Subsequently, enzyme inhibition assay was carried out for our five antimicrobial hits to confirm their biological target and determine their IC₅₀ values. Three of these tested compounds exhibited inhibition activity for the FabI enzyme where our best hit MN02 had an IC₅₀ value of 7.8 μM. Furthermore, MN02 is a small bisphenolic compound that is predicted to have all required features to firmly bind with the target enzyme. To sum up, hits discovered in this work can act as a good starting point for the future development of new and potent antimicrobial agents.     

2‐Prenylated m‐dimethoxybenzenes as potent inhibitors of 15‐lipo‐oxygenase: inhibitory mechanism and SAR studies

15‐lipo‐oxygenases are one of the iron‐containing proteins capable of performing peroxidation of unsaturated fatty acids in animals and plants. The critical role of enzymes in the formation of inflammations, sensitivities, and some cancers has been demonstrated in mammals. The importance of enzymes has led to the development of mechanistic studies, product analysis, and synthesis of inhibitors. In this study, a series of allyl and prenyl dimethoxybenzenes were synthesized and their inhibitory potency against soybean 15‐Lipo‐oxygenase (L1; EC 1,13,11,12) was determined. Among the synthetic compounds, 2,6‐dimethoxy‐1‐isopentenyl‐4‐methylbenzene, 2,6‐dimethoxy‐1‐geranyl‐4‐methylbenzene, and 2,6‐dimethoxy‐1‐farnesyl‐4‐methylbenzene showed the most potent inhibitory activity with IC₅₀ values of 7.6, 5.3, and 0.52 μm , respectively. For some of the compounds, SAR studies showed acceptable relationship between inhibitory potency and enzyme–ligand interactions. Radical scavenging assessment results apart from the SAR studies indicate that electronic properties are the major factors for lipo‐oxygenase inhibition potency of the mentioned compounds. Based on the theoretical studies, it was suggested that CH…O intramolecular hydrogen bond between ortho‐methoxy oxygen and methine hydrogen atoms is one of the major factors in the stability of 2,6‐dimethoxyallyl(or prenyl)benzenes radical via the planarity fixation between phenyl and allyl (or prenyl) pi orbitals.     

Discovery of 2‐(3,4‐dialkoxyphenyl)‐2‐(substituted pyridazin‐3‐yl)acetonitriles as phosphodiesterase 4 inhibitors with anti‐neuroinflammation potential based on three‐dimensional quantitative structure–activity relationship study

Phosphodiesterase 4 (PDE4) inhibitors with potential activities for CNS disorders provide a new therapeutic strategy for depression. To discover PDE4 inhibitors with anti‐neuroinflammation activities, reliable three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) models on our previous reported catecholic PDE4 inhibitors was built with a statistically significant cross‐validated coefficient (q²), conventional coefficient (r²), and good predictive capabilities based on the molecular docking results, using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods. Based on the analysis of CoMFA and CoMSIA contour maps, a series of 2‐(3,4‐dialkoxyphenyl)‐2‐(substituted pyridazin‐3‐yl) acetonitriles 16a–i was designed and synthesized. Among these compounds, compound 16a exhibited good inhibitory activities toward PDE4B1 and PDE4D7 with mid‐nanomolar IC₅₀ values and potential anti‐neuroinflammation activity in BV‐2 cells. Docking simulation of compound 16a in the PDE4 catalytic domain activity pocket revealed that compound 16a maybe assumed a “V‐shaped” conformation, extending the side chain to S‐pocket.     

