The nylon oligomer biodegradation system ofFlavobacterium andPseudomonas

This review article is a compendium of the available information on the degradation of a man-made compound, 6-aminohexanoate-oligomer, inFlavobacterium andPseudomonas strains, and discusses the molecular basis for adaptation of microorganisms toward these xenobiotic compounds. Three plasmid-encoded enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endo-type 6-aminohexanoate-oligomer hydrolase (EIII) are responsible for the degradation of the oligomers. Two repeated sequences, designated RS-I and RS-II, are found on plasmid pOAD2, which is involved in 6-aminohexanoate degradation inFlavobacterium. RS-I appears 5 times on the pOAD2, and all copies have the same sequences as insertion sequence IS6100. RS-II appears twice on the plasmid. RS-IIA contains the gene encoding EII, while RS-IIB contains the gene for the analogous EII' protein. Both EII and EII' are polypeptides of 392 amino acids, which differ by 46 amino acid residues. The specific activity of the EII enzyme is 200-fold higher than that of EII'. Construction of various hybrid genes demonstrated that only the combination of two amino acid residues in the EII' enzyme can enhance the activity of the EII' to the same level as that of EII enzyme.Abbreviations EI 6-aminohexanoate-cyclic-dimer hydrolase - EII 6-aminohexanoate-dimer hydrolase - EIII endo-type 6-aminohexanoate-oligomer hydrolase - F-EI EI fromFlavobacterium - F-EII EII fromFlavobacterium - P-EI EI fromPseudomonas - P-EII EII fromPseudomonas - EII' a protein having 88% homology to the EII encoded on the RS-IIB region of pOAD2 - nylA gene for the EI enzyme - nylB gene for the EII enzyme - nylC gene for the EIII enzyme - nylB' gene for the EII' protein - kb kilo-base-pairs     

Mutational analysis of 6-aminohexanoate-dimer hydrolase: relationship between nylon oligomer hydrolytic and esterolytic activities

Carboxylesterase (EII') from Arthrobacter sp. KI72 has 88% homology to 6-aminohexanoate-dimer hydrolase (EII) and possesses ca. 0.5% of the level of 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity of EII. To study relationship between Ald-hydrolytic and esterolytic activities, random mutations were introduced into the gene for Hyb-24 (an EII/EII' hybrid with the majority of the sequence deriving for EII' and possessing an EII'-like level of Ald-hydrolytic activity). Either a G181D or a D370Y substitution in Hyb-24 increased the Ald-hydrolytic activity ca. 10-fold, and a G181D/D370Y double substitution increased activity ca. 100-fold. On the basis of kinetic studies and the three-dimensional structure of the enzyme, we suggest that binding of Ald is improved by these mutations. D370Y increased esterolytic activity for glycerylbutyrate ca. 30-50-fold, whereas G181D decreased the activity to 30% of the parental enzyme.     

Amino acid alterations essential for increasing the catalytic activity of the nylon-oligomer-degradation enzyme of Flavobacterium sp

The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Gly181----Asp (EII type) and His266----Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold.     

Characterization of hybrid enzymes between 6-aminohexanoate-Dimer hydrolase and its analogous protein

6-Aminohexanoate-dimer hydrolase (EII) and its analogous protein (EII′), of Flavobacterium sp. K172 are composed of 392 amino acids, in which 47 are different. The enzyme activity of EII′ toward 6-aminohexanoate dimer is approximately 0.5% of that of EII. We have constructed various hybrids of the two genes by exchanging fragments flanked by conserved restriction sites such as PvuII, BglII, SalI, and BamHI (respectively 74, 483, 771, and 1,141 bp downstream of the initiation codon), and purified their gene products to homogeneity. Hyb-12 protein, which was obtained by the replacement of the BglII-SalI region of the EII′ with the corresponding region of EII, had 12 times higher specific activity towards the 6-aminohexanoate dimer and its related substrates than EII′ protein. Hyb-10, which was composed of the N-terminal -BglII regions of EII′ and the BglII-C terminal region of EII, had activity toward these substrates nearly equal to the activity of the EII enzyme. Comparisons of the activity toward 6-aminohexanoate dimer and its analogues has demonstrated that EII, EII′, and their hybrid enzymes are highly active only toward the substrates that contain 6-aminohexanoate as the N-terminal residue, while the recognition of the C-terminal residue in the substrate was not stringent. The substrate specificity, pH-activity profile, and heat stability of these enzymes varied slightly.     

Biodegradation of nylon oligomers

This mini-review is a compendium of the degradation of a man-made compound, 6-aminohexanoate-oligomer, in Flavobacterium strains. The results are summarized as follows: 1. Three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endotype 6-aminohexanoate-oligomer hydrolase (EIII) were responsible for degradation of the oligomers. 2. The genes coding these enzymes were located on pOAD2, one of three plasmids harbored in Flavobacterium sp. KI72, which comprised 45,519 bp. 3. The gene coding the EII′ protein (a protein having 88% homology with EII) and five IS6100 elements were identified on pOAD2. 4. The specific activity of EII was 200-fold higher than that of EII′. However, altering two amino acid residues in the EII′ enzyme enhanced the activity of EII′ to the same level as that of the EII enzyme. 5. The deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (encoding oligopeptide permease), ftsX (filamentation temperature sensitivity), penDE (isopenicillin N-acyltransferase) and rep (plasmid replication). 6. The EI and EII genes of Pseudomonas sp. NK87 (another nylon oligomer-degrading bacterium) were also located on plasmids. 7. Through selective cultivation using nylon oligomers as a sole source of carbon and nitrogen, two strains which initially had no metabolic activity for nylon oligomers, Flavobacterium sp. KI725 and Pseudomonas aeruginosa PAO1, were given the ability to degrade xenobiotic compounds. A molecular basis for the adaptation of microorganisms toward xenobiotic compounds was described. Received: 25 February 2000 / Received revision: 22 May 2000 / Accepted: 26 May 2000     

Studies on cellulolytic enzymes I: The use of 3,4-dinitrophenyl glycosides as substrates

The syntheses of 3,4-dinitrophenyl β-d-glucoside, β-cellobioside, β-cellotrioside, and β-cellotetraoside and their use to monitor the purification of two enzymes from a crude commercial cellulase preparation from Trichoderma viride are described. The enzymes isolated are an endo-β-1,4-d-glucan glucanohydrolase (EI) of molecular weight ca. 12 000 which catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenol-β-cellotetraoside, and an enzyme of molecular weight about 76 000 which catalysed the hydrolysis of 3,4-dinitrophenyl β-d-glucoside (EII) and is probably a cellobiase or exo-β-1,4-d-glucan glucohydrolase. Kinetic parameters are reported for the hydrolyses of 3,4-dinitrophenyl β-cellobioside, β-cellotrioside, and β-cellotetraoside catalysed by enzyme EI. In the presence of cellotriose, cellotetraose, or cellopentaose 3,4-dinitrophenyl β-d-glucoside underwent induced hydrolyses by EI. Similar but faster induced hydrolyses were shown by 3,4-dinitrophenyl β-d-xyloside and 3,4-dinitrophenyl β-d-6-deoxyglucoside; 3,4-dinitrophenyl 6-chloro-6-deoxy-β-d-glucoside and 3,4-dinitrophenyl 6-O-methyl-β-d-glucoside underwent slower induced hydrolyses than the glucoside. p-Nitrophenyl β-d-glucoside also underwent an induced hydrolysis in the presence of cellopentaose and the enzyme EI, but p-nitrophenyl 2-deoxy-β-d-glucoside did not. These results are discussed and compared with the results obtained previously on induced hydrolyses found with lysozyme. Kinetic parameters are reported for the hydrolysis of 3,4-dinitrophenyl and p-nitrophenyl β-d-glucosides catalysed by the enzyme EII. 3,4-Dinitrophenyl 6-deoxy-β-d-glucoside, β-d-xyloside, 6-chloro-6-deoxy-β-d-glucoside, 6-O-methyl-β-d-glucoside and p-nitrophenyl-β-d-galactopyranoside and 2-deoxy-β-d-glucopyranoside were hydrolysed 10² to 10³ times slower by EII than the corresponding glucosides, but 3,4-dinitrophenyl 2-acetamido-2-deoxy-β-d-glucoside was only hydrolysed about 25 times slower than 3,4-dinitrophenyl β-d-glucoside. The significance of these results is discussed. EII catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenyl β-cellobioside, β-cellobioside, and β-cellotetraoside, but these reactions showed induction periods which are consistent with stepwise removal of glucose residues from the oligosaccharide chains before release of the phenol.     