Development of 2‐(Substituted Benzylamino)‐4‐Methyl‐1, 3‐Thiazole‐5‐Carboxylic Acid Derivatives as Xanthine Oxidase Inhibitors and Free Radical Scavengers

A series of 2‐(substituted benzylamino)‐4‐methylthiazole‐5‐carboxylic acid was designed and synthesized as structural analogue of febuxostat. A methylene amine spacer was incorporated between the phenyl ring and thiazole ring in contrast to febuxostat in which the phenyl ring was directly linked with the thiazole moiety. The purpose of incorporating methylene amine was to provide a heteroatom which is expected to favour hydrogen bonding within the active site residues of the enzyme xanthine oxidase. The structure of all the compounds was established by the combined use of FT‐IR, NMR and MS spectral data. All the compounds were screened in vitro for their ability to inhibit the enzyme xanthine oxidase as per the reported procedure along with DPPH free radical scavenging assay. Compounds 5j, 5k and 5l demonstrated satisfactory potent xanthine oxidase inhibitory activities with IC₅₀ values, 3.6, 8.1 and 9.9 μm , respectively, whereas compounds 5k , 5n and 5p demonstrated moderate antioxidant activities having IC₅₀ 15.3, 17.6 and 19.6 μm , respectively, along with xanthine oxidase inhibitory activity. Compound 5k showed moderate xanthine oxidase inhibitory activity as compared with febuxostat along with antioxidant activity. All the compounds were also studied for their binding affinity in active site of enzyme (PDB ID‐1N5X).     

Design,synthesis, and biological evaluation of novel 1,2‐diaryl‐4‐substituted‐benzylidene‐5(4H)‐imidazolone derivatives as cytotoxic agents and COX‐2/LOX inhibitors

A new series of 1,2‐diaryl‐4‐substituted‐benzylidene‐5(4H)‐imidazolone derivatives 4a–l was synthesized. Their structures were confirmed by different spectroscopic techniques (IR, ¹H NMR, DEPT‐Q NMR, and mass spectroscopy) and elemental analyses. Their cytotoxic activities in vitro were evaluated against breast, ovarian, and liver cancer cell lines and also normal human skin fibroblasts. Cyclooxygenase (COX)‐1, COX‐2 and lipoxygenase (LOX) inhibitory activities were measured. The synthesized compounds showed selectivity toward COX‐2 rather than COX‐1, and the IC₅₀ values (0.25–1.7 µM) were lower than that of indomethacin (IC₅₀ = 9.47 µM) and somewhat higher than that of celecoxib (IC₅₀ = 0.071 µM). The selectivity index for COX‐2 of the oxazole derivative 4e (SI = 3.67) was nearly equal to that of celecoxib (SI = 3.66). For the LOX inhibitory activity, the new compounds showed IC₅₀ values of 0.02–74.03 µM, while the IC₅₀ of the reference zileuton was 0.83 µM. The most active compound 4c (4‐chlorobenzoxazole derivative) was found to have dual COX‐2/LOX activity. All the synthesized compounds were docked inside the active site of the COX‐2 and LOX enzymes. They linked to COX‐2 through the N atom of the azole scaffold, while C?O of the oxazolone moiety was responsible for the binding to amino acids inside the LOX active site.

     

Design,microwave‐assisted synthesis,biological evaluation and molecular modeling studies of 4‐phenylthiazoles as potent fatty acid amide hydrolase inhibitors

Endocannabinoids, anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), are endogenous lipids that activate cannabinoid receptors. Activation of these receptors produces anti‐inflammatory and analgesic effects. Fatty acid amide hydrolase (FAAH) is a membrane enzyme that hydrolases endocannabinoids; thus, inhibition of FAAH represents an attractive approach to develop new therapeutics for treating inflammation and pain. Previously, potent rat FAAH inhibitors containing 2‐naphthyl‐ and 4‐phenylthiazole scaffolds were identified, but up to the present time, very little structure–activity relationship studies have been performed on these moieties. We designed and synthesized several analogs containing these structural motifs and evaluated their inhibition potencies against human FAAH enzyme. In addition, we built and validated a homology model of human FAAH enzyme and performed docking experiments. We identified several inhibitors in the low nanomolar range and calculated their ADME predicted values. These FAAH inhibitors represent promising drug candidates for future preclinical in vivo studies.