Replacement of tyrosine 181 by phenylalanine in gentisate 1,2-dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 enhances catalytic activities

xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k_cat/K_m) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.     

Analysis of a promoter-like region in Flavobacterium which controls 6-aminohexanoic acid linear oligomer hydrolase gene expression in Escherichia coli

The region located 6.0–6.2 kb upstream from a structural gene for 6-aminohexanoic acid linear oligomer hydrolase (EII) in Flavobacterium was found to be responsible for EII expression in Escherichia coli. In this region there existed a 50 bp long inverted repeat containing a sequence homologous to the E. coli promoter −35 and −10 regions. Introduction of deletions in this region resulted in decreases in EII activity to various levels.     

Purification and characterization of two β-N-acetylhexosaminidases from the tobacco hornworm,Manduca sexta (L.) (Lepidoptera: Sphingidae)

β-N-Acetylhexosaminidases were detected in 10 insects including species of Lepidoptera, Coleoptera, Hemiptera, and Orthoptera. Two enzymes were purified from the tobacco hornworm, Manduca sexta (L.). EI was detected in larval and pharate pupal molting fluid, integument, and pupal hemolymph while EII was found in larval and pupal hemolymphs. They are acidic hydrolases with similar molecular weights (6.1 × 10⁴), molar extinction coefficients at 280 nm (1.9 × 10⁵ liters mol^?1 cm^?1), and pH optima (pH 6). They differ in the number of polypeptide chains per molecule (EI is a single chain and EII consists of two polypeptide chains), amino acid composition, extent of glycosylation (EII is probably a glycoprotein), isoelectric point (pI_EI = 5.9 and pI_EII ～- 5.1), tissue distribution, and reactivities toward nitrophenylated N-acetylglucosamine (k_cat,I = 328 s^?1 and k_cat,II = 103 s^?1) and N,N′-diacetylchitobiose (k_cat,I = 307 s^?1 and k_cat,II = 3 s^?1). These results suggest that EI is a chitinase and that EII may function as a hexosaminidase in vivo.     

New pyrazolopyrimidine derivatives as Leishmania amazonensis arginase inhibitors

Arginase performs the first enzymatic step in polyamine biosynthesis in Leishmania and represents a promising target for drug development. Polyamines in Leishmania are involved in trypanothione synthesis, which neutralize the oxidative burst of reactive oxygen species (ROS) and nitric oxide (NO) that are produced by host macrophages to kill the parasite. In an attempt to synthesize arginase inhibitors, six 1-phenyl-1H-pyrazolo[3,4-d]pyrimidine derivatives with different substituents at the 4-position of the phenyl group were synthesized. All compounds were initially tested at 100 µM concentration against Leishmania amazonensis ARG (LaARG), showing inhibitory activity ranging from 36 to 74%. Two compounds, 1 (R=H) and 6 (R=CF₃), showed arginase inhibition >70% and IC₅₀ values of 12 µM and 47 µM, respectively. Thus, the kinetics of LaARG inhibition were analyzed for compounds 1 and 6 and revealed that these compounds inhibit the enzyme by an uncompetitive mechanism, showing K_is values, and dissociation constants for ternary complex enzyme-substrate-inhibitor, of 8.5 ± 0.9 µM and 29 ± 5 µM, respectively. Additionally, the molecular docking studies proposed that these two uncompetitive inhibitors interact with different LaARG binding sites, where compound 1 forms more H-bond interactions with the enzyme than compound 6. These compounds showed low activity against L. amazonensis free amastigotes obtained from mice lesions when assayed with as much as 30 µM. The maximum growth inhibition reached was between 20 and 30% after 48 h of incubation. These results suggest that this system can be promising for the design of potential antileishmanial compounds.     

Discovery of human lactate dehydrogenase 5 inhibitors (hLDH5) with anti-lung cancer activity through an in silico method and biological validation

Human lactate dehydrogenase 5 (hLDH5) is an important metabolic enzyme playing critical roles in the anaerobic glycolysis. Herein, we employed an in silico method and biological validation to identify a novel hLDH5 inhibitor with a promising cellular activity under hypoxia condition. The identified compound 9 bound to hLDH5 with a K_d value of 1.02 µM, and inhibited the enzyme with an EC₅₀ value of 0.7 µM. Compound 9 exhibited a weak potency against NCI-H1975 cell proliferation under normal condition (IC₅₀ = 36.5 µM), while dramatically increased to 5.7 µM under hypoxia condition. In line with the observation, hLDH5 expression in NCI-H1975 cell under hypoxia condition is much higher as compared to the normal oxygenated condition, indicating the hLDH5 inhibition may contribute to the cancer cell death.     

Antioxidant and angiotensin-converting enzyme (ACE) inhibitory activity of thymosin alpha-1 (Thα1) peptide

In this research, the antioxidant property of thymosin alpha-1 (Thα1) peptide was investigated through various antioxidant methods. Thα1 showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC₅₀ = 20 µM) and its 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging reached 45.33% at 80 µM (IC₅₀ = 85 µM). In addition, hydroxyl and superoxide radical scavenging of Thα1 peptide exhibited a concentration-depended manner. The IC₅₀ values of hydroxyl and superoxide radical scavenging were estimated to be 82 µM and 20 µM, respectively. The effect of Thα1 on eliminating superoxide radicals was higher (62.23%) than other antioxidant assays. Moreover, the antioxidant activity of Thα1 peptide was evaluated by measuring cellular reactive oxygen species (ROS). Results indicated that Thα1 decreased the generation of ROS level in 1321 N1 human neural asterocytoma cells. The inhibitory effect of Thα1 on angiotensin-converting enzyme (ACE) was determined. The kinetic parameters (K_m and V_max) and the inhibition pattern were examined. Based on the Lineweaver-Burk plot, Thα1 displayed a mixed inhibition pattern. The IC₅₀ and K_i values of Thα1 were 0.8 µM and 3.33 µM, respectively. Molecular modeling suggested that Thα1 binds to ACE-domains with higher affinity binding to N-domain with the binding energy of −22.87 kcal/mol. Molecular docking indicated that Thα1 interacted with ACE enzyme (N- and C-domains) due to electrostatic, hydrophobic, and hydrogen forces. Our findings suggested that Thα1 possess a multifunctional peptide with dual antioxidant and ACE-inhibitory properties. Further researches are needed to investigate the antioxidant and anti-hypertensive effect of Thα1 both in vitro and in vivo.     

Synthesis,anticancer assessment on human breast,liver and colon carcinoma cell lines and molecular modeling study using novel pyrazolo[4,3-c]pyridine derivatives

The key intermediate 3-aminopyrazolo[4,3-c]pyridine-4,6-dione (2) is considered as a precursor for some novel pyrazolo[4,3-c]pyridines 4a-c, arylhydrazopyrazolo[4,3-c]pyridines 8a-e, pyrazolo[4,5,1-ij][1,6]naphthyridines 11a-e and pyrido[4′,3′:3,4]pyrazolo[1,5-a]-pyrimidines 15a-d through Knovenegal condensation, coupling reaction and Michael addition. Some of the newly synthesized pyrazolo[4,3-c]pyridine derivatives were investigated for anticancer activity. The results of the cytotoxic activity revealed that compound 6b was the most active compound against the breast and liver carcinoma cell lines which gives IC₅₀ values of 1.937 and 3.695 µg/mL, respectively compared to reference drug (doxorubicin) with IC₅₀ values of 2.527 and 4.749 µg/ml, respectively. Moreover, compound 6c was potent compound against the colon carcinoma cell line which gives the value of IC₅₀ = 2.914 µg/ml compared to doxorubicin with IC₅₀ value of 3.641 µg/ml. Some selected of the novel synthesized compounds were docked inside the active site of ERK2 enzyme and were found display a suitable binding with the active site amino acids according to their bond lengths, angles and conformational energy.     

Design,molecular docking and synthesis of novel 5,6-dichloro-2-methyl-1H-benzimidazole derivatives as potential urease enzyme inhibitors

A novel series of 5,6-dichloro-2-methyl-1H-benzimidazole derivatives was synthesized and then screened for their urease inhibitory activity. All compounds showed more potent inhibitory activity in the range of IC₅₀ = 0.0294 ± 0.0015–0.1494 ± 0.0041 µM than thiourea (IC₅₀ = 0.5117 ± 0.0159 µM), as a reference inhibitor. Among all the tested compounds, the compound 15 (IC₅₀ = 0.0294 ± 0.0015 µM) having strong electron-withdrawing nitro group on the phenyl ring was recorded as the most potent inhibitor of urease. All compounds were docked at the active sites of the Jack bean urease enzyme to investigate the reason of the inhibitory activity and the possible binding interactions of enzyme-ligand complexes.     

Synthesis of 1H-1,2,3-triazole derivatives as new α-glucosidase inhibitors and their molecular docking studies

Inhibition of α-glucosidase is an effective strategy for controlling the post-prandial hyperglycemia in diabetic patients. For the identification of new inhibitors of this enzyme, a series of new (R)-1-(2-(4-bromo-2-methoxyphenoxy) propyl)-4-(4-(trifluoromethyl) phenyl)-1H-1,2,3-triazole derivatives were synthesized (8a–d and 10a–e). The structures were confirmed by NMR, mass spectrometry and, in case of compound 8a, by single crystal X-ray crystallography. The α-glucosidase inhibitory activities were investigated in vitro. Most derivatives exhibited significant inhibitory activity against α-glucosidase enzyme. Their structure-activity relationship and molecular docking studies were performed to elucidate the active pharmacophore against this enzyme. Compound 10b was the most active analogue with IC₅₀ value of 14.2 µM, while compound 6 was found to be the least active having 218.1 µM. A preliminary structure-activity relationship suggested that the presence of 1H-1,2,3-triazole ring in 1H-1,2,3-triazole derivatives is responsible for this activity and can be used as anti-diabetic drugs. The molecular docking studies of all active compounds were performed, in order to understand the mode of binding interaction and the energy of this class of compounds.     

A new series of aryl sulfamate derivatives: Design,synthesis, and biological evaluation

Steroid sulfatase (STS) has recently emerged as a drug target for management of hormone-dependent malignancies. In the present study, a new series of twenty-one aryl amido-linked sulfamate derivatives 1a-u was designed and synthesized, based upon a cyclohexyl lead compound. All members were evaluated as STS inhibitors in a cell-free assay. Adamantyl derivatives 1h and 1p-r were the most active with more than 90% inhibition at 10 µM concentration and, for those with the greatest inhibitory activity, IC₅₀ values were determined. These compounds exhibited STS inhibition within the range of ca 25–110 nM. Amongst them, compound 1q possessing a o-chlorobenzene sulfamate moiety exhibited the most potent STS inhibitory activity with an IC₅₀ of 26 nM. Furthermore, to assure capability to pass through the cell lipid bilayer, compounds with low IC₅₀ values were tested against STS activity in JEG-3 whole-cell assays. Consequently, 1h and 1q demonstrated IC₅₀ values of ca 14 and 150 nM, respectively. Thus, compound 1h is 31 times more potent than the corresponding cyclohexyl lead (IC₅₀ value = 421 nM in a JEG-3 whole-cell assay). Furthermore, the most potent STS inhibitors (1h and 1p-r) were evaluated for their antiproliferative activity against the estrogen-dependent breast cancer cell line T-47D. They showed promising activity with single digit micromolar IC₅₀ values (ca 1–6 µM) and their potency against T-47D cells was comparable to that against STS enzyme. In conclusion, this new class of adamantyl-containing aryl sulfamate inhibitor has potential for further development against hormone-dependent tumours.     

Two alternative modes for optimizing nylon‐6 byproduct hydrolytic activity from a carboxylesterase with a β‐lactamase fold: X‐ray crystallographic analysis of directly evolved 6‐aminohexanoate‐dimer hydrolase

Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a β-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (K_m = 15 mM) and turnover (k_cat = 3.1 s⁻¹) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald—COOH and Tyr370, a hydrogen-bonding network from Ser187 to , and interaction between and Gln27-O_ɛ derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a β-lactamase fold.     

Anti-inflammatory drug approach: Synthesis and biological evaluation of novel pyrazolo[3,4-d]pyrimidine compounds

In this study, the acid chlorides of pyrazolo[3,4-d]pyrimidine compounds were prepared and reacted with a number of nucleophiles. The novel compounds were experimentally tested via enzyme assay and they showed cyclooxygenase-2 inhibition activity in the middle micro molar range (4b had a COX-1 IC₅₀ of 26 µM and a COX-2 IC₅₀ of 34 µM, 3b had a COX-1 IC₅₀ of 19 µM and a COX-2 IC₅₀ of 31 µM, 3a had a COX-2 IC₅₀ of 42 µM). These compounds were analyzed via docking and were predicted to interact with some of the COX-2 key residues. Our best hit, 4d (COX-1 IC₅₀ of 28 µM, COX-2 IC₅₀ of 23 µM), appears to adopt similar binding modes to the standard COX-2 inhibitor, celecoxib, proposing room for possible selectivity. Additionally, the resultant novel compounds were tested in several in vivo assays. Four compounds 3a (COX-2 IC₅₀ of 42 µM), 3d, 4d and 4f were notable for their anti-inflammatory activity that was comparable to that of the clinically available COX-2 inhibitor celecoxib. Interestingly, they showed greater potency than the famous non-steroidal anti-inflammatory drug, Diclofenac sodium. In summary, these novel pyrazolo[3,4-d]pyrimidine analogues showed interesting anti-inflammatory activity and could act as a starting point for future drugs.     

Novel nalidixic acid derivatives targeting topoisomerase II enzyme; Design,synthesis, anticancer activity and effect on cell cycle profile

AimDesign and synthesis of novel nalidixic acid derivatives of potent anticancer and topoisomerase II inhibitory activities were our major aim.Materials & methodsAll the newly synthesized nalidixic acid derivatives were submitted to the National Cancer Institute (NCI), Bethesda, USA and were accepted for single dose screening. Further investigation via IC₅₀ determination of the most potent compound 6a against K-562 and SR leukemia cell lines. Finally, the topoisomerase II inhibitory activity, the cell cycle analysis and molecular docking of 6a were performed in order to identify the possible mechanism of the anticancer activity.ResultsCompound 6a showed interesting selectivity against leukemia especially K-562 and SR subpanels with IC₅₀ 35.29 µM and 13.85 µM respectively. Moreover, compound 6a revealed potent topoisomerase IIα and topoisomerase IIβ inhibitory activity compared with known topoisomerase inhibitors such as doxorubicin and topotecan with IC₅₀ 1.30 µM and 0.017 µM respectively. Cell cycle analysis indicated that compound 6a induced cell cycle arrest at G2-M phase leading to inhibition of cell proliferation and apoptosis. Molecular modeling demonstrated that the potent topoisomerase inhibitory activity of 6a was due to the interaction with the topoisomerase II enzyme through coordinate bonding with the magnesium ion Mg²⁺, hydrogen bonding with Asp 545 and arene cation interaction with His 759.     

Synthesis,in-vitro antiprotozoal activity and molecular docking study of isothiocyanate derivatives

Novel isothiocyanate derivatives were synthesized starting from noscapine, bile acids, amino acids, and some aromatic compounds. Antiparasitic activities of the synthesized derivatives were tested against four unicellular protozoa, i.e., Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium falciparum. Interestingly, seven isothiocyanate analogues displayed promising antiparasitic activity against Leishmania donovani with IC₅₀ values between 0.4 and 1.0 µM and selectivity index (SI) ranged from 7.8 to 18.4, comparable to the standard drug miltefosine (IC₅₀ = 0.7 μM). Compound 7h demonstrated the best antileishmanial activity with an IC₅₀ value of 0.4 µM. Seven products exhibited inhibition activity against T. brucei rhodesiense with IC₅₀s below 2.0 μM and SI between 2.7 and 29.3. Four primary amine derivatives of noscapine and five isothiocyanate derivatives exhibited antiplasmodial activity with IC₅₀s in the range of 1.1–2.7 µM and SI values between 1.1 and 14.5. The isothiocyanate derivative 7c showed against T. cruzi with an IC₅₀ value of 1.9 µM and SI 4. Molecular docking and ADMET studies were performed to investigate the interaction between active ligands and T. brucei trypanothione reductase active site. The docking studies showed significant binding affinity of noscapine derivatives to enzyme active site and good compatibility with experimental data